Heme induces rapid endothelial barrier dysfunction via the MKK3/p38MAPK axis.

Several studies demonstrate that hemolysis and free heme in circulation cause endothelial barrier dysfunction and are associated with severe pathological conditions such as acute respiratory distress syndrome, acute chest syndrome, and sepsis. However, the precise molecular mechanisms involved in the pathology of heme-induced barrier disruption remain to be elucidated. In this study, we investigated the role of free heme in the endothelial barrier integrity and mechanisms of heme-mediated intracellular signaling of human lung microvascular endothelial cells (HLMVECs). Heme, in a dose-dependent manner, induced a rapid drop in the endothelial barrier integrity of HLMVECs. An investigation into barrier proteins revealed that heme primarily affected the tight junction proteins zona occludens-1, claudin-1, and claudin-5, which were significantly reduced after heme exposure. The p38MAPK/HSP27 pathway, involved in the regulation of endothelial cytoskeleton remodeling, was also significantly altered after heme treatment, both in HLMVECs and mice. By using a knockout (KO) mouse for MKK3, a key regulator of the p38MAPK pathway, we showed that this KO effectively decreased heme-induced endothelial barrier dysfunction. Taken together, our results indicate that targeting the p38MAPK pathway may represent a crucial treatment strategy in alleviating hemolytic diseases.

Single Mutation in the NFU1 Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells.

OBJECTIVE: NFU1 is a mitochondrial iron-sulfur scaffold protein, involved in iron-sulfur assembly and transfer to complex II and LAS (lipoic acid synthase). Patients with the point mutation NFU1G208C and CRISPR/CAS9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-generated rats develop mitochondrial dysfunction leading to pulmonary arterial hypertension. However, the mechanistic understanding of pulmonary vascular proliferation due to a single mutation in NFU1 remains unresolved. Approach and Results: Quantitative proteomics of isolated mitochondria showed the entire phenotypic transformation of NFU1G206C rats with a disturbed mitochondrial proteomic landscape, involving significant changes in the expression of 208 mitochondrial proteins. The NFU1 mutation deranged the expression pattern of electron transport proteins, resulting in a significant decrease in mitochondrial respiration. Reduced reliance on mitochondrial respiration amplified glycolysis in pulmonary artery smooth muscle cell (PASMC) and activated GPD (glycerol-3-phosphate dehydrogenase), linking glycolysis to oxidative phosphorylation and lipid metabolism. Decreased PDH (pyruvate dehydrogenase) activity due to the lipoic acid shortage is compensated by increased fatty acid metabolism and oxidation. PASMC became dependent on extracellular fatty acid sources due to upregulated transporters such as CD36 (cluster of differentiation 36) and CPT (carnitine palmitoyltransferase)-1. Finally, the NFU1 mutation produced a dysregulated antioxidant system in the mitochondria, leading to increased reactive oxygen species levels. PASMC from NFU1 rats showed apoptosis resistance, increased anaplerosis, and attained a highly proliferative phenotype. Attenuation of mitochondrial reactive oxygen species by mitochondrial-targeted antioxidant significantly decreased PASMC proliferation. CONCLUSIONS: The alteration in iron-sulfur metabolism completely transforms the proteomic landscape of the mitochondria, leading toward metabolic plasticity and redistribution of energy sources to the acquisition of a proliferative phenotype by the PASMC.

Glucose-6-phosphate dehydrogenase deficiency contributes to metabolic abnormality and pulmonary hypertension.

We have previously reported that several patients with idiopathic pulmonary hypertension (PH) had different types of G6PD deficiency. However, the role of G6PD in PH is multifactorial because G6PD is involved in controlling oxidative stress, metabolic switch, and red blood cell fragility. To delineate the contribution of G6PD to PH pathogenesis, we utilized a mouse line with decreased expression of G6PD (10% from wild-type level). We confirmed that mice with G6PD deficiency develop spontaneous pulmonary hypertension with pulmonary artery and right heart remodeling. G6PD deficiency resulted in increased free hemoglobin and activation of the p38 pathway, which we recently reported induces the development of PH in the sugen/hypoxia model via endothelial barrier dysfunction. Metabolomics analysis of G6PD deficient mice indicates the switch to alternative metabolic fluxes that feed into the pentose phosphate pathway (PPP), resulting in the upregulation of oxidative stress, fatty acid pathway, and reduction in pyruvate production. Thus, G6PD deficiency did not reduce PPP flux that is important for proliferation but activated collateral pathways at the cost of increased oxidative stress. Indeed, we found the upregulation of myo-inositol oxidase, reduction in GSH/GSSG ratio, and increased nitration in the lungs of G6PD-deficient mice. Increased oxidative stress also results in the activation of PI3K, ERK1/2, and AMPK that contribute to the proliferation of pulmonary vasculature. Therefore, G6PD deficiency has a multimodal effect, including hemolysis, metabolic reprogramming, and oxidative stress leading to the PH phenotype in mice.

Rats with a Human Mutation of NFU1 Develop Pulmonary Hypertension.

NFU1 is a mitochondrial protein that is involved in the biosynthesis of iron-sulfur clusters, and its genetic modification is associated with disorders of mitochondrial energy metabolism. Patients with autosomal-recessive inheritance of the NFU1 mutation G208C have reduced activity of the respiratory chain Complex II and decreased levels of lipoic-acid-dependent enzymes, and develop pulmonary arterial hypertension (PAH) in ∼70% of cases. We investigated whether rats with a human mutation in NFU1 are also predisposed to PAH development. A point mutation in rat NFU1G206C (human G208C) was introduced through CRISPR/Cas9 genome editing. Hemodynamic data, tissue samples, and fresh mitochondria were collected and analyzed. NFU1G206C rats showed increased right ventricular pressure, right ventricular hypertrophy, and high levels of pulmonary artery remodeling. Computed tomography and angiography of the pulmonary vasculature indicated severe angioobliterative changes in NFU1G206C rats. Importantly, the penetrance of the PAH phenotype was found to be more prevalent in females than in males, replicating the established sex difference among patients with PAH. Male and female homozygote rats exhibited decreased expression and activity of mitochondrial Complex II, and markedly decreased pyruvate dehydrogenase activity and lipoate binding. The limited development of PAH in males correlated with the preserved levels of oligomeric NFU1, increased expression of ISCU (an alternative branch of the iron-sulfur assembly system), and increased complex IV activity. Thus, the male sex has additional plasticity to overcome the iron-sulfur cluster deficiency. Our work describes a novel, humanized rat model of NFU1 deficiency that showed mitochondrial dysfunction similar to that observed in patients and developed PAH with the same sex dimorphism.

Greater high-mobility group box 1 in male compared with female spontaneously hypertensive rats worsens renal ischemia-reperfusion injury.

Renal ischemia is the most common cause of acute kidney injury. Damage-associated molecular patterns (DAMPs) initiate an inflammatory response and contribute to ischemia-reperfusion (IR) injury in males, yet the contribution of DAMPs to IR injury in females is unknown. The goal of the current study was to test the hypothesis that males have greater increases in the DAMP high-mobility group box 1 (HMGB1), worsening injury compared with females. Thirteen-week-old male and female spontaneously hypertensive rats (SHR) were subjected to sham or 45-min warm bilateral ischemia followed by 24 h of reperfusion before measurement of HMGB1 and renal function. Additional SHR were pre-treated with control (IgG) or HMGB1 neutralizing antibody (300 µg/rat) 1 h prior to renal ischemia. Blood, urine and kidneys were harvested 24 h post-IR for histological and Western blot analyses. Initial studies confirmed that IR resulted in greater increases in renal HMGB1 in male SHR compared with females. Greater renal HMGB1 in male SHR post-IR resulted in greater increases in serum TNF-α and renal IL-1ß, neutrophil infiltration and tubular cell death. Neutralization of HMGB1 attenuated IR-induced increases in plasma creatinine, blood urea nitrogen (BUN), inflammation, tubular damage and tubular cell death only in male SHR. In conclusion, our data demonstrate that there is a sex difference in the contribution of HMGB1 to IR-induced injury, where males exhibit greater increases in HMGB1-mediated renal injury in response to IR compared with females.

Complex III Inhibition-Induced Pulmonary Hypertension Affects the Mitochondrial Proteomic Landscape.

The mitochondria play a vital role in controlling cell metabolism and regulating crucial cellular outcomes. We previously demonstrated that chronic inhibition of the mitochondrial complex III in rats by Antimycin A (AA) induced sustained pulmonary vasoconstriction. On the metabolic level, AA-induced mitochondrial dysfunction resulted in a glycolytic shift that was reported as the primary contributor to pulmonary hypertension pathogenesis. However, the regulatory proteins driving this metabolic shift with complex III inhibition are yet to be explored. Therefore, to delineate the mechanisms, we followed changes in the rat lung mitochondrial proteome throughout AA treatment. Rats treated with AA for up to 24 days showed a disturbed mitochondrial proteome with significant changes in 28 proteins (p < 0.05). We observed a time-dependent decrease in the expression of key proteins that regulate fatty acid oxidation, the tricarboxylic acid cycle, the electron transport chain, and amino acid metabolism, indicating a correlation with diminished mitochondrial function. We also found a significant dysregulation in proteins that controls the protein import machinery and the clearance and detoxification of oxidatively damaged peptides via proteolysis and mitophagy. This could potentially lead to the onset of mitochondrial toxicity due to misfolded protein stress. We propose that chronic inhibition of mitochondrial complex III attenuates mitochondrial function by disruption of the global mitochondrial metabolism. This potentially aggravates cellular proliferation by initiating a glycolytic switch and thereby leads to pulmonary hypertension.

Inositol monophosphatase 1 as a novel interacting partner of RAGE in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vascular remodeling. The receptor for advanced glycation end products (RAGE) plays an important role in PAH by promoting proliferation of pulmonary vascular cells. RAGE is also known to mediate activation of Akt signaling, although the particular molecular mechanism remains unknown. This study aimed to identify the interacting partner of RAGE that could facilitate RAGE-mediated Akt activation and vascular remodeling in PAH. The progressive angioproliferative PAH was induced in 24 female Sprague-Dawley rats ( n = 8/group) that were randomly assigned to develop PAH for 1, 2, or 5 wk [right ventricle systolic pressure (RVSP) 56.5 ± 3.2, 63.6 ± 1.6, and 111.1 ± 4.5 mmHg, respectively, vs. 22.9 ± 1.1 mmHg in controls]. PAH triggered early and late episodes of apoptosis in rat lungs accompanied by RAGE activation. Mass spectrometry analysis has identified IMPA1 as a novel PAH-specific interacting partner of RAGE. The proximity ligation assay (PLA) confirmed the formation of RAGE/IMPA1 complex in the pulmonary artery wall. Activation of IMPA1 in response to increased glucose 6-phosphate (G6P) is known to play a critical role in inositol synthesis and recycling. Indeed, we confirmed a threefold increase in G6P ( P = 0.0005) levels in lungs of PAH rats starting from week 1 that correlated with accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), membrane translocation of PI3K, and a threefold increase in membrane Akt levels ( P = 0.02) and Akt phosphorylation. We conclude that the formation of the newly discovered RAGE-IMPA1 complex could be responsible for the stimulation of inositol pathways and activation of Akt signaling in PAH.

Hemolysis-induced Lung Vascular Leakage Contributes to the Development of Pulmonary Hypertension.

Although hemolytic anemia-associated pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH) are more common than the prevalence of idiopathic PAH alone, the role of hemolysis in the development of PAH is poorly characterized. We hypothesized that hemolysis independently contributes to PAH pathogenesis via endothelial barrier dysfunction with resulting perivascular edema and inflammation. Plasma samples from patients with and without PAH (both confirmed by right heart catheterization) were used to measure free hemoglobin (Hb) and its correlation with PAH severity. A sugen (50 mg/kg)/hypoxia (3 wk)/normoxia (2 wk) rat model was used to elucidate the role of free Hb/heme pathways in PAH. Human lung microvascular endothelial cells were used to study heme-mediated endothelial barrier effects. Our data indicate that patients with PAH have increased levels of free Hb in plasma that correlate with PAH severity. There is also a significant accumulation of free Hb and depletion of haptoglobin in the rat model. In rats, perivascular edema was observed at early time points concomitant with increased infiltration of inflammatory cells. Heme-induced endothelial permeability in human lung microvascular endothelial cells involved activation of the p38/HSP27 pathway. Indeed, the rat model also exhibited increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling, there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF-1a during the first 2 weeks of PH regardless of hypoxic conditions. Our data suggest that hemolysis may play a significant role in PAH pathobiology.

Recurrent inhibition of mitochondrial complex III induces chronic pulmonary vasoconstriction and glycolytic switch in the rat lung.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease; however, the mechanisms directly involved in triggering and the progression of PAH are not clear. Based on previous studies that demonstrated a possible role of mitochondrial dysfunction in the pathogenesis of PAH, we investigated the effects of chronic inhibition of mitochondrial function in vivo in healthy rodents. METHODS: Right ventricle systolic pressure (RVSP) was measured in female rats at baseline and up to 24 days after inhibition of mitochondrial respiratory Complex III, induced by Antimycin A (AA, 0.35 mg/kg, given three times starting at baseline and then days 3 and 6 as a bolus injection into the right atrial chamber). RESULTS: Rodents exposed to AA demonstrated sustained increases in RVSP from days 6 through 24. AA-exposed rodents also possessed a progressive increase in RV end-diastolic pressure but not RV hypertrophy, which may be attributed to either early stages of PAH development or to reduced RV contractility due to inhibition of myocardial respiration. Protein nitration levels in plasma were positively correlated with PAH development in AA-treated rats. This finding was strongly supported by results obtained from PAH humans where plasma protein nitration levels were correlated with markers of PAH severity in female but not male PAH patients. Based on previously reported associations between increased nitric oxide production levels with female gender, we speculate that in females with PAH mitochondrial dysfunction may represent a more deleterious form, in part, due to an increased nitrosative stress development. Indeed, the histological analysis of AA treated rats revealed a strong perivascular edema, a marker of pulmonary endothelial damage. Finally, AA treatment was accompanied by a severe metabolic shift toward glycolysis, a hallmark of PAH pathology. CONCLUSIONS: Chronic mitochondrial dysfunction induces the combination of vascular damage and metabolic reprogramming that may be responsible for PAH development. This mechanism may be especially important in females, perhaps due to an increased NO production and nitrosative stress development.

NADPH oxidase 4 is expressed in pulmonary artery adventitia and contributes to hypertensive vascular remodeling.

OBJECTIVE: Pulmonary hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PAs) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PAs and determine the functional relevance of Nox4 in PH. APPROACH AND RESULTS: Elevated expression of Nox4 was detected in hypertensive PAs from 3 rat PH models and human PH using qualititative real-time reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia, and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of reactive oxygen species production. Small-molecule inhibitors of Nox4 reduced adventitial reactive oxygen species generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and noninvasive indices of PA stiffness in monocrotaline-treated rats as determined by morphometric analysis and high-resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PAs. In fibroblasts, Nox4 overexpression stimulated migration and proliferation and was necessary for matrix gene expression. CONCLUSION: These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling, and development of PH.

Endothelin-1 induces a glycolytic switch in pulmonary arterial endothelial cells via the mitochondrial translocation of endothelial nitric oxide synthase.

Recent studies have indicated that, during the development of pulmonary hypertension (PH), there is a switch from oxidative phosphorylation to glycolysis in the pulmonary endothelium. However, the mechanisms underlying this phenomenon have not been elucidated. Endothelin (ET)-1, an endothelial-derived vasoconstrictor peptide, is increased in PH, and has been shown to play an important role in the oxidative stress associated with PH. Thus, in this study, we investigated whether there was a potential link between increases in ET-1 and mitochondrial remodeling. Our data indicate that ET-1 induces the redistribution of endothelial nitric oxide synthase (eNOS) from the plasma membrane to the mitochondria in pulmonary arterial endothelial cells, and that this was dependent on eNOS uncoupling. We also found that ET-1 disturbed carnitine metabolism, resulting in the attenuation of mitochondrial bioenergetics. However, ATP levels were unchanged due to a compensatory increase in glycolysis. Further mechanistic investigations demonstrated that ET-1 mediated the redistribution of eNOS via the phosphorylation of eNOS at Thr495 by protein kinase C δ. In addition, the glycolytic switch appeared to be dependent on mitochondrial-derived reactive oxygen species that led to the activation of hypoxia-inducible factor signaling. Finally, the cell culture data were confirmed in vivo using the monocrotaline rat model of PH. Thus, we conclude that ET-1 induces a glycolytic switch in pulmonary arterial endothelial cells via the redistribution of uncoupled eNOS to the mitochondria, and that preventing this event may be an approach for the treatment of PH.

Asymmetric dimethylarginine induces endothelial nitric-oxide synthase mitochondrial redistribution through the nitration-mediated activation of Akt1.

We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation of endothelial nitric-oxide synthase (eNOS) to the mitochondrion via a mechanism that requires protein nitration. Thus, the goal of this study was elucidate how eNOS redistributes to mitochondria and to identify the nitrated protein responsible for this event. Our data indicate that exposure of pulmonary arterial endothelial cells to ADMA enhanced eNOS phosphorylation at the Akt1-dependent phosphorylation sites Ser(617) and Ser(1179). Mutation of these serine residues to alanine (S617A and S1179A) inhibited nitration-mediated eNOS translocation to the mitochondria, whereas the phosphormimic mutations (S617D and S1179D) exhibited increased mitochondrial redistribution in the absence of ADMA. The overexpression of a dominant-negative Akt1 also attenuated ADMA-mediated eNOS mitochondrial translocation. Furthermore, ADMA enhanced Akt1 nitration and increased its activity. Mass spectrometry identified a single nitration site in Akt1 located at the tyrosine residue (Tyr(350)) located within the client-binding domain. Replacement of Tyr(350) with phenylalanine abolished peroxynitrite-mediated eNOS translocation to mitochondria. We also found that in the absence of ADMA, eNOS translocation decreased mitochondrial oxygen consumption and superoxide production without altering cellular ATP level. This suggests that under physiologic conditions, eNOS translocation enhances mitochondria coupling. In conclusion, we have identified a new mechanism by which eNOS translocation to mitochondria is regulated by the phosphorylation of eNOS at Ser(617) and Ser(1179) by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr(350).

Female sex hormones protect against salt-sensitive hypertension but not essential hypertension.

Initial studies found that female Dahl salt-sensitive (DS) rats exhibit greater blood pressure (BP) salt sensitivity than female spontaneously hypertensive rats (SHR). On the basis of the central role played by NO in sodium excretion and BP control, we further tested the hypothesis that blunted increases in BP in female SHR will be accompanied by greater increases in renal inner medullary nitric oxide synthase (NOS) activity and expression in response to a high-salt (HS) diet compared with DS rats. Gonad-intact and ovariectomized (OVX) female SHR and DS rats were placed on normal salt (NS; 0.4% salt) or HS (4% salt) diet for 2 wk. OVX did not alter BP in SHR, and HS diet produced a modest increase in BP. OVX significantly increased BP in DS rats on NS; HS further increased BP in all DS rats, although OVX had a greater increase in BP. Renal inner medullary NOS activity, total NOS3 protein, and NOS3 phosphorylated on serine residue 1177 were not altered by salt or OVX in either strain. NOS1 protein expression, however, significantly increased with HS only in SHR, and this corresponded to an increase in urinary nitrate/nitrite excretion. SHR also exhibit greater NOS1 and NOS3 protein expression than DS rats. These data indicate that female sex hormones offer protection against HS-mediated elevations in BP in DS rats but not SHR. We propose that the relative resistance to HS-mediated increases in BP in SHR is related to greater NOS expression and the ability to increase NOS1 protein expression compared with DS rats.

Novel Populations of Lung Capillary Endothelial Cells and Their Functional Significance.

The role of the lung's microcirculation and capillary endothelial cells in normal physiology and the pathobiology of pulmonary diseases is unequivocally vital. The recent discovery of molecularly distinct aerocytes and general capillary (gCaps) endothelial cells by single-cell transcriptomics (scRNAseq) advanced the field in understanding microcirculatory milieu and cellular communications. However, increasing evidence from different groups indicated the possibility of more heterogenic structures of lung capillaries. Therefore, we investigated enriched lung endothelial cells by scRNAseq and identified five novel populations of gCaps with distinct molecular signatures and roles. Our analysis suggests that two populations of gCaps that express Scn7a(Na+) and Clic4(Cl-) ion transporters form the arterial-to-vein zonation and establish the capillary barrier. We also discovered and named mitotically-active "root" cells (Flot1+) on the interface between arterial, Scn7a+, and Clic4 + endothelium, responsible for the regeneration and repair of the adjacent endothelial populations. Furthermore, the transition of gCaps to a vein requires a venous-capillary endothelium expressing Lingo2. Finally, gCaps detached from the zonation represent a high level of Fabp4, other metabolically active genes, and tip-cell markers showing angiogenesis-regulating capacity. The discovery of these populations will translate into a better understanding of the involvement of capillary phenotypes and their communications in lung disease pathogenesis.

Dimethyl Sulfoxide Induces Hemolysis and Pulmonary Hypertension.

Vascular and lung injury are well established complications associated with hemolytic disorders, and hemolysis associated pulmonary hypertension (PH) has emerged as the most serious complication of sickle cell disease. The causal relationship between intravascular hemolysis and the development of PH is still under investigation. Previously we have shown that repetitive administration of hemolyzed autologous blood causes PH in rats. Dimethyl sulfoxide (DMSO), a widely used solvent and anti-inflammatory agent, induces hemolysis in vivo. We hypothesized that repetitive administration of DMSO would induce PH in rats. We also examined hemolysis-induced release of adenosine deaminase (ADA) and arginase from red blood cells, which may amplify hemolysis-mediated vascular injury. Acute administration of DMSO (1.5ml/30 min into the right atrium) induced intravascular hemolysis and pulmonary vasoconstriction. DMSO-induced increase in right ventricular peak systolic pressure (RVPSP) was associated with increased release of ADA. Notably, the acute increase in RVPSP was attenuated by administration of an adenosine A2A receptor agonist or by pretreatment of animals with ADA inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). Repetitive administration of DMSO for 10 days produced anemia, hemoglobinuria, hemoglobinemia, splenomegaly, and development of PH. Histopathological analysis revealed pulmonary vascular remodeling. The presented data describe a new model of hemolysis induced PH, suggesting that hemolysis is mechanistically related to pulmonary hypertension, and pointing to a potential pathogenic role that adenosine deaminase and accelerated adenosine metabolism may play in hemolysis associated pulmonary hypertension.

Cytokine profiling in pulmonary arterial hypertension: the role of redox homeostasis and sex.

Pulmonary arterial hypertension (PAH) is a fatal disease with a well-established sexual dimorphism. Activated inflammatory response and altered redox homeostasis, both known to manifest in a sex-specific manner, are implicated in the pathogenic mechanisms involved in PAH development. This study aimed to evaluate the impact of sex and plasma redox status on circulating cytokine profiles. Plasma oxidation-reduction potential (ORP), as a substitute measure of redox status, was analyzed in male and female Group 1 PAH and healthy subjects. The profiles of 27 circulating cytokines were compared in 2 PAH groups exhibiting the highest and lowest quartile for plasma ORP, correlated with clinical parameters, and used to predict patient survival. The analysis of the PAH groups with the highest and lowest ORP revealed a correlation between elevated cytokine levels and increased oxidative stress in females. In contrast, in males, cytokine expressions were increased in the lower oxidative environment (except for IL-1b). Correlations of the increased cytokine expressions with PAH severity were highly sex-dependent and corresponded to the increase in PAH severity in males and less severe PAH in females. Machine learning algorithms trained on the combined cytokine and redox profiles allowed the prediction of PAH mortality with 80% accuracy. We conclude that the profile of circulating cytokines in PAH patients is redox- and sex-dependent, suggesting the vital need to stratify the patient cohort subjected to anti-inflammatory therapies. Combined cytokine and/or redox profiling showed promising value for predicting the patients' survival.

Endothelial eNAMPT drives EndMT and preclinical PH: rescue by an eNAMPT-neutralizing mAb.

Pharmacologic interventions to halt/reverse the vascular remodeling and right ventricular dysfunction in pulmonary arterial hypertension (PAH) remains an unmet need. We previously demonstrated extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a DAMP (damage-associated molecular pattern protein) contributing to PAH pathobiology via TLR4 ligation. We examined the role of endothelial cell (EC)-specific eNAMPT in experimental PH and an eNAMPT-neutralizing mAb as a therapeutic strategy to reverse established PH. Hemodynamic/echocardiographic measurements and tissue analyses were performed in Sprague Dawley rats exposed to 10% hypoxia/Sugen (three weeks) followed by return to normoxia and weekly intraperitoneal delivery of the eNAMPT mAb (1 mg/kg). WT C57BL/6J mice and conditional EC-cNAMPTec-/- mice were exposed to 10% hypoxia (three weeks). Biochemical and RNA sequencing studies were performed on rat PH lung tissues and human PAH PBMCs. Hypoxia/Sugen-exposed rats exhibited multiple indices of severe PH (right ventricular systolic pressure, Fulton index), including severe vascular remodeling, compared to control rats. PH severity indices and plasma levels of eNAMPT, IL-6, and TNF-α were all significantly attenuated by eNAMPT mAb neutralization. Compared to hypoxia-exposed WT mice, cNAMPTec-/- KO mice exhibited significantly reduced PH severity and evidence of EC to mesenchymal transition (EndMT). Finally, biochemical and RNAseq analyses revealed eNAMPT mAb-mediated rectification of dysregulated inflammatory signaling pathways (TLR/NF-κB, MAP kinase, Akt/mTOR) and EndMT in rat PH lung tissues and human PAH PBMCs. These studies underscore EC-derived eNAMPT as a key contributor to PAH pathobiology and support the eNAMPT/TLR4 inflammatory pathway as a highly druggable therapeutic target to reduce PH severity and reverse PAH.

Pulmonary Arterial Hypertension Induces a Distinct Signature of Circulating Metabolites.

Pulmonary arterial hypertension (PAH) is an incurable, progressive disorder, and the early diagnosis and treatment of PAH are associated with increased survival [...].