Antioxidants and Reactive Oxygen Species in PH – Do Antioxidants Help or Hurt PH?

How antioxidant works

While it may not be the cause, evidence from several studies that supports the fact that increased oxidative stress and reactive oxygen species (ROS) together with decreased antioxidant activity can contribute to enhanced pulmonary vasoconstriction, vascular remodeling, and right heart dysfunction in pulmonary hypertension. Despite this evidence however, it is still unknown whether or not an oxidant/antioxidant imbalance contributes directly to the development of severe PAH. Answering this question is the aim of the recent paper reviewed in today’s post by Jernigan et al., “Contribution of reactive oxygen species to the pathogenesis of pulmonary arterial hypertension”.


Many studies have shown that there is an imbalance of oxidant production and antioxidant capacity in the pulmonary vasculature of PH patients. The authors of the present study, as well as several others, have previously shown that superoxide levels are elevated (NADPH oxidases, xanthine oxidases, and the mitochondria) and are a main source of ROS in the pulmonary vasculature and circulation under chronic hypoxia conditions. Other ROS species like hydrogen peroxide (H2O2) are also implicated in PH, although some studies show elevation in H2O2 and some studies show a decrease.

Antioxidants in PH

Antioxidant activity is decreased in both animal and human PH, as measured by the absence or decreased expression of the antioxidant enzyme superoxide dismutase (SOD), which acts to neutralize the harmful superoxide species: “SOD1 and SOD3 expression and activity are decreased in chronic hypoxia-induced pulmonary hypertensive mice, rats, calves, and piglets and in a lamb model of persistent pulmonary hypertension of the newborn. Furthermore, fawn-hooded rats, which have an epigenetic silencing of SOD2 expression/activity, and SOD1 knockout mice develop spontaneous pulmonary hypertension. These animal studies further correlate with evidence of significantly lower SOD mRNA and SOD activity in patients with idiopathic PAH compared to healthy individuals. Interestingly, SOD-1 immunoreactivity is markedly absent in neointimal lesions of hypoxia/SU5416 rats, suggesting a potential role for oxidative stress in the development of angioproliferative PAH.”

If internal antioxidant activity is decreased, and there are imbalances in ROS within the body during PH, than it makes sense to conclude that antioxidants (e.g. antioxidant supplements or drugs) may have a therapeutic impact on PH. Indeed, the therapeutic impact of some antioxidants on PH has actually already been studied. Most results indicate that antioxidants are effective for treating right heart failure associated with severe PAH, but whether they help alleviate the pathological pulmonary arterial remodeling associated with PAH is unknown, either because it has yet to be studied, or because no distinctive conclusions can be drawn on the studies that have already been done. A few examples of the antioxidants already studied in PH:

  • N-acetylcysteine (NAC) – NAC attenuates PH in both chronic hypoxia and monocrotaline-induced pulmonary hypertension animal models.
  • SOD – In addition to experiments mentioned previously whereby SOD is decreased or removed, studies have been done where SOD is overexpressed. When overexpressed, SOD reduces pulmonary hypertension in chronic hypoxia, monocrotaline, and lamb models of PH.
  • Protandim – Protandim is a dietary supplement and a Nrf2 “activator”. It increases the expression of the antioxidant enzymes SOD and heme-oxygenase-1. In an animal model of severe PH, SUGEN/Hypoxia, Protandim prevented right ventricular hypertrophy and preserved right ventricular function.

Study & Results

In the study by Jernigan et al., the investigators tested the theory of the role of ROS and an oxidant/antioxidant imbalance in contributing to PAH by studying the effects of the antioxidant TEMPOL on two different animal model forms of PH: 1) the SUGEN/Hypoxia animal model (which represents Group I PAH), and 2) the chronic hypoxia rat animal model (which represents Group III PH). The SUGEN/Hypoxia model represents Group I PAH because it is the animal model that most closely resembles the human form of PH, since it is the only animal model that produces plexiform-like lesions in the lung vasculature. Plexiform lesions are the hallmark of human PAH, and other animal models, like the hypoxia only animal model, do not recapitulate the plexiform lesions observed in human PH.

Why use TEMPOL in this study? TEMPOL acts as a SOD mimetic, and neutralizes superoxide radicals, facilitates hydrogen peroxide metabolism, and limits formation of toxic hydroxyl radicals from Fenton reactions.

In brief, here is what the study found…

  • TEMPOL effects on Right Ventricular Systolic Pressure (RVSP): TEMPOL prevented increases in RVSP in hypoxic rats. It also attenuated RVSP in the SUGEN/Hypoxia rats, but to a lesser degree.
  • TEMPOL effects on Right Ventricular Hypertrophy: RVH in animal models is typically measured by assessing heart weight as well as the Fulton index, which is defined as [right/(left + septum) ventricular weight] or [RV/(LV+S)]. It essentially measures the ratio of the right ventricle to the left ventricle. Increases in this ratio indicate RVH. In the study, TEMPOL had no effect on RVH in either test group, the hypoxia treated rats, or the SUGEN/Hypoxia treated rats.
  • TEMPOL effects on Pulmonary Arterial Remodeling – Medial Hypertrophy: A common hallmark of pulmonary arterial remodeling in PAH is hypertrophy of the medial layer of the blood vessel. In the hypoxia only animal model group, hypertrophy of the medial layer of the pulmonary artery was observed. In the SUGEN/Hypoxia group, this was also observed, but the extent of the hypertrophy was not as great in this group compared to hypoxia only rats. TEMPOL treatment had no effect on this hypertrophy in the hypoxia only group. Surprisingly, however, treatment with TEMPOL in the SUGEN/Hypoxia group resulted in an increase in hypertrophy in the medial layer.
  • TEMPOL effects on Pulmonary Arterial Remodeling – Plexiform Lesions: Another hallmark of pulmonary arterial remodeling in PAH is the presence of plexiform lesions in the vasculature. In SUGEN/Hypoxia rats, two types of plexiform lesions were observed: 1) “plexiform-like neointimal lesions demonstrating Von Willebrand factor immunoreactivity combined with medial collagen deposition” and 2) “hypercellular lesions projecting outward from the medial and adventitial layers… extending into the adjacent lung parenchyma.” This second type of lesion was found to consist largely of myofibroblasts. As expected, no lesions were observed in hypoxia only treated rats. Again, surprisingly, TEMPOL treatment was found to increase the size and number of the type 2 lesions in the SUGEN/Hypoxia rat group.
  • TEMPOL effects on Vasoconstriction: TEMPOL decreased the ET-1 mediated vasoconstriction in both hypoxia only rats and SUGEN/Hypoxia rats.
  • TEMPOL effects on ROS: The amount of superoxide production in both hypoxic rats and SUGEN/Hypoxia rats were similar. However, TEMPOL treatment only decreased superoxide levels in hypoxic rats. TEMPOL also was shown to increase H2O2 induced oxidative stress, but it did so across all test groups, including the control normoxic rats.


Since the authors observed that TEMPOL attenuated RVSP in SUGEN/Hypoxia rats, and decreased vasoconstriction, it is tempting to conclude that ROS does indeed contribute to both pulmonary artery remodeling and vasoconstriction, respectively, in PH. Since TEMPOL is an antioxidant and scavenges ROS, and since RVSP is a function of pulmonary vascular resistance, which is a function of BOTH hypertrophy, blood vessel overgrowth, and vasoconstriction, and increases in RVSP and vasoconstriction could be indicative of pathological pulmonary arterial remodeling.

However, at odds with this is the fact that TEMPOL failed to reduce hypoxia-induced pulmonary arterial muscularization and right heart hypertrophy. Increases in these parameters are also indicative of pathological pulmonary arterial remodeling. Rather, “scavenging of ROS in hypoxia/SU5416-treated rats [by TEMPOL] caused an unexpected increase in arterial muscularization, vimentin, and HSP-47 expression, and severity of adventitial fibrotic lesions.” The fact that TEMPOL failed to reduce the parameters of medial hypertrophy and RVH, and actually increased muscularization and fibrotic lesions indicates that the antioxidants may actually promote pulmonary arterial remodeling.

How could TEMPOL possibly do this? As the authors explain, “ROS are essential signaling molecules that are tightly regulated to maintain physiological homeostasis… low levels (submicromolar) induce growth but higher concentrations induce apoptosis. It is possible that TEMPOL decreases superoxide levels thereby disrupting oxidative regulation of proliferation and host defense resulting in excessive proliferation and fibroblast activation.” If this is true, then the mechanism by which TEMPOL lowers RVSP in SUGEN/Hypoxia rats is via a reduction in vasoconstrictor reactivity, not because it decreases pulmonary arterial remodeling.

H2O2 is another ROS that is essential for physiological homeostasis, and the verdict is by no means out on H2O2… Small amounts are necessary to induce vasodilation (H2O2 binds to the potassium ion channel in the pulmonary artery smooth muscle cells, causing them to stay open, which causes relaxation/vasodilation in the cells), but large amounts, especially in the presence of iron, can be toxic due to formation of dangerous hydroxyl radicals. Furthermore, both contraction and relaxation have been observed in the presence of H2O2.

Since the antioxidant SOD neutralizes toxic superoxide into H2O2, by its very nature, reduced SOD activity, as observed in animal and human PH, leads to reduced H2O2. Under reduced SOD activity or hypoxic conditions, decreased H2O2 could contribute to proproliferative and antiapoptotic effects that are mediated by hypoxia-inducible factor 1α (HIF-1α).

However, other studies show that “H2O2 stimulates cell migration, proliferation, and differentiation in the pulmonary circulation.” And since TEMPOL is an SOD mimetic, it technically can lead to increased H2O2 levels and leading to excessive proliferation. The authors are not keen to accept this version of the theory however, since “TEMPOL induction of H2O2-specific oxidative stress was independent of chronic hypoxia or SU5416.”

Overall, the authors conclude that “despite a dramatic effect of the antioxidant, TEMPOL, to limit vasoconstrictor responsiveness and increases in RVSP in each rat model, we observed a paradoxical effect of TEMPOL to exacerbate both medial and adventitial remodeling in animals with severe PAH. Furthermore, TEMPOL had no effect to reduce RV hypertrophy. Together, these studies support a major role for ROS in mediating the vasoconstrictor component of PAH, however there may be therapeutic limitations of using TEMPOL in severe PAH due to exacerbation of medial remodeling and adventitial lesion formation.” The authors also stress that there may be therapeutic limitations to using SOD mimetics in general.

Study Limitations

The limitations of this study are twofold:

  1. It relies on animal models. Even though the SUGEN/Hypoxia model closely resembles the human form of PH, it does not fully recapitulate the same exact types of features and lesions observed in human PAH. Nevertheless, useful information can still be gained.
  2. It only studied the effect of one antioxidant. Even though superoxide dismutase (SOD) is implicated in PH, and a key antioxidant enzyme, studying this effects of this one compound on ROS does not allow us to make comprehensive conclusive statements about the role of ROS in PAH. Furthermore, while TEMPOL may have promoted pulmonary arterial remodeling in an animal model, another antioxidant may not have the same effects.


  1. Contribution of reactive oxygen species to the pathogenesis of pulmonary arterial hypertension
  2. Inflammation in Pulmonary Hypertension – A Scientific Perspective, with a focus on Hypoxic PH

Inflammation in Pulmonary Hypertension – A Scientific Perspective, with a focus on Hypoxic PH

Chronic Inflammation

What is inflammation?

Inflammation is a complex biological response of the body to remove foreign objects like pathogens (bacteria, virus, fungus), damaged cells, or irritants. It involves cells of the immune system, blood cells, tissue cells, and chemical mediators such as cytokines, chemokines, and reactive oxygen species. We typically have negative connotations associated with inflammation, which is justified, but not all cases of inflammation are negative. We need inflammatory processes to remove harmful pathogens, damaged cells or irritants. The problem arises when this inflammation goes unresolved, and becomes a chronic condition.

There is another common harmful side of the immune system, however. One with negative connotations that are well justified. That is, our immune system can be activated to “attack” our own cells. Cells of the immune system can be thought of as guard dogs. Early in the course of the growth and development of each individual, a process takes place that “conditions” the guard dogs of the immune system to attack only foreign objects, and not our healthy cells. Sometimes, however, this process goes awry, and this gives rise to the guard dogs sniffing our own cells and mistaking them for invaders, thus giving rise to autoimmunity. The presence of autoimmunity can also occur, however, via interaction with foreign objects (typically proteins on cell surfaces) that look very similar to our own proteins. Thus when the guard dog encounters this look-alike protein, it senses something different, and attacks it, but since it is similar to our proteins, the guard dog eventually has a hard time differentiating between the healthy cells and the similar looking foreign objects. That is how autoimmunity can arise.

In sum, inflammation is a protective mechanism, but when unresolved or when directed at the wrong culprit (i.e. our healthy cells), it can lead to chronic inflammation and unwanted conditions such as heart disease, lung disease, cancer, and autoimmunity.

What about in the context of a rare syndrome such as pulmonary hypertension (PH)?

PH takes on many different forms (hypoxia induced, lung disease induced, etc.), and in most cases such as idiopathic PH (IPAH), the underlying cause is unknown. It is well known that there is a large component of chronic inflammation observed in PH. Whether this inflammation is present as a cause or consequence of PH has yet to be determined. We do know however, that a large portion of patients with autoimmune conditions like Rheumatoid Arthritis and Scleroderma, eventually develop PH, or have a higher likelihood of developing PH than normal. This clues us in to the fact that there may be an autoimmune component, or at least some type of inflammatory process, that is central to PH.

Markers of inflammation, such as activated immune cells (both innate and adaptive), cytokines, chemokines, as well as the presence of reactive oxygen species (ROS) are all present in both the systemic circulation and within the tissue and vasculature of the lung. While each case of PH is different, and the extent and type of inflammation in each form may differ (as well as from patient to patient), there are common underlying features of inflammation in PH that may give us clues to whether inflammation is a cause or consequence of PH. In this article, we will review inflammation in PH as it occurs in the context of hypoxic pulmonary hypertension. The review compiles information from the excellent review article “The Effects of Chronic Hypoxia on Inflammation and Pulmonary Vascular Function” by Stenmark et al. in “Pulmonary Hypertension: Basic Science to Clinical Medicine.

Inflammation in PH

Regardless of the cause, chronic inflammation is present and is a staple of PH. As discussed above, chronic inflammation could be the result of PH, but it could also be the trigger that causes PH to develop. I believe it is a trigger, and recent evidence (described below) points to this being the case. However, more studies need to be done to confirm this. It also may be the case that, in one form of PH, inflammation is the cause, but in another form, it may be a result of PH as the underlying condition (thus inflammation as a cause or consequence may be “situational” or “categorical” in nature).

Data from both human and animal studies indicate that early and persistent inflammation contributes to pulmonary vascular disease and that the amount of inflammatory infiltrates in the perivascular region (i.e. the area situated or occurring around the blood vessel) correlates with vascular remodeling and hemodynamic parameters in PH. In this case, it is a positive correlation, i.e. you can expect increased inflammatory infiltrates when the markers of vascular remodeling, vascular resistance, vascular pressure, etc. are all high.

One recent and very interesting theory posits that inflammation contributes to pulmonary vascular disease via an “outside-in” mechanism, whereby there is an influx of leukocytes (inflammatory cells) into the adventitia (the outermost layer of the blood vessel) during vascular injuries and/or sustained inflammatory states such as those which occur in PH. We do know this influx occurs, but the origin of these sustained inflammatory states and/or vascular injuries causing this influx could occur in any portion of the blood vessel, the inside (intima) or the outside (adventitia). Regardless, the influx itself in this outside-in fashion (and resulting cross-talk with resident stromal and adventitial cells) helps to contribute to a perpetual increase in pulmonary vascular dysfunction and remodeling, leading to the pathological development of PH.

The sustained inflammatory states mentioned above, creating the outside-in influx of inflammatory cells, can arise due to a hypoxic environment, cytokine release, or persistent immune and vascular cell activation perhaps due to an “off-switch” failure in these cells preventing them from turning off. The influxed leukocytes, along with adventitial fibroblasts, can then penetrate inward into the media (middle) and intima (innermost region) of the blood vessel. Hypoxia, cytokine release, and vascular injury activates these immune and adventitial cells, which can then go on to initiate and perpetuate inflammatory responses within the adventitia, media and intima of the blood vessel. In light of this, it could perhaps be the case that persistent autoimmunity in the lung vasculature or lung tissue creates these sustained inflammatory states and thus attracts leukocytes and fibroblasts from the adventitia all the way into the media. This would occur because autoimmunity could trigger either cytokine release or vascular injury. Either way, if this were the case, than inflammation would be a “cause”. However, in the case of hypoxia and/or other forms of vascular injury, than the sustained inflammatory state is clearly a consequence, thus being sustained only as long as hypoxia (from low-altitudes, or from lung disease) and/or vascular injury persists.

Aside from the cause of the sustained inflammatory state, the mechanism by which this sustained state occurs is thought to be due to a positive feedback loop between A) resident stromal cells (i.e. connective tissue cells) and mesenchymal cells (i.e. stromal stem cells that can differentiate into bone cells, fat cells, muscle cells, etc.) and B) resident/infiltrating cells like macrophages. This feedback loop is thought to be key in driving the persistence of inflammation in diseases such as cancer and PH. What’s more is that once present, inflammation can drive epigenetic changes in innate immune cells, locking them in a state where they experience a loss of functional plasticity and failure to respond to regulatory signals. In particular, inflammation can promote epigenetic marks in fibroblasts and macrophages specifically, switching them into a pro-fibrogenic and pro-remodeling phenotype.

Below we will look into how hypoxia in particular can induce inflammatory states in different portions of the vasculature. As you will see, each section of the vasculature can contribute to inflammation, although it typically is triggered via the very first layer inside the lumen of the blood vessel, or the “intima”.

The Intima: Endothelial Cells (EC’s)

Under hypoxic conditions, pulmonary artery endothelial cells (PAEC’s) obtain a vasoconstrictive phenotype via 1) decreased activity/production of prostacyclin and nitric oxide (NO), and 2) increased production of endothelin, serotonin, and leukotrienes. Hypoxia also causes the release of the following molecules from PAEC’s:

  • Pro-inflammatory mediators – IL1, IL6, IL8
  • Pro-mitogenic mediators – VEGF-1, Endothelin-1, Thromboxane, PDGF-B, CXCL1
  • Anti-thrombic mediators – increased tissue factor, decreased thrombomodulin
  • Inflammatory cell adhesion molecules – ICAM, VCAM, P-selectin

It is clear from the above, that a multitude of inflammatory mediators and molecules are released from PAEC’s under hypoxic conditions. But if this is the case, then why do we observe inflammatory cells aggregating into adventitia (the “outside-in” hypothesis mentioned earlier) as opposed to directly through PAEC’s? One reason could be that the EC’s of the vasa vasorum, which feeds the adventitia, have the highest expression of inflammatory adhesion cells compared to the other cells in the vasculature. Indeed new evidence is beginning to support this fact, showing that there are distinct interactions between EC’s and inflammatory cells in the EC’s of different organs. This means that it is the EC’s of the blood vessels supplying the outermost layer (i.e. the vasa vasorum) that could be the funnel for the infiltrating leukocytes, in an “outside-in” fashion.

The Media: Smooth Muscle Cells

One of the main reasons for the increased pulmonary vascular resistance that is observed during PH is due to thickening and remodeling of the small pulmonary arteries. The small pulmonary arteries account for the majority of the total cross sectional area in the entire pulmonary vasculature. Thus, any changes in these arteries yields a significant change in resistance to blood flow. The portion of the blood vessel responsible for the thickening of the pulmonary artery? The media layer of the blood vessel which contains the smooth muscle cells.

A hallmark of hypoxic PH is the thickening of the media, the section of the vasculature that comprises the pulmonary artery smooth muscle cells (PASMC’s). From in vivo studies, we know that in large hypoxic animals, there are subsets of undifferentiated resident smooth muscle cells with high proliferative potential in all branches of the pulmonary artery. In normal animals (normoxic animals), this does not occur; the resident smooth muscle cells are well-differentiated and have a low proliferative potential. Typically, at least in terms of hypoxia induced PH, it is the least differentiated cells that have the highest proliferative potential. This is a common observation. In humans with PAH, we observe the presence of these types of undifferentiated/proliferative cells. However, as an interesting side note, it has also been found that there are well-differentiated PASMC’s that appear to be hyperproliferative at baseline compared to PASMC’s from control patients. So markers of cell differentiation may not be a good marker of proliferative capacity of the PASMC.

PASMC’s are typically activated by the EC’s in the intima. Specifically, PASMC’s are activated by endothelin-1 (ET-1), the vasoconstrictive and pro-inflammatory mediator produced by EC’s (however, ET-1 can also be secreted by PASMC’s). Once activated, via the unfolded protein response, PASMC’s release pro-inflammatory and chemotactic mediators.

An interesting feature about resident PASMC’s is that once they are activated and release pro-inflammatory and pro-mitogenic factors, they can then create a feedback loop where those same cells respond to their own signals by increased proliferation/inflammation. One example of this is the HIF1/ET-1 axis: “…there is evidence that hypoxia induces PASMC ET-1 in a HIF1 dependent manner which induces a feed forward loop whereby ET-1 further stimulates HIF1-alpha protein and HIF1 gene expression.”1

There is also evidence that recruited proinflammatory cells (perhaps via the outside-in method, coming in from the adventitia) can induce PASMC proliferation.

The Adventitia: Resident Fibroblasts and Immune Cells

And now to the adventitia… the key player in our “outside-in” hypothesis. The adventitia is the outermost layer of the blood vessel and acts as a supporting framework for the extracellular matrix. It contains conduits for nutrient supply and removal and for circulating cells (the vasa vasorum and lymphatic vessels). And, as we have discussed above, it may play a role in the initial steps of vascular inflammation and remodeling. The adventitia is home to resident macrophages, dendritic cells (DCs), progenitor cells, and fibroblasts (which have recently been found to be capable of exerting immune functions).

So how does the adventitia contribute to inflammation in the vasculature, specifically under hypoxia? Under hypoxic conditions, the vasa vasorum supplying the adventitia begins to expand and import recruited circulating immune and progenitor cells into the adventitia. The adventitia itself also begins to thicken due to extracellular matrix protein and collagen deposition. Furthermore, resident macrophages and fibroblasts become activated and proliferate. The adventitial fibroblast itself acts as a sentinel cell, being the first cell type to respond to vascular stresses (such as hypoxia and mechanical stress) by becoming activated. Once activated the adventitial fibroblast sentinel cell can upregulate contractile and ECM proteins, recruit inflammatory cells, and release compounds that affect PASMC tone and growth in the medial layer. At least in the context of hypoxia, where adventitial fibroblasts act as first responders, the inflammation really does originate completely from the “outside” (with the exception of the sensing of hypoxia, which likely occurs in the smooth muscle cells which act as “oxygen sensors”) and can progress inward, as opposed to being triggered by internal inflammatory states or vascular injuries.

Fibroblasts, macrophages, and dendritic cells (DC’s) present in the adventitia all posses machinery that, once activated, can potently respond to exogenous and endogenous danger signals. This machinery includes toll-like receptors (TLRs) and inflammasome components (NLRs). Once activated, each cell can release a variety of cytokines, chemokines, ROS and tissue remodeling proteins such as MMPS and TIMPS. In the setting of PH, there are increased numbers of these activated fibroblasts, macrophages, dendritic cells (DC’s) present in the pulmonary arteries.

Macrophages have a wide variety of functions due to their functional plasticity, and thus may be key in initiation, propagation, and resolution of immune responses: “macrophages can promote or resolve fibrosis, promote insulin resistance and obesity, are essential in thermoregulation through generation of catecholamines, are essential for wound healing, can promote and restrict T cell responses, promote angiogenesis, promote or suppress tumor growth, fight pathogens, and control homeostasis in local immune networks.” It is now evident that certain tissue resident macrophages like pleural macrophages are even able to renew and proliferate independently from the bone marrow. This means that the macrophages in the adventitia of the pulmonary arteries may be able to renew and proliferate on their own, being a primary source of perpetuating inflammation (local danger signals can trigger them to proliferate rapidly on the spot).

DC’s, on the other hand, encounter self and non-self (environmental) antigens at epithelial surfaces in the pulmonary vasculature, and coordinate innate and acquired immune responses.  In PH, as well as other diseases like asthma and COPD, DC’s exhibit a preference for residing in the adventitia. Here, the DC’s may be able to modulate inflammatory and immunological processes.

Interestingly, it appears that macrophages and dendritic cells may be the “same cell” but operating on different locations of a functional continuum, i.e. macrophages are the result of state A, carrying out function A, while dendritic cells are the same cell in a different state B, carrying out function B: “…macrophages and DCs, based on the fact that no surface or functional marker definitively distinguishes macrophages from dendritic cells, do not represent separate entities but rather two extremes of regulated functional activation states on a continuum of a yet unknown number of functional activities. Chief among these are the capability of macrophages to mount strong proinflammatory cytokine responses (initiating innate immune responses) and DCs to be strong antigen presenters and inducers of T cell responses (initiating adaptive immune responses). However, as pointed out, both cell types can perform both functions in response to adequate stimulation.”

Activated adventitial fibroblasts also appear “to exert a functional plasticity reminiscent of that of macrophages/DCs in that they have been shown to express a combination of functional phenotypes including generation of proinflammatory cytokines and molecules necessary for antigen presentation and T-cell stimulation. This functional plasticity of the activated adventitial fibroblast may therefore play a key role in initiating and propagating adventitial inflammation through generation of numerous cytokines and chemokines that create a microenvironment tailored to fine-tuning the activation of tissue resident macrophages and DCs as well as promoting recruitment of blood derived inflammatory monocytes.”

It seems that, in general, a proinflammatory environment in the adventitia may be caused by the presence of antigens, in which case the dendritic cell may be the primary driver, or by hypoxia, vascular injury, or other causes (such as those that occur in hypoxia induced or idiopathic PH), in which case the macrophage would be the primary driver. However, a third cause may be due to epigenetic factors (caused by hypoxia, inflammation from autoimmune process, or other processes), which can lock immune cells into a specific state rendering them unable to respond to regulatory signals. This would then lead to a sustained pro-inflammatory phenotype in the adventitial fibroblast which would further sustain and lock macrophages and DCs into proinflammatory, pro-fibrotic, and pro-remodeling phenotype. Regardless of the cause, it is evident that “…an environment is created in chronically inflamed tissues, whereby the adventitia acts as a foster home for leukocytes leading to their inappropriate/pathologic retention and survival.”

More on Macrophages and Chronic Inflammation

So what about the persistence of inflammation in PH? Before we answer that, let’s briefly define the parenchyma and non-parenchyma. Parenchymal cells are cells that are part of the parenchyma, which is the functional part an organ. Non-parenchymal cells refer to cells that are part of the stroma, or the structural tissue (connective tissue) of an organ.

It appears that resident tissue macrophages may communicate with local lung (parenchymal) and connective tissue (non-parenchymal) cells to maintain homeostasis. Furthermore, the non-parenchymal cells could play a vital role in providing turn off signals to resident and recruited cells, such as resident tissue macrophages, resident fibroblasts, and recruited macrophages, which could thus promote resolution or inflammation: “…intricate cross-talk between resident and recruited macrophages with their [non-parenchymal connective tissue cells] is key in maintaining tissue homeostasis, coordinating an appropriate inflammatory response tailored to the inciting noxious agent and finally providing signals that allow for resolution when the inflammatory trigger has been removed. Malfunctioning of this cross-talk is thus hypothesized to result in aberrant permanent activation of macrophages and [non-parenchymal connective tissue cells] with subsequent progression to chronic non-resolving inflammation as the driver of pathologic tissue remodeling.”

There is evidence of this macrophage/connective tissue cell inflammatory crosstalk occurring in adipose tissue, as well as in conditions like cancer and rheumatoid arthritis. It is then plausible that this can also occur in PH.

Macrophages also have very plastic (i.e. “malleable”) phenotype. As part of their “duty”, they constantly survey the local tissue status and can change their phenotype based on the signals received from the local tissue microenvironment. Thus, an activated pro-inflammatory macrophage phenotype can transform into an anti-inflammatory pro-resolution phenotype if the appropriate local signals are present. This is important for therapeutic purposes because, if chronic non-resolving inflammatory processes are present, one could theoretically target the local tissue signals that are driving the macrophages to stay in a pro-inflammatory phenotype. For example, what if altering the metabolic microenvironment by metformin or AMPK activation, and other means, could help switch macrophages to anti-inflammatory phenotype?

The Role of Extracellular ATP and Nucleotides in Inflammation (and Support for Metabolic Therapies for Inflammation?)

Targeting the metabolic microenvironment could make sense seeing that extracellular ATP, other nucleotides (ADP, UTP, and UDP) and adenosine, all have been known to regulate vascular function, controlling blood flow, cell proliferation, migration, inflammation (ATP acts synergistically with cytokines and integrins), and chemotaxis. Purine homeostasis in particular is an important factor for proper vascular endothelial function.

It is known that the extracellular ATP in tumor microenvironments are 1000 times higher than normal. Extracellular nucleotides may even contribute to vascular diseases like PH. Extracellular ATP is released from adventitial fibroblasts, vasa vasorum endothelial cells, and inflammatory cells in response to hypoxia, inflammation, oxidative stress, and mechanical forces, all of which are experienced in PH. In addition, it is known that extracellular ATP induces a pro-inflammatory phenotype in immune cells like monocytes and macrophages by regulating cytokine and chemokine production.

Extracellular ATP is also released by adventitial fibroblasts and the vasa vasorum as a result of hypoxia or oxidative stress, which can thus act to activate macrophages. Stenmark et al. showed “that pulmonary artery adventitial fibroblasts and vasa vasorum endothelial cells (VVECs) are a potent source of extracellular ATP, which acts as an autocrine/paracrine factor augmenting hypoxia-induced VVEC angiogenesis.” In this case, metabolic dysregulation of the adventitia, leading to high extracellular ATP concentrations, could be a trigger for inflammatory processes in PH.

Adenosine, however, appears to be protective and anti-inflammatory. Extracellular adenosine inhibits the chemotactic response of immune cells to ATP, thus potentially preventing the excessive accumulation of inflammatory cells in the adventitia. It also prevents endothelial cell permeability via its receptor A1R. Animal model studies have indicated that A1R can attenuate endotoxin induced lung injury, pulmonary edema, and alveolar destruction. Studies have also shown “a significant attenuation of TNF-alpha induced VVEC permeability upon adenosine treatment, indicative of the barrier-protective effect of adenosine.” TNF-alpha is one of the most potent inflammatory mediators and regulates endothelial cell permeability, and its expression is increased under hypoxia, inflammation, and PH. Both macrophages and perivascular adipocytes are potent sources of TNF-alpha.

However, data indicate that A1R may be downregulated under conditions of chronic hypoxia, which may contribute to pulmonary vascular remodeling and inflammation. Stenmark et al. gives a great mechanistic explanation of the interplay of extracellular ATP and adenosine and how they are involved in pulmonary vascular remodeling under hypoxic conditions: “Endogenously released ATP, by acting on P2 purinergic receptors (P2R) results in angiogenic activation of the vasa vasorum, characterized by increased proliferation and dysregulated barrier properties. Elevated extracellular ATP is subsequently hydrolyzed by ectoenzymes… to adenosine. In turn, extracellular adenosine by acting on P1 purinergic receptors (P1R) induces a phenotypic switch of the vasa vasorum endothelial cell to a more quiescent state, characterized by low proliferation rate and improved barrier function. Inhibition of [ectoenzymes] by hypoxia and oxidative stress results in consistently elevated levels of extracellular ATP and lower levels of adenosine that eventually exacerbate pathological vascular remodeling.”

Aside from extracellular ATP, Stenmark et al. mention that “mediators produced downstream of glycolysis, which occurs in hypoxic PH and PAH, are able to directly affect pulmonary vascular remodeling.”

All of this evidence supports the idea that metabolic interventions could be implemented (e.g. in order to alter the status of extracellular ATP and adenosine, or glycolysis) to promote an anti-inflammatory and non-proliferative microenvironment.

The Role of ROS in Inflammation in PH

Reactive oxygen species (ROS) are increased in all forms of PH, and are thought to contribute to both vasoconstriction and vascular remodeling. ROS are produced by inflammation, and their production further activates inflammatory pathways. Thus, ROS act as a positive feedback loop for promoting inflammation once inflammation occurs. However, as with inflammation, it is not clear that all ROS are “bad”, especially in the context of PH (as we’ll describe soon). For example, ROS are necessary signals for repairing and building muscle during weight lifting. After you lift weights, you want ROS present to help rebuild and grow stronger.

The primary ROS in the vasculature are superoxide and hydrogen peroxide, the latter of which is involved in both physiological and pathophysiological cell signalling. Superoxide is degraded by the antioxidant enzyme superoxide dismutase (SOD) to yield oxygen and hydrogen peroxide. If there is not enough SOD present, superoxide reacts with local bioavailable nitric oxide (NO), thus reducing the local concentration of NO, and producing toxic peroxynitrite as a byproduct.

Hydrogen peroxide, on the other hand, is scavenged by antioxidant enzymes like glutathione peroxidase and catalase. Hydrogen peroxide, especially that generated by the mitochondria (mitochondrial ROS), is necessary for physiological cell signaling, but too much can be detrimental. Additionally, in the presence of iron, hydrogen peroxide decomposes into a toxic hydroxyl radical.

In the pulmonary vasculature, the primary sources of superoxide and hydrogen peroxide are NADPH oxidases (abundant in the adventitia), uncoupled eNOS, the mitochondrial electron transport chain (mitochondrial ROS), and xanthine oxidase. In diseased states, eNOS is uncoupled and produces superoxide. eNOS uncoupling can occur via: “deficiencies in L-arginine substrate; increased ADMA. an L-arginine analog; increased arginase activity; or BH4 deficiency due to low production or oxidation.”

Xanthine oxidase produces superoxide. “…XO-derived ROS contribute to injury in a number of processes associated with inflammation including ischemia-reperfusion injury, acute lung injury, COPD and cigarette exposure, and cancer.” XO also “promotes the inflammatory state of pulmonary mononuclear phagocytes through effects on HIF1alpha.”

Mitochondrial ROS, however, are controversial. Numerous studies implicate mitochondrial ROS and mitochondrial dysfunction in pulmonary vascular disease. However, mitochondria also act as oxygen sensors for the cell, producing mitochondrial ROS in the presence of oxygen which then goes to keep potassium channels open, and thus keep the vasculature in a vasodilated state. When oxygen levels decrease, so does mitochondrial ROS, and thus the potassium channels in the SMCs close causing vasoconstriction. In fawn hood rats that spontaneously develop PH, mitochondrial ROS is decreased. Also, under the setting of hypoxia, mitochondrial ROS is decreased. Thus it is not quite clear that hydrogen peroxide/mitochondrial ROS is either beneficial or detrimental. We know that it is necessary for vasodilation and proper cell signaling, but it can also initiate inflammatory pathways and cell damage.

The sum of all ROS produced in the lung microvasculature is likely higher than normal in PH however and probably causes net harm, since they are produced by inflammation, and go on to further promote inflammation. ROS produced by all of the above mentioned sources “can be activated by pro-inflammatory cytokines, including interleukins and tumor necrosis factor-alpha, and conversely, the ROS generated in the vessel wall can augment inflammation by activating redox sensitive targets including key transcription factors, NF-kB, AP-1, and HIF. In addition, ROS can modulate a wide range of other signaling molecules that impact inflammation, proliferation, migration, differentiation, and matrix production.”

Chronic Inflammation induces Epigenetic Changes

Evidence continues to emerge to support the idea that chronic inflammation leads to stable (and heritable) epigenetic changes in gene expression and cell function (leaving the underlying base DNA composition unchanged). There are three overall mechanisms of epigenetic regulation: 1) DNA methylation, 2) histone modifications, and 3) gene silencing (via microRNA’s).

A primary example of DNA methylation occurring in PH is that the SOD2 gene in the pulmonary arteries and plexiform lesions of PH patients is hypermethylated. When this hypermethylation is reversed, SOD2 is reduced. SOD2 is responsible for neutralizing the harmful superoxide radical.

Regarding HDAC’s, increased HDAC expression and activity is associated with increased vascular cell proliferation, and is known to contribute to the pathological remodeling of the pulmonary arteries in PH.

All of these epigenetic changes (methylation, HDAC activity, and microRNA activity) can occur in the setting of hypoxia and inflammation. Furthermore, HDAC’s in particular, once activated can regulate microRNA activity to create a feedback loop. This can explain the constitutively active phenotype of PH: “long term adaptation to chronic hypoxia involves significant modification of chromatin structure in order to maintain the hypoxic phenotype, even in the absence of HIF1.” Thus, chronic hypoxia is capable of inducing epigenetic changes in gene expression that are independent of the classical HIF pathway.

Concluding Remarks

Inflammation can either be a cause or consequence of PH. Regardless of this, inflammation operating via an “outside-in” mechanism, where infiltrating immune cells penetrate inward into the blood vessel, helps to contribute to a perpetual increase in pulmonary vascular dysfunction and remodeling, leading to the pathological development of PH. Emerging evidence indicates a growing role for the adventitia and resident immune cells in the adventitia in the inflammatory processes that lead to PH, and it appears that the adventitia is a perfect microenvironment for hosting and promoting pathological inflammatory responses. If sustained for long enough, chronic inflammation can induce epigenetic changes that further lock-in this inflammatory state.

Extracellular ATP, nucleotides, ROS, macrophages, and adventitial fibroblasts all are mediators in the inflammatory processes mentioned above, and thus could be therapeutic targets. Furthermore, since it is plausible that autoimmunity and metabolic dysfunction contribute to inflammation, therapeutically targeting these areas may also help resolve inflammation and PH. Metabolic therapies can take the form of a drug like metformin, or even an anti-inflammatory diet (removing foods that trigger allergic reactions or autoimmune responses, e.g. removing wheat for celiac disease) like Paleo or AIP, or a combination of those. I’m obsessed with diet and metabolism, and strongly believe that what you put in your body can either greatly help you or harm you. Your stomach is after all one of the only areas where the outside world comes in contact with the “inside” world. As such, it harnesses the largest concentration of immune cells in your body. Foods that induce gut permeability can thus potentially create autoimmune responses. So, in the end, food could be a great therapy, in combination with modern medicine for resolving inflammation, and could maybe even help to reverse PH.


  1. “The Effects of Chronic Hypoxia on Inflammation and Pulmonary Vascular Function” by Stenmark et al. in “Pulmonary Hypertension: Basic Science to Clinical Medicine”.
  2. Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities
  3. Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP In Vitro and In Vivo Using Multiple Endocytic Mechanisms
  4. Rethinking The Role Of Antioxidants in Sports
  5. Histone deacetylation inhibition in pulmonary hypertension: therapeutic potential of valproic acid and suberoylanilide hydroxamic acid
  6. Hormonal, Metabolic, and Signaling Interactions in PAH
  7. What Is The Paleo Diet?
  8. The Autoimmune Protocol 

The Cholesterol Paradox, Part 3 – LDL in Heart Disease & Pulmonary Hypertension

LDL Cholesterol

There is more to LDL than meets the eye… While it is true that LDL is an established marker of cardiovascular disease risk, we need to be clear just how and why exactly this is so.

First things first. LDL is a means for delivering cholesterol to tissues that need it (and all cells need cholesterol to survive). Cholesterol (and thus LDL) is necessary for life: it is involved in hormone synthesis (including sex hormones), cell wall synthesis and maintenance, and the synthesis of vitamin D and bile acids. Additionally, it is involved in the process of repairing cellular injury. Now to tackle a few myths…

Myth #1 – LDL is the cause of Heart Disease

While LDL is involved in the atherosclerosis process, it isn’t the driving force, the immune system is… Atherosclerosis is the result of an immune response, by definition you need a macrophage deposited in an arterial wall to initiate the process of atherosclerosis. Thus, elevated non-esterified lipids, triacylglycerols, or cholesterol, resulting in increased risk of atherosclerosis is only one part of the equation; the other part is that you need elevated inflammation (macrophage immune cells, cytokines, etc.).1

Now while LDL is necessary, it is true you don’t want too high of an amount. More specifically, you don’t want a large number of small LDL particles. What does that mean and how does that relate to standard cholesterol testing? Firstly, a standard lipid panel done by your doctor does not provide the information necessary for accurate atherosclerosis risk assessment. To really understand your risk, you really need to know the LDL particle size and number. Small LDL particles, and increased numbers of those particles, are the real risk factors for atherosclerosis and heart disease. Testing can be done by labs like Quest Diagnostics which use NMR to analyze the LDL particle size and particle count for you.2,3,4 

Myth #2 – Very low levels of LDL are beneficial

While you don’t want too much LDL (in the form of high numbers of small LDL particles), you also don’t want too little. Recently, Kopeć et al. found low levels of LDL in PAH patients and that low circulating cholesterol is a marker of increased mortality in PAH and may accelerate disease progression: “While high LDL-C levels are associated with worse prognosis in the general population, there are some populations with chronic diseases including diabetes, heart failure, chronic kidney disease, and rheumatoid arthritis where low LDL-C levels have been linked to increased mortality. In PAH, low LDL-C levels might accelerate disease progression by several mechanisms including exacerbation of inflammation and direct effects on the arterial wall. In fact, we have recently shown that low LDL-C levels are associated with increased stiffness of pulmonary arteries. Conversely, as observed in other diseases, chronic inflammation, hepatic congestion, and low nutritional status, might be associated with reduced LDL-C levels in individuals with PAH.”5

They go on to say that the “traditional interpretation of hypercholesterolemia as a risk factor for increased mortality may not apply in PAH population. Instead, our finding of the deleterious signal of low LDL-C levels in PAH corresponds with the concept of cholesterol paradox and reverse epidemiology whereby lower levels of traditional risk factors are associated with worse prognosis. The evidence of a survival advantage associated with higher cholesterol levels has been provided for several populations with debilitating disorders such as heart failure, rheumatoid arthritis, acute myocardial infarction and in the elderly.”

They also mention that PAH treatment (vasodilator therapy, etc.) improves LDL-C.

Question: Is a low-fat diet or a high-fat diet good for PH?

My personal theory is that a high-fat, low carbohydrate diet may be very beneficial for PH, but this is a tough question and warrants serious investigation. For example, it is known that PH patients have a dysregulated fatty acid metabolism.6 Normally, the heart’s metabolism derives ~70-80% of its energy from fatty acid oxidation, but in PH this changes and glycolysis is the favored metabolic pathway. Whether this change is pathological or due to physiological adaptation is not certain. For example, it is known that a switch to aerobic glycolysis in tissues that normally rely on glucose and fatty acid oxidation is usually a pathological switch (cancer cells switch their metabolism to aerobic glycolysis).7 But perhaps glycolysis (which is a rapid but inefficient mechanism of obtaining energy) is needed by the right ventricle in PAH so it can quickly adapt to increased pulmonary vascular resistance?

Whether the PAH patient has a dysregulated fat metabolism as a result of insulin resistance and/or metabolic syndrome (which is known to be present in a majority of PAH patients), or as a result of underlying genetics or the PAH disease itself (e.g. a result of the BMPR2 mutation, or from the phenotype of proliferative PASMCs, and/or the chronic inflammation that occurs along with PAH), is also unknown. If dysregulated fat metabolism was a result of insulin resistance or metabolic syndrome than in theory dietary interventions could significantly improve the body’s metabolism and hopefully PAH (low-carb diets like The Paleo Diet and Ketogenic Diet have been shown to improve insulin resistance, improve inflammation, and improve cardiac function for a variety of individuals, more on this later…).8,9,10

Regardless of the underlying mechanism of dysregulated fat metabolism, this does not necessarily imply that individuals with or without PAH should decrease their dietary fat intake to absolute zero. Again, I personally suspect that specifically high-fat low-carb diets can improve PH, but I also admit that if dysregulated fat metabolism is a driver of PH (which it isn’t proven that it is), then high-fat diets may not be so beneficial.

However, additional observations by Kopeć et al. provide support in favor of my theory of high-fat, low-carb diets research potentially improving PAH: “In rabbits, it has been demonstrated that hypercholesterolemia can increase pulmonary artery relaxation in response to methacholine. Another experimental study in rats with monocrotaline induced PAH showed that high-fat diet and hypercholesterolemia was associated with better prognosis as compared with standard diet.” 


  1. http://searchingphoracure.com/2016/08/13/pulmonary-hypertension-atherosclerosis-lungs/
  2. http://www.questdiagnostics.com/home/physicians/testing-services/condition/cardiovascular/cardio-iq-report.html
  3. http://eatingacademy.com/how-low-carb-diet-reduced-my-risk-of-heart-disease
  4. http://eatingacademy.com/cholesterol-2/the-straight-dope-on-cholesterol-part-ix
  5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309849/
  6. http://searchingphoracure.com/2016/08/24/fatty-acid-metabolism-pulmonary-arterial-hypertension-role-right-ventricular-dysfunction-hypertrophy-review/
  7. https://en.wikipedia.org/wiki/Warburg_effect
  8. http://robbwolf.com/what-is-the-paleo-diet/
  9. http://thepaleodiet.com/research/
  10. http://www.ketonutrition.org/

The Cholesterol Paradox, Part 2

This is part of a series of posts where I share and dissect information about cholesterol, the science behind cholesterol, and common fallacies surrounding cholesterol. Most of these posts are going to be short… where I just share bits of information that I’ve archived or that I’ve recently found, and some thoughts surrounding them. For Part I of this series, click here.

For this post, I want to reflect on a quote from Metabolic Regulation: A Human Perspective by Keith N. Frayn (my emphasis added):

Perhaps surprisingly, the amount of cholesterol in the diet is not a major factor affecting the blood cholesterol concentration. The amount of cholesterol we eat is not large in comparison with the body pool: we eat less than 1g per day whereas the amount of cholesterol in the body is more like 140g, of which about 8g is present in the plasma. Contrast this with glucose, where we eat several “plasma’s-worth” in a single meal. And cholesterol is not rapidly absorbed like glucose: it enters the plasma slowly, even more so than triacylglycerol. Further, cholesterol intake leads to cholesterol entering cells, which effectively suppresses cholesterol synthesis. The blood cholesterol concentration is related far more closely to the dietary intake of particular fatty acids, especially the ratio of saturated to polyunsaturated fatty acids.” 

So, the cholesterol that we eat pales in comparison to that stored in our bodies. As mentioned in this video by Dr. Peter Attia, cholesterol synthesis and transport in the body is a highly regulated process and is not that influenced by the cholesterol that we eat in food. And any cholesterol that is indeed present blood is carefully controlled by the cholesterol transport system. But keep in mind it’s not the presence of cholesterol in the blood that matters, it’s the amount and type of that cholesterol that matters (specifically the size and number of LDL particles are what matters, but we will dive into that in another post). Cholesterol is vital, and cells need cholesterol to function (cholesterol is actually part of the structural makeup of cells). “Cholesterol” in the blood only becomes “dangerous” if the LDL, which is a protein/cholesterol complex, increases in number, and decreases in particle size.

As Keith states, the way cholesterol is processed from our diet significantly contrasts with that of glucose (sugar). When we eat a typical meal, which usually contains large amounts of sugar and/or carbohydrate (which breaks down into sugar), our blood glucose levels rise and insulin is released in order to control this. It is well known that elevated blood sugar and/or chronically elevated insulin lead to a variety of health problems including diabetes, cancer, heart disease, and chronic inflammatory disease. In light of this, we should most likely be concerned with the amount of sugar/carbohydrate that we consume as opposed to the cholesterol that we consume.

Keep in mind that cholesterol is not the same as fat, and as Keith mentions above, the quality, type and amount of fat that we consume influences the cholesterol levels in our body. But more importantly, and what he doesn’t mention in that particular quote, is that dietary fat only becomes a “concern” if it is consumed in the presence of sugar and/or carbohydrate: when dietary fat is consumed along with sugar/carbohydrate, fat is preferntially stored and the sugar is preferentially “burned”, this being due to the fact that elevated blood sugar and insulin lead to a switching of a cells fuel preference… leading to sugar burning and fat storing, and eventually a dysregulated fat metabolism (as opposed to normal/healthy fat metabolism). This sustained reliance of cells on sugar consumption and fat storage is what leads to problems (via sustained elevation of insulin and eventual development of insulin resistance). In this case, the fat isn’t what causes the issue, it is the “sugar” that causes a cascade of issues (this is why populations consuming “Western” diets of high carb and high fat have higher rates of disease incidence).

There is plenty evidence that shows that dietary fat is not the enemy we once thought, and I’ll conclude this post with some links supporting this:

Notes regarding Pulmonary Hypertension:

  • I strongly believe that metabolic and dietary interventions can improve health conditions and heal individuals with health issues. But, while it is evident from the above links that high fat/low carb diets (like ketogenic diets) are healthy and show promise for cancer, it is NOT evident that this is healthy for Pulmonary Hypertension. I do suspect that low carb only diets (meaning diets low in carb but no elevation in fat content) are healthy for PH, since any dietary intervention that lowers blood glucose and insulin improves health outcomes. And since PH patients typically show insulin resistance and cellular dependence on glycolysis (in pulmonary vasculature) this is further evidence that low carb dietary interventions could be beneficial. However, PH patients have a dysregulated fat metabolism as well, and in light of this, a high fat/low carb diet could be a potential problem. I personally don’t suspect this to be the case, but there is more research to be done… If anything, if ketogenic diets do prove to be unhealthy, I believe the reason for this is only that PH patients can’t process “fat” in the same way that normal individuals do. However, the byproduct of fat metabolism, ketones, could still be very beneficial and there may be a way to bypass fat metabolism but still get the benefits of fat by taking exogenous ketones. I’ll write more about this later…

NOTE: Nothing in this post is written or intended to be medical advice. These are my own thoughts and opinions based on my research. I am not a doctor. I am merely a scientist with a passion for Pulmonary Hypertension.


Can Diet Help Improve Pulmonary Hypertension? An Insight from Inflammatory Bowel Disease Research

Paleo diet

While reading a recent paper entitled “Endothelial dysfunction in inflammatory bowel diseases: Pathogenesis, assessment and implications” I experienced a feeling a remarkable familiarity… I felt as if I was reading a paper about endothelial dysfunction in Pulmonary Hypertension.

It appears that endothelial cell (EC) dysfunction in both inflammatory bowel disease (IBD) and pulmonary hypertension (PH) are quite similar: the same mechanisms of dysfunction abound, the same proinflammatory molecules are released. There is proliferation, smooth muscle cell tone activation, platelet aggregation, hypoxia, eNOS downregulation, imbalance between vasodilators and vasoconstrictors, etc. 

Even though this kind of makes sense (that pathology in a cell such as an endothelial cell could be quite similar regardless of what tissue/organ it occurs in), it nevertheless is quite interesting. And why is it interesting to me?

Because, as some of you may know, I’m a big believer that nutrition and diet, specifically low carbohydrate diets, can help prevent and alleviate many chronic diseases. In addition to low carbohydrate diets (like a Paleo Diet or Ketogenic Diet) making sense from an evolutionary context, since foods were scarce when we evolved (and there were no highly refined grains), these diets have also shown remarkable results; people with autoimmune conditions experience significant improvements on a Paleo diet (and even put them into remission), and ketogenic diets show great promise for treating brain tumors. There are a variety of other reasons why I think low-carb diets are beneficial, but I’ll save those for a later post, and direct you to some of the research that’s been done on Paleo and Ketogenic Diets, both of which, again, show great promise.

So what does this have to do with EC dysfunction and IBD and PH? Well, Ulcerative Colitis and Crohn’s disease are classic cases of IBD and have been put into remission several times by following a Paleo type diet. If endothelial dysfunction is a primary cause and/or symptom of IBD, and endothelial dysfunction is similar in IBD and PH, then if diet can put IBD into remission, can it potentially put PH into remission? It’s a large jump. It could be true, but the opposite could be true as well, e.g. what if these diets are good for IBD but harmful for PH? There is much more research to be done, and many more points to be discussed (PH is a complex pathology involving multiple organs in addition to lungs), but it is interesting to think about nonetheless…

NOTE: Nothing in this post is written or intended to be medical advice. These are my own thoughts and opinions based on my research. I am not a doctor. I am merely a scientist with a passion for Pulmonary Hypertension.


  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4716020/“Endothelial dysfunction in inflammatory bowel diseases: Pathogenesis, assessment and implications”
  2. http://robbwolf.com/Biochemist Robb Wolf’s website & Paleo Diet Testimonials
  3. http://thepaleodiet.com/research/Dr. Loren Cordain’s website on Paleo Diet Research
  4. http://www.ketonutrition.org/Dr. Dominic D’Agostino’s website on Ketogenic Diet Research & Resources
  5. https://www.charliefoundation.org/ketogenic-therapy/therapies-2/brain-tumor-cancerThe Charlie Foundation website on Ketogenic Diet Therapies for Brain Tumors/Cancer

“Aldehyde dehydrogenase 2 protects against oxidative stress associated with pulmonary arterial hypertension” – A Review of Lipid Peroxidation in PH

Lipid Peroxidation

A key underpinning in the pathological development of PAH is thought to be the abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs), and it is well known that oxidative stress plays a key role in this process. One oxidative stress pathway is lipid peroxidation, which has been found to contribute to abnormal PASMC growth. A major end product of lipid peroxidation processes, specifically from omega-6 peroxidation, is the compound 4-hydroxynonenal (HNE). [For those of a chemistry bent, 4-HNE is an aldehyde]. Of interest is the fact that 4-HNE has been found to contribute to PASMC growth and this compound has also been found in excess in the pulmonary arteries of PH patients.

Normally, Aldehyde dehydrogenase 2 (ALDH2) is the enzyme that is responsible for conversion of 4-HNE into harmless byproducts. However, any alterations in ALDH2 activity could result in improper removal of 4-HNE.

Furthermore, 4-HNE is typically a byproduct of oxidative stress (and thus a marker of oxidative stress), 4-HNE can also contribute to disease by binding to proteins to form protein adducts which can cause dysregulation in cellular signaling pathways.

In this paper, the role of 4-HNE and ALDH2 in development of PH was investigated further to determine if 4-HNE contributes to abnormal medial remodeling in the pulmonary vasculature (i.e., stimulates PASMC growth and migration] and if ALDH2 agonists help to improve PH.


  • After injection of monocrotaline (MCT), which is a toxin used to induce PH, 4-HNE accumulates in rat PASMCs
  • 4-HNE was found to stimulate growth of cultured human PASMCs.
  • 4-HNE increased expression of the matrix metalloproteinases MMP-2 and MMP-9 in human PASMCs in vitro. Since MMPs are involved in degrading ECM components to facilitate cell migration, this suggests that 4-HNE mediates PASMC migration within the ECM.
  • NF-kB is a signaling pathway involved in oxidative stress. 4-HNE was found to decrease the presence of the NF-kB inhibitor, IκBα, and increase the presence of NF-kB. It was also found to stimulate the MMPs via NF-kB.
  • 4-HNE decreases the presence of ALDH2 in human PASMCs in vitro. When ALDH2 is increased (via treatment with an agonist), it prevented the 4-HNE induced proliferation in PASMCs as well as prevented the oxidative stress pathway triggered by NF-kB (by preventing NF-kB translocation to nucleus, which occurs because ALDH2 removes 4-HNE which is was found to activate NF-kB as indicated above).
  • In MCT rat lungs, 4-HNE accumulated and ALDH2 activity was decreased.
  • ALDH2 agonists improve RV hypertrophy and RV systolic pressure, but does not improve pulmonary arterial pressure. The agonist also increased presence of NF-kB inhibitor and decreased the activity of NF-kB.


The study was only performed on one animal model, the MCT mouse model: It should be noted that we only examined the role of ALDH2 in MCT-induced PAH in rats, without evaluating using other animal models of PAH, such as hypoxia-induced PAH or Sugen-5416/hypoxia-induced PAH. Another, MCT-induced PAH is only a pro-inflammatory and oxidative stress relevant animal model, whereas PAH in humans is considered to develop by multiple pathogenic factors.” Additionally, cultured PASMCs from humans were used, and studies done on cultured cells should be interpreted with caution.

Furthermore, the action of 4-HNE is complex. While 4-HNE was shown to induce PASMC proliferation, it does so at low levels. High levels of 4-HNE, however, might do something different: “high concentrations of HNE initiated apoptosis by inducing endoplasmic reticulum stress and mitochondrial dysfunction in human colon carcinoma cells and neuroblastoma cells… These findings indicated that the function of HNE is complex; whether it promotes proliferation, differentiation or cell apoptosis depends upon its concentration and the cell type involved.”


It was convincingly shown in this article that 4-HNE can stimulate PASMC growth and stimulate oxidative stress, indicating that even if oxidative stress produces 4-HNE, the 4-HNE product can contribute back to oxidative stress creating a potential feedback loop. The reasons for 4-HNE accumulation most likely have to due with a decrease in ALDH2 enzyme activity. However, it could still also be the case that even if ALDH2 expression remains the same, an overall increase in oxidative stress can still increase 4-HNE (if 4-HNE production surpasses its degradation by ALDH2).


Why is ALDH2 activity decreased? According to the paper, a few reasons could be polymorphisms in ALDH2 gene as well as reactive oxygen species (ROS), which has been shown to decrease ALDH2 activity. However, what about metabolism? Does an altered cellular metabolism affect ALDH2 activity?



Pulmonary Edema in PH & PVOD – Potential Causes of PVOD Misdiagnosis

*DISCLAIMER: These are my own thoughts and opinions based on my research and are not meant to be taken as medical advice.*

Below is a potential explanation for how edema could occur in Pulmonary Hypertension (PH) patients receiving vasodilator therapy, thus leading to them being potentially misdiagnosed for Pulmonary Veno-Occlusive Disease (PVOD)…

It makes sense to think that because a PH patient does not respond to vasodilator therapy well, or experiences edema due to vasodilator therapy, it may be an indication that they have PVOD. The theory goes that vasodilators can “open” up the vasculature (even in PH patients) and if the veins are “occluded” or non-responsive/rigid, then there is a large pressure difference that occurs when the blood flows from the “open” to “closed/rigid” section causing backpressure and edema: “PAH-specific vasodilator therapies may cause an augmentation of pulmonary arteriolar blood flow against the fixed resistance of occluded pulmonary venules and veins. The resultant increase in the transcapillary hydrostatic pressure gradient, manifest initially as subpleural interlobular septal line thickening, may progress to severe pulmonary oedema.” 1

However, instead of it being PVOD, there could be a multiple number of other reasons for why edema occurs in these patients who are subject to vasodilators. For example, inflammation could be a sole factor in pushing a patient into edema following vasodilator therapy:

“Aberrations of endothelial barrier function lead to an abnormal extravasation of fluid and macromolecules, resulting in edema and tissular dysfunction. In the course of inflammation, this is the first recognized step occurring predominantly not only at the level of post-capillary venules but also, for instance, in response to VEGF at the levels of arterioles and capillaries. Edema develops when plasma extravasation exceeds its re-absorption and the capacity of the lymphatic system to remove fluids from the interstitial space.” 2 If you have an overactive immune system, vasodilators can enhance extravasation of vasculature… thus exceeding the capacity for things to be taken back up into the bloodstream! Hence edema! 

Other reasons for edema following vasodilator therapy could be due to the following:

  • Increased capillary permeability: “Analysis of the fluid from high altitude pulmonary edema shows that it contains high-molecular weight proteins, which indicates that the edema is caused by increased capillary permeability. The increased capillary permeability may result from capillary stress failure caused by high pulmonary artery pressure and blood flow and by altered release of cytokines or other mediators.” 3 Perhaps the PH patient has increased capillary permeability? PH has a strong inflammatory component, and inflammation is known to cause endothelial cell permeability. If patients have increased capillary permeability, why would you give vasodilators to these patients?
  • High “interstitial” colloid osmotic pressure: “The colloid osmotic pressure of the [blood] plasma proteins normally exceeds the pulmonary capillary hydrostatic pressure. This tends to pull fluid from the alveoli into the pulmonary capillaries and keep the alveolar surface free of liquids other than pulmonary surfactant.” 4 What if you have a high colloid osmotic pressure outside of the blood vessel, in the interstitium, due to an overactive immune system depositing proteins/debris in interstitium, or due to a lymphatic drainage problem causing buildup of proteins in interstitium? This combined with a high capillary hydrostatic pressure (from the elevated pressure due to PH) can technically push fluid into interstitium and then into alveoli, especially if you administer vasodilators which can just increase the vascular permeability even further. Again, it is well known that lymph drainage problems, leukocyte recruitment, and inflammation in general, can increase sucseptability to edema 

Furthermore, it must be remembered that not all PH patients respond well to vasodilator therapy. Since these patients don’t have PVOD, and do not respond well to vasodilators, this should be a red flag that patient response to vasodilator therapy should not be a determining factor if they have PVOD or not. We need more accurate and non-invasive ways measures of identifying whether or not someone has PVOD to prevent this type of potential misdiagnosis.


  1. http://www.sciencedirect.com/science/article/pii/S0954611110001320
  2. https://www.ncbi.nlm.nih.gov/books/NBK57148/
  3. Levitzky, Michael G. Pulmonary Physiology, 8th edition
  4. http://www.ncbi.nlm.nih.gov/books/NBK53445/

Appearances Can Be Deceiving – The Problem With Genetic Knockout Mice Studies

Appearances Can Be Deceiving – The Problem With Genetic Knockout Mice Studies


When scientists want to study the effect that a gene has on an organism, they perform what is called a “knockout” experiment. The knockout method is an experimental method, usually employed in mice, whereby the expression of a specific gene under study is blocked. The goal of this method is to learn about what effect the absence of the gene has on the animal.

Due to the inevitable rise in the importance of genetic targeting in therapeutics, the knockout model has become a very important staple in studying the functions and endogenous expression patterns of single genes in vivo. While knockout experiments can produce valuable information about the gene of interest, as explained below, this is not always the case.  Thus, results from knockout experiments should be interpreted with caution.

There are three overall categories of problems, or “shortcomings”, with knockout studies. The first results from the limitations inherent in the knockout method technology and experimental method employed, the second results from the phenomena of biological robustness, and the third results from the complexity of the organism and constant interplay of a myriad of genes and proteins in vivo.

First Shortcoming

The first problem is that original genetic material from the embryonic stem cells used for the knockout study can remain in the genome, despite attempts to breed it out. These genetic confounding factors in turn impact the phenotype observed and thus the results obtained, since this residual genetic material can contribute to the observed phenotype; you don’t know whether the phenotype is due to single gene ablation or background genetic effects from residual genes.

There are three ways to improve this experimental shortcoming: 1) improve the gene targeting technology itself, 2) improve breeding techniques employed, or 3) rescue the knocked out gene during experimentation. Rescue is the most common method. If a gene is inactivated, and you reactivate it (i.e. “rescue” it), if the observed phenotype changes there’s a good chance it could be due to that gene.

According to Eisener-Dorman et al., for “any assessment of whether the ablation of a gene is, in fact, responsible for a phenotypic trait, the basic question should always be asked: is the observed phenotype relevant to what is known of the protein function? If the function of a gene is unknown, or if the observed phenotype deviates from what is reasonably anticipated, then the potential influence of [background stem cell] genes should be evaluated.”1

Second Shortcoming

Robustness is classified as a lack of variation in phenotype to genetic or environmental changes. As Dear et al. explain, “central metabolic pathways appear to have more alternatives than other pathways. This might reflect intrinsic robustness where central metabolic pathways must function under variable physiological and environmental conditions. However, it may also be adaptive—central metabolic pathways are critical for the organism’s survival and a back-up mechanism may be advantageous. It has been found in an analysis of transcriptional and signal transduction networks that parallel pathways connecting a regulator to a regulated molecule are not, as is commonly perceived, rare but are actually quite common…”.2

Genetic robustness can occur through “(i) genetic buffering—where alternative pathways for a process exist in the organism, or (ii) functional complementation—where genes are to some extent redundant in function. Two genes are considered to be redundant if they can fully or partially substitute each others functions.”

A common example of biological robustness is that often the same mutations that are studied between human and mouse in a specific gene result in diffrent phenotypes. That is, mouse mutations in gene X do not give the same result as human mutations in gene X (this is the primary problem in Pulmonary Hypertension… animal models, including knockout mice studies, don’t fully recapitulate the human form of PH): “Consider, for example, the OCRL1 gene, which encodes a phosphatidylinositol 4,5-bisphosphate 5-phosphatase. This gene is mutated in Lowe syndrome, a rare genetic disorder in humans that results in serious physical and mental problems. Yet, the mouse Ocrl knockout appears unaffected. However, mice have a related gene, Inpp5b, which is not present in humans. Inactivation of this gene results in only a mild phenotype, while the Ocrl-Inpp5b double knockout is embryonic lethal. Thus Inpp5b may be able to protect mice from any deleterious effects that would normally result from the absence of Ocrl.”

Finally, consider the example of the Hsp90 gene in Drosophila: “When this gene is mutated, widespread phenotypic variation results from other mutations, previously silent in the presence of the wild type Hsp90. Thus, Hsp90 is able to buffer against genetic mutations that would normally have a phenotypic effect.”

Third Shortcoming

The third and final shortcoming results from the complexity of the genome; there are a myriad of potential interactions between genes, between genes and the environment, and between proteins, and feedback loops exists between all of these…

In short, a gene might be doing something “X”, but that “X” combined with the product/activity of another gene “Y” produces an effect “Z”, which we will refer to here is a phenotype or microphenotype (disease, or a microstate like a malignancy or a lesion). Let’s consider two scenarios here…

Scenario 1: X is thought to be bad, Y is unknown, and Z is the bad resulting phenotype (a disease, etc.)… thus delete X and improve Z

In this scenario, Y is the unknown variable. Since Y is the factor you are unaware of, and you only see Z which is the combined effect of X and Y, you may think that Z is really caused ONLY by X. So, when you take away X, if Z disappears, you would be led to the conclusion that the gene you knocked out caused Z.

What’s the problem with this? You don’t really know that the gene product X is causing Z by itself; you don’t know it works in tandem with the product Y of another gene to produce the observed phenotype. Thus, in this case, therapies that target the gene that produces X will be unsuccessful because of the confounding factor Y that you are unaware of. What’s more is that these therapies may even have unintended side effects due to the fact that X may have other crucial functions critical to the organism’s survival and homeostasis.

Scenario 2: Loss of X is thought to be bad, Y is unknown, and Z is the bad phenotype… thus add/increase X to Improve Z

In this scenario, let’s use an example from PH. In PH, knockout of the BMPR2 gene can induce PH… and there are strong suggestions that loss of BMPR2 function is a principal factor in PH pathogenesis. To PH researchers credit, however, they do understand that to induce PH you usually need both BMPR2 loss of function AND an external stimulus like inflammation. However, just because there is an external stimulus doesn’t mean there isn’t another gene that is working in synergy with BMPR2 to prevent PH. That is, is BMPR2 really THE gene responsible for PH? Perhaps it is both the lack of BMPR2 and the presence of HERV-K genes? This would explain why inflammation can act as a coactivator in inducing PH, since HERV-K is a endogenous retrovirus responsible for releasing cytokines and inflammatory factors.

In this example, therapy for PH would include inducing BMPR2. But my point is, that even if you induce BMPR2, there could be another factor Y, silently working away to induce PH. This explains why people without BMPR2 mutations still can get PH. When you took out BMPR2 function, you saw the PH phenotype because you didn’t know about Y and that it was working to induce PH. Thus, an incorrect conclusion is that BMPR2 loss induces PH. However, even with BMPR2 working, Y is also working… and all you see is Z, which is PH.

In both of the above scenarios, Y can be any one of the myriad of human genes… This is why we should be wary of interpreting knockout mice studies. Analyzing one variable is important linear systems and simple systems… but in complex nonlinear systems like biology, it can lead to mistaken conclusions. As a result, results from knockout studies should be interpreted with caution, and researchers should fully understand all of the shortcomings associated with such experiments.

Hopefully, improvements in technology will improve the shortcomings of knockout mice studies. However, even with improvements in technology, scientists still face the daunting task of teasing out causal factors in experiments that involve manipulation of a complex environment such as an organism’s genome.

References & technical explanations of knockout methods:

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2746382/
  2. http://bfg.oxfordjournals.org/content/6/2/91.full

“A proteomic approach to altered innate and adaptive immunity in the pathogenesis of PAH” – A Synopsis

Below is a synopsis of A proteomic approach to altered innate and adaptive immunity in the pathogenesis of PAH, a talk by Marlene Rabinovitch (MD, Stanford) given at the Vera Moulton Wall Center for Pulmonary Vascular Disease (video below). It is one of my favorite talks so far in this series of lectures…

  • Hypothesis: there is an abnormal immune response (both innate and adaptive) affecting pulmonary arteries that is common in all forms of PAH

Altered adaptive immunity in PAH pathogenesis

  • What if antigens produced in lung are the site for autoantibody formation and immune complex deposition directly in the lung and in the perivascular area?
  • SAMHD1 is an antiviral nuclear protein that suppresses HIV replication, and this is part of an innate immune response
  • Using Western immunoblot analysis, SAMHD1 protein expression was found to be increased in IPAH lung
  • Via Immunohistochemistry analysis:
    1. In controls, SAMHD1 expression is present in some of the inflammatory cells situated in the periphery
    2. In IPAH, abundant expression of SAMHD1 was found in occluded lesions and in perivascular region
  • Via confocal immunohistochemistry (immunofluorescence microscopy):
    1. Abundant expression of SAMHD1 observed in nuclei of endothelial cells of occluded lesions
    2. Colocalization of SAMHD1 was found in CD11 cells (dendritic cells), and in CD68+ (monocyte/macrophages)
  • How is SAMHD1 induced?
    1. SAMHD1 is induced in pulmonary arterial endothelial cells by TNF-alpha
  • Since SAMHD1 is an antiviral protein, is increased SAMHD1 an innate immune response to a virus localized to the pulmonary artery tissue?
  • Analyzed the virome of the IPAH lung tissues and found not much difference between IPAH and controls, with the exception of amplified expression of endogenous retroviruses from the HERV-K family, specifically HERV-KII
  • Why is a retrovirus expressed in PAH?
  • In zebrafish that are prone to cancer due to genomic instability, endogeneous retroviruses are enhanced.
  • In patients with PAH, there is genomic instability due to BMPR2 mutations. This could lead to HERV-K2 amplifications. As a consequence of this, HERV-K2 can induce cytokines that are implicated in PAH, such as TNF-alpha, IL-6, and SAMHD1, in macrophages, endothelial cells, and tertiary lymphoid tissue.
  • In embryonic stem cells, there is transient amplification of HERV-K2 to protect the embryonic stem cells. But as they differentiate, the HERV-K2 amplification disappears. Thus, it appears that HERV-K2 (the message/protein) can be amplified and can be repressed, but the DNA is present either way. So, perhaps in PAH patients, amplification of the HERV-K2 mRNA from the DNA never really quiets down or is re-activated by inflammatory stimuli.
  • HERV-K does indeed induce IL-6 in PAECs, and TNF-alpha in PBMCs
  • In IPAH lungs, HERV-K can be seen being expressed in CD68+ Adventitial Macrophages in the perivascular region, and this is not seen in control lung tissue
  • HERV-K has also been implicated in autoimmunity and in the activation of B Cells
  • In tertiary lymph nodes in close proximity to occluded vessels, SAMHD1 antibodies were detected.
  • In summary: HERV-K2 activation leads to a) endothelial and inflammatory cell activation which leads to increased SAMHD1 and b) activation of B cells in tertiary lymph nodes to produce SAMHD1 antibodies. The interaction of SAMHD1 antibodies and SAMHD1 leads to the formation of immune complexes followed by complement activation which leads to chronic inflammation and PAH. What can fuel this whole system is reduced BMPR2 which 1) enhances cytokine production and 2) provides some genomic instability that could amplify HERV-K

Altered innate immunity in PAH

  • When cells are under fire or there is tissue damage, elastases are released by neutrophils
  • Regardless of PH etiology, circulating elastase levels are elevated in PAH. That is, they are elevated in all forms of PH. You also see naturally occurring inhibitors like elafin, which are usually produced in abundance when there is heightened elastase activity, are suppressed in all forms of PH.
  • Increased neutrophil elastase is linked to autoimmunity in diabetes.
  • Neutrophils are recruited to site of injury, and it produces elastase. And elastase can condition dendritic cells to activate Treg cells to come in to protect the tissue. But if there is heightened elastase, there is transdifferentiation of anti-inflammatory Treg cells to proinflammatory Th17 cells.
  • The good news is you can control elastase levels by using elafin. If you recognize an elafin deficit, you can add recombinant elafin (given in Europe in clinical trials)
  • Elafin suppresses PMN (polymorphonuclear cell) elastase, PMN adhesion, netosis, cytokines, and autoimmunity, and improves BMPR2 function.
  • An alternative to controlling elastase is using Treg therapy. Treg therapy suppresses PMN and Monocyte activation and autoimmunity, as well as increases BMPR2
  • Using SPADE analysis with Flow Cytometry Time of Flight techniques, it was found that the SDF1 receptor (or CD184, or CXCR4) is increased in IPAH circulating dendritic cells, T cells, and B cells. This increase in SDF1 could explain why some of these cells can be recruited to the lung, and specifically the perivascular region.
  • Elevated pSTAT1 activity across all PH groups (IPAH, Scleroderma, and Drug & Toxin) in CD4+ CD25hi T cells (which include Treg cells)
  • It was found that Treg transdifferentiation into to proinflammatory STAT1 positive cells is implicated in Rheumatoid Arthritis
  • What may be happening here is that you no longer have a protective Treg cell, but rather a cell that can be contributing to the disease… if it can get to the “disease” location
  • The CD4+ CD25hi T cells were found to have elevated levels of GM-CSF receptor across all PH patients (IPAH, Scleroderma, and Drug & Toxin). Furthermore, since there is increased GM-CSF in IPAH pulmonary arteries in the intima, media, and adventitia (normal control pulmonary arteries do not have elevated GM-CSF in any of these regions)… this allows for these proinflammatory pSTAT1 positive CD4+ CD25hi T cells to target and be recruited to the pulmonary arteries.
  • Loss of BMPR2 + TNF-alpha stimulation enhances GM-CSF pulmonary arterial endothelial cells. The enhancement was on the translation side as opposed to the transcription side (i.e. GM-CSF mRNA translated more into protein, as opposed to GM-CSF gene being transcribed more into mRNA). This is interesting because when cells are under stress/assault… they undergo a protective ER stress response where they produce stress granules that keep mRNA’s from being very actively translated. But what is happening here is abnormally increased translation of cytokines… increased GM-CSF + IL6 + IL8
  • Heightened GM-CSF causes recruitment of all cells that have the GM-CSF receptor… including monocytes and macrophages… as well as the aforementioned proinflammatory Treg cells.

Promising Therapies to Reverse Abnormal Innate and Adaptive Immunity:

  • Tacrolimus – activates BMPR2 and is immunosuppressive, and can low doses can increase and improve function of T reg cells
  • Elafin – a natural elastase inhibitor, activates BMPR2
  • Bestatin – a leukotriene B4 inhibitor
  • IL-6 Receptor Antagonist
  • T reg Immunotherapy
  • All these have shown positive results in pre-clinical studies


  • Amplification of HERVK occurs in PAH and can induce a chronic innate immune antiviral response leading to the deposition of SAMHD1 immune complexes which causes inflammation and…
  • …heightened elastase activity in PAH (reflecting the abnormal innate immunity) that could lead to abnormal adaptive immunity and autoimmunity by switching T regs to proinflammatory cells which are…
  • ….recruited to pulmonary arteries to induce PH due to heightened GM-CSF in pulmonary arteries that are caused by 1) BMPR2 mutations 2) heightened inflammation

“Matrix metalloproteinases and their inhibitors in pulmonary hypertension” – A Review

This past week, I delved into the arena of Matrix Metalloproteinases and their role in disease and Pulmonary Hypertension. One of the first papers that I read with regards to this is by S.S. Pullamsetti et al. entitled “Matrix metalloproteinases and their inhibitors in pulmonary hypertension”. My review and summary of this paper is below.


ECs = Endothelial Cells
SMCs = Smooth Muscle Cells
ECM = Extracellular Matrix
PAH = Pulmonary Arterial Hypertension
MMPs = Matrix Metalloproteinases
TIMPs = Tissue Inhibitors of Matrix Metalloproteinases

In PAH, complex remodeling is observed in the pulmonary vasculature that involves all components of the blood vessel. In addition to this complex vascular remodeling, there is enhanced synthesis and degradation of ECM components, imbalanced turnover, as well as accumulation of ECM components. It is believed that these processes within the ECM (imbalanced turnover, etc.) may contribute to the pathogenic vascular remodeling seen in PAH.

Overview of Blood Vessel and ECM Physiology

The blood vessel in the pulmonary artery is comprised of the following distinct layers: ECs (innermost layer of blood vessel, or intima), SMCs (middle layer of blood vessel, or media), adventitia (the outermost layer of the blood vessel), and the ECM (layer that is interspersed between all other layers).

The ECM of the vasculature is a complex matrix interspersed between all other layers of the blood vessel, and provides blood vessels with mechanical strength, elasticity, and compressibility. It also functions to maintain homeostasis within the vasculature since interactions and crosstalk between cells of the ECM, and between cells and the ECM components, tightly regulate cell migration, proliferation and differentiation.

The innermost layer of the blood vessels is comprised of EC’s, which line the pulmonary arteries and rest on a basement membrane for support. They are tightly regulated and altogether form a semi-permeable membrane. The EC’s are responsible for synthesizing vasoactive compounds, as well as components of the basal membrane on which it lies.

The middle layer, or media, contains several layers of SMCs in a concentric fashion, together with fenestrated elastic lamina. In muscular arteries, diffusion can occur between EC and SMCs due to the fenestrated layer of elastin separating the intima and media beneath the ECs.

The SMC’s in the pulmonary arteries can exist in multiple states: quiescent, synthetic, or intermediate. Normally, SMCs are quiescent and exhibit a differentiated contractile phenotype, regulating vascular tone in response to vasoactive compounds, maintaining contractility, and possessing low rates of proliferation and synthesis. However, the synthetic phenotype, which is usually observed in PH, exhibits increased proliferation, cell migration, synthesis (synthesizing and secreting multiple ECM components such as elastin, fibronectin and collagens), and ECM turnover.

It has been found that certain ECM components can play a role in determining the SMC phenotype. For example, “elastin, one of the major constituents in the ECM of the medial layer, is a potent autocrine regulator of vascular SMC activity. Besides rendering resilience and elasticity to the arteries, elastin is critical for stabilisation of the arterial structure by inducing a quiescent contractile state. Several in vitro studies indicate an inverse correlation between elastin expression and SMC proliferation.”

The adventitia is the thick outermost layer of the vasculature, of which fibroblasts are the most prominent cell. “Fibroblasts also regulate synthesis and secretion of adventitial ECM components such as collagen types I and III, which constitute the chief ECM content of the adventitia. Besides rendering structural integrity to the vasculature, adventitia has resident immunomodulatory and progenitor cell populations that contribute to the growth and repair processes of the vessel wall.”

Proteases of the ECM, and their inhibitors

ECM turnover and homeostasis is controlled by the balance between the proteolytic enzymes matrix metalloproteinases (MMPs) and serine elastases, as well as their respective inhibitors. There are a vast number of MMPs and they are grouped together and categorized into six groups according to their respective substrates as follows: “1) interstitial collagenases (MMP-1, MMP-8, MMP-13 and MMP-18); 2) type IV collagenases or gelatinases (MMP-2 and MMP-9); 3) stromelysins (MMP-3, MMP-10, MMP-11 and MMP-19); 4) matrilysins (MMP-7 and MMP-26); 5) transmembrane MMPs (membrane type (MT)-MMPs: MMP-14, MMP-15, MMP-16, MMP-17, MMP-24 and MMP-25); and 6) other MMPs (MMP-12, MMP-20, MMP-21, MMP-22, MMP-23, MMP-27 and MMP-28).”

MMPs are initially inactive (they are zymogens), and become active through proteolytic cleavage. After activation, cleavage can still occur, either from the MMP cleaving itself, or via exogeneous means, each of which results in smaller forms of MMP.

Once active, MMPs can degrade many components of the ECM and in doing so mediate a wide range of physiological processes including embryogenesis, angiogenesis, cellular reproduction, bone remodeling and tissue repair. They are also implicated in a variety pathological settings, such as PAH.

MMPs are controlled by transcription, translation, and posttranslational mechanisms, as well as by the activity of their endogenous inhibitors. There are two primary endogenous inhibitors of MMPs: 1) the tissue inhibitors of MMPs (TIMPs), and 2) Alpha 2-macroglobulin.

The TIMP family tightly regulates MMPs via reversible inhibition, and has four members, TIMP-1 through TIMP-4, which form complexes with MMPs in a 1:1 stoichiometric ratio through binding of the TIMP to the MMP active site. Of all the TIMPs, TIMP-3 is unique “due to its direct binding to ECM proteins, and thus can provide a means for stabilising MMP-TIMP complexes within the interstitial space. TIMP-3 has also been demonstrated to inhibit ADAM (a disintegrin and metalloproteinase), notably ADAM-17.”

Αlpha2-macroglobulin is an abundant plasma protein and is the other major endogenous inhibitor of MMP activity, specifically in the plasma. Alpha2-macroglobulin binds to MMP complexes, which are then recognized by macrophages/monocytes and removed via scavenger receptor-mediated endocytosis. Thus, inhibition of MMPs via alpha2-macroglobulin is an irreversible process.

Another enzyme present in the ECM that is similar to MMP is ADAM (A Disintegrin And Metalloproteinase). There are two forms of this enzyme: membrane bound (ADAMs) and secreted (ADAMTSs). “The domain structure of the ADAMs consists of a pro-peptide domain, a metalloprotease domain, a disintegrin domain, a cysteine- rich domain, an endothelial growth factor (EGF)-like domain, a transmembrane domain, and a cytoplasmic tail. Like most proteases, the ADAMs are initially synthesised as enzymatically inactive precursor proteins… [Upon activation, the] metalloprotease domain hydrolyses protein substrates such as cytokines and growth factors, as well as their respective receptors. This process is termed ectodomain shedding and ADAM is often termed “sheddases”. The disintegrin domain binds integrin receptors such as α9β1 and, therefore, mediates cell–cell and cell–matrix interactions.”

The different domains within the ADAM and ADAMTS enzymes implicate that these enzymes can participate in proliferation, migration, and angiogenesis processes. For example, the “EGF receptor ligands (amphiregulin and heparin-binding EGF) released/shed by ADAM-17 enhance cell proliferation of cancer cells and induce angiogenesis. In accordance, altered expression of specific ADAMs has been implicated in different diseases; their best documented role is in cancer formation and progression. However, to date, no data are available about the roles of those proteins in the pathogenesis of PH.”

Serine proteases are the last form of proteolytic enzymes within the ECM that are involved in degrading the ECM. Serine proteases are enzymes that cleave peptide bonds in proteins. “Among several serine elastases, neutrophil elastases secreted by neutrophils and… endogenous vascular elastase (EVE) have been strongly implicated in vascular diseases. EVE is produced by vascular SMCs and degrades elastin and several other ECM proteins. Regulatory control of serine elastase activity is accomplished by the concomitant production of endogenous inhibitors like α1-antitrypsin, α2-macroglobulin and elafin, with the most relevant inhibitor in vasculature being elafin.”

Expression and Regulation of MMPs

The major sources of MMPs in the pulmonary vasculature during physiological and pathological states are the endothelium, SMCs, and fibroblasts and immune cells that infiltrate the vessel wall. In both physiological and pathological states, biological, mechanical, haemodynamic and/or neurohormonal stimuli can modulate MMP expression and activity.

During either angiogenesis or wound-healing, the endothelium can be a major source of MMP expression and activity. Basal MMP expression is typically low in ECs, but MMP expression in ECs can be activated by angiogenic factors, pro-inflammatory molecules, as well as organic compounds such as plant-derived phorbol esters. VEGF, and angiogenic compound, was found to increase MMP-2 and reduce TIMP-1 and TIMP-2 in ECs. In addition to inducing MMP-2, the downregulation of TIMP by VEGF allows for activation of other pre-existing collagenases within the ECM. This most likely contributes to the invasive migration of endothelial cells into the basement membrane and interstitial matrix.

During pathological states, growth factors and cytokines can also modulate MMPs within the medial layer. “Exposure of cultured SMCs to pro-inflammatory molecules like interleukin (IL)-1α, tumour necrosis factor (TNF)-α, and oxidised low-density protein are shown to significantly increase the expression and activity of MT1-MMPs…”. In a separate study, it was found that “autocrine expression and activation of transforming growth factor (TGF)-β1 antagonises the platelet-derived growth factor (PDGF)-BB… induced upregulation of MMP-2 in SMCs during phenotypic modulation, suggesting TGF-β1 promotes contractile phenotype by modulating MMPs.” This is interesting since, while TGF-β1 is profibrotic and plays an active role in PH pathogenesis, it also plays a role in promoting differentiation of SMCs into quiescent phenotypes.

Within the adventitia, fibroblasts, which are typically quiescent, may become activated during growth/proliferation and can thus affect MMP activity. For example, during hypoxia, there is a significant increase in active MMP-2, TIMP-1 and TIMP-2 production in fibroblasts when compared to normoxia. MMP activity can also affect fibroblast activity: oxidative stress via reactive oxygen species (ROS) can increase MMP activity, and thus in turn modulate fibroblast proliferation and collagen synthesis.

Inflammatory cells are also a major source of MMPs and other proteases, as well as cytokines and growth factors within the vasculature. Major players typically are monocyte/macrophages, dendritic cells, neutrophils, mast cells, T-cells and B-cells, all of which are capable of infiltrating the vessel wall.

Exposure of human alveolar macrophages “to native or denatured collagen type I and III selectively stimulated the expression of interstitial collagenase and TIMPs, suggesting that ECM components can directly influence macrophage-mediated MMP secretion…”  Furthermore, immune cells can regulate MMPs via cytokines and other factors. For example, when “granulocyte-macrophage colony-stimulating factor is added in combination with either TNF-α or IL-1β-induced MMP-1 synthesis it synergistically enhanced MMP-9 and TIMP-1 expression. Whereas MMP-9 production is negatively regulated by IL-4, IL-10, IFN-β, IFN-γ and TGF-β in monocytes… Similarly, MMP-12 expression in human peripheral blood-derived macrophages is induced by several cytokines and growth factors including IL-1β, TNF-α, M-CSF, VEGF and PDGF-BB.”

Moreover, there are distinct patterns of MMP expression in circulating leukocytes and monocytes depending on their state of activation. These distinct patterns of MMP expression in monocytes can thus increase transmigratory capacity across certain barriers (e.g. the blood-brain barrier). In addition, T-cell extravasation into the ECM and perivascular tissue during inflammation is mediated by induction and localisation of MMP-2 on the surface of T-cells via interaction with vascular cell adhesion molecule-1 on ECs.

Neutrophils are another significant source of MMPs and other proteolytic enzymes. Neutrophils “participate in different inflammatory process by releasing enzymatically active neutrophil elastase, and other proteases including cathepsin G, proteinase 3, MMP-1, MMP-8, MMP-9 and MMP-12. In addition to the innate immune functions of neutrophils, several studies indicate the role of neutrophil-derived proteases in trans-endothelial neutrophil migration and in the initiation of the angiogenic switch. Specifically, neutrophil-derived MMP-9 is shown to play an important role in basal lamina type IV collagen degradation and in catalysing the angiogenic switch by facilitating MMP-9-dependent VEGF mobilisation and consequent pro-angiogenic signalling.”

Finally, mast cells are also players that can induce proteases in the ECM. While they are typically localized in the adventitia, they have also be found in the media of pulmonary arteries. Mast cells “release several factors including MMPs, neutral and serine proteinases, heparin, heparinase, tryptase, chymase, histamine, and angiogenic growth factors such as basic fibroblast growth factor (bFGF), VEGF and cytokines. Mast cell-derived neutral proteases like tryptase and chymase potentially mediate activation of latent MMPs in human carotid arteries and subsequently enhance angiogenic phenotypes and also degrade different components of BM.”

Physiological Functions of MMPs

MMPs have many functions in the vasculature. Aside from proteolysis, “MMPs play a crucial role in multiple physiological process like morphogenesis, angiogenesis, tissue remodelling and tissue repair, as well as modulating fundamental cellular processes like proliferation, migration, differentiation, apoptosis, permeability, host defence, release of ECM-bound chemotactic factors and chemokine processing.”


A complex process involving multiple steps that are regulated both in space and time, physiological angiogenesis typically involves the interaction of “multiple growth factors that modulate the complex cell–cell and cell–matrix interactions through activation of different proteolytic systems in the vascular microenvironment. In response to an angiogenic stimulus, the endothelial-derived MMPs mediate proteolytic degradation of endothelial cell–cell and EC–matrix interactions and facilitate ECs to acquire a proliferative and migratory or invasive phenotype. In particular, angiogenic factor-induced secretion of MMP-2, MMP-9 and MT1-MMP in vascular ECs is critical for EC proliferation, migration and vascular ECM invasion associated with sprouting angiogenesis.” MMP-2 and MMP-9 are critical for sprouting angiogenesis because they degrade type IV collagen, which is a major component of the basement membrane of the intima.

Furthermore, the process of proteolysis itself “liberates ECM-sequestered pro-angiogenic factors, exposes cryptic pro-angiogenic integrin binding sites in the vascular ECM and also generates pro-angiogenic fragments, which collectively trigger the angiogenic switch.”

Pathological angiogenesis, on the other hand, is a dysregulated process. While their role isn’t entirely clear, it appears that MMPs are also major players in this process as evidenced by studies done on knockout mice: “MMP-2 deficient mice displayed reduced rates of tumour neovascularisation, total vascular area, number of vessels and tumour progression, as well as normal embryonic development of the vascular system. However, the combined deficiency of MMP-2/MMP-9 in the experimental model of tumour angiogenesis displayed an impaired angiogenic and invasive phenotype with strong reduction of gelatinolytic activity… Similarly, FGF-2-induced angiogenic response was lacking in the MT1-MMP deficient mice, suggesting that MT1-MMP might be important for initiation of angiogenesis. Furthermore, studies with MMP inhibitors revealed strong inhibition of angiogenic responses both in vitro and in vivo.”

Cell Proliferation & Migration

MMPs both directly and indirectly regulate proliferation, migration, and apoptosis of ECs and SMCs. For example, MMP-2 and MMP-9 act in synergy with MT1-MMPs to promote EC migration by proteolytically remodelling the BM, controlling the dissolution of ECM components, and releasing ECM derived chemotactic factors. It has been observed that when new vessels are being formed, at the leading edge of the developing vessel, there is EC proliferation, MT1-MMP-dependent activation of MMP-2, and MT1-MMP-dependent collagenolysis.

Arterial injury can induce medial hyperplasia that involves proliferation and migration of SMCs, as well as ECM remodelling. This process is regulated by various cytokines and growth factors, e.g. PDGF. In medial explants, it was found that migration of SMCs induced by PDGF-BB or -AB “was mediated by MMP-2, whereas the stimulatory effect of bFGF on medial SMC migration was mediated by both MMP-2 and MMP-9. Administration of potent MMP inhibitor (BB94) dose-dependently suppressed the PDGF-BB-induced migration of cultured SMCs and also suppressed the intimal thickening and medial SMC proliferation after arterial injury… Moreover, in situ and in vitro studies performed on pulmonary arteries and pulmonary artery smooth muscle cells (PASMCs) from patients showed increased MMP expression, suggesting a critical role for MMPs in different vascular remodelling processes that involves SMC proliferation, migration and intimal thickening.”

Cell Differentiation

As mentioned, SMCs can exist in multiple states. They can transition from a quiescent/contractile phenotype to a synthetic phenotype during either physiological or pathological conditions. Typically, the proliferative phenotype is migratory in nature and exhibits increased proteolytic activity and ECM turnover. This phenotype also undergoes a loss of cellular differentiation markers like myosin bundles and α-actin.

MMP-2 upregulation has been associated with the process of PDGF-BB induced SMC dedifferentiation from a quiescent to a synthetic phenotype. Although, as mentioned, TGF-β antagonizes this upregulation. It was also found that TGF-β upregulates TIMP-1 mRNA levels, further showing how TGF-β can promote a contractile SMC phenotype by altering MMP/TIMP ratios.

Moreover, both MMPs and serine elastases can produce elastin peptides within the ECM which stimulates the production of fibronectin. Fibronectin also influences the SMC phenotypic, switching it from a contractile to a synthetic state.

Interestingly, with regards to fibroblasts, “the homeostatic relationship between resident fibroblasts and the collagen matrix keeps them in a quiescent, undifferentiated state. However, normal fibroblasts subjected to hypoxia display hypoxia-induced phenotypic switching to myofibroblasts, through the MMP-2/TIMP mediated pathway. Specifically, PDGF, TGF-β1, tenascin-C (TN-C), fibronectin and ET-1 exhibit mitogenic activity and induce the myofibroblast phenotype both in vitro and in vivo, probably by regulating the MMP/TIMP balance.”

Vascular Permeability and Cell Adhesion

The integrity and permeability of the EC barrier, as well as the quiescent EC phenotype, is usually sustained through cell-matrix and cell-cell interactions. However, pro-angiogenic factors such as VEGF and angiopoietins also regulate EC barrier, permeability, and phenotype: “VEGF promotes vascular permeability by uncoupling inter-endothelial junctions, inducing the formation of endothelial fenestrae and an increase in modification of caveolae. A recent study revealed that the hypoxia-induced vascular leakage in vivo and the associated rearrangement of tight junction protein occludin and its diminished expression is mediated by hypoxia-induced MMP-9 activation. However, VEGF inhibition attenuated vascular leakage, hypoxia-induced MMP-9 activation and dependent gelatinolytic activity. Also, MMP inhibition attenuated vascular hyperpermeability, and prevented gap formation and tight junction rearrangement.”

Integrins, cadherins, selectins and the immunoglobin family of endothelial cell adhesion molecules also play a role in normal cell–cell and cell–matrix interactions, as well as in pathophysiological events: “In particular, the αvβ3 and αvβ5 integrins are described to regulate endothelial cell adhesion and migration during angiogenesis and vasculogenesis. In addition, αvβ3 integrins interact with an array of ECM ligands such as vitronectin, fibronectin, collagen, laminin, von Willebrand factor, fibrinogen, osteopontin, thrombospondin and RGD-containing peptides, which are potential MMP substrates… Moreover, during endothelial sprouting, surface expression and MT1-MMP dependent activation of MMP-2 in ECs were found to be associated with integrin αvβ3 and TIMPs at basolateral focal contacts and mediate focal degradation of ECM.”

Cadherins are also known to play an important role in tissue cohesion and cell migration, as there is typically a correlation between tissue cohesion and E-cadherin expression. However, cells that exist in an environment with intermediary tissue cohesion but high MMP expression become migratory in nature. From MMP inhibition studies, it has been found “that the decrease in the expression of E-cadherin and increase in type IV collagenase activity (MMP-2 and MMP-9) would enhance detachment of tumour cells and facilitate invasion. In contrast, in ECs, an increase in VE-cadherin was observed in connection with the downregulated MMP production and pericellular proteolysis during stabilisation and maturation of the neovessel. This recent study confirms the mechanism of cell–cell contact dependent regulation of pericellular proteolysis in angiogenesis. This correlates with the finding that MMP inhibitor treatment enhanced colocalisation of cadherin/β-catenin at cell–cell contacts and promoted stabilisation of cadherin-mediated cell–cell adhesion in fibroblasts suggesting a possible feedback loop between MMP and cadherin/β-catenin systems.”

Mobilization of Growth Factors and Cytokine Processing

Aside from being a structural support for the vasculature, the ECM is a repository for a variety of compounds such as VEGF, bFGF, hepatocyte growth factor, insulin-like growth factor-1, TGF-β1 and connective tissue growth factor (CTGF). During digestion/proteolysis of the ECM, MMPs liberate these compounds: “VEGF is sequestered as an inactive form by the ECM components like CTGF, pleiotrophin, etc., resulting in reduced bioavailability. Several MMPs (MMP-1, -2, -7, -9, -16 and -19) mediate proteolytic cleavage of this inhibitory complex and increase the bioavailability of VEGF… Mice with targeted disruption of MMP-9 exhibited reduced hypertrophic cartilage vascularisation due to the lack of mobilisation of ECM-bound VEGF, confirming the essential role of MMP-9 in bioavailability of growth factors. In addition, in cultured SMCs, serine elastase (EVE) was shown to mediate degradation of elastin, which subsequently promotes liberation of ECM-bound bFGF.”

Proteolysis of ECM components by MMPs also release matrikines. Matrikines are fragmented matrix peptides that can regulate cell activities. For instance, proteolysis of the ECM components collagen and perlecan by MMPs generates anti-angiogenic matrikines (arrestin, canstatin, tumstatin, metastatin, endostatin, neostatin, vastatin, restin and endorepellin), whereas proteolysis of fibronectin, laminin, osteonectin and elastin liberates matrikines that promote cell proliferation, migration and angiogenesis.

MMPs also play a role in processing cytokines, such as TGF-β, which regulates cellular processes such as proliferation, migration, differentiation, cell survival and synthesis of vascular cells in the ECM. In the ECM, TGF-β isoforms are secreted and maintained in an inactive state by binding with elastic microfibrils within the ECM. Proteolytic enzymes such as MMP-9, MT1-MMP and plasmin degrade these microfibrils thus releasing the active form of TGF-β.

MMPs also influence inflammatory responses by mediating chemokines through alteration of their chemotactic properties: “MMP-2 mediated processing of CCL7/MCP-3 generated a cleaved MCP-3 that acts as a general chemokine antagonist and attenuates chemotaxis and the host inflammatory response in vivo.”

MMP Role in PAH Pathogenesis

MMP Expression in PAH

MMP/TIMP expression is altered/dysregulated in PAH:

  • MT1-MMP is present in myofibroblasts and endothelial cells in onion-skin lesions and cellular plexiform lesions
  • MT1-MMP and MMP-2 detected in myofibroblasts and endothelial cells in the cellular plexiform lesions
  • Discontinuous type IV collagen and focal thinning was observed in onion-skin lesions and mature plexiform lesions
  • MMP-2 expression and activity increased in PASMCs from IPAH patients
  • MMP-3 is decreased and TIMP-1 is increased IPAH PASMCs, which favors ECM accumulation
  • Circulating MMP-9 and TIMP-1 levels are increased in plasma of PH patients
  • MMP-2, MMP-9, neutrophil collagenase (MMP-8), stromelysins (MMP-10, MMP-11), macrophage metalloelastase (MMP-12), MMP-20, PLAT (tPA) and SERPINB2 are upregulated in MCT-treated lungs.
  • Transgenic expression of human MMP-9 in MCT-treated lungs resulted in extensive infiltration of macrophages. However, another study demonstrated that “macrophage-specific transgenic expression of human MMP-1 in a mouse model of MCT-induced PH resulted in attenuation of collagen deposition, SMC proliferation, infiltration of macrophages and consequently medial thickening.”
  • MMP-2 expression is enhanced in both hypoxia- and MCT- induced PH in animal models
  • Increased MMP-13 and collagenolytic activity was found in the pulmonary arteries of hypoxic rats.
  • Increased serine elastase activity and elastin fragmentation was observed in pulmonary arteries of MCT-induced PAH rats.
  • Preceding the development of MCT-induced PH, there is increased activity of endogenous vascular elastase (EVE)

However, despite the above, it appears that not all MMPs and proteolytic activity are “bad”. For example, there is a transient increase of MMP-1 and MMP-3 during normoxic recovery from hypoxia-induced PH. Even more interesting, there was a “significant increase in stromelysins and total proteolytic, collagenolytic and gelatinolytic activities was predominantly found in the media and adventitia of pulmonary arteries during normoxic recovery of hypertensive vessels. This observed increase in protease activity correlated with the rapid reduction in collagen and elastin content in pulmonary arteries, thus indicating a correlation between decreased vascular remodelling and increased MMP activity during early reversal of hypoxia-induced PH. In a similar study by Tozzi et al., increased activation of mast cell-derived interstitial collagenase was shown to mediate restoration of vascular architecture by facilitating collagen breakdown in remodelled pulmonary arteries during the early recovery phase from chronic hypoxia. Poiani et al. also reported accumulation of collagen and elastin in the main pulmonary arteries of rats during chronic hypoxia, while the normoxic recovery of hypertensive vessels were associated with decreased collagen and elastin content. Cumulatively, these experimental studies suggest an essential role of MMPs in reabsorption of vascular collagen during the de-remodelling process that occurs in the post-hypoxic recovery phase.”

Consequences of MMP dysregulation in PAH

  • SMC phenotype switching that is associated with SMC hyperplasia, hypertrophy and migration are regulated by the homeostasis and turnover of ECM components such as elastin, collagen, and fibronectin within the medial layer. An increased deposition of collagen and elastin responsible for the medial thickening during PAH pathogenesis, and this thickening is responsible for the stiffening of the pulmonary arteries observed in PAH.
  • Fragmentation and gaps in the internal elastic lamina have been observed in lung tissue from PAH patients and in patients with PAH and neointimal lesions. Animal studies have also shown that elastin fragmentation precedes the development of changes in vascular morphology. The production of fibronectin within the ECM, which switches the phenotype of the SMC to a proliferative/migratory state, is stimulated by serine elastases and MMPs.
  • When MMP and elastase activity increases, TN-C is increased which enhances PASMC proliferation via positive regulation. Furthermore, increased “expression of the TN-C is associated with progression of clinical and experimental PH. In support, treatment of organ cultures with either MMP-2 or an elastase inhibitor resulted in suppression of TN-C expression, and regression of medial hypertrophy associated with PASMC apoptosis. Subsequent studies in cultured PASMCs documented that TN-C amplifies the mitogenic response to FGF-2 and is a prerequisite for EGF-dependent SMC proliferation.”
  • Crosstalk between MMPs/serine elastases, and growth factors and pro-hypertensive molecules has been observed. For example, MMP activity is affected by BMP: “Knockdown of BMPR1A in human PASMCs reduced MMP-2 and MMP-9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. Furthermore, knockdown of MMP-2 or MMP-9 recapitulated these abnormalities…”.
  • Serine elastases, through degrading the ECM, release bFGF, which may be amplified by MMPs.
  • “MMPs also positively regulate EGF-dependent SMC proliferation by promoting TN-C induced clustering of αvβ3 integrin receptor. In addition, studies from genetic or pharmacological ablation of serotonin receptor 5-HT2BR suggests that 5-HT2BR-dependent increased elastase activity and MMP/TIMP imbalance, subsequently leading to latent growth factor release, including TGF-β. Vice versa, evidence suggests that TGF-β1 stimulates expression of pro-MMP-9 in IL-1β treated PASMCs. IL-1β, a major cytokine associated with PH, was shown to markedly increase MMP-2 and MMP-9 gelatinase activity.”

MMP Inhibitors in Experimental Models of PH

MMP inhibition studies have mostly been performed on the hypoxia- and MCT- experimental animal models of PH. Elastase and MMP inhibitor therapy has proven effective in the MCT-induced PH model by apparently reducing TN-C, suppressing proliferation of SMCs and by inducing apoptosis. Furthermore, “adenovirus-mediated overexpression of human TIMP-1 gene in the lungs of rats exposed to MCT reduced pulmonary vascular remodelling, right ventricular hypertrophy, gelatinase activity and muscularisation of peripheral pulmonary arteries, suggesting that balancing the MMP/TIMP ratio can reverse the disease.”

However, MMP inhibition studies yield mixed results when performed on the hypoxia-induced PH model. In hypoxia induced PH, there is increased synthesis and accumulation of collagen and elastin in the pulmonary arteries. Since MMPs can also degrade collagen, inhibiting their activity could result in increased collagen deposition. Furthermore, “using inhibitors for collagen synthesis (cis-4-hydroxy-L-proline) and crosslinking (β-aminopropionitrile) in the rats exposed to chronic hypoxia, demonstrated reduction of excess vascular collagen and attenuation of PH. However, in contrast, Vieillard-Baron et al. demonstrated that intratracheal instillation of the adenovirus-mediated overexpression of human TIMP-1 gene or administration of a broad-spectrum MMP blocker (doxycycline) in rats subjected to chronic hypoxia was associated with increased muscularisation and periadventitial collagen accumulation in distal arteries.”

Serine elastase inhibitors have also shown promise in reversing PH. Serine elastase inhibitors have been shown to reverse advanced PH in the MCT-induced PH rat model as well as attenuate PH in the hypoxia-induced animal model. “Furthermore, transgenic mice that overexpress the serine elastases inhibitor elafin when exposed to chronic hypoxia demonstrated reduced serine elastase and MMP activity compared to the non-transgenic mice. Importantly, elafin-transgenic mice displayed reduced right ventricular pressure, reduced muscularisation and preservation of the number of distal vessels as compared with control or non-transgenic mice.”

*Side note: In this talk by Marlene Rabinovitch, it is mentioned that 1) circulating elastase levels are significantly elevated in all forms of PAH, and 2) elafin levels, which are natural elastase inhibitors and are usually elevated with elevated levels of elastase to match activity/maintain homeostasis, are decreased in all forms of PAH.*

What’s interesting is that pharmacological agents responsible for regression/reversal of established PH appear to function by modulating the MMP/TIMPs ratio in the ECM as evidenced by the following:

  • “…inhalation of a combined selective PDE3/4 inhibitor (tolafentrine) exhibited anti-proliferative, anti-migratory and anti-remodelling effects, and consequently reversed MCT-induced PH in rats… due to downregulation or even normalisation of the deregulated profile of several MMPs and adhesion molecules…”.
  • “Lercanidipine, a vasoselective dihydropyridine calcium channel blocker, demonstrated beneficial effects in patients with PH by decreasing elevated circulating MMP-9 levels. This lercanidipine-induced effect was associated with a significant decrease in MMP-9 activity without affecting proMMP-2 activity and TIMP-1 concentration…”
  • “Similarly, administration of a third-generation calcium channel blocker, amlodipine, immediately followed by MCT treatment suppressed the MCT-induced increase in MMP-2 activity, platelet activation, EC damage and SMC proliferation, and consequently inhibited the development of PH.”
  • “Administration of the FDA-approved drug for PH, bosentan… also attenuated the MCT-induced upregulation of MMP-2, TIMP-1, endothelial NO synthase expression and MCT-induced PH.”
  • “Amelioration of MCT-induced PH by fluoxetine (selective serotonin reuptake inhibitor) treatment was associated with suppression of MMP-2, MMP-9, TIMP-1 and TIMP-2 expression.”
  • “…captopril (an angiotensin-converting enzyme inhibitor) and losartan (angiotensin II type 1 receptor antagonist) administration attenuated pulmonary vascular remodelling, probably associated with the regulation of the expressions of MMP-2, MMP-9 and TIMP-1, in pneumonectomy plus MCT injection-induced severe PAH.”

Overall, selective or complete MMP inhibition can either attenuate or enhance vascular remodelling and PH, depending on the circumstance and how the PH is initiated. As a result, using selective or complete MMP inhibition is not a currently effective strategy for treating PH.

MMP inhibitor therapy may be a viable treatment for PH once we understand more about MMPs, their functions, as well as their natural inhibitors. Increasing the selectivity of MMP inhibitors, as well as honing in on primary MMP culprits in PH is most likely the key in establishing MMP inhibitor therapy as an effective treatment for PH.

Furthermore, since there are multiple subgroups of PAH, it is most likely critical to obtain the MMP/TIMP profile of each subgroup.


  • Elastin is crucial for inducing a quiescent/differentiated SMC state
  • VEGF downregulates TIMP levels and may allow activation of collagenases and contribute to the invasion of ECs into the basement membrane and interstitial matrix.
  • TGF-β1 is profibrotic, but promotes contractile phenotype in SMCs by decreasing PDGF-BB induced increase in MMP-2. It also attenuates expression of MMP-9 in mast cells. However, TGF-β activates fibroblasts to become myofibroblasts.
  • During inflammation, T-cells can “leak” into perivascular tissue due to expression of MMP-2 on surface of T-cell via interaction with VCAM-1 on ECs.
  • Neutrophil-derived proteases (specifically MMP-9) are responsible for trans-endothelial neutrophil migration and initiating angiogenesis.
  • During hypoxia the tight junction protein occludin is rearranged and its presence is diminished, all due to MMP-9 activation
  • Pericellular proteolysis due to increased MMPs is associated with angiogenesis and dysregulated angiogenesis. VE-cadherin expression is associated with decreased MMPs and proteolysis, as well as with stabilization and maturation of blood vessels.  
  • MMP-9 (and others) enhances the bioavailability of growth factors in the vasculature via degradation of ECM components.
  • MMP-2 can attenuate inflammation via altering properties of chemokines
  • Proteolysis of collagen and perlecan by MMPs generates anti-angiogenic matrikines (arrestin, canstatin, tumstatin, metastatin, endostatin, neostatin, vastatin, restin and endorepellin), whereas proteolysis of fibronectin, laminin, osteonectin and elastin liberates matrikines that promote cell proliferation, migration and angiogenesis.
  • MMP-1 may be responsible for attenuation of collagen deposition, SMC proliferation, infiltration of macrophages and thickening of the media.
  • Not all MMPs and proteolytic activity are “bad”. MMP-1 and MMP-3 increases during recovery from hypoxia induced PH. There is also increased proteolytic activity to reduce collagen and elastin content, as well as activation of mast cell-derived interstitial collagenase (to help facilitate collagen breakdown) in pulmonary arteries during the early recovery from chronic hypoxia.
  • It appears that both excess proteolytic activity due to excess MMP activity, as well as excess collagen synthesis due to low MMP activity contribute to PH pathogenesis. Thus, this provides weight to the case that there must be a delicate balance of MMP/TIMP (proteases and their inhibitors) within the ECM.
  • In PAH, there is a deregulated expression and activity profile of MMP/TIMP
  • Overall, selective or complete MMP inhibition can either attenuate or enhance vascular remodelling and PH, depending on the circumstance. Thus, using selective or complete MMP inhibition is not a currently effective strategy for treating PH, and more studies are warranted. This may be due to the fact that there are particular MMP/TIMP profiles for both physiological and pathological states; certain MMPs may be “good” in one scenario but “bad” in another.
  • Most clinical trials of MMP inhibitors fail to demonstrate efficacy since they induce severe side effects (e.g. musculoskeletal syndrome) most likely due to their lack of selectivity and poor oral bioavailability.