An Inducible and Vascular Smooth Muscle Cell-Specific Pink1 Knockout Induces Mitochondrial Energetic Dysfunction during Atherogenesis.

DNA damage and mitochondrial dysfunction are defining characteristics of aged vascular smooth muscle cells (VSMCs) found in atherosclerosis. Pink1 kinase regulates mitochondrial homeostasis and recycles dysfunctional organelles critical for maintaining energetic homeostasis. Here, we generated a new vascular-specific Pink1 knockout and assessed its effect on VSMC-dependent atherogenesis in vivo and VSMC energetic metabolism in vitro. A smooth muscle cell-specific and MHC-Cre-inducible flox'd Pink1f/f kinase knockout was made on a ROSA26+/0 and ApoE-/- C57Blk6/J background. Mice were high fat fed for 10 weeks and vasculature assessed for physiological and pathogical changes. Mitochondrial respiratory activity was then assessed in wild-type and knockout animals vessels and isolated cells for their reliance on oxidative and glycolytic metabolism. During atherogenesis, we find that Pink1 knockout affects development of plaque quality rather than plaque quantity by decreasing VSMC and extracellular matrix components, collagen and elastin. Pink1 protein is important in the wild-type VSMC response to metabolic stress and induced a compensatory increase in hexokinase II, which catalyses the first irreversible step in glycolysis. Pink1 appears to play an important role in VSMC energetics during atherogenesis but may also provide insight into the understanding of mitochondrial energetics in other diseases where the regulation of energetic switching between oxidative and glycolytic metabolism is found to be important.

Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype.

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox'd Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. METHODS: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. RESULTS: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. CONCLUSIONS: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.

Role of the Aryl Hydrocarbon Receptor in Sugen 5416-induced Experimental Pulmonary Hypertension.

Rats dosed with the vascular endothelial growth factor inhibitor Sugen 5416 (Su), subjected to hypoxia, and then restored to normoxia have become a widely used model of pulmonary arterial hypertension (PAH). However, the mechanism by which Su exacerbates pulmonary hypertension is unclear. We investigated Su activation of the aryl hydrocarbon receptor (AhR) in human pulmonary artery smooth muscle cells (hPASMCs) and blood outgrowth endothelial cells (BOECs) from female patients with PAH. We also examined the effect of AhR on aromatase and estrogen levels in the lung. Protein and mRNA analyses demonstrated that CYP1A1 was very highly induced in the lungs of Su/hypoxic (Su/Hx) rats. The AhR antagonist CH223191 (8 mg/kg/day) reversed the development of PAH in this model in vivo and normalized lung CYP1A1 expression. Increased lung aromatase and estrogen levels in Su/Hx rats were also normalized by CH223191, as was AhR nuclear translocator (ARNT [HIF-1ß]), which is shared by HIF-1α and AhR. Su reduced HIF-1α expression in hPASMCs. Su induced proliferation in BOECs and increased apoptosis in human pulmonary microvascular ECs and also induced translocation of AhR to the nucleus in hPASMCs. Under normoxic conditions, hPASMCs did not proliferate to Su. However, when grown in hypoxia (1%), Su induced hPASMC proliferation. In combination with hypoxia, Su is proliferative in hPASMCs and BOECs from patients with PAH, and Su/Hx-induced PAH in rats may be facilitated by AhR-induced CYP1A1, ARNT, and aromatase. Inhibition of AhR may be a novel approach to the treatment of pulmonary hypertension.

The Role of Sex in the Pathophysiology of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterised by increased pulmonary vascular resistance and pulmonary artery remodelling as result of increased vascular tone and vascular cell proliferation, respectively. Eventually, this leads to right heart failure. Heritable PAH is caused by a mutation in the bone morphogenetic protein receptor-II (BMPR-II). Female susceptibility to PAH has been known for some time, and most recent figures show a female-to-male ratio of 4:1. Variations in the female sex hormone estrogen and estrogen metabolism modify FPAH risk, and penetrance of the disease in BMPR-II mutation carriers is increased in females. Several lines of evidence point towards estrogen being pathogenic in the pulmonary circulation, and thus increasing the risk of females developing PAH. Recent studies have also suggested that estrogen metabolism may be crucial in the development and progression of PAH with studies indicating that downstream metabolites such as 16α-hydroxyestrone are upregulated in several forms of experimental pulmonary hypertension (PH) and can cause pulmonary artery smooth muscle cell proliferation and subsequent vascular remodelling. Conversely, other estrogen metabolites such as 2-methoxyestradiol have been shown to be protective in the context of PAH. Estrogen may also upregulate the signalling pathways of other key mediators of PAH such as serotonin.

Sex affects bone morphogenetic protein type II receptor signaling in pulmonary artery smooth muscle cells.

RATIONALE: Major pulmonary arterial hypertension (PAH) registries report a greater incidence of PAH in women; mutations in the bone morphogenic protein type II receptor (BMPR-II) occur in approximately 80% of patients with heritable PAH (hPAH). OBJECTIVES: We addressed the hypothesis that women may be predisposed to PAH due to normally reduced basal BMPR-II signaling in human pulmonary artery smooth muscle cells (hPASMCs). METHODS: We examined the BMPR-II signaling pathway in hPASMCs derived from men and women with no underlying cardiovascular disease (non-PAH hPASMCs). We also determined the development of pulmonary hypertension in male and female mice deficient in Smad1. MEASUREMENTS AND MAIN RESULTS: Platelet-derived growth factor, estrogen, and serotonin induced proliferation only in non-PAH female hPASMCs. Female non-PAH hPASMCs exhibited reduced messenger RNA and protein expression of BMPR-II, the signaling intermediary Smad1, and the downstream genes, inhibitors of DNA binding proteins, Id1 and Id3. Induction of phospho-Smad1/5/8 and Id protein by BMP4 was also reduced in female hPASMCs. BMP4 induced proliferation in female, but not male, hPASMCs. However, small interfering RNA silencing of Smad1 invoked proliferative responses to BMP4 in male hPASMCs. In male hPASMCs, estrogen decreased messenger RNA and protein expression of Id genes. The estrogen metabolite 4-hydroxyestradiol decreased phospho-Smad1/5/8 and Id expression in female hPASMCs while increasing these in males commensurate with a decreased proliferative effect in male hPASMCs. Female Smad1(+/-) mice developed pulmonary hypertension (reversed by ovariectomy). CONCLUSIONS: We conclude that estrogen-driven suppression of BMPR-II signaling in non-PAH hPASMCs derived from women contributes to a pro-proliferative phenotype in hPASMCs that may predispose women to PAH.

Direct Delivery of MicroRNA96 to the Lungs Reduces Progression of Sugen/Hypoxia-Induced Pulmonary Hypertension in the Rat.

The 5HT1B receptor (5HT1BR) contributes to the pathogenic effects of serotonin in pulmonary arterial hypertension. Here, we determine the effect of a microRNA96 (miR96) mimic delivered directly to the lungs on development of severe pulmonary hypertension in rats. Female rats were dosed with sugen (30 mg/kg) and subjected to 3 weeks of hypobaric hypoxia. In normoxia, rats were dosed with either a 5HT1BR antagonist SB216641 (7.5 mg/kg/day for 3 weeks), miR96, or scramble sequence (50 µg per rat), delivered by intratracheal (i.t) administration, once a week for 3 weeks. Cardiac hemodynamics were determined, pulmonary vascular remodeling was assessed, and gene expression was assessed by qRT-PCR, and in situ hybridization and protein expression were assessed by western blot and ELISA. miR96 expression was increased in pulmonary arteries and associated with a downregulation of the 5HT1BR protein in the lung. miR96 reduced progression of right ventricular systolic pressure, pulmonary arterial remodeling, right ventricular hypertrophy, and the occurrence of occlusive pulmonary lesions. Importantly, miR96 had no off-target effects and did not affect fibrotic markers of liver and kidney function. In conclusion, direct delivery of miR96 to the lungs was effective, reducing progression of sugen/hypoxia-induced pulmonary hypertension with no measured off-target effects. miR96 may be a novel therapy for pulmonary arterial hypertension, acting through downregulation of 5HT1BR.

Influence of 2-Methoxyestradiol and Sex on Hypoxia-Induced Pulmonary Hypertension and Hypoxia-Inducible Factor-1-α.

Background Women are at greater risk of developing pulmonary arterial hypertension, with estrogen and its downstream metabolites playing a potential role in the pathogenesis of the disease. Hypoxia-inducible factor-1-α (HIF 1α) is a pro-proliferative mediator and may be involved in the development of human pulmonary arterial hypertension . The estrogen metabolite 2-methoxyestradiol (2 ME 2) has antiproliferative properties and is also an inhibitor of HIF 1α. Here, we examine sex differences in  HIF 1α signaling in the rat and human pulmonary circulation and determine if 2 ME 2 can inhibit HIF 1α in vivo and in vitro. Methods and Results HIF 1α signaling was assessed in male and female distal human pulmonary artery smooth muscle cells ( hPASMC s), and the effects of 2 ME 2 were also studied in female hPASMC s. The in vivo effects of 2 ME 2 in the chronic hypoxic rat (male and female) model of pulmonary hypertension were also determined. Basal HIF 1α protein expression was higher in female hPASMC s compared with male. Both factor-inhibiting HIF and prolyl hydroxylase-2 (hydroxylates HIF leading to proteosomal degradation) protein levels were significantly lower in female hPASMC s when compared with males. In vivo, 2 ME 2 ablated hypoxia-induced pulmonary hypertension in male and female rats while decreasing protein expression of HIF 1α. 2 ME 2 reduced proliferation in hPASMC s and reduced basal protein expression of HIF 1α. Furthermore, 2 ME 2 caused apoptosis and significant disruption to the microtubule network. Conclusions Higher basal HIF 1α in female hPASMC s may increase susceptibility to developing pulmonary arterial hypertension . These data also demonstrate that the antiproliferative and therapeutic effects of 2 ME 2 in pulmonary hypertension may involve inhibition of HIF 1α and/or microtubular disruption in PASMC s.

Impaired mitochondrial respiration in human carotid plaque atherosclerosis: A potential role for Pink1 in vascular smooth muscle cell energetics.

BACKGROUND AND AIMS: DNA damage and mitochondrial dysfunction are thought to play an essential role in ageing and the energetic decline of vascular smooth muscle cells (VSMCs) essential for maintaining plaque integrity. We aimed to better understand VSMCs and identify potentially useful compensatory pathways that could extend their lifespan. Moreover, we wanted to assess if defects in mitochondrial respiration exist in human atherosclerotic plaques and to identify the appropriate markers that may reflect a switch in VSMC energy metabolism. METHODS: Human plaque tissue and cells were assessed for composition and evidence of DNA damage, repair capacity and mitochondrial dysfunction. Fresh plaque tissue was evaluated using high resolution oxygen respirometry to assess oxidative metabolism. Recruitment and processing of the mitochondrial regulator of autophagy Pink1 kinase was investigated in combination with transcriptional and protein markers associated with a potential switch to a more glycolytic metabolism. RESULTS: Human VSMC have increased nuclear (nDNA) and mitochondrial (mtDNA) damage and reduced repair capacity. A subset of VSMCs within plaque cap had decreased oxidative phosphorylation and expression of Pink1 kinase. Plaque cells demonstrated increased glycolytic activity in response to loss of mitochondrial function. A potential compensatory glycolytic program may act as energetic switch via AMP kinase (AMPK) and hexokinase 2 (Hex2). CONCLUSIONS: We have identified a subset of plaque VSMCs required for plaque stability that have increased mitochondrial dysfunction and decreased oxidative phosphorylation. Pink1 kinase may initiate a cellular response to promote a compensatory glycolytic program associated with upregulation of AMPK and Hex2.

Lin28A induces energetic switching to glycolytic metabolism in human embryonic kidney cells.

BACKGROUND: Loss of a cell's capacity to generate sufficient energy for cellular functions is a key hallmark of the ageing process and ultimately leads to a variety of important age-related pathologies such as cancer, Parkinson's disease and atherosclerosis. Regenerative medicine has sought to reverse these pathologies by reprogramming somatic cells to a more juvenile energetic state using a variety of stem cell factors. One of these factors, Lin28, is considered a candidate for modification in the reprogramming of cellular energetics to ameliorate the ageing process while retaining cell phenotype. RESULTS: Over-expression of Lin28A resulted in key changes to cellular metabolism not observed in wild-type controls. Extracellular pH flux analysis indicated that Lin28A over expression significantly increased the rate of glycolysis, whilst high resolution oxygen respirometry demonstrated a reduced oxygen consumption. Western blot and real-time PCR analysis identified Hexokinase II as one of the key modulators of glycolysis in these cells which was further confirmed by increased glucose transport. A metabolic switching effect was further emphasised by Western blot analysis where the oxygen consuming mitochondrial complex IV was significantly reduced after Lin28A over expression. CONCLUSIONS: Results from this study confirm that Lin28A expression promotes metabolic switching to a phenotype that relies predominantly on glycolysis as an energy source, while compromising oxidative phosphorylation. Mechanisms to augment regulated Lin28A in age related pathologies that are characterised by mitochondria dysfunction or in differentiated and aged post-mitotic cells is the future goal of this work.