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.

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 NFU1 ^G208C 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 NFU1 ^G206C 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.

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.

Vascular remodeling is considered a key event in the pathogenesis of pulmonary arterial hypertension (PAH). However, mechanisms of gaining the proliferative phenotype by pulmonary vascular cells are still unresolved. Due to well-established pyruvate dehydrogenase (PDH) deficiency in PAH pathogenesis, we hypothesized that the activation of another branch of pyruvate metabolism, anaplerosis, via pyruvate carboxylase (PC) could be a key contributor to the metabolic reprogramming of the vasculature. In sugen/hypoxic PAH rats, vascular proliferation was found to be accompanied by increased activation of Akt signaling, which upregulated membrane Glut4 translocation and caused upregulation of hexokinase and pyruvate kinase-2, and an overall increase in the glycolytic flux. Decreased PDH activity and upregulation of PC shuttled more pyruvate to oxaloacetate. This results in the anaplerotic reprogramming of lung vascular cells and their subsequent proliferation. Treatment of sugen/hypoxia rats with the PC inhibitor, phenylacetic acid 20 mg/kg, starting after one week from disease induction, significantly attenuated right ventricular systolic pressure, Fulton index, and pulmonary vascular cell proliferation. PC inhibition reduced the glycolytic shift by attenuating Akt-signaling, glycolysis, and restored mitochondrial pyruvate oxidation. Our findings suggest that targeting PC mediated anaplerosis is a potential therapeutic intervention for the resolution of vascular remodeling in PAH.

Impaired pulmonary angiogenesis plays a pivotal role in the progression of pulmonary arterial hypertension (PAH) and patient mortality, yet the molecular mechanisms driving this process remain enigmatic. Our study uncovered a striking connection between mitochondrial dysfunction (MD), caused by a humanized mutation in the NFU1 gene, and severely disrupted pulmonary angiogenesis in adult lungs. Restoring the bioavailability of the NFU1 downstream target, lipoic acid (LA), alleviated MD and angiogenic deficiency and rescued the progressive PAH phenotype in the NFU1G206C model. Notably, significant NFU1 expression and signaling insufficiencies were also identified in idiopathic PAH (iPAH) patients’ lungs, emphasizing this study’s relevance beyond NFU1 mutation cases. The remarkable improvement in mitochondrial function of PAH patient-derived pulmonary artery endothelial cells (PAECs) following LA supplementation introduces LA as a potential therapeutic approach. In conclusion, this study unveils a novel role for MD in dysregulated pulmonary angiogenesis and PAH manifestation, emphasizing the need to correct MD in PAH patients with unrecognized NFU1/LA deficiency.

Pulmonary arterial hypertension (PAH) is a chronic cardiopulmonary disorder instigated by pulmonary vascular cell proliferation. Activation of Akt was previously reported to promote vascular remodeling. Also, the irreversible nitration of Y350 residue in Akt results in its activation. NitroAkt was increased in PAH patients and the SU5416/Hypoxia (SU/Hx) PAH model. This study investigated whether the prevention of Akt nitration in PAH by Akt targeted nitroxide-conjugated peptide (NP) could reverse vascular remodeling and metabolic reprogramming. Treatment of the SU/Hx model with NP significantly decreased nitration of Akt in lungs, attenuated right ventricle (RV) hypertrophy, and reduced RV systolic pressure. In the PAH model, Akt-nitration induces glycolysis by activation of the glucose transporter Glut4 and lactate dehydrogenase-A (LDHA). Decreased G6PD and increased GSK3β in SU/Hx additionally shunted intracellular glucose via glycolysis. The increased glycolytic rate upregulated anaplerosis due to activation of pyruvate carboxylase in a nitroAkt-dependent manner. NP treatment resolved glycolytic switch and activated collateral pentose phosphate and glycogenesis pathways. Prevention of Akt-nitration significantly controlled pyruvate in oxidative phosphorylation by decreasing lactate and increasing pyruvate dehydrogenases activities. Histopathological studies showed significantly reduced pulmonary vascular proliferation. Based on our current observation, preventing Akt-nitration by using an Akt-targeted nitroxide-conjugated peptide could be a useful treatment option for controlling vascular proliferation in PAH.