In pulmonary hypertension, there is pathological remodeling of the blood vessels due to a pathological hypertensive environment within the vasculature. This hypertensive environment influences how all cells of the vasculature (endothelial cells and smooth muscle cells), as well as fibroblasts and immune cells behave. The environment typically activates fibroblasts and immune cells, causes de-differentiation in smooth muscle cells, induces the contractile phenotype in smooth muscle cells, and may even pushes endothelial cells to failure. How this pathological hypertensive environment occurs is unknown. For example, it could arise from underlying inflammation, from hypoxia, or an apoptosis process gone haywire… There are many theories. One theory in particular, however, posits that overproduction of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) can induce these hypertensive changes.
It is well documented that there is altered production of both ROS and RNS in pulmonary hypertension. There is also evidence that this altered ROS/RNS plays a role in PH pathology. However, should we target ROS/RNS for treatment of PH? To answer this we need to answer the following questions first:
- Is there a net increase or decrease in ROS/RNS in PH? Or a net increase or decrease in certain ROS/RNS species relative to others?
- Which ROS/RNS species are detrimental and which are beneficial?
- Do altered levels of ROS/RNS cause PH or contribute to PH pathophysiology?
- Are altered ROS/RNS species a compensatory mechanism of the vasculature in response to PH? Does PH worsen when you remove certain sources of ROS/RNS?
- Do altered ROS/RNS species induce a cascade of cell signaling, whereby the ROS/RNS species triggers angiogenesis, altered immune responses, and fibrosis via a variety of cell receptors and enzyme activation etc.?
Answers to these will give us clues to whether ROS/RNS is a byproduct of PH, is actively contributing to PH, or is attempting to attenuate PH in some odd manner. The recent article entitled “Reactive Oxygen and Nitrogen Species in the Development of Pulmonary Hypertension” by Fulton et al., attempts to answer some of these questions, and is the subject of today’s post.
Blood vessels in the body react to levels of oxygen throughout the body in different ways. Low oxygen (hypoxia) will cause the systemic blood vessels (vessels that supply whole body circulation, i.e. arms, legs, etc.), to vasodilate in order to increase perfusion. If there is low oxygen, the body wants to make sure it is efficient at transferring all available oxygen to all parts of the body equally. If the blood vessels are dilated, then this increases perfusion and blood delivery throughout every area of the body, to ensure what little oxygen there is available is equally distributed. However, the lung vasculature behaves in an opposite manner. In response to low oxygen, the pulmonary circulation will constrict in certain areas, in order to redirect blood flow to areas of the lung with the greatest oxygen concentration. This is done in order to increase the efficiency of oxygen take-up by the lungs. If only certain areas of the lungs are receiving oxygen, blood should be directed to those regions in order to pick it up.
How do the lungs do this? The answer is ROS. It is well known that ROS induce this physiological vasoconstriction in the pulmonary arteries during hypoxia. While the mechanism is complicated, and typically occurs in three stages – acute, sustained, and chronic – each of which are mediated by different mechanisms, the general gist is this: When oxygen levels are reduced (e.g. under hypoxia conditions), so are the ROS, and this acts to constrict the arteries in the areas of the lungs affected by low oxygen in order to maximize blood flow to other higher oxygen regions (to maximize blood oxygen uptake). As Fulton et al. explain, mitochondria respond to low oxygen “by altering the production of ROS which compromises potassium channel function, leading to the depolarization of smooth muscle cells, activation of voltage-sensitive calcium channels, and an influx of calcium that can initiate and amplify smooth muscle contraction.”
To complicated matters further, RNS released from the endothelium opposes and/or tames this hypoxic vasoconstriction caused by ROS. What are RNS? Primarily Nitric Oxide (NO) as well as compounds produced upon reaction with NO.
NO is a potent vasodilator in the pulmonary circulation. It is synthesized in the vascular endothelium by endothelial nitric oxide synthase. Once formed, NO then diffuses through the vasculature to bind to and activate soluble guanylate cyclase (sGC) in the smooth muscle cells. Activation of sGC produces cGMP which then goes on to stimulate protein kinase G (PKG) to induce smooth muscle relaxation.
ROS/RNS and Cell Signaling
ROS and RNS are implicated in both physiological and pathophysiological cell signaling. ROS include the following reactive molecules: superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radical (•OH-), and hypochlorite (OCl-). Hydrogen peroxide is important for proper physiological functioning of the blood vessels (H2O2 helps to keep the pulmonary arteries vasodilated), but it also can be damaging. All of the other ROS are mostly damaging in nature.
RNS include the free radical NO, a necessary species for proper physiological functioning of the blood vessels (especially in the case of pulmonary hypertension, where nitric oxide levels are reduced). However, nitric oxide can also go on to form the toxic radical peroxynitrite (ONOO-) through interaction with superoxide species.
As Fulton et al. explain, ROS and RNS function by influencing “cellular behavior through a variety of post-translational mechanisms. They can bind to proteins via susceptible iron centers, cysteine or tyrosine residues in addition to lipids and DNA… Post-translational changes in proteins include (but are not limited to) NO binding to soluble guanylate cyclase (sGC) and the production of cyclic guanosine monophosphate (cGMP), NO binding to cytochrome C oxidase, NO binding to cysteine residues (S-nitrosylation), oxidation of cysteine and methionine residues (disulfides, cystine), and ONOO− induced tyrosine nitration.”
Sources of ROS
The amount of ROS present at any time is a function of the rate and amount of enzymes synthesizing ROS, as well as the rate and amount of enzymes or antioxidants which neutralize or break down the ROS. Sources of superoxide, for example, include the mitochondrial electron transport chain, NADPH oxidases (Nox1-5), lipid oxygenases (cyclooxygenase, lipoxygenase and cytochrome P450), nitric oxide synthases, and xanthine oxidase. Let’s take a look at each of these sources in a bit more detail…
Nox are enzymes that reside within the membranes of the cell or the membranes of internal cell organelles. Once activated, they transfer electrons from NADPH to oxygen, which occurs on the inside of the cell and/or organelle. The enzyme then transfers the product, superoxide, outside of the cell and/or organelle. Nox activation is diverse and typically requires other transmembrane protein subunits, such as p22phox, p67phox, p40phox, Rac, NOXO1, and NOXA1. In depth explanations and references can be found in the article by Fulton.
The one interesting Nox, Nox4, is regulated a bit differently than the others. It only requires p22phox, and it is constitutively activated and continuously produces ROS, specifically hydrogen peroxide. Nox1-3 and 5 all produce a mixture of superoxide and H2O2, but Nox4 primarily converts superoxide into H2O2. Nox4 activity is most likely regulated by changes in the local oxygen concentration since Nox4 has an very high Km for oxygen (which means that it does not function optimally in low oxygen concentrations). Nox5 is different. It is activated by elevated intracellular calcium levels, and its activity can be regulated by calmodulin and Hsp90 and Hsp70.
The mitochondria are the powerhouses of the cell. They are solely responsible for providing the energy and ATP needed for life for oxygen breathing mammals. The electron transport chain is the mechanism in the mitochondria by which electrons are transferred to oxygen. In this process, there is a buildup of chemical potential energy between the inner and outer walls of the mitochondria that is eventually utilized to generate ATP. During this whole process, electrons are shuttled around. Specifically, they are shuttled around by different transmembrane protein complexes in the mitochondria. Mishandling, malfunctioning, or simple randomness can result in one of these electron species being released. In fact, because mitochondria deal with electrons so much, they harbor a specific antioxidant enzyme, superoxide dismutase (SOD) to scavenge common ROS like superoxide in the electron transport chain. Superoxide leakage typically occurs at both complex I and III, but the highest leakage occurs at complex I.
In order to minimize ROS, you need efficient mitochondria. As Fulton et al. explain, inside the mitochondria “the respiratory chain can assemble into higher molecular weight supramolecular structures called supercomplexes. Supercomplexes provide kinetic efficiency and are thought to limit the production of ROS. On the other hand, impaired mitochondrial structure and function as seen with aging, diabetes, and ischemia reperfusion results in greater ROS production from the mitochondria.”
However, another factor for ROS production is the level of oxygen. As with the Nox enzymes, ROS produced from the mitochondria have a positive linear correlation with oxygen concentration: the higher the oxygen concentration, the higher the amount of ROS produced within the mitochondria.
Other Sources of ROS
In addition to the above-mentioned sources, the pulmonary circulation also harbors enzymes that are capable of ROS production, namely the arachidonicoxygenases like cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450, as well as xanthine oxidase, and uncoupled endothelial nitric oxide synthases (eNOS). How ROS production occurs with each of these enzymes:
- COX, LOX, and Cytochrome P450: oxygen and arachidonic acid combine, and due to inefficient enzyme activity, superoxide can form.
- Xanthine oxidase: xanthine oxidase is involved in the catabolism of purines. It catalyzes the oxidation of hypoxanthine to xanthine, and then to uric acid, all producing H2O2 and superoxide along the way.
- eNOS: normally eNOS synthesizes vasoactive nitric oxide for vasodilation. However, when the cofactor for this reaction, tetrahydrobiopterin (BH4), is low or depleted, eNOS will uncouple to form superoxide.
- Vascular peroxidase 1 (VPO1 or PXDN): this enzyme is expressed in smooth muscle cells, and converts H2O2 to hypochlorite (OCl−).
Sources of RNS
In the vasculature, eNOS enzymes consume L-arginine together with oxygen and NADPH to form NO. In the presence of superoxide, NO can form the potent oxidant peroxynitrite ONOO-, and in the presence of molecular oxygen, NO can form the potent nitrogen oxides oxidants NO2 and N2O3.
ROS/RNS in PH
In PH, pathological remodeling of the pulmonary arteries is accompanied by altered ROS and RNS production and removal. In both human and experimental models of PH, there is ample evidence of ROS levels being increased relative to normal. The reason for this increase is due to either 1) increased production of ROS relative to removal, or 2) decreased removal rates relative to production. Which of these mechanisms is responsible for increased ROS depends on the circumstance. In order to get a better idea of how the altered ROS/RNS in PH comes about, we need to look at each source of ROS/RNS individually, and how the sources are affected in PH.
There is conflicting evidence on whether or not Nox1 plays a role in PH. Nox1 is expressed in both endothelial cells and smooth muscle cells. It’s expression is increased in the pig hypoxia model of PH and the rat MCT model of PH. However, in the fawn hooded rat PH model and the SUGEN/Hypoxia model (the animal model most closely resembling the human PH form due to the presence of plexiform like lesions in the animal model), Nox1 expression is unchanged. Additional conflicting results are as follows:
- Nox1 knockout in mice promotes PH under normoxic conditions (possibly via a mechanism involving reduced apoptosis and decreased smooth muscle cell expression of the Kv1.5 potassium channel, according to Fulton et al.)
- Hypoxia induced PH is reduced in Nox1 knockout mice
Odd right? However, Fulton et al. posit that the reasons for these diverse results could be due to differences in sex of the organism studied, as well as to studying isolated blood vessels versus whole lung blood vessels. More importantly, changes in Nox1 expression may not represent changes in enzyme activity since Nox1 (unlike Nox 4 below) must be activated via post-translational means in order to produce ROS. That is, just because Nox1 is expressed doesn’t mean it is active. Something else has to activate it.
Nox 2 produces large amounts of ROS. It is expressed primarily in immune cells, and to a lesser extent in endothelial cells and fibroblasts. Nox 2 expression is increased in the rat MCT animal model of PH and the pig hypoxia animal model of PH, but not the mouse hypoxia model of PH.
In the MCT rat model, as well as the hypoxic mice, rat, and pig animal model, the Nox inhibitor apocynin (which inhibits p47phox-dependent activation of Nox2) helps to ameliorate PH. Additionally, genetic deletion of p47phox (NCF1) also reduces PH. We may be led to believe by this evidence that inhibiting Nox2 would be a beneficial therapy for PH. We should use caution however when considering Nox2 knockout therapies for humans since Nox2 is necessary for the immune system to fight infections.
Nox 3 is expressed in the media and adventitia of the pulmonary blood vessels, however, changes in its expression are not seen in the development of PH.
Nox4 is expressed in all layers of the pulmonary blood vessel wall, but appears to have a slightly higher abundance in the intima and adventitia of the vessel. As Fulton et al. explain, the “relative abundance and location of Nox4 in the respective cell types is likely to vary and to be influenced by factors such as the species, the type of vascular bed, blood flow and pressure, inflammation, growth factors and oxygen concentrations.” This is interesting because this may hint that Nox4 may play a more prominent role in PH compared to other Nox enzymes if its expression is influenced by factors like hypoxia and blood pressure and flow. For example, what if factors that cause PH, like hypoxia, alter Nox4 which contributes to PH development? Or what if high blood pressure in the lungs as a result of PH alters Nox4 which worsens PH?
Indeed, Nox4 does appear to be most influential to the development and course of PH. Nox4 expression is increased in both human PH and mouse and rat models of PH. Furthermore, inhibition or loss of Nox4 reduces PH in many animal models. While recent experiments do show that increases or decreases in Nox4 don’t alter PH in hypoxia mouse models of PH, it is hypothesized that these results are due to the fact that Nox4 changes are minimal in hypoxia models of PH. Moreover, high oxygen concentrations are needed for Nox4 activity.
Like Nox3, Nox5 is expressed in the media and adventitia of the pulmonary blood vessels, however, changes in its expression are not seen in the development of PH.
The role of each Nox enzyme in PH is summarized in the following table.
[pdf-embedder url=”http://searchingphoracure.com/wp-content/uploads/2017/09/Role-of-Nox-enzymes-in-Pulmonary-Hypertension-PH-LIT-REVIEW-11-Sheet1-2.pdf” title=”Review #4 Supplemental Chart”]
In PH, several mitochondrial abnormalities are observed which affect ROS levels:
- SOD2, the mitochondrial enzyme for scavenging superoxide ROS, is decreased
- Electron transport chain protein levels are decreased
- Enhanced mitochondrial fission and decreased mitochondrial fusion is observed
- Decreased glucose oxidation and enhanced glycolysis is observed
These observations suggest that ROS levels are increased in PH. However, again, it is unclear if increases in ROS play a causative role in PH. Perhaps whether ROS is causative or not depends on the species of ROS involved? Or the ratio of ROS/RNS?
Recent experiments in mouse hypoxia PH models have shown that reducing superoxide via enhancement of SOD2 gene expression exacerbated PH, whereas reducing hydrogen peroxide via enhancement of catalase expression reversed PH. However, other research indicates that mice lacking SOD1 exhibit exaggerated chronic hypoxic PH.
This indicates that effects of ROS may depend on species, as well as the type of antioxidant enzymes expressed to reduce those enzymes, e.g. in chronic hypoxia mice models, SOD2 expression = “not good” but SOD1 expression = “good”. But this may not hold for other species. Fawn Hooded Rats, known for spontaneously developing PH, have reduced expression of SOD2. Humans with PH show decreased SOD2 expression in hypertensive regions of pulmonary arteries, and in plexiform lesions. Furthermore, humans with polymorphisms in SOD2 show increased risk for PAH.
Other sources of ROS
Among the other sources of ROS (xanthine oxidase, COX, VPO1, and cytochrome-p450), xanthine oxidase is the source that plays the most prominent role in PH. Under hypoxia, xanthine oxidase expression and activity is increased. Inhibiting xanthine oxidase with allopurinol or other inhibitors stops the pulmonary vascular remodeling process.
Removal of ROS is just as important as its generation. Some of the major anti-oxidant enzymes include SOD (described at length above), heme oxgenase (HO), glutathione peroxidase, and thioredoxin. Genetic deletion of HO enhances hypoxia induced PH, whereas increasing expression of HO prevents the development of PH. Furthermore, reduced expression and activity of glutathione peroxidase and thioredoxin have also been reported in PH.
TGF Beta Receptor Family
TGF-beta1, BMPR2, and ALK1 are all receptors that belong to the TGF-beta superfamily of receptors. Each can influence ROS in different ways. TGF-beta1 is a potent inducer of Nox expression in vascular cells. Additionally, BMPR2 mutations, which are known to make individuals susceptible to PH, can induce ROS via activation of the immune system as well as changes in Hsp90 protein expression (as discussed above, Hsp90 protein is necessary for Nox activation). ALK1 can induce ROS formation via uncoupled eNOS.
RNS in PH
Any situation or condition that chronically alters NO levels can influence one’s susceptibility to get PH. Conditions that can alter NO levels include a loss in the ability to synthesize NO, or decrease in NO synthesis rate (e.g a decrease or loss of eNOS activity), or blood disorders like hemoglobinopathies. Hemoglobinopathies can cause an increase in free hemoglobin in the plasma. Free hemoglobin is a potent scavenger of free NO. Hemoglobinopathies also typically increase plasma levels of arginase 1, an enzyme that metabolizes L-arginine thus reducing its availability for NO synthesis.
It is well known that in PH, there is reduced NO bioavailability, most likely due to reduced eNOS expression or activity, dysfunctional eNOS activity, or enhanced scavenging of NO by either free hemoglobin or by ROS. Since hemoglobinopathies only occur in a subset of PH patients, eNOS and ROS appear to be the main culprits responsible for reduced NO bioavailability in all forms of PH.
eNOS expression can either be increased, decreased, or unchanged, depending on the form of PH. Regardless of this however, there is an overall net decrease in eNOS function in all forms of PH due to compromised post-translational regulation (i.e., after the eNOS protein is made, its regulation is altered such that its activity is decreased). As a result of this, either eNOS activity can decrease, or its function can be impaired, as in the case of eNOS uncoupling. eNOS uncoupling occurs when the levels of its cofactors or substrates are altered. Decreased levels of tetrahydrobiopterin (BH4) and L-arginine, as well as increased levels of asymmetric dimethylarginine (ADMA) can uncouple eNOS. Decreased BH4, L-arginine, and increased ADMA are all observed in PH. Once uncoupled, eNOS begins to produce superoxide instead of NO.
There is ample evidence that eNOS uncoupling contributes directly to PH:
- Decreased BH4 levels increase the severity of PH
- Enhancing the expression of enzymes that synthesize BH4 protects mice from developing PH
- Supplementation with BH4 increases bioavailable NO and decreases PH
Reduced NO bioavailability also occurs by reaction of NO with ROS like superoxide, forming peroxynitrite as a product. Once formed, peroxynitrite goes on to alter tyrosine residues in proteins in a process called protein nitration. In PH, there is ample evidence of increased protein nitration, indicating a large portion of bioavailable NO is being scavenged by ROS to produce peroxynitrite.
eNOS is actually a multi-protein complex, and several binding factors regulate its activity and influence its ability to produce NO vs. superoxide. For example, calmodulin and caveolin-1 are two proteins that bind to eNOS and influence its activity. Caveolin-1 binding to eNOS inhibits eNOS synthesis. Interestingly, caveolin-1 is reduced in human and experimental PH. If this were there case, one would suspect that eNOS activity should be improved in PH. But others have found that this loss of caveolin-1 leads to a hyperactive eNOS which paradoxically can lead to impaired vasodilator activity. How? Because the hyperactive eNOS leads to excess peroxynitrite formation due to either 1) excess NO from hyperactive eNOS combining with increased local levels of superoxide, and 2) excess superoxide derived from the hyperactive uncoupled eNOS population reacting with local levels of NO. In either case, the excess peroxynitrite produced from hyperactive eNOS activity (uncoupled or not) goes on to nitrate PKG proteins which impairs vasodilation. They proved this because they found elevated PKG nitration in PH, which only occurs with elevated peroxynitrite activity. Furthermore, both superoxide scavengers and eNOS inhibitors reversed this induced PH, respectively, in mice lacking caveolin-1.
How does lack of caveolin-1 induce hyperactive eNOS? This is not clear. But we do know that caveolin-1 serves to specifically inhibit eNOS that lacks BH4, thus reducing its capacity to become uncoupled. Caveolin-1 also represses Nox and Mitochondrial ROS. In light of these findings, it can be thought of that caveolin-1 is a regulator of eNOS, serving to keep eNOS in check, and preventing it from being overactive. Whether it’s normal overactivity or uncoupled overactivity, overactive eNOS can lead to excess peroxynitrite through the mechanisms described above.
Interestingly, superoxide and Nox enzymes also regulate eNOS. Elevated superoxide levels paradoxically stimulate eNOS to produce NO, but this NO is not bioactive as it quickly reacts with superoxide to form peroxynitrite. Nox4 (which remember is highly expressed in the endothelium) also stimulates eNOS via gene expression. However, peroxynitrite formation isn’t as much of a concern here since Nox4 primarily produces H2O2. Due to this, as well as the fact that Nox4 is in a prime position to stimulate eNOS (because eNOS and Nox4 are both localized in the endothelium), it is possible that Nox4 inhibition could be detrimental. However, it could also be the case that in PH, since there are reduced eNOS cofactors like BH4, Nox4 inhibition could be beneficial as this would prevent uncoupled eNOS stimulation.
In PH, the ability of the pulmonary arteries to vasodilate in response to NO from endothelial sources is compromised. To recap, once formed in the endothelium, NO traverses to the smooth muscle cells to activate sGC, which then goes on to produce cGMP which then activates PKG to induce vasodilation. However, elevated ROS and RNS can oxidize the sGC receptor, rendering it immune to the effects of NO. Other aspects that hinder vasodilation of the media layer include elevated PDE5 levels (which consume cGMP preventing it from activating PKG), as well as altered PKG activity due to nitration of tyrosine residues on the protein due to elevated ROS and RNS. An interesting side note is that in PH, there is elevated sGC as well as elevated plasma and urine levels of cGMP. If NO/smooth muscle cell activation is compromised, then we would expect the opposite, i.e. lower cGMP levels. The authors posit that this could be due to the fact that natriuretic peptides like ANP and BNP, which are also elevated in PH, stimulate sGC activity.
Paradoxically, NO derived from eNOS, as well as Nox4 activity, contribute to angiogenesis. Dysfunctional angiogenesis leads to plexiform lesions in PH. Oddly, elevated eNOS and eNOS activating factors (PI3K, Akt, and Src) have been found in plexiform lesions.
Current Treatments Targeting ROS/RNS
Current FDA approved treatments of PH include prostacyclin analogues, PDE5 inhibitors, calcium channel blockers, and endothelin receptor antagonists, which all aim to improve vasodilation of the pulmonary arteries. No treatments currently directly target ROS/RNS levels, but one class of treatments, the PDE5 inhibitors, indirectly target RNS signaling pathways. PDE5 inhibitors prevent the degradation of cGMP species. Thus PDE5 inhibitors influence the downstream effects of RNS signaling.
Why do this? Why not supplement with NO directly? And do any of these drugs help? Direct use of NO is tricky as it is a gas and there are complications with dosing, drug delivery, and tolerance. Also, not all patients respond equally to these medicines; some are non-responsive. Furthermore, these medicines don’t reverse pulmonary hypertension, but help to improve symptoms and slow progression. Therefore, it appears then that targeting vasodilation is not effective at treating the underlying cause of PH. Treatments that target metabolism, the immune system, angiogenesis, cell proliferation, as well as ROS/RNS, are perhaps more promising strategies to treat PH.
For example, instead of targeting the byproduct of NO (i.e. vasodilation), why not target the sources of ROS/RNS. Perhaps supplemental BH4, antioxidants, or even Nox inhibitors are viable treatment strategies? We must proceed with caution here however. As Fulton et al. explain: “Approaches to bolster broad spectrum antioxidant pathways using chemical or genetic approaches have been efficacious in attenuating PH in animal models, but equivalent antioxidant strategies in the treatment of cancer and atherosclerosis have not proven effective in humans. This may relate to the luxury of timing in animal models, the use of optimal doses and the simple nature of preclinical models versus the complexity of human disease, as well as the multiple sources of ROS which may be both beneficial and detrimental.”
Thus, more careful studies are needed since we’ve seen conflicting results in animal studies regarding ROS/RNS treatments. Overall, we still need to have a better understanding of which species of ROS and RNS contribute to PH pathology and ensure that by targeting pathological ROS/RNS levels, we are not removing/altering other sources of physiological ROS and/or the body’s protective defense mechanisms (for example, if we inhibit Nox4, we may inhibit the expression of physiological eNOS).
Among the all of the sources that produce or influence ROS levels, Nox4 is the one that most likely plays a role in PH. Nox2 may also play a role, but this role is secondary as it is most likely a byproduct of a dysregulated/overactive immune system (and thus the immune system may be the primary agent contributing to PH). Other enzymes that also play a role are xanthine oxidase and hemeoxygenase.
Among the sources of RNS, a dysregulated and hyperactive eNOS most likely contributes to PH due to enhancing production of superoxide as well as peroxynitrite.
Thus some “potentially” good treatment strategies might be to:
- Decrease Nox4, only if Nox4 stimulated eNOS is uncoupled: since Nox4 can stimulate eNOS, inhibition could be beneficial only if the eNOS is uncoupled. Otherwise, Nox4 inhibition may impact eNOS expression and impair vascular homeostasis.
- Use caution when implementing a strategy that alters NO, eNOS, and Nox4, since these influence angiogenesis, and dysfunctional angiogenesis can lead to plexiform lesions.
- Decrease Xanthine Oxidase, which can be done via therapeutic drugs or via diets: diets like the paleo diet can reduce xanthine oxidase activity. As a side note, diets high in fructose, as well as vegan diets, can lead to elevated uric acid plasma levels. Diets high in protein can decrease serum uric acid levels and gout.
- Increase Heme Oxygenase
- Improve mitochondrial efficiency, which will minimize damaging ROS
- Decrease hydrogen peroxide levels, only if H2O2 is detrimental. It appears that in some animal studies, removing H2O2 is beneficial. However, H2O2 is also necessary for pulmonary vasodilation. Decreasing H2O2 can be achieved perhaps via glutathione supplementation since glutathione scavenges hydrogen peroxide.
- Stabilize eNOS, perhaps by using supplemental BH4, or by targeting its cofactors.
- Increase or stabilize Caveolin-1: since Caveolin-1 inhibits hyperactive eNOS and represses Nox and Mitochondrial ROS.
Questions That Remain
- Is xanthine oxidase activity elevated in other forms of PH (not just hypoxia)?
- Are heme oxygenase levels decreased in all forms of PH?
- Are Nox enzymes really causative? Or are they triggered by altered TGF-beta and/or BMPR2 activity?
- What average levels of each ROS and RNS species in each tissue compartment of the pulmonary artery vasculature during physiological functioning and during PH progression.
- Are elevated NO and eNOS in plexiform lesions a compensatory mechanism? Or are elevated eNOS in plexiform lesions uncoupled eNOS?
- How does oxygen therapy affect ROS species in PH. ROS from Nox and mitochondria increase and decrease in proportion to oxygen concentration (e.g. the higher the oxygen concentration, the higher the amount of ROS produced within the mitochondria).