HIPPO components mammalian Ste20-like protein kinases 1 and 2 (MST1/2) are well described growth suppressors. However, in pulmonary arterial hypertension (PAH), MST1/2 switch their roles and become pro-proliferative and pro-survival molecules, supporting hyper-proliferation of pulmonary artery (PA) smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs), remodeling of small PAs, and pulmonary hypertension. Here, we report that MST1/2 promotes hyper-proliferation and apoptosis resistance of human PAH PASMCs and PAAFs by up-regulating cell division cycle protein 20 (CDC20), establishing novel link between HIPPO-MST1/2 and cell cycle regulation in PAH. Authors Contributions:conception and design of the work (EAG, SSP, TVK); acquisition, analysis, and interpretation of data (TD, IZ, LJ, SOO, AP, TA, DL, DG, JRG, PJW, HD, TK); drafting and editing the manuscript (EAG, SSP, TVK, JRG, PJW).

Background: Pulmonary Arterial Hypertension (PAH) is a progressive disease defined by a mean pulmonary arterial pressure exceeding 20 mmHg at rest. PAH is characterized by pulmonary vascular remodeling caused by increased proliferation and survival of resident PA cells. Cell Division Cycle 20 (CDC20) promotes cell cycle progression via activating the anaphase-promoting complex (APC/C) ubiquitin ligase and consequent ubiquitination and degradation of securin and cyclin B. The role of CDC20 in PAH is not known. Goal: To investigate the role of CDC20 in PAH. Methods: RNA sequencing, immunoblotting, immunohistochemistry, proliferation and apoptosis assays, SU5416/Hypoxia pulmonary hypertension (PH) rodent models. Results: CDC20 was overexpressed in distal pulmonary arteries (PAs) from human PAH and rat SU546/hypoxia PH lungs and in human PAH PA smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs), but not endothelial cells, compared to controls. This was associated with increased proliferation and deficiency of pro-apoptotic protein Bcl-2 interacting mediator of cell death (BIM). Knockdown of CDC20 reduced proliferation, up-regulated BIM, and induced apoptosis in human PAH PASMCs and PAAFs, while the pan-APC/C inhibitor proTAME had no effect, suggesting APC/C-independent mechanism of CDC20 action. Interestingly, overexpression of CDC20 in PAH PASMCs and PAAFs was accompanied by over-accumulation of canonical CDC20-APC/C substrate securin. Immunocytochemical and immunoprecipitation experiments revealed that securin co-localizes and directly interacts with CDC20.CDC20 knockdown or treatment with apcin (blocks CDC20 interaction with its substrates, including securin) reduced securin protein levels. To test potential mechanisms of CDC20 up-regulation in PAH, we treated non-diseased PASMCs and PAAFs with pro-PH factors, including CCL2, PDGF-BB, 10 ng/ml TGFβ, and TNFα. Interestingly, only TNFα induced accumulation of CDC20 and securin in control cells. Furthermore, significantly higher levels of TNFα were detected in media conditioned by PAH PASMCs (PAH PASMC-CM), but not PAAFs. PAH PASMC-CM induced hyper-proliferation and CDC20 over-accumulation in non-diseased PASMCs and PAAFs. CM from PAH PASMCs transfected with siRNA CDC20 was unable to induce PASMC proliferation, suggesting that CDC20 over-accumulation is self-supported via TNFα feed-forward loop. Treatment with CDC20-specific degrader PROTAC CP5V effectively reduced CDC20 and securin protein content, inhibited proliferation, restored BIM, and induced apoptosis in PAH PASMCs and PAAFs, without affecting control cells. CP5V treatment reversed SuHx-induced PA remodeling, PH, and right ventricular hypertrophy in male and female mice. Conclusions: CDC20 promotes PASMC and PAAF hyper-proliferation and survival, remodeling, and PH through a non-canonical up-regulation of Securin. Targeting CDC20 may represent a promising strategy to treat PAH.

Background: Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by excessive proliferation and reduced apoptosis of pulmonary arterial smooth muscle cells (PASMCs) and pulmonary vascular remodeling. In PAH, PASMCs undergo a metabolic shift to glycolysis, resulting in lactate over-production. Lactate promotes tumor progression through a post-translational protein modification called lysine lactylation. Protein arginine methyltransferase 5 (PRMT5), the primary enzyme responsible for symmetric dimethylarginine (SDMA) modifications of histone and non-histone proteins, is implicated in cancer. However, the role of PRMT5 and its potential interplay with lactate in PAH remains unknown. Goal: To determine the role of PRMT5 in PA remodeling and PAH. Methods: Immunohistochemical, immunoblot, proteomic analyses, proliferation, apoptosis assays; SU5416/Hypoxia (SuHx) mouse and rat models of PH. Results: PRMT5 was over-accumulated in SMα-actin-positive areas of small muscular PAs from PAH patients and rats with SuHx PH, and distal human PAH PASMCs compared to non-diseased controls, supporting increased protein SDMA modification, PASMC hyper-proliferation, and apoptosis resistance. Proteomic analysis of nuclear fraction anti-Kla immunoprecipitates identified PRMT5 as one of most significantly hyper-lactylated nuclear proteins in PAH PASMCs compared to controls. siRNA-induced depletion of lactate dehydrogenase (LDHA), an enzyme converting pyruvate into lactate, downregulated PRMT5, inhibited proliferation and induced apoptosis in human PAH PASMCs. Treatment of control PASMCs with lactate or pro-PH factor PDGF-BB upregulated PRMT5 and cell proliferation. Importantly, siRNA PRMT5 reduced P-S6 and P-S473-Akt, inhibited proliferation and induced apoptosis of human PAH PASMCs. Furthermore, pharmacological inhibition of PRMT5 with GSK3326595 significantly inhibited proliferation and induced apoptosis of human PAH PASMCs, and significantly decreased PA remodeling and PH, as assessed by medial wall thickness (PA MT) and systolic RV pressure (sRVP), in mice with SuHx-induced PH compared to vehicle-treated group. Conclusions: Collectively, our data suggest that LDHA/lactate and PDGF-BB-driven PRMT5 up-regulation promotes PASMC hyper-proliferation, survival, PA remodeling, and PH via activating Akt/mTOR. Targeting PRMT5 signaling could represent potentially attractive strategy to treat PAH.

Pulmonary arterial hypertension (PAH) is a progressive and potentially a rapidly fatal disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PA) leading to increased pulmonary vascular resistance and right heart failure. Central to the remodeling process is a switch of the smooth muscle cells in small PAs (PASMC) to a proliferative, apoptosis-resistant phenotype. There is reason to suspect that the plasminogen activator system may play an important role in the remodeling program in PAH based on its roles in vascular post-injury restenosis, fibrosis, angiogenesis and tumorigenesis. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of the plasminogen activators - urokinase-type and tissue-type (uPA and tPA, respectively). Immunohisto- chemical and immunoblot analyses revealed that PAI-1 was deficient in smooth muscle areas of small remodeled PAs and early-passage PASMC from subjects with PAH compared to non-PAH controls. PAI1-/- male and female mice developed spontaneous pulmonary vascular remodeling and pulmonary hypertension (PH) as evidenced by significant increase in PA medial thickness, systolic right ventricular pressure, and right ventricular hypertrophy. Lastly, the uPA inhibitors upamostat (WX-671) and amiloride analog BB2-30F down-regulated mTORC1 and SMAD3, restored PAI-1 levels, reduced proliferation, and induced apoptosis in human PAH PASMC. We examined the effect of inhibition of uPA catalytic activity by BB2-30F on the development of SU5416/Hypoxia (SuHx)-induced PH in mice. Vehicletreated SuHx-exposed mice had up-regulated mTORC1 in small PAs, developed pulmonary vascular remodeling and PH, as evidenced by significant increase of PA MT, sRVP, RV hypertrophy, and a significant decrease in the pulmonary artery acceleration time/pulmonary ejection time (PAAT/PET) ratio compared to age- and sex-matched normoxia controls, whereas BB2-30F-treated group was protected from all these pathological changes. Taken together, our data strongly suggest that PAI-1 down- regulation in PASMC from human PAH lungs promotes PASMC hyper-proliferation, remodeling, and spontaneous PH due to unopposed uPA activation. Further studies are needed to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate the progression and/or reverse pulmonary vascular remodeling and PH.

Background: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs) due to hyper-proliferation and apoptosis resistance of resident PA cells, the mechanisms of which are not completely understood. Specifically, the role of cell division cycle protein 20 (CDC20), a key regulator of cell cycle, canonically acting as an APC/C E3 ubiquitin ligase activator and substrate recruiter, is not known. Goals: To determine the role of CDC20 in PA remodeling in PAH. Methods: RNAseq, immunoblot, immunohistochemistry, proliferation, apoptosis assays; SU5416/Hypoxia (SuHx) mouse model of PH. Results: CDC20 was over-expressed in lungs, small PAs, isolated PA smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs), but not endothelial cells from PAH patients compared to non-diseased controls. siRNA CDC20 significantly reduced proliferation and induced apoptosis in PAH PASMCs and PAAFs. This effect was re-capitulated by CDC20 inhibitor apcin (blocks binding of CDC20 with substrates); however, pan-APC inhibitor proTAME had no effect. Over-expression of CDC20 in PAH PASMCs and PAAFs was correlated with over-accumulation of its substrate, pro-proliferative protein securin, and deficiency of pro-apoptotic protein Bcl-2 interacting mediator of cell death (BIM). Depletion of CDC20 with specific siRNA, but not treatment with proTAME, dramatically reduced securin and restored BIM protein levels in PAH PASMCs and PAAFs. Apcin restored BIM protein content in human PAH PASMCs, and siRNA BIM protected PAH PASMCs from apcin-induced apoptosis. Pharmacological targeting of CDC20 with specific PROTAC degrader CP5V down-regulated CDC20 and securin, restored BIM, inhibited hyper-proliferation, and induced apoptosis in PAH, but not in control PASMCs, and reversed SuHx-induced pulmonary vascular remodeling, PH, and right ventricular hypertrophy in male and female mice. Conclusions: Collectively, our data demonstrate that CDC20 is overexpressed and supports proliferative, apoptosis resistant PASMC/PAAF phenotype in PAH via non-canonical APC/C independent upregulation of pro-proliferative securin and downregulation of pro-apoptotic BIM. Our data also suggest that targeting CDC20 could represent a novel potentially attractive strategy to suppress PA resident cells proliferation and reverse established PAH.

Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by a progressive increase in pulmonary vascular resistance leading to right ventricular failure and often death. Here we report that deficiency of transcription factor GATA6 is a shared pathological feature of PA endothelial (PAEC) and smooth muscle cells (PASMC) in human PAH and experimental PH, which is responsible for maintenance of hyper-proliferative cellular phenotypes, pulmonary vascular remodeling and pulmonary hypertension. We further show that GATA6 acts as a transcription factor and direct positive regulator of anti-oxidant enzymes, and its deficiency in PAH/PH pulmonary vascular cells induces oxidative stress and mitochondrial dysfunction. We demonstrate that GATA6 is regulated by the BMP10/BMP receptors axis and its loss in PAECs and PASMC in PAH supports BMPR deficiency. In addition, we have established that GATA6-deficient PAEC, acting in a paracrine manner, increase proliferation and induce other pathological changes in PASMC, supporting the importance of GATA6 in pulmonary vascular cell communication. Treatment with dimethyl fumarate resolved oxidative stress and BMPR deficiency, reversed hemodynamic changes caused by endothelial Gata6 loss in mice, and inhibited proliferation and induced apoptosis in human PAH PASMC, strongly suggesting that targeting GATA6 deficiency may provide a therapeutic advance for patients with PAH.

Background: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of small pulmonary arteries (PA) due to hyper-proliferation of resident PA cells, leading to elevated right ventricular (RV) afterload and heart failure. PA smooth muscle (SM) cells (PASMC) in PAH have metabolic shift to glycolysis and lactate over-production. Goals: To investigate the role of lactate over-production in PA remodeling and PAH. Methods: RT-PCR, immunohistochemical, immunoblot, proteomic analyses; proliferation (Ki67), lactate, apoptosis assays; SU5416/Hypoxia (SuHx) rat and mouse models of PH. Results: Lactate dehydrogenase A (LDHA) was over-expressed in SMα-actin-positive areas of small muscular PAs from PAH subjects and rodents with SuHx PH, and distal human PAH PASMC compared to controls, promoting lactate over-production, consequent up-regulation of Yap/Taz, Akt-mTOR, increased proliferation and survival. SM22α-Ldha-/- mice were protected from SuHx-induced PH and RV hypertrophy. Lysine lactylation (Kla) was increased in small PAs and PASMC from human PAH lungs. Proteomic analysis identified 12 non-histone proteins that were hyper-lactylated in PAH PASMC. Following validation revealed that hyper-lactylation induced over-accumulation of TOP1 and deficiency of EMILIN1 in small PAs and PASMC from PAH lungs, leading to up-regulation of Yap/Taz, Akt-mTOR, TGFβ1, and increased proliferation. Lactate self-supported its over-production by up-regulating glycolysis and LDHA overexpression through EMILIN1-TGFβ1-HIF1α loop. Human PAH PASMC had elevated lactate secretion; exogenous lactate induced proliferation of control PAEC and PAAF. Pharmacological inhibition of LDHA-lactate reduced proliferation and selectively promoted apoptosis in human PAH PASMC, reversed SuHx-induced PA remodeling, PH, and RV hypertrophy in mice. Conclusions: LDHA-driven lactate over-production promotes proliferative, apoptosis-resistant PASMC phenotype, pulmonary vascular remodeling, and PH via hyper-lactylation and over-accumulation of TOP1, deficiency of EMILIN1, and consequent activation of Yap/Taz, Akt/mTOR, and TGFβ1. Targeting LDHA-lactate network could represent potentially attractive strategy to treat PAH.

Rationale: The MSTs (mammalian Ste20-like kinases) 1/2 are members of the HIPPO pathway that act as growth suppressors in adult proliferative diseases. Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small PAs, pulmonary vascular remodeling, and the rise of pulmonary arterial pressure. The role of MST1/2 in PAH is currently unknown. Objective: To investigate the roles and mechanisms of the action of MST1 and MST2 in PAH. Methods and Results: Using early-passage pulmonary vascular cells from PAH and nondiseased lungs and mice with smooth muscle-specific tamoxifen-inducible Mst1/2 knockdown, we found that, in contrast to canonical antiproliferative/proapoptotic roles, MST1/2 act as proproliferative/prosurvival molecules in human PAH pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts and support established pulmonary vascular remodeling and pulmonary hypertension in mice with SU5416/hypoxia-induced pulmonary hypertension. By using unbiased proteomic analysis, gain- and loss-of function approaches, and pharmacological inhibition of MST1/2 kinase activity by XMU-MP-1, we next evaluated mechanisms of regulation and function of MST1/2 in PAH pulmonary vascular cells. We found that, in PAH pulmonary arterial adventitial fibroblasts, the proproliferative function of MST1/2 is caused by IL-6-dependent MST1/2 overexpression, which induces PSMC6-dependent downregulation of forkhead homeobox type O 3 and hyperproliferation. In PAH pulmonary arterial vascular smooth muscle cells, MST1/2 acted via forming a disease-specific interaction with BUB3 and supported ECM (extracellular matrix)- and USP10-dependent BUB3 accumulation, upregulation of Akt-mTORC1, cell proliferation, and survival. Supporting our in vitro observations, smooth muscle-specific Mst1/2 knockdown halted upregulation of Akt-mTORC1 in small muscular PAs of mice with SU5416/hypoxia-induced pulmonary hypertension. Conclusions: Together, this study describes a novel proproliferative/prosurvival role of MST1/2 in PAH pulmonary vasculature, provides a novel mechanistic link from MST1/2 via BUB3 and forkhead homeobox type O to the abnormal proliferation and survival of pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.

Introduction: Pulmonary arterial hypertension (PAH), a deadly disease with no cure, manifests by small PA remodeling, leading to increased PA pressure and right ventricular (RV) afterload, heart failure, and death. Pulmonary vascular remodeling is characterized by increased proliferation and survival of resident PA vascular cells, the mechanisms of which are not fully understood. Class III phosphatidylinositol 3-kinase vacuolar protein sorting 34 (Vps34) promotes cell hyper-proliferation in cancer. The status and mechanisms of regulation and function of Vps34 in PA vascular cells in PAH are not clear. Methods/Results: Immunohistochemical analysis showed that inhibitory Ser164 phosphorylation of Vps34 (P-Ser164-Vps34) was significantly decreased in smooth muscle alpha-actin (SMA)-positive areas of small remodeled PAs from PAH patients compared to PAs from non-diseased donors. Immunoblot (IB) analysis of human PA vascular smooth muscle cells (PAVSMC) from small PAs further demonstrated that P-Ser164-Vps34 is significantly decreased in human PAH PAVSMC. Similar to human PAH, we detected a significant decrease of P-Ser164-Vps34 in SMA-positive areas of small remodeled PAs from mice and rats with SU5416/hypoxia-induced PH, which was associated with increased PA medial thickness, RV pressure, and RV hypertrophy. IB analysis of PAH PAVSMC with Akt inhibitor VIII treatment or Akt-knockdown by specific siRNA demonstrated that Vps34 activation is Akt-dependent, which is associated with TSC2 deficiency, Vps15 over-accumulation, and increased proliferation and survival. Pharmacological inhibition of Vps34 in PAH PAVSMC by selective inhibitors SAR405 and VPS34-IN1 significantly decreased proliferation (Ki67) and induced apoptosis (TUNEL). Two-week treatment of SAR405 in mice with SU5416/hypoxia-induced PH attenuated pulmonary vascular remodeling. The above results suggest a therapeutic potential of Vps34 inhibition to reduce PAH PAVSMC hyper-proliferation and attenuate pulmonary vascular remodeling in PAH. Conclusions: Akt-dependent Vps34 activation supports proliferation and survival of PAH PAVSMC. Further studies are needed to evaluate the therapeutic potential of Vps34 inhibition against PAH.

Hyper-proliferation of pulmonary arterial vascular smooth muscle cells (PAVSMC) is an important pathological component of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Lipogenesis is linked to numerous proliferative diseases, but its role in PAVSMC proliferation in PAH remains to be elucidated. We found that early-passage human PAH PAVSMC had significant up-regulation of key fatty acids synthesis enzymes ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN), and increased unstimulated proliferation compared to control human PAVSMC. Treatment with an allosteric ACC inhibitor 5-tetradecyloxy-2-furoic acid (TOFA) significantly decreased proliferation and induced apoptosis of human PAH PAVSMC. Intracellular lipid content and proliferation of PAH PAVSMC were not reduced by incubation in lipid-depleted media but suppressed by a non-metabolizable analog of glucose 2-Deoxy-D-glucose (2-DG) and partially restored by addition of pyruvate. Protein kinase Akt was upregulated in human PAH PAVSMC in a sirtuin 7 (SIRT7)- and c-Jun N-terminal kinase (JNK)-dependent manner. Pharmacological inhibition of Akt down-regulated ACLY and ACC, significantly reduced intracellular lipid content, inhibited proliferation and induced apoptosis of human PAH PAVSMC. Taken together, these data demonstrate that human PAH PAVSMC have up-regulated lipogenesis, which is supported in an Akt- and glycolysis-dependent manner and is required for increased proliferation and survival. Our data suggest that there is a mechanistic link between glycolysis, lipogenesis, and the proliferation of human PAH PAVSMC and call for further studies to determine the potential attractiveness of a SIRT7/JNK-Akt-lipogenesis axis as a target pathway to inhibit PAVSMC hyper-proliferation in PAH.

Whether fragile X mental retardation protein (FMRP) target mRNAs and neuronal activity contributing to elevated basal neuronal protein synthesis in fragile X syndrome (FXS) is unclear. Our proteomic experiments reveal that the de novo translational profile in FXS model mice is altered at steady state and in response to metabotropic glutamate receptor (mGluR) stimulation, but the proteins expressed differ under these conditions. Several altered proteins, including Hexokinase 1 and Ras, also are expressed in the blood of FXS model mice and pharmacological treatments previously reported to ameliorate phenotypes modify their abundance in blood. In addition, plasma levels of Hexokinase 1 and Ras differ between FXS patients and healthy volunteers. Our data suggest that brain-based de novo proteomics in FXS model mice can be used to find altered expression of proteins in blood that could serve as disease-state biomarkers in individuals with FXS.