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.

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.

Early progression of pulmonary hypertension in the monocrotaline model in males is associated with increased lung permeability.

BACKGROUND: The mechanisms involved in pulmonary hypertension (PH) development in patients and pre-clinical models are poorly understood. PH has a well-established sex dimorphism in patients with increased frequency of PH in females, and more severe disease with poor survival prognosis in males. Previously, we found that heme signaling plays an essential role in the development phase of the Sugen/Hypoxia (SU/Hx) model. This study is focused on the elucidation of sex differences in mechanisms of PH development related to heme action at the early stage of the monocrotaline (MCT) PH model. METHODS: Rats received MCT injection (60 mg/kg, i.p.) and followed for 14 days to investigate early disease changes. Hemodynamic parameters were recorded at the end of the study; plasma, lung homogenates, and nuclear fractions were used for the evaluation of protein levels. RESULTS: Our data indicate that on day 14, rats did not show any significant increase in the Fulton index due to the early disease phase. However, the right ventricular systolic pressure was significantly increased in male rats, while female rats showed only a trend. Interestingly, only males demonstrated an increased lung-to-bodyweight ratio that indicated lung edema. Indeed, lung histology confirmed severe perivascular edema in males. Previously, we have reported that the increased perivascular edema in SU/Hx model correlated with intravascular hemolysis and activated heme signaling. Here, we found that elevated free hemoglobin levels and perivascular edema were increased, specifically in males showing more rapid progress of PH. A high level of heme carrier protein 1 (HCP-1), which is involved in heme uptake from the bloodstream into the cells, was also found elevated in the lungs of males. The upregulation of heme oxygenase in males indicated increased intracellular heme catabolism. Increased heme signaling resulted in the activation of heme-mediated barrier-disruptive mechanisms. Thus, hemolysis in males can be responsible for increased permeability of the lungs and early disease development. CONCLUSIONS: Our study indicates the importance of barrier-disruptive mechanisms as an earlier event in the induction of pulmonary hypertension. Importantly, males are more susceptible to hemolysis and develop PH earlier than females.

Inhibition of Anaplerosis Attenuated Vascular Proliferation in Pulmonary Arterial 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.

Antioxidant-Conjugated Peptide Attenuated Metabolic Reprogramming in Pulmonary Hypertension.

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 GSK3ß 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.

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.

New cases of Glucose-6-Phosphate Dehydrogenase deficiency in Pulmonary Arterial Hypertension.

Pulmonary Arterial Hypertension (PAH) is a fatal disorder with limited treatment options and reduced life expectancy after diagnosis. Complex genetic backgrounds in PAH complicates identification of causative mutations that is essential for an understanding of the disease diagnostics and etiology especially for idiopathic PAH (iPAH). Hemolysis has been implicated as contributing to the pathobiology of PAH. Glucose-6-Phosphate Dehydrogenase (G6PD) expression and activity define erythrocyte's antioxidant capacity, and its decrease contributes to erythrocyte fragility. As G6PD deficiency was previously reported in a limited number of PAH cases, we tested whether iPAH patients exhibit underlying G6PD alterations in erythrocytes. A cohort of 22 PAH patients and 8 non-PAH patients were recruited for this study. DNA isolated from Peripheral Blood Mononuclear Cells (PBMC) was used for detection of mutations in the coding region of the G6PD gene. RNA isolated from PBMC was used for determination of G6PD mRNA expression level. G6PD activity was measured in Red Blood Cell (RBC) pellets. Three patients had missense mutations in G6PD (Val291Met, Asn126Asp, Asp194Glu), however, only one mutation (Val291Met) results in a severe G6PD deficiency. A single patient with mutation (Asn126Asp) showed a 21% decrease in G6PD activity, two subjects showed G6PD deficiency without mutations, and one patient had a decreased level of G6PD mRNA and reduced enzyme levels. This study demonstrates that a moderate decrease in G6PD activity is associated with PAH. Screening for G6PD activity and mutations in the G6PD gene may provide early detection of individuals predisposed to PAH.