This is a review and summary of a recent paper entitled “Metabolic Changes Precede the Development of Pulmonary Hypertension in the Monocrotaline Exposed Rat Lung”. In this study, metabolomic profiling was used in an animal model of PH to try to identify biomarkers of early stage PH, in hopes of identifying a process to diagnose PH earlier.

At the end of this post, I’ve included a chart summarizing all of the biomarkers found in the study.

Some terms:
MCT = Monocrotaline
SMC = Smooth Muscle Cell
EC = Endothelial Cell

Summary & Key Findings

  • In the MCT animal model, they observed metabolic changes in “multiple pathways associated with the development of PH, including activated glycolysis, increased markers of proliferation, disruptions in carnitine homeostasis, increased inflammatory and fibrosis biomarkers, and a reduction in glutathione biosynthesis.”
  • While less pronounced, the metabolic data derived from this study compared favorably with prior work carried out in humans with PH: “In support of our data in the MCT-exposed rat, PH patient metabolomic data also observed a significant elevation of glucose and fructose 6-phosphate levels [7]. However, the changes in PH patients were less pronounced [7].” Also, a “decrease in fatty acid beta-oxidation was also observed in humans with PH [7]. In this study we also found that several conjugated carnitine-fatty acid intermediates were decreased in the pre-PH lung suggesting that decreased beta-oxidation may be involved in the development of PH. As restoring carnitine homeostasis has been shown to resolve endothelial dysfunction [10, 18, 19] this may also be a potential therapeutic target in PH.”
  • There are a variety of PH animal models but they all differ from the human form of PH in terms of how the disease is physically manifested in the later stages. In the common MCT model, you see primarily 1) SMC proliferation instead of endothelial cell proliferation, and 2) concentric lesions in the lung as opposed to plexiform lesions in the human form. However, since early changes in the MCT model closely resemble changes in human PH, the MCT model may provide insight into biomarkers for predicting early stage PH.
  • They found that “suggesting that despite its failure to recapitulate all the structural characteristics of human PH, the MCT model recapitulates much of the metabolic changes occurring during the development of PH.”
  • How the MCT animal model works: “Following injection, MCT is metabolically activated to a pyrrolizidine alkaloid in the liver; this compound exhibits extensive toxicity toward pulmonary endothelial cells, resulting in decreased barrier function, edema and eventually fibrosis. As a result, pulmonary vascular resistance increases and the right ventricle of the heart compensates by hypertrophy, leading to its eventual failure.”
  • To test metabolic markers, blood was removed from pulmonary vessels.
  • Induction and validation of early stage PH: “Our hemodynamic data, collected after 14 days of MCT treatment, demonstrate a significant increase in right ventricle peak systolic pressure (RVPSP), and indicate that at this early time point there is already active pulmonary vasoconstriction… Thus, 14 days of MCT exposure induces a very early stage of PH (pre-PH) that exhibits only pulmonary vasoconstriction with no significant changes in the primary determinants of developed PH, RV work load and hypertrophy.”
  • They found that “compared to control group, there are significant elevations in glucose (~10-fold), glycolytic intermediates (glucose 6-phosphate and fructose 6-phosphate) and glycolytic products (pyruvate and lactate).” While this is not surprising to find in PH in general, it is interesting to find that this shift to glycolysis takes place in pre-PH development. They also found elevated levels of pentose phosphate pathway metabolites which indicates an increase in nucleic acid synthesis and amino acid synthesis, as well as an increase in NADPH. NADPH can be used for both lipid synthesis as well as the reduction of oxidized glutathione. Since NADPH is itself a reducing agent, the authors suggest that perhaps this pathway is activated in response to increased oxidative stress.
  • It is known that there is a disruption in carnitine homeostasis in pulmonary hypertension. What they found was that while “long chain fatty acids were not significantly changed as a class… significantly decreased levels of long-chain acylcarnitines (palmitoylcarnitine, stearoylcarnitine, linoleoylcarnitine, and oleoylcarnitine) [were observed] in the pre-PH lung.” This is interesting as another paper observed an elevation of acylcarnitines in the peripheral blood. Perhaps this is due to the difference between peripheral blood and pulmonary arterial blood?
  • Overall, they concluded that the decrease in acylcarnitines “suggests that there is a reduced utilization of fatty acids for beta-oxidation, though the ketone body 3-hydroxybutyrate (BHBA) was significantly increased (3.02 fold, data not shown) pointing again on increased glucose metabolism that can produce BHBA. Taken together, these data are again indicative that fatty acid beta-oxidation is altered prior to the development of PH and, in conjunction with increased glycolytic products, are consistent with a Warburg-like metabolic shift.” I don’t quite understand the BHBA glucose link. I didn’t think glucose metabolism produced BHBA… Ketone bodies are produced from Acetyl-CoA. Acetyl-CoA can come from beta-oxidation of fatty acids or from pyruvate entry into the TCA cycle (pyruvate is converted into Acetyl-CoA via PDH enzyme in the mitochondria). However, in PH, both beta-oxidation and glucose oxidation (TCA cycle), are disrupted, and glycolysis is favored. Thus where is the elevated BHBA coming from, if both pathways that produce Acetyl-CoA are disrupted?
  • Regarding inflammatory markers, they found that “Omega-6 fatty acids (for example, arachidonate, docosadienoate and dihomo-linoleate), which are precursor compounds for prostaglandin biosynthesis, were increased in the pre-PH lung (Fig 5A). These metabolites can be further processed by lipoxygenase (LOX) and cyclooxygenase (COX) enzymes to generate inflammatory eicosanoids, such as prostaglandin E2, prostaglandin D2, prostaglandin J2, and leukotriene B5, all of which were increased in pre-PH lungs (Fig 5B).” They also noticed increases in kynurenine/kynurenate, tryptophan, serotonin and histamine.
  • They noticed elevations in markers of phospholipid degradation and membrane remodeling. They also noticed an increase in methylhistidines, which are “produced by methylation of actin and myosin in muscle, [and] provide an index of the rate of muscle protein breakdown.” Additionally, they found “increased 1- and 2-stearoylglycerophosphoglycerols (derived from the mitochondrial inner membrane component cardiolipin) and N-formylmethionine, a breakdown product of mitochondrially-encoded/ synthesized proteins, which may reflect mitochondrial damage resulting from stress or the induction of cell death.” This is interesting, since a current postulate of PH initiation is that endothelial cell damage causes cell death and then a corresponding outgrowth of a pro-proliferative anti-death endothelial cell. If cell death is indeed causing the release of these mitochondrial damage markers, then this contraindicates the hypothesis that as PH develops, it is due to an EC population that resists cell death only. Instead, it gives weight to the alternate hypothesis that EC death occurs cyclically throughout PH with corresponding intervals of proliferation.
  • Furthermore, it is known that following damage caused by MCT in lungs, hyaluronic acid in the extracellular matrix is degraded, and consistent with this, they observed increases in metabolites of hyaluronic acid in the pre-PH lung. They also observed that markers of collagen breakdown, e.g. trans-4 hydroxyproline, were increased.
  • Arginine can be converted into NO via Nitric Oxide Synthase or via arginase in the urea cycle. The urea cycle is typically carried out in the liver, but the endothelium of the lung also contains arginase and other enzymes necessary for urea cycle operation. Arginine utilization via the urea cycle produces ornithine. They found that “in the PH lung there is a significant increase in urea suggesting that L-arginine is mainly utilized by arginase. The arginase product, ornithine, required for polyamine and proline biosynthesis, is also increased in the PH lung”. Ornithine metabolites are necessary both to support cellular growth and proliferation, and the ornithine metabolite spermidine, was found to be increased in PH lung. Also, as mentioned above, ornithine is used for proline biosynthesis, and proline metabolites are believed to play a role in fibrosis. In the pre-PH lung, the found that the proline metabolites 4-hydroxyproline and proline-hydroxyproline were increased.
  • They also found evidence for disturbances in redox homeostasis, as reduced glutathione, “cystine (an oxidative product of cysteine), methionine sulfoxide and N-acetylmethionine sulfoxide (products of methionine oxidation) were all increased in pre-PH rats.” They also found an increase in S-adenosylmethionine and a “decrease in S-adenosylhomocysteine suggestive of a high demand for glutathione.”
  • Further evidence of increased glutathione demand as well as increases in oxidative stress come from the following: “Increased levels of 2-hydroxybutyrate (AHB), which is produced as a byproduct when cystathionine is converted to cysteine in times of high glutathione demand (such as in response to an oxidative environment), were also identified in the pre-PH lung (Fig 10), while increases in ophthalmate and norophthalmate, a tripeptide analogue of GSH produced by glutathione synthase in which cysteine has been replaced by 2-aminobutyrate, are also consistent with increased demand for glutathione synthesis. Elevated levels of gamma-glutamyl amino acids (AA, Fig 10B) were accompanied by increases in 5-oxoproline (Fig 10A) and may reflect increased gamma-glutamyl AA exchange to replenish GSH. Finally, trends in increased tocopherols and carnosine (a histidine-derived dipeptide with anti-oxidative capacity), and significant increases in 12-HETE (which is generated by free radical oxidation of arachidonate, Fig 10) are consistent with increased oxidative stress.”
  • Thus, while glutathione recycling was found to be increased, “as indicated by increases in the 5-oxoproline metabolite, both reduced and oxidized glutathione are significantly decreased in the pre-PH, indicative of increased oxidative stress.” Also, the significant increases in gamma-glutamyl amino acids observed are a result of activation of the gammaglutamyl cycle, which is the cycle that is usually associated with increased inflammation.

Other things learned:

  • “The pro-inflammatory damage associated molecular pattern (DAMPs) protein HMGB1 was also found to be a contributing factor in the development of PH through its ability to activate the TLR4 receptor.”
  • Evidence is consistent with “TLR4 receptor and fibroblast/macrophage activations in the pathogenesis of PH.”
  • “The balance between omega 3 and 6 fatty acids is very important for inflammatory response and cytokine production in lungs [36]. Although, both are polyunsaturated fatty acids, omega 3 exhibits anti-inflammatory properties [37, 38] whereas omega 6 generally promotes inflammation…” They found that “there is disruption of the 6:3 ratio with an increase in total omega 6 pro-inflammatory fatty acids, specifically arachidonic acid. The formation of prostaglandins from arachidonic acid via cyclooxygenase results in an inflammatory microenvironment that attracts and stimulates immune cells [41–43]. Interestingly, treatment with a lipid emulsion containing omega-3 fatty acid in newborns with persistent PH increased left pulmonary blood flow by 30% and decreased pulmonary vascular resistance by 28% [44]. This suggests that restoring the balance between omega 6 and 3 fatty acids could be a potential new therapeutic target in PH.”
  • Glucosamine and hydroxyproline contribute to ECM remodeling, and the “glucosamine pathway starts from elevated levels of fructose-6-phosphate, which is upregulated in PH lungs due to a glycolytic switch in the cell’s metabolism. Thus, the glycolytic switch in PH can alter the proliferation of vascular cells, leading to the activation of inflammatory cells and ECM remodeling producing both fibrosis and plexiform lesions.”

Questions for further understanding:

  • Ketone bodies are produced from Acetyl-CoA. Acetyl-CoA can come from beta-oxidation of fatty acids or from pyruvate entry into the TCA cycle (pyruvate is converted into Acetyl-CoA via PDH enzyme in the mitochondria). However, in PH, both beta-oxidation and glucose oxidation (TCA cycle), are disrupted, and glycolysis is favored. Thus where is the elevated BHBA coming from, if both pathways that produce Acetyl-CoA are disrupted?

Biomarker Summary:

Review #4 Supplemental Chart

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