Histone deacetylase inhibitors synergize with sildenafil to suppress purine metabolism and proliferation in pulmonary hypertension

RATIONALE: Sildenafil, a well-known vasodilator known to interfere with purinergic signaling through effects on cGMP, is a mainstay in the treatment of pulmonary hypertension (PH). However, little is known regarding its effects on the metabolic reprogramming of vascular cells, which is a hallmark of PH. Purine metabolism, especially intracellular de novo purine biosynthesis is essential for vascular cell proliferation. Since adventitial fibroblasts are critical contributors to proliferative vascular remodeling in PH, in this study we aimed to investigate if sildenafil, beyond its well-known vasodilator role in smooth muscle cells, impacts intracellular purine metabolism and proliferation of fibroblasts derived from human PH patients. METHODS: Integrated omics approaches (plasma and cell metabolomics) and pharmacological inhibitor approaches were employed in plasma samples and cultured pulmonary artery fibroblasts from PH patients. MEASUREMENTS AND MAIN RESULTS: Plasma metabolome analysis of 27 PH patients before and after treatment with sildenafil, demonstrated a partial, but specific effect of sildenafil on purine metabolites, especially adenosine, adenine, and xanthine. However, circulating markers of cell stress, including lactate, succinate, and hypoxanthine were only decreased in a small subset of sildenafil-treated patients. To better understand potential effects of sildenafil on pathological changes in purine metabolism (especially purine synthesis) in PH, we performed studies on pulmonary fibroblasts from PAH patients (PH-Fibs) and corresponding controls (CO-Fibs), since these cells have previously been shown to demonstrate stable and marked PH associated phenotypic and metabolic changes. We found that PH-Fibs exhibited significantly increased purine synthesis. Treatment of PH-Fibs with sildenafil was insufficient to normalize cellular metabolic phenotype and only modestly attenuated the proliferation. However, we observed that treatments which have been shown to normalize glycolysis and mitochondrial abnormalities including a PKM2 activator (TEPP-46), and the histone deacetylase inhibitors (HDACi), SAHA and Apicidin, had significant inhibitory effects on purine synthesis. Importantly, combined treatment with HDACi and sildenafil exhibited synergistic inhibitory effects on proliferation and metabolic reprogramming in PH-Fibs. CONCLUSIONS: While sildenafil alone partially rescues metabolic alterations associated with PH, treatment with HDACi, in combination with sildenafil, represent a promising and potentially more effective strategy for targeting vasoconstriction, metabolic derangement and pathological vascular remodeling in PH

Extracellular ATP is a pro-angiogenic factor for pulmonary artery vasa vasorum endothelial cells

Expansion of the vasa vasorum network has been observed in a variety of systemic and pulmonary vascular diseases. We recently reported that a marked expansion of the vasa vasorum network occurs in the pulmonary artery adventitia of chronically hypoxic calves. Since hypoxia has been shown to stimulate ATP release from both vascular resident as well as circulatory blood cells, these studies were undertaken to determine if extracellular ATP exerts angiogenic effects on isolated vasa vasorum endothelial cells (VVEC) and/or if it augments the effects of other angiogenic factors (VEGF and basic FGF) known to be present in the hypoxic microenvironment. We found that extracellular ATP dramatically increases DNA synthesis, migration, and rearrangement into tube-like networks on Matrigel in VVEC, but not in pulmonary artery (MPAEC) or aortic (AOEC) endothelial cells obtained from the same animals. Extracellular ATP potentiated the effects of both VEGF and bFGF to stimulate DNA synthesis in VVEC but not in MPAEC and AOEC. Analysis of purine and pyrimidine nucleotides revealed that ATP, ADP and MeSADP were the most potent in stimulating mitogenic responses in VVEC, indicating the involvement of the family of P2Y1-like purinergic receptors. Using pharmacological inhibitors, Western blot analysis, and Phosphatidylinositol-3 kinase (PI3K) in vitro kinase assays, we found that PI3K/Akt/mTOR and ERK1/2 play a critical role in mediating the extracellular ATP-induced mitogenic and migratory responses in VVEC. However, PI3K/Akt and mTOR/p70S6K do not significantly contribute to extracellular ATP-induced tube formation on Matrigel. Our studies indicate that VVEC, isolated from the sites of active angiogenesis, exhibit distinct functional responses to ATP, compared to endothelial cells derived from large pulmonary or systemic vessels. Collectively, our data support the idea that extracellular ATP participates in the expansion of the vasa vasorum that can be observed in hypoxic conditions

SIRT1 Overexpression Antagonizes Cellular Senescence with Activated ERK/S6k1 Signaling in Human Diploid Fibroblasts

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated β-galactosidase (SA-β-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan.. Western blot results showed that SIRT1 reduced the expression of p16INK4A and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16INK4A and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca2+ concentration ([Ca2+]i) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca2+ signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca2+]i via Ca2+ influx through plasma membrane channels as well as Ca2+ mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca2+ responses in both cytosolic and nuclear compartments. An increase in [Ca2+]i was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPβS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αβMeATP, and βγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca2+ responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion

Pulmonary Artery Adventitial Fibroblasts Cooperate with Vasa Vasorum Endothelial Cells to Regulate Vasa Vasorum Neovascularization : A Process Mediated by Hypoxia and Endothelin-1

The precise cellular and molecular mechanisms regulating adventitial vasa vasorum neovascularization, which occurs in the pulmonary arterial circulation in response to hypoxia, remain unknown. Here, using a technique to isolate and culture adventitial fibroblasts (AdvFBs) and vasa vasorum endothelial cells (VVECs) from the adventitia of pulmonary arteries, we report that hypoxia-activated pulmonary artery AdvFBs exhibited pro-angiogenic properties and influenced the angiogenic phenotype of VVEC, in a process of cell-cell communication involving endothelin-1 (ET-1). We demonstrated that AdvFBs, either via co-culture or conditioned media, stimulated VVEC proliferation and augmented the self-assembly and integrity of cord-like networks that formed when VVECs where cultured on Matrigel. In addition, hypoxia-activated AdvFBs produced ET-1, suggesting a paracrine role for this pro-angiogenic molecule in these processes. When co-cultured on Matrigel, AdvFBs and VVECs self-assembled into heterotypic cord-like networks, a process augmented by hypoxia but attenuated by either selective endothelin receptor antagonists or oligonucleotides targeting prepro-ET-1 mRNA. From these observations, we propose that hypoxia-activated AdvFBs exhibit pro-angiogenic properties and, as such, communicate with VVECs, in a process involving ET-1, to regulate vasa vasorum neovascularization occurring in the adventitia of pulmonary arteries in response to chronic hypoxia