Yen-Chun Lai

PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis

Although aging is a known risk factor for idiopathic pulmonary fibrosis (IPF), the pathogenic mechanisms that underlie the effects of advancing age remain largely unexplained. Some age-related neurodegenerative diseases have an etiology that is related to mitochondrial dysfunction. Here, we found that alveolar type II cells (AECIIs) in the lungs of IPF patients exhibit marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities in AECIIs of IPF lungs were associated with upregulation of ER stress markers and were recapitulated in normal mice with advancing age in response to stimulation of ER stress. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of PTEN-induced putative kinase 1 (PINK1). Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization and expression of profibrotic factors. Moreover, young PINK1-deficient mice developed similarly dysmorphic, dysfunctional mitochondria in the AECIIs and were vulnerable to apoptosis and development of lung fibrosis. Our data indicate that PINK1 deficiency results in swollen, dysfunctional mitochondria and defective mitophagy, and promotes fibrosis in the aging lung.

Pulmonary Arterial Hypertension The Clinical Syndrome

Pulmonary arterial hypertension is a progressive disorder in which endothelial dysfunction and vascular remodeling obstruct small pulmonary arteries, resulting in increased pulmonary vascular resistance and pulmonary pressures. This leads to reduced cardiac output, right heart failure, and ultimately death. In this review, we attempt to answer some important questions commonly asked by patients diagnosed with pulmonary arterial hypertension pertaining to the disease, and aim to provide an explanation in terms of classification, diagnosis, pathophysiology, genetic causes, demographics, and prognostic factors. Furthermore, important molecular pathways that are central to the pathogenesis of pulmonary arterial hypertension are reviewed, including nitric oxide, prostacyclin, endothelin-1, reactive oxygen species, and endothelial and smooth muscle proliferation.

SIRT3–AMP-Activated Protein Kinase Activation by Nitrite and Metformin Improves Hyperglycemia and Normalizes Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction

Background— Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is an increasingly recognized clinical complication of metabolic syndrome. No adequate animal model of PH-HFpEF is available, and no effective therapies have been identified to date. A recent study suggested that dietary nitrate improves insulin resistance in endothelial nitric oxide synthase null mice, and multiple studies have reported that both nitrate and its active metabolite, nitrite, have therapeutic activity in preclinical models of pulmonary hypertension. Methods and Results— To evaluate the efficacy and mechanism of nitrite in metabolic syndrome associated with PH-HFpEF, we developed a 2-hit PH-HFpEF model in rats with multiple features of metabolic syndrome attributable to double-leptin receptor defect (obese ZSF1) with the combined treatment of vascular endothelial growth factor receptor blocker SU5416. Chronic oral nitrite treatment improved hyperglycemia in obese ZSF1 rats by a process that requires skeletal muscle SIRT3-AMPK-GLUT4 signaling. The glucose-lowering effect of nitrite was abolished in SIRT3-deficient human skeletal muscle cells, and in SIRT3 knockout mice fed a high-fat diet, as well. Skeletal muscle biopsies from humans with metabolic syndrome after 12 weeks of oral sodium nitrite and nitrate treatment (IND#115926) displayed increased activation of SIRT3 and AMP-activated protein kinase. Finally, early treatments with nitrite and metformin at the time of SU5416 injection reduced pulmonary pressures and vascular remodeling in the PH-HFpEF model with robust activation of skeletal muscle SIRT3 and AMP-activated protein kinase. Conclusions— These studies validate a rodent model of metabolic syndrome and PH-HFpEF, suggesting a potential role of nitrite and metformin as a preventative treatment for this disease.

Nitrate–Nitrite–Nitric Oxide Pathway in Pulmonary Arterial Hypertension Therapeutics

Pulmonary arterial hypertension (PAH) is a disorder characterized by elevated vascular resistance in pulmonary arterioles. Progressive increases in pulmonary vascular resistance and pulmonary artery pressures result in right heart failure and reduced cardiac output. Patients experience progressive exertional dyspnea, right heart failure, syncope, and ultimately death. The common pathophysiological features of PAH include pulmonary vasoconstriction, intimal and smooth muscle proliferation, in situ thrombosis, and pathological remodeling of pulmonary arterial circulation. Although the origin of PAH is multifactorial, impairments in vasodilator (nitric oxide [NO] and prostaglandin signaling) and vasoconstrictor (endothelin-1, reactive oxygen species, angiotensin II) pathways underlie the evolution of early disease.1 Based on this knowledge, drugs that enhance the NO signaling pathways (phosphodiesterase 5 inhibitors), the prostenoids, and endothelin receptor blockers, have been developed and approved for PAH-specific therapy. Article see p 2922 Inhaled NO gas can alleviate vasoconstriction and may modulate cellular proliferative responses, but NO therapy is limited by the need for continuous inhalation, NO reactions with oxygen to form nitrogen dioxide, and special delivery devices. It is now appreciated that inorganic nitrite and nitrate are bio-transformed to NO via the nitrate-to-nitrite-to-NO pathway,2 leading to studies with inhaled nitrite as an alternative to NO gas inhalation.3,4 In this issue of Circulation , Baliga and colleagues5 investigate the nitrate-to-nitrite-to-NO pathway by studying the effects of oral nitrite and nitrate on preclinical mouse and rat models of PAH, and then attempt to characterize the enzymes that regulate bioconversion of nitrite to NO. They find that both nitrate and nitrite delivered in drinking water can prevent and reverse experimental PAH in the hypoxic and bleomycin mouse models, which is consistent with published models for in vivo conversion of nitrite to NO.6–8 They also provide unexpected evidence that eNOS may have nitrite reductase …

Nitrite-Mediated S-Nitrosylation of Caspase-3 Prevents Hypoxia-Induced Endothelial Barrier Dysfunction

Rationale: Hypoxia is a significant perturbation that exacerbates endothelial barrier dysfunction, contributing to the disruption of vascular homeostasis and the development of various diseases such as atherosclerosis and metastasis of tumors. To date, it is not known what strategy might be used to counter the effect of hypoxia on endothelial permeability. Objective: This study investigated the role of nitrite in regulating vascular integrity under hypoxic conditions. Methods and Results: We found denitrosylation and the resulting activation of caspase-3 to be critical for hypoxia-induced endothelial permeability. Nitrite treatment led to S-nitrosylation and the inactivation of caspase-3, suppressing the barrier dysfunction of endothelia caused by hypoxia. This process required the conversion of nitrite to bioactive nitric oxide in a nitrite reductase-dependent manner. Using primary human umbilical vein endothelial cells as a model, we showed that in the presence of nitrite, the S-nitrosylated and inactivated form of caspase-3 was unable to cleave &bgr;-catenin, a key component in the VE-cadherin complex. Therefore, nitrite treatment led to the maintenance of VE-cadherin–mediated adherens junctions under hypoxic conditions. In in vivo experiments using a zebrafish model, nitrite was found to protect blood vessels from hypoxia-induced vascular leakage. Conclusions: These results are the first to demonstrate that nitrite plays a critical role in the protection of endothelial barrier function against hypoxic insult. Our findings show that nitrite holds great potential for the treatment of diseases associated with hypoxia-induced disorder of vascular homeostasis.

Novel Targets of Drug Treatment for Pulmonary Hypertension

Biomedical advances over the last decade have identified the central role of proliferative pulmonary arterial smooth muscle cells (PASMCs) in the development of pulmonary hypertension (PH). Furthermore, promoters of proliferation and apoptosis resistance in PASMCs and endothelial cells, such as aberrant signal pathways involving growth factors, G protein-coupled receptors, kinases, and microRNAs, have also been described. As a result of these discoveries, PH is currently divided into subgroups based on the underlying pathology, which allows focused and targeted treatment of the condition. The defining features of PH, which subsequently lead to vascular wall remodeling, are dysregulated proliferation of PASMCs, local inflammation, and apoptosis-resistant endothelial cells. Efforts to assess the relative contributions of these factors have generated several promising targets. This review discusses recent novel targets of therapies for PH that have been developed as a result of these advances, which are now in pre-clinical and clinical trials (e.g., imatinib [phase III]; nilotinib, AT-877ER, rituximab, tacrolimus, paroxetine, sertraline, fluoxetine, bardoxolone methyl [phase II]; and sorafenib, FK506, aviptadil, endothelial progenitor cells (EPCs) [phase I]). While substantial progress has been made in recent years in targeting key molecular pathways, PH still remains without a cure, and these novel therapies provide an important conceptual framework of categorizing patients on the basis of molecular phenotype(s) for effective treatment of the disease.

Development of a Mouse Model of Metabolic Syndrome, Pulmonary Hypertension, and Heart Failure with Preserved Ejection Fraction

&NA; Pulmonary hypertension (PH) associated with heart failure with preserved ejection fraction (PH‐HFpEF; World Health Organization Group II) secondary to left ventricular (LV) diastolic dysfunction is the most frequent cause of PH. It is an increasingly recognized clinical complication of the metabolic syndrome. To date, no effective treatment has been identified, and no genetically modifiable mouse model is available for advancing our understanding for PH‐HFpEF. To develop a mouse model of PH‐HFpEF, we exposed 36 mouse strains to 20 weeks of high‐fat diet (HFD), followed by systematic evaluation of right ventricular (RV) and LV pressure‐volume analysis. The HFD induces obesity, glucose intolerance, insulin resistance, hyperlipidemia, as well as PH, in susceptible strains. We observed that certain mouse strains, such as AKR/J, NON/shiLtJ, and WSB/EiJ, developed hemodynamic signs of PH‐HFpEF. Of the strains that develop PH‐HFpEF, we selected AKR/J for further model validation, as it is known to be prone to HFD‐induced metabolic syndrome and had low variability in hemodynamics. HFD‐treated AKR/J mice demonstrate reproducibly higher RV systolic pressure compared with mice fed with regular diet, along with increased LV end‐diastolic pressure, both RV and LV hypertrophy, glucose intolerance, and elevated HbA1c levels. Time course assessments showed that HFD significantly increased body weight, RV systolic pressure, LV end‐diastolic pressure, biventricular hypertrophy, and HbA1c throughout the treatment period. Moreover, we also identified and validated 129S1/SvlmJ as a resistant mouse strain to HFD‐induced PH‐HFpEF. These studies validate an HFD/AKR/J mouse model of PH‐HFpEF, which may offer a new avenue for testing potential mechanisms and treatments for this disease.

Scavenger Receptor Function of Mouse Fcγ Receptor III Contributes to Progression of Atherosclerosis in Apolipoprotein E Hyperlipidemic Mice

Recent studies showed loss of CD36 or scavenger receptor-AI/II (SR-A) does not ameliorate atherosclerosis in a hyperlipidemic mouse model, suggesting receptors other than CD36 and SR-A may also contribute to atherosclerosis. In this report, we show that apolipoprotein E (apoE)-CD16 double knockout (DKO; apoE-CD16 DKO) mice have reduced atherosclerotic lesions compared with apoE knockout mice. In vivo and in vitro foam cell analyses showed apoE-CD16 DKO macrophages accumulated less neutral lipids. Reduced foam cell formation in apoE-CD16 DKO mice is not due to change in expression of CD36, SR-A, and LOX-1. This led to a hypothesis that CD16 may have scavenger receptor activity. We presented evidence that a soluble form of recombinant mouse CD16 (sCD16) bound to malondialdehyde-modified low-density lipoprotein (MDALDL), and this binding is blocked by molar excess of MDA- modified BSA and anti-MDA mAbs, suggesting CD16 specifically recognizes MDA epitopes. Interestingly, sCD16 inhibited MDALDL binding to macrophage cell line, as well as soluble forms of recombinant mouse CD36, SR-A, and LOX-1, indicating CD16 can cross-block MDALDL binding to other scavenger receptors. Anti-CD16 mAb inhibited immune complex binding to sCD16, whereas it partially inhibited MDALDL binding to sCD16, suggesting MDALDL binding site may be in close proximity to the immune complex binding site in CD16. Loss of CD16 expression resulted in reduced levels of MDALDL-induced proinflammatory cytokine expression. Finally, CD16-deficient macrophages showed reduced MDALDL-induced Syk phosphorylation. Collectively, our findings suggest scavenger receptor activity of CD16 may, in part, contribute to the progression of atherosclerosis.

Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor

&NA; Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high‐fat diet (HFD) in some inbred mouse strains. We conducted a genome‐wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD‐induced PH. Mice from 36 inbred and wild‐derived strains were fed with regular diet or HFD for 20 weeks beginning at 6‐12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end‐systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest‐path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH‐related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high‐fat feeding. Network‐based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest‐path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high‐fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network‐based approach adds evidence for a role for Egfr in murine PH.

Emerging therapeutics in pulmonary hypertension

Pulmonary hypertension (PH) is a progressive and often fatal illness presenting with nonspecific symptoms of dyspnea, lower extremity edema, and exercise intolerance. Pathologically, endothelial dysfunction leads to abnormal intimal and smooth muscle proliferation along with reduced apoptosis, resulting in increased pulmonary vascular resistance and elevated pulmonary pressures. PH is subdivided into five World Health Organization groups based on the disease pathology and specific cause. While there are Food and Drug Administration-approved medications for the treatment of pulmonary arterial hypertension (PAH; Group 1 PH), as well as for chronic thromboembolic PH (Group 4 PH), the morbidity and mortality remain high. Moreover, there are no approved therapies for other forms of PH (Groups 2, 3, and 5) at present. New research has identified molecular targets that mediate vasodilation, anti-inflammatory, and antifibrotic changes within the pulmonary vasculature. Given that PAH is the most commonly studied form of PH worldwide and because recent studies have led to better mechanistic understanding of this devastating disease, in this review we attempt to provide an updated overview of new therapeutic approaches under investigation for the treatment of PH, with a particular focus on PAH, as well as to offer guidelines for future investigations.