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Background: In 2012, mutations in Cav1 were found to be the driving mutation in several cases of heritable pulmonary arterial hypertension (PAH). These mutations replaced the last 21 amino acids of Cav1 with a novel 22-amino-acid sequence. Because previously only Cav1 knockouts had been studied in the context of PAH, examining the in vivo effects of this novel mutation holds promise for new understanding of the role of Cav1 in disease etiology. Methods: The new 22 amino acids created by the human mutation were knocked into the native mouse Cav1 locus. The mice underwent hemodynamic, energy balance, and inflammatory measurements, both at baseline and after being stressed with either a metabolic or an inflammatory challenge [low-dose lipopolysaccharide (LPS)]. To metabolically challenge the mice, they were injected with streptozotocin (STZ) and fed a high-fat diet for 12 weeks. Results: Very little mutant protein was found in vivo (roughly 2% of wild-type by mass spectrometry), probably because of degradation after failure to traffic from the endoplasmic reticulum. The homozygous mutants developed a mild, low-penetrance PAH similar to that described previously in knockouts, and neither baseline nor metabolic nor inflammatory stress resulted in pressures above normal in heterozygous animals. The homozygous mutants had increased lean mass and worsened oral glucose tolerance, as previously described in knockouts. Novel findings include the preservation of Cav2 and accessory proteins in the liver and the kidney, while they are lost with homozygous Cav1 mutation in the lungs. We also found that the homozygous mutants had a significantly lower tolerance to voluntary spontaneous exercise than the wild-type mice, with the heterozygous mice at an intermediate level. The mutants also had higher circulating monocytes, with both heterozygous and homozygous animals having higher pulmonary MCP1 and MCP5 proteins. The heterozygous animals also lost weight at an LPS challenge level at which the wild-type mice continued to gain weight. Conclusions: The Cav1 mutation identified in human patients in 2012 is molecularly similar to a knockout of Cav1. It results in not only metabolic deficiencies and mild pulmonary hypertension, as expected, but also an inflammatory phenotype and reduced spontaneous exercise.

The increased afterload of pulmonary arterial hypertension (PAH) impairs right ventricular function and ultimately leads to failure, as the RV struggles to adapt to increased pressure with remodeling and fibrosis. During PAH, cardiomyocytes upregulate cell-surface expression of the G protein-coupled thromboxane/prostanoid receptor (TPr). Increased myofibroblast and immune cell populations may also contribute to the enhanced TPr expression seen in the PAH RV. Activation of the cardiomyocyte TPr increases intracellular calcium via G αq /IP 3 ; activation of the receptor in other cells leads to fibrosis and vasoconstriction. Preventing signaling through the TPr prevents RV fibrosis in murine models of PAH without affecting arterial pressure. Because infusion of TPr agonist can cause arrhythmia in anesthetized rabbits, and we have previously found that RV pressure overload causes sustained increases in end-diastolic calcium in RV cardiomyocytes that is blocked with TPr antagonist, we hypothesized that endogenous TPr activation can lead to conduction abnormalities in RV pressure overload. Here, we used pulmonary arterial banding (PAB) of female mice to induce fixed pressure overload of the RV. Sham-operated or PAB mice were treated with normal drinking water or water containing 25 mg/kg/day of the TPr antagonist ifetroban and were evaluated at 4 weeks past PAB. RV ejection fraction was similarly depressed in vehicle- and antagonist-treated mice, although spontaneous running, RV fibrosis, and RV relaxation time were improved in PAB mice given ifetroban. ECG abnormalities in PAB mice confirmed a prolonged relaxation and suggested delays in repolarization. These were abolished with TPr antagonism. PAB altered RV expression and localization of connexin-43 (Cx43) in vehicle-treated, but not ifetroban-treated mice. Cx43 derangement is associated with impaired cell-to-cell electrical conduction and impulse propagation. Compiled, our findings suggest that endogenous TPr activation produces alterations in RV calcium handling, signaling, and cell-cell junctions that contribute to early failure in pressure overload. Therapeutic TPr antagonism may prevent this deleterious remodeling and prolong survival in patients with PAH.

Inflammation is a major cause of morbidity and mortality in Western societies. Despite use of multiple drugs, both chronic and acute inflammation still represent major health burdens. Inflammation produces highly reactive dicarbonyl lipid peroxidation products such as isolevuglandins which covalently modify and cross-link proteins via lysine residues. Mitochondrial dysfunction has been associated with inflammation; however, its molecular mechanisms and pathophysiological role are still obscure. We hypothesized that inflammation-induced isolevuglandins contribute to mitochondrial dysfunction and mortality. To test this hypothesis, we have (a) investigated the mitochondrial dysfunction in response to synthetic 15-E2-isolevuglandin (IsoLG) and its adducts; (b) developed a new mitochondria-targeted scavenger of isolevuglandins by conjugating 2-hydroxybenzylamine to the lipophilic cation triphenylphosphonium, (4-(4-aminomethyl)-3-hydroxyphenoxy)butyl)-triphenylphosphonium (mito2HOBA); (c) tested if mito2HOBA protects from mitochondrial dysfunction and mortality using a lipopolysaccharide model of inflammation. Acute exposure to either IsoLG or IsoLG adducts with lysine, ethanolamine or phosphatidylethanolamine inhibits mitochondrial respiration and attenuates Complex I activity. Complex II function was much more resistant to IsoLG. We confirmed that mito2HOBA markedly accumulates in isolated mitochondria and it is highly reactive with IsoLGs. To test the role of mitochondrial IsoLGs, we studied the therapeutic potential of mito2HOBA in lipopolysaccharide mouse model of sepsis. Mito2HOBA supplementation in drinking water (0.1 g/L) to lipopolysaccharide treated mice increased survival by 3-fold, improved complex I-mediated respiration, and histopathological analyses supported mito2HOBA-mediated protection of renal cortex from cell injury. These data support the role of mitochondrial IsoLG in mitochondrial dysfunction and inflammation. We conclude that reducing mitochondrial IsoLGs may be a promising therapeutic target in inflammation and conditions associated with mitochondrial oxidative stress and dysfunction., Graphical abstract Image 1, Highlights • Isolevuglandins and their adducts inhibit mitochondrial respiration and attenuates Complex I activity. • 2-Hydroxybenzylamine conjugated to triphenylphosphonium, mito2HOBA, accumulates in mitochondria. • Mito2HOBA in drinking water improves complex I-mediated respiration in LPS model of sepsis. • Mitochondria-targeted scavenger of isolevuglandins mito2HOBA reduces mortality in LPS model.

In pulmonary arterial hypertension (PAH), the right ventricle undergoes remodeling and fibrosis as it struggles to adapt to the increased pressure overload. RV dysfunction and failure is the primary cause of death in PAH patients. The G protein-coupled thromboxane/prostanoid receptor (TPr) is expressed in vascular smooth muscle, myofibroblasts, and immune cells, and is upregulated in cardiomyocytes following PAH. Activation of the cardiomyocyte TPr increases intracellular calcium via G αq /IP 3 ; activation of the receptor in other cells leads to fibrosis and vasoconstriction. The TPr is activated by isoprostane as well as thromboxane, which suggests that the receptor could contribute to deleterious remodeling during cardiac stress. Our previous studies demonstrate that TPr antagonism prevents RV fibrosis and dilatation in murine models of PAH, without affecting pressures. Because the TPr can signal through Gq, we hypothesized that its activation in PAH causes changes in cardiomyocyte calcium-handling proteins which contribute to remodeling and failure. In this study, we used pulmonary arterial banding (PAB) to induce fixed pressure overload of the RV. Mice were treated for 4 weeks past PAB with normal drinking water or water containing 25 mg/kg/day of the TPr antagonist ifetroban, and either underwent pressure-volume catheterization and whole RV evaluation, or cardiomyocytes were isolated for calcium handling and protein. PAB caused an increase in cardiomyocyte resting (end-diastolic) intracellular calcium, which was ameliorated in mice given TPr antagonist. The increased intracellular calcium following PAB was associated with increased activity of the calcium-activated protease calpain, also blocked with TPr antagonism. There was no decrease in caffeine-mediated release of calcium from the sarcoplasmic reticulum (SR) at 4 weeks past PAB, and phosphorylation of phospholamban was increased, suggesting compensation to drive calcium into the SR. Our findings suggest that TPr activation produces alterations in RV calcium handling, signaling, and calpain activity that contribute to deleterious remodeling and early failure in pressure overload. Therapeutic TPr antagonism may help preserve RV function in patients with PAH.

Loss of function KCNK3 mutation is one of the gene variants driving hereditary pulmonary arterial hypertension (PAH). KCNK3 is expressed in several cell and tissue types on both membrane and endoplasmic reticulum and potentially plays a role in multiple pathological process associated with PAH. However, the role of various stressors driving the susceptibility of KCNK3 mutation to PAH is unknown. Hence, we exposed kcnk3fl/fl animals to hypoxia, metabolic diet and low dose lipopolysaccharide (LPS) and performed molecular characterization of their tissue. We also used tissue samples from KCNK3 patients (skin fibroblast derived inducible pluripotent stem cells, blood, lungs, peripheral blood mononuclear cells) and performed microarray, immunohistochemistry (IHC) and mass cytometry time of flight (CyTOF) experiments. Although a hypoxic insult did not alter vascular tone in kcnk3fl/fl mice, RNASeq study of these lungs implied that inflammatory and metabolic factors were altered, and the follow-up diet study demonstrated a dysregulation of bone marrow cells in kcnk3fl/fl mice. Finally, a low dose LPS study clearly showed that inflammation could be a possible second hit driving PAH in kcnk3fl/fl mice. Multiplex, IHC and CyTOF immunophenotyping studies on human samples confirmed the mouse data and strongly indicated that cell mediated, and innate immune responses may drive PAH susceptibility in these patients. In conclusion, loss of function KCNK3 mutation alters various physiological processes from vascular tone to metabolic diet through inflammation. Our data suggests that altered circulating immune cells may drive PAH susceptibility in patients with KCNK3 mutation.

Background: Pulmonary hypertension (PH) is a progressive cardiovascular disease, characterized by endothelial and smooth muscle dysfunction and vascular remodeling, followed by right heart failure. Group III PH develops secondarily to chronic lung disease such as idiopathic pulmonary fibrosis (IPF), and both hastens and predicts mortality despite of all known pharmacological interventions. Thus, there is urgent need for development of newer treatment strategies. Objective: Angiotensin converting enzyme 2 (ACE2), a member of the renin angiotensin family, is therapeutically beneficial in animal models of pulmonary vascular diseases and is currently in human clinical trials for primary PH. Although previous studies suggest that administration of ACE2 prevents PH secondary to bleomycin-induced murine IPF, it is unknown whether ACE2 can reverse or treat existing disease. Therefore, in the present study, we tested the efficacy of ACE2 in arresting the progression of group 3 PH. Methods: To establish pulmonary fibrosis, we administered 0.018 U/g bleomycin 2x/week for 4 weeks in adult FVB/N mice, and sacrificed 5 weeks following the first injection. ACE2 or vehicle was administered via osmotic pump for the final 2 weeks, beginning 3 weeks after bleomycin. Echocardiography and hemodynamic assessment was performed prior to sacrifice and tissue collection. Results: Administration of bleomycin significantly increased lung collagen expression, pulmonary vascular remodeling, and pulmonary arterial pressure, and led to mild right ventricular hypertrophy. Acute treatment with ACE2 significantly attenuated vascular remodeling and increased pulmonary SOD2 expression without measurable effects on pulmonary fibrosis. This was associated with nonsignificant positive effects on pulmonary arterial pressure and cardiac function. Conclusion: Collectively, our findings enumerate that ACE2 treatment improved pulmonary vascular muscularization following bleomycin exposure, concomitant with increased SOD2 expression. Although it may not alter the pulmonary disease course of IPF, ACE2 could be an effective therapeutic strategy for the treatment of group 3 PH.

Pulmonary fibrosis is often complicated by pulmonary hypertension (PH), and previous studies have shown a potential link between bone morphogenetic protein receptor II (BMPR2) and PH secondary to pulmonary fibrosis. We exposed transgenic mice expressing mutant BMPR2 and control mice to repetitive intraperitoneal injections of bleomycin for 4 weeks. The duration of transgene activation was too short for mutant BMPR2 mice to develop spontaneous PH. Mutant BMPR2 mice had increased right ventricular systolic pressure compared to control mice, without differences in pulmonary fibrosis. We found increased hypoxia-inducible factor (HIF)1-α stabilization in lungs of mutant-BMPR2-expressing mice compared to controls following bleomycin treatment. In addition, expression of the hypoxia response element protein connective tissue growth factor was increased in transgenic mice as well as in a human pulmonary microvascular endothelial cell line expressing mutant BMPR2. In mouse pulmonary vascular endothelial cells, mutant BMPR2 expression resulted in increased HIF1-α and reactive oxygen species production following exposure to hypoxia, both of which were attenuated with the antioxidant TEMPOL. These data suggest that expression of mutant BMPR2 worsens secondary PH through increased HIF activity in vascular endothelium. This pathway could be therapeutically targeted in patients with PH secondary to pulmonary fibrosis.