Restoration of Foxp3+ Regulatory T Cells by HDAC-Dependent Epigenetic Modulation Plays a Pivotal Role in Resolving Pulmonary Arterial Hypertension Pathology.

Rationale: Immune dysregulation is a common feature of pulmonary arterial hypertension (PAH). Histone deacetylase (HDAC)-dependent transcriptional reprogramming epigenetically modulates immune homeostasis and is a novel disease-oriented approach in modern times. Objectives: To identify a novel functional link between HDAC and regulatory T cells (Tregs) in PAH, aiming to establish disease-modified biomarkers and therapeutic targets. Methods: Peripheral blood mononuclear cells were isolated from patients with idiopathic PAH (IPAH) and rodent models of pulmonary hypertension (PH): monocrotaline rats, Sugen5416-hypoxia rats, and Treg-depleted mice. HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) was used to examine the immune modulatory effects in vivo, ex vivo, and in vitro. Measurements and Main Results: Increased HDAC expression was associated with reduced Foxp3+ Tregs and increased PD-1 (programmed cell death-1) signaling in peripheral blood mononuclear cells from patients with IPAH. SAHA differentially modified a cluster of epigenetic-sensitive genes and induced Foxp3+ Treg conversion in IPAH T cells. Rodent models recapitulated these epigenetic aberrations and T-cell dysfunction. SAHA attenuated PH phenotypes and restored FOXP3 transcription and Tregs in PH rats; interestingly, the effects were more profound in female rats. Selective depletion of CD25+ Tregs in Sugen5416-hypoxia mice neutralized the effects of SAHA. Furthermore, SAHA inhibited endothelial cytokine/chemokine release upon stimulation and subsequent immune chemotaxis. Conclusions: Our results indicated HDAC aberration was associated with Foxp3+ Treg deficiency and demonstrated an epigenetic-mediated mechanism underlying immune dysfunction in PAH. Restoration of Foxp3+ Tregs by HDAC inhibitors is a promising approach to resolve pulmonary vascular pathology, highlighting the potential benefit of developing epigenetic therapies for PAH.

Macrophage Xanthine Oxidoreductase Links LPS Induced Lung Inflammatory Injury to NLRP3 Inflammasome Expression and Mitochondrial Respiration.

Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) remain poorly treated inflammatory lung disorders. Both reactive oxygen species (ROS) and macrophages are involved in the pathogenesis of ALI/ARDS. Xanthine oxidoreductase (XOR) is an ROS generator that plays a central role in the inflammation that contributes to ALI. To elucidate the role of macrophage-specific XOR in endotoxin induced ALI, we developed a conditional myeloid specific XOR knockout in mice. Myeloid specific ablation of XOR in LPS insufflated mice markedly attenuated lung injury demonstrating the essential role of XOR in this response. Macrophages from myeloid specific XOR knockout exhibited loss of inflammatory activation and increased expression of anti-inflammatory genes/proteins. Transcriptional profiling of whole lung tissue of LPS insufflated XOR fl/fl//LysM-Cre mice demonstrated an important role for XOR in expression and activation of the NLRP3 inflammasome and acquisition of a glycolytic phenotype by inflammatory macrophages. These results identify XOR as an unexpected link between macrophage redox status, mitochondrial respiration and inflammatory activation.

Local Complement Contributes to Pathogenic Activation of Lung Endothelial Cells in SARS-CoV-2 Infection.

Endothelial dysfunction and inflammation contribute to the vascular pathology of coronavirus disease (COVID-19). However, emerging evidence does not support direct infection of endothelial or other vascular wall cells, and thus inflammation may be better explained as a secondary response to epithelial cell infection. In this study, we sought to determine whether lung endothelial or other resident vascular cells are susceptible to productive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and how local complement activation contributes to endothelial dysfunction and inflammation in response to hypoxia and SARS-CoV-2-infected lung alveolar epithelial cells. We found that ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) mRNA expression in lung vascular cells, including primary human lung microvascular endothelial cells (HLMVECs), pericytes, smooth muscle cells, and fibroblasts, was 20- to 90-fold lower compared with primary human alveolar epithelial type II cells. Consistently, we found that HLMVECs and other resident vascular cells were not susceptible to productive SARS-CoV-2 infection under either normoxic or hypoxic conditions. However, viral uptake without replication (abortive infection) was observed in HLMVECs when exposed to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells. Furthermore, we demonstrated that exposure of HLMVECs to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells and hypoxia resulted in upregulation of inflammatory factors such as ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), and IL-6 (interleukin 6) as well as complement components such as C3 (complement C3), C3AR1 (complement C3a receptor 1), C1QA (complement C1q A chain), and CFB (complement factor B). Taken together, our data support a model in which lung endothelial and vascular dysfunction during COVID-19 involves the activation of complement and inflammatory signaling and does not involve productive viral infection of endothelial cells.

Functional and molecular determinants of right ventricular response to severe pulmonary hypertension in a large animal model.

Right ventricular (RV) failure is the major determinant of outcome in pulmonary hypertension (PH). Calves exposed to 2-wk hypoxia develop severe PH and unlike rodents, hypoxia-induced PH in this species can lead to right heart failure. We, therefore, sought to examine the molecular and structural changes in the RV in calves with hypoxia-induced PH, hypothesizing that we could identify mechanisms underlying compensated physiological function in the face of developing severe PH. Calves were exposed to 14 days of environmental hypoxia (equivalent to 4,570 m/15,000 ft elevation, n = 29) or ambient normoxia (1,525 m/5,000 ft, n = 25). Cardiopulmonary function was evaluated by right heart catheterization and pressure volume loops. Molecular and cellular determinants of RV remodeling were analyzed by cDNA microarrays, RealTime PCR, proteomics, and immunochemistry. Hypoxic exposure induced robust PH, with increased RV contractile performance and preserved cardiac output, yet evidence of dysregulated RV-pulmonary artery mechanical coupling as seen in advanced disease. Analysis of gene expression revealed cellular processes associated with structural remodeling, cell signaling, and survival. We further identified specific clusters of gene expression associated with 1) hypertrophic gene expression and prosurvival mechanotransduction through YAP-TAZ signaling, 2) extracellular matrix (ECM) remodeling, 3) inflammatory cell activation, and 4) angiogenesis. A potential transcriptomic signature of cardiac fibroblasts in RV remodeling was detected, enriched in functions related to cell movement, tissue differentiation, and angiogenesis. Proteomic and immunohistochemical analysis confirmed RV myocyte hypertrophy, together with localization of ECM remodeling, inflammatory cell activation, and endothelial cell proliferation within the RV interstitium. In conclusion, hypoxia and hemodynamic load initiate coordinated processes of protective and compensatory RV remodeling to withstand the progression of PH.NEW & NOTEWORTHY Using a large animal model and employing a comprehensive approach integrating hemodynamic, transcriptomic, proteomic, and immunohistochemical analyses, we examined the early (2 wk) effects of severe PH on the RV. We observed that RV remodeling during PH progression represents a continuum of transcriptionally driven processes whereby cardiac myocytes, fibroblasts, endothelial cells, and proremodeling macrophages act to coordinately maintain physiological homeostasis and protect myocyte survival during chronic, severe, and progressive pressure overload.

Potential long-term effects of SARS-CoV-2 infection on the pulmonary vasculature: a global perspective.

The lungs are the primary target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, with severe hypoxia being the cause of death in the most critical cases. Coronavirus disease 2019 (COVID-19) is extremely heterogeneous in terms of severity, clinical phenotype and, importantly, global distribution. Although the majority of affected patients recover from the acute infection, many continue to suffer from late sequelae affecting various organs, including the lungs. The role of the pulmonary vascular system during the acute and chronic stages of COVID-19 has not been adequately studied. A thorough understanding of the origins and dynamic behaviour of the SARS-CoV-2 virus and the potential causes of heterogeneity in COVID-19 is essential for anticipating and treating the disease, in both the acute and the chronic stages, including the development of chronic pulmonary hypertension. Both COVID-19 and chronic pulmonary hypertension have assumed global dimensions, with potential complex interactions. In this Review, we present an update on the origins and behaviour of the SARS-CoV-2 virus and discuss the potential causes of the heterogeneity of COVID-19. In addition, we summarize the pathobiology of COVID-19, with an emphasis on the role of the pulmonary vasculature, both in the acute stage and in terms of the potential for developing chronic pulmonary hypertension. We hope that the information presented in this Review will help in the development of strategies for the prevention and treatment of the continuing COVID-19 pandemic.

Complement-containing small extracellular vesicles from adventitial fibroblasts induce proinflammatory and metabolic reprogramming in macrophages.

Pulmonary hypertension (PH) is a severe cardiopulmonary disease characterized by complement-dependent, fibroblast-induced perivascular accumulation and proinflammatory activation of macrophages. We hypothesized that, in PH, nanoscale-sized small extracellular vesicles (sEVs), released by perivascular/adventitial fibroblasts, are critical mediators of complement-dependent proinflammatory activation of macrophages. Pulmonary adventitial fibroblasts were isolated from calves with severe PH (PH-Fibs) and age-matched controls (CO-Fibs). PH-Fibs exhibited increased secretion of sEVs, compared with CO-Fibs, and sEV biological activity was tested on mouse and bovine bone marrow-derived macrophages (BMDMs) and showed similar responses. Compared with sEVs derived from CO-Fibs, sEVs derived from PH-Fibs (PH-Fib-sEVs) induced augmented expression of proinflammatory cytokines/chemokines and metabolic genes in BMDMs. Pharmacological blockade of exosome release from PH-Fibs resulted in significant attenuation of proinflammatory activation of BMDMs. "Bottom-up" proteomic analyses revealed significant enrichment of complement and coagulation cascades in PH-Fib-sEVs, including augmented expression of the complement component C3. We therefore examined whether the PH-Fib-sEV-mediated proinflammatory activation of BMDMs was complement C3 dependent. Treatment of PH-Fibs with siC3-RNA significantly attenuated the capacity of PH-Fib-sEVs for proinflammatory activation of BMDMs. PH-Fib-sEVs mediated proglycolytic alterations and complement-dependent activation of macrophages toward a proinflammatory phenotype, as confirmed by metabolomic studies. Thus, fibroblast-released sEVs served as critical mediators of complement-induced perivascular/microenvironmental inflammation in PH.

Mechanisms of SARS-CoV-2-induced lung vascular disease: potential role of complement.

The outbreak of COVID-19 disease, caused by SARS-CoV-2 beta-coronovirus, urges a focused search for the underlying mechanisms and treatment options. The lung is the major target organ of COVID-19, wherein the primary cause of mortality is hypoxic respiratory failure, resulting from acute respiratory distress syndrome, with severe hypoxemia, often requiring assisted ventilation. While similar in some ways to acute respiratory distress syndrome secondary to other causes, lungs of some patients dying with COVID-19 exhibit distinct features of vascular involvement, including severe endothelial injury and cell death via apoptosis and/or pyroptosis, widespread capillary inflammation, and thrombosis. Furthermore, the pulmonary pathology of COVID-19 is characterized by focal inflammatory cell infiltration, impeding alveolar gas exchange resulting in areas of local tissue hypoxia, consistent with potential amplification of COVID-19 pathogenicity by hypoxia. Vascular endothelial cells play essential roles in both innate and adaptive immune responses, and are considered to be "conditional innate immune cells" centrally participating in various inflammatory, immune pathologies. Activated endothelial cells produce cytokines/chemokines, dynamically recruit and activate inflammatory cells and platelets, and centrally participate in pro-thrombotic processes (thrombotic microangiopathies). Initial reports presented pathological findings of localized direct infection of vascular endothelial cells with SARS-CoV-2, yet emerging evidence does not support direct infection of endothelial or other vascular wall cell and thus widespread endothelial cell dysfunction and inflammation may be better explained as secondary responses to epithelial cell infection and inflammation. Endothelial cells are also actively engaged in a cross-talk with the complement system, the essential arm of innate immunity. Recent reports present evidence for complement deposition in SARS-CoV-2-damaged lung microcirculation, further strengthening the idea that, in severe cases of COVID-19, complement activation is an essential player, generating destructive hemorrhagic, capillaritis-like tissue damage, clotting, and hyperinflammation. Thus, complement-targeted therapies are actively in development. This review is intended to explore in detail these ideas.

Microenvironmental Regulation of Macrophage Transcriptomic and Metabolomic Profiles in Pulmonary Hypertension.

The recruitment and subsequent polarization of inflammatory monocytes/macrophages in the perivascular regions of pulmonary arteries is a key feature of pulmonary hypertension (PH). However, the mechanisms driving macrophage polarization within the adventitial microenvironment during PH progression remain unclear. We previously established that reciprocal interactions between fibroblasts and macrophages are essential in driving the activated phenotype of both cell types although the signals involved in these interactions remain undefined. We sought to test the hypothesis that adventitial fibroblasts produce a complex array of metabolites and proteins that coordinately direct metabolomic and transcriptomic re-programming of naïve macrophages to recapitulate the pathophysiologic phenotype observed in PH. Media conditioned by pulmonary artery adventitial fibroblasts isolated from pulmonary hypertensive (PH-CM) or age-matched control (CO-CM) calves were used to activate bone marrow derived macrophages. RNA-Seq and mass spectrometry-based metabolomics analyses were performed. Fibroblast conditioned medium from patients with idiopathic pulmonary arterial hypertension or controls were used to validate transcriptional findings. The microenvironment was targeted in vitro using a fibroblast-macrophage co-culture system and in vivo in a mouse model of hypoxia-induced PH. Both CO-CM and PH-CM actively, yet distinctly regulated macrophage transcriptomic and metabolomic profiles. Network integration revealed coordinated rewiring of pro-inflammatory and pro-remodeling gene regulation in concert with altered mitochondrial and intermediary metabolism in response to PH-CM. Pro-inflammation and metabolism are key regulators of macrophage phenotype in vitro, and are closely related to in vivo flow sorted lung interstitial/perivascular macrophages from hypoxic mice. Metabolic changes are accompanied by increased free NADH levels and increased expression of a metabolic sensor and transcriptional co-repressor, C-terminal binding protein 1 (CtBP1), a mechanism shared with adventitial PH-fibroblasts. Targeting the microenvironment created by both cell types with the CtBP1 inhibitor MTOB, inhibited macrophage pro-inflammatory and metabolic re-programming both in vitro and in vivo. In conclusion, coordinated transcriptional and metabolic reprogramming is a critical mechanism regulating macrophage polarization in response to the complex adventitial microenvironment in PH. Targeting the adventitial microenvironment can return activated macrophages toward quiescence and attenuate pathological remodeling that drives PH progression.

Inflammation, immunity, and vascular remodeling in pulmonary hypertension; Evidence for complement involvement?

Pulmonary (arterial) hypertension (PH/PAH) is a life-threatening cardiopulmonary disorder. Experimental evidence suggests involvement of inflammatory and autoimmune processes in pathogenesis of PH/PAH, however the triggering and disease-promoting mechanisms remain unknown. The complement system is a key arm of innate immunity implicated in various pro-inflammatory and autoimmune diseases, yet, surprisingly little is known about the role of complement in PH/PAH pathogenesis. The preponderance of the existing data associates complement with PH/PAH via analysis of plasma and does not study the lung directly. Therefore, we aimed to resolve this by analyzing both the mechanisms of local lung-specific complement activation and the correlation of dysregulated plasma complement to clinical outcome in PAH patients. In our recent studies, reviewed herein, we show, for the first time, that  immunoglobulin-driven activation of the complement cascade, specifically its alternative pathway, in the pulmonary perivascular areas, is a key mechanism initiating pro-inflammatory processes in the early stage of experimental hypoxic PH (a form of "sterile inflammation"). In human patients with end-stage PAH, we have demonstrated that perivascular deposition of immunoglobulin G (IgG) and activation of the complement cascade are "longitudinally" persistent in the disease. We also showed, using unbiased network analysis, that plasma complement signaling, including again the Alternative pathway, is a prognostic factor of survival in patients with idiopathic PAH (IPAH). Based on these initial findings, we suggest that vascular-specific, immunoglobulin-driven dysregulated complement signaling triggers and maintains pulmonary vascular remodeling and PH. Future experiments in this area would facilitate discoveries on whether complement signaling can serve both as a biomarker and therapeutic target in PH/PAH.

Immunoglobulin-driven Complement Activation Regulates Proinflammatory Remodeling in Pulmonary Hypertension.

Rationale: Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder in which inflammation and immunity have emerged as critical early pathogenic elements. Although proinflammatory processes in PH and pulmonary arterial hypertension (PAH) are the focus of extensive investigation, the initiating mechanisms remain elusive.Objectives: We tested whether activation of the complement cascade is critical in regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and can serve as a prognostic biomarker of outcome in human PAH.Methods: We used immunostaining of lung tissues from experimental PH models and patients with PAH, analyses of genetic murine models lacking specific complement components or circulating immunoglobulins, cultured human pulmonary adventitial fibroblasts, and network medicine analysis of a biomarker risk panel from plasma of patients with PAH.Measurements and Main Results: Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH and PAH in experimental animal models and humans. In experimental hypoxic PH, proinflammatory and pro-proliferative responses were dependent on complement (alternative pathway and component 5), and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identified Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of patients with PAH, we demonstrated that complement signaling can serve as a prognostic factor for clinical outcome in PAH.Conclusions: This study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and demonstrates complement signaling as a critical determinant of clinical outcome in PAH.

A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remodelling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efficacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH.

Diagnostic pitfalls in vitamin B6-dependent epilepsy caused by mutations in the PLPBP gene.

Vitamin B6-responsive epilepsies are a group of genetic disorders including ALDH7A1 deficiency, PNPO deficiency, and others, usually causing neonatal onset seizures resistant to treatment with common antiepileptic drugs. Recently, biallelic mutations in PLPBP were shown to be a novel cause of vitamin B6-dependent epilepsy with a variable phenotype. The different vitamin B6-responsive epilepsies can be detected and distinguished by their respective biomarkers and genetic analysis. Unfortunately, metabolic biomarkers for early detection and prognosis of PLPBP deficiency are currently still lacking. Here, we present data from two further patients with vitamin B6-dependent seizures caused by variants in PLPBP, including a novel missense variant, and compare their genotype and phenotypic presentation to previously described cases. Hyperglycinemia and hyperlactatemia are the most consistently observed biochemical abnormalities in pyridoxal phosphate homeostasis protein (PLPHP) deficient patients and were present in both patients in this report within the first days of life. Lactic acidemia, the neuroradiological, and clinical presentation led to misdiagnosis of a mitochondrial encephalopathy in two previously published cases with an early fatal course. Similarly, on the background of glycine elevation in plasma, glycine encephalopathy was wrongly adopted as diagnosis for a patient in our report. In this regard, lactic acidemia as well as hyperglycinemia appear to be diagnostic pitfalls in patients with vitamin B6-responsive epilepsies, including PLPHP deficiency. SYNOPSIS: In vitamin B6-responsive epilepsies, including PLPHP deficiency, there are several diagnostic pitfalls, including lactic acidemia as well as hyperglycinemia, highlighting the importance of a pyridoxine trial, and genetic testing.

Suppression of HIF2 signalling attenuates the initiation of hypoxia-induced pulmonary hypertension.

Most published studies addressing the role of hypoxia inducible factors (HIFs) in hypoxia-induced pulmonary hypertension development employ models that may not recapitulate the clinical setting, including the use of animals with pre-existing lung/vascular defects secondary to embryonic HIF ablation or activation. Furthermore, critical questions including how and when HIF signalling contributes to hypoxia-induced pulmonary hypertension remain unanswered.Normal adult rodents in which global HIF1 or HIF2 was inhibited by inducible gene deletion or pharmacological inhibition (antisense oligonucleotides (ASO) and small molecule inhibitors) were exposed to short-term (4 days) or chronic (4-5 weeks) hypoxia. Haemodynamic studies were performed, the animals euthanised, and lungs and hearts obtained for pathological and transcriptomic analysis. Cell-type-specific HIF signals for pulmonary hypertension initiation were determined in normal pulmonary vascular cells in vitro and in mice (using cell-type-specific HIF deletion).Global Hif1a deletion in mice did not prevent hypoxia-induced pulmonary hypertension at 5 weeks. Mice with global Hif2a deletion did not survive long-term hypoxia. Partial Hif2a deletion or Hif2-ASO (but not Hif1-ASO) reduced vessel muscularisation, increases in pulmonary arterial pressures and right ventricular hypertrophy in mice exposed to 4-5 weeks of hypoxia. A small molecule HIF2 inhibitor (PT2567) significantly attenuated early events (monocyte recruitment and vascular cell proliferation) in rats exposed to 4 days of hypoxia, as well as vessel muscularisation, tenascin C accumulation and pulmonary hypertension development in rats exposed to 5 weeks of hypoxia. In vitro, HIF2 induced a distinct set of genes in normal human pulmonary vascular endothelial cells, mediating inflammation and proliferation of endothelial cells and smooth muscle cells. Endothelial Hif2a knockout prevented hypoxia-induced pulmonary hypertension in mice.Inhibition of HIF2 (but not HIF1) can provide a therapeutic approach to prevent the development of hypoxia-induced pulmonary hypertension. Future studies are needed to investigate the role of HIFs in pulmonary hypertension progression and reversal.

Hypoxia-induced pulmonary hypertension and chronic lung disease: caveolin-1 dysfunction an important underlying feature.

Caveolin-1 (cav-1) has been shown to play a significant role in the pathogenesis of pulmonary hypertension (PH). In the monocrotaline model of PH, the loss of endothelial cav-1 as well as reciprocal activation of proliferative and anti-apoptotic pathways initiate the disease process and facilitate its progression. In order to examine the role of cav-1 in hypoxia-induced PH, we exposed rats and neonatal calves to hypobaric hypoxia and obtained hemodynamic data and assessed the expression of cav-1 and related proteins eNOS, HSP90, PTEN, gp130, PY-STAT3, ß-catenin, and Glut1 in the lung tissue. Chronic hypoxic exposure in rats (48 h-4 weeks) and calves (two weeks) did not alter the expression of cav-1, HSP90, or eNOS. PTEN expression was significantly decreased accompanied by PY-STAT3 activation and increased expression of gp130, Glut1, and ß-catenin in hypoxic animals. We also examined cav-1 expression in the lung sections from steers with chronic hypoxic disease (Brisket disease) and from patients with chronic lung disease who underwent lung biopsy for medical reasons. There was no cav-1 loss in Brisket disease. In chronic lung disease cases, endothelial cav-1 expression was present, albeit with less intense staining in some cases. In conclusion, hypoxia did not alter the cav-1 expression in experimental models. The presence of cav-1, however, did not suppress hypoxia-induced activation of PY-STAT3 and ß catenin, increased gp130 and Glut1 expression, or prevent the PTEN loss, indicating cav-1 dysfunction in hypoxia-induced PH.

Dynamic and diverse changes in the functional properties of vascular smooth muscle cells in pulmonary hypertension.

Pulmonary hypertension (PH) is the end result of interaction between pulmonary vascular tone and a complex series of cellular and molecular events termed 'vascular remodelling'. The remodelling process, which can involve the entirety of pulmonary arterial vasculature, almost universally involves medial thickening, driven by increased numbers and hypertrophy of its principal cellular constituent, smooth muscle cells (SMCs). It is noted, however that SMCs comprise heterogeneous populations of cells, which can exhibit markedly different proliferative, inflammatory, and extracellular matrix production changes during remodelling. We further consider that these functional changes in SMCs of different phenotype and their role in PH are dynamic and may undergo significant changes over time (which we will refer to as cellular plasticity); no single property can account for the complexity of the contribution of SMC to pulmonary vascular remodelling. Thus, the approaches used to pharmacologically manipulate PH by targeting the SMC phenotype(s) must take into account processes that underlie dominant phenotypes that drive the disease. We present evidence for time- and location-specific changes in SMC proliferation in various animal models of PH; we highlight the transient nature (rather than continuous) of SMC proliferation, emphasizing that the heterogenic SMC populations that reside in different locations along the pulmonary vascular tree exhibit distinct responses to the stresses associated with the development of PH. We also consider that cells that have often been termed 'SMCs' may arise from many origins, including endothelial cells, fibroblasts and resident or circulating progenitors, and thus may contribute via distinct signalling pathways to the remodelling process. Ultimately, PH is characterized by long-lived, apoptosis-resistant SMC. In line with this key pathogenic characteristic, we address the acquisition of a pro-inflammatory phenotype by SMC that is essential to the development of PH. We present evidence that metabolic alterations akin to those observed in cancer cells (cytoplasmic and mitochondrial) directly contribute to the phenotype of the SM and SM-like cells involved in PH. Finally, we raise the possibility that SMCs transition from a proliferative to a senescent, pro-inflammatory and metabolically active phenotype over time.

Development of an electrospun biomimetic polyurea scaffold suitable for vascular grafting.

The optimization of biomechanical and biochemical properties of a vascular graft to render properties relevant to physiological environments is a major challenge today. These critical properties of a vascular graft not only regulate its stability and integrity, but also control invasion of cells for scaffold remodeling permitting its integration with native tissue. In this work, we have synthesized a biomimetic scaffold by electrospinning a blend of a polyurea, poly(serinol hexamethylene urea) (PSHU), and, a polyester, poly-ε-caprolactone (PCL). Mechanical properties of the scaffold were varied by varying polymer blending ratio and electrospinning flow rate. Mechanical characterization revealed that scaffolds with lower PSHU content relative to PCL content resulted in elasticity close to native mammalian arteries. We also found that increasing electrospinning flow rates also increased the elasticity of the matrix. Optimization of elasticity generated scaffolds that enabled vascular smooth muscle cells (SMCs) to adhere, grow and maintain a SMC phenotype. The 30/70 scaffold also underwent slower degradation than scaffolds with higher PSHU content, thereby, providing the best option for in vivo remodeling. Further, Gly-Arg-Gly-Asp-Ser (RGD) covalently conjugated to the polyurea backbone in 30/70 scaffold resulted in significantly increased clotting times. Reducing surface thrombogenicity by the conjugation of RGD is critical to avoiding intimal hyperplasia. Hence, biomechanical and biochemical properties of a vascular graft can be balanced by optimizing synthesis parameters and constituent components. For these reasons, the optimized RGD-conjugated 30/70 scaffold electrospun at 2.5 or 5 mL/h has great potential as a suitable material for vascular grafting applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 278-290, 2018.

Identification of MicroRNA-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 (Polypyrimidine Tract Binding Protein) and Pyruvate Kinase M2.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified. METHODS: Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. RESULTS: Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1, and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo. CONCLUSIONS: Pulmonary vascular and circulating progenitor endothelial cells isolated from patients with PAH demonstrate downregulation of miR-124, leading to the metabolic and proliferative abnormalities in PAH ECs via PTPB1 and PKM1/PKM2. Therefore, the manipulation of this miRNA or its targets could represent a novel therapeutic approach for the treatment of PAH.

Metabolic and Proliferative State of Vascular Adventitial Fibroblasts in Pulmonary Hypertension Is Regulated Through a MicroRNA-124/PTBP1 (Polypyrimidine Tract Binding Protein 1)/Pyruvate Kinase Muscle Axis.

BACKGROUND: An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments. METHODS: Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry. RESULTS: We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1ß expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results. CONCLUSIONS: In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

A Time- and Compartment-Specific Activation of Lung Macrophages in Hypoxic Pulmonary Hypertension.

Studies in various animal models suggest an important role for pulmonary macrophages in the pathogenesis of pulmonary hypertension (PH). Yet, the molecular mechanisms characterizing the functional macrophage phenotype relative to time and pulmonary localization and compartmentalization remain largely unknown. In this study, we used a hypoxic murine model of PH in combination with FACS to quantify and isolate lung macrophages from two compartments over time and characterize their programing via RNA sequencing approaches. In response to hypoxia, we found an early increase in macrophage number that was restricted to the interstitial/perivascular compartment, without recruitment of macrophages to the alveolar compartment or changes in the number of resident alveolar macrophages. Principal component analysis demonstrated significant differences in overall gene expression between alveolar and interstitial macrophages (IMs) at baseline and after 4 and 14 d hypoxic exposure. Alveolar macrophages at both day 4 and 14 and IMs at day 4 shared a conserved hypoxia program characterized by mitochondrial dysfunction, proinflammatory gene activation, and mTORC1 signaling, whereas IMs at day 14 demonstrated a unique anti-inflammatory/proreparative programming state. We conclude that the pathogenesis of vascular remodeling in hypoxic PH involves an early compartment-independent activation of lung macrophages toward a conserved hypoxia program, with the development of compartment-specific programs later in the course of the disease. Thus, harnessing time- and compartment-specific differences in lung macrophage polarization needs to be considered in the therapeutic targeting of macrophages in hypoxic PH and potentially other inflammatory lung diseases.

Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1.

BACKGROUND: Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1). METHODS: RNA sequencing, quantitative polymerase chain reaction,13C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U-13C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD+ ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice. RESULTS: We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD+ ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated proliferation, and suppressed macrophage numbers and remodeling in the distal pulmonary vasculature. CONCLUSIONS: CtBP1 is a critical factor linking changes in cell metabolism to cell phenotype in hypoxic and other forms of PH and a therapeutic target.