There is strong evidence supporting inflammatory and autoimmune processes in the pathogenesis of pulmonary arterial hypertension (PAH), although the initiating and disease-sustaining mechanisms remain unclear. Studies in arthritis, kidney disease, and cancer have demonstrated that dysregulation of the complement system can drive inflammation-mediated tissue injury. We have shown that activation of the complement cascade, particularly the alternative pathway, within the pulmonary vasculature is a key driver of proinflammatory responses in pulmonary hypertension (PH). Single-cell spatial transcriptomic analysis of PAH lungs further revealed complement-rich adventitial fibroblasts, granzyme K (GZMK)+ CD8 T cells, and activated macrophages forming proinflammatory niches within the pulmonary artery adventitia in the different pulmonary vascular lesions in PAH. Our recent work highlights the role of both extracellular and intracellular complement activation in fibroblast-mediated pulmonary vascular inflammation. Specifically, activation of the alternative pathway within fibroblasts, through complement factors D (CFD) and B (CFB), promotes production of anaphylatoxins (C3a and C5a), cytokines, and complement-containing extracellular vesicles (EVs). These mediators drive macrophage and T-cell recruitment and activation, contributing to disease progression. Additionally, a fourth pathway of complement activation mediated by CD8+ T cell-derived GZMK, which triggers extracellular complement cascades in fibroblasts and macrophages, has recently been identified. These findings support the hypotheses that local complement production by pulmonary artery adventitial fibroblasts, activated intracellularly by CFD and CFB and extracellularly by GZMK+, and its secretion in soluble form and within EVs promotes macrophage chemotaxis and activation. These complement-driven proinflammatory niches in PAH pulmonary arteries represent promising therapeutic targets in PH.

Background: Pulmonary hypertension (PH) is a progressive disorder marked by persistently activated cell types within pulmonary vessels, such as adventitial fibroblasts. PH-associated fibroblasts show altered gene expression linked to proliferation, metabolism, and inflammation. The complement system is traditionally viewed as a serum-based immune effector. However, recent findings suggest a role of fibroblast-specific, local, and intracellular-produced complement (complosome) proteins in driving vascular inflammation and remodeling in PH. This study investigates fibroblast-specific complement (CFD) in regulating glycolysis, mitochondrial function, and inflammation and assesses the therapeutic potential of CFD inhibitors in PH. Methods and Results: In vivo observations in humans and animals demonstrated high C3, CFB, and especially CFD signals in fibroblasts of PH lesions. To understand the role of complements fibroblasts. Fibroblasts were isolated from distal pulmonary arteries of patients with idiopathic pulmonary arterial hypertension (IPAH-Fibs) and calves with severe PH (PH-Fibs), and control fibroblasts (CO-Fibs) from healthy donors and age-matched bovine controls. Transcriptomic and protein analyses using RNAseq/qRT-PCR and immunoblotting revealed that IPAH/PH-Fibs showed significantly higher C3, C5, CFB, and CFD expression than CO-Fibs. This increased expression was accompanied by elevated levels of C3 and C5 activation fragments (C3a) both intracellularly and in extracellular medium, suggesting intracellular and local complement activation. CFD, a key enzyme in the alternative complement pathway, is crucial for C3 activation, as downregulating CFD in PH-Fibs led to reduced C3a production, suggesting a link between CFD activity and C3a generation. C3a receptor (C3aR1) was detected on isolated mitochondria and cell membranes of adventitial fibroblasts. C3a signal was also detected in fibroblast conditioned media and mitochondrial fraction, supporting the hypothesis that C3a-induced metabolic and proinflammatory reprogramming occurs via C3aR1. CFD knockdown normalized expression of elevated metabolic genes (GLUT1, HK2, GPI, ENO1, and ACO1) and proinflammatory genes (MCP1, SDF1, IL-6, IL-13, and IL-33) in PH-Fibs. PH-fibs exhibited increased activation of glycolysis, TCA, and fatty acid metabolism pathways, which were significantly reduced by CFD knockdown. Further in this study, we repurposed commercially available CFD inhibitors, particularly Danicopan and Vimircopan. We used isolated primary adventitial fibroblasts and precision-cut lung slices (PCLS) to test therapeutic potential of CFD inhibitors. Treatment with CFD inhibitors effectively reduced intracellular and local complement activation, particularly of CFB and C3 in PH Fibs. Conclusion: This study highlights the crucial role of local and intracellular complement proteins, particularly CFD, in PH fibroblasts. Targeting CFD significantly reduced production of C3-activated fragments (C3a) and metabolites and genes associated with metabolic and pro-inflammatory reprogramming.

Hypoxia is a major cause of pulmonary hypertension (PH) worldwide, and it is likely that interstitial pulmonary macrophages contribute to this vascular pathology. We observed in hypoxia-exposed mice an increase in resident interstitial macrophages, which expanded through proliferation and expressed the monocyte recruitment ligand CCL2. We also observed an increase in CCR2+ macrophages through recruitment, which express the protein thrombospondin-1, which functionally activates TGF-β to cause vascular disease. Blockade of monocyte recruitment with either CCL2-neutralizing antibody treatment or CCR2 deficiency in the bone marrow compartment suppressed hypoxic PH. These data were supported by analysis of plasma samples from humans who traveled from low (225 m) to high (3500 m) elevation, revealing an increase in thrombospondin-1 and TGF-β expression following ascent, which was blocked by dexamethasone prophylaxis. In the hypoxic mouse model, dexamethasone prophylaxis recapitulated these findings by mechanistically suppressing CCL2 expression and CCR2+ monocyte recruitment. These data suggest a pathologic cross talk between 2 discrete interstitial macrophage populations, which can be therapeutically targeted.

The complement system is central to the innate immune response, playing a critical role in proinflammatory and autoimmune diseases such as pulmonary hypertension (PH). Recent discoveries highlight the emerging role of intracellular complement, or the “complosome,” in regulating cellular processes such as glycolysis, mitochondrial dynamics, and inflammatory gene expression. This study investigated the hypothesis that intracellular complement proteins C3, CFB, and CFD are upregulated in PH fibroblasts (PH-Fibs) and drive their metabolic and inflammatory states, contributing to PH progression. Our results revealed a pronounced upregulation of CFD, CFB, and C3 in PH-Fibs from human samples and bovine models, both in vivo and in vitro. The finding of elevated levels of C3 activation fragments, including C3b, C3d, and C3a, emphasized enhanced C3 activity. PH-Fibs exhibited notable metabolic reprogramming and increased levels of proinflammatory mediators such as MCP1, SDF1, IL-6, IL-13, and IL-33. Silencing CFD via shRNA reduced CFB activation and C3a production, while normalizing glycolysis, tricarboxylic acid (TCA) cycle activity, and fatty acid metabolism. Metabolomic and gene expression analyses of CFD-knockdown PH-Fibs revealed restored metabolic and inflammatory profiles, underscoring CFD’s crucial role in these changes. This study emphasizes the crucial role of intracellular complement in PH pathogenesis, highlighting the potential for complement-targeted therapies in PH.

Rationale: Pulmonary hypertension (PH) is characterized by a mean pulmonary arterial pressure above 20 mm Hg with no pharmacological cures currently. While rodent models are widely used to explore molecular mechanisms, their inherent limitations hinder the accurate recapitulation of human disease complexity, resulting in low drug translation success rates. Precision-cut lung slices (PCLS) preserve lung tissue structure, enabling ex vivo studies of PH pathogenesis and drug discovery. Recent studies, like Jandl et al. (PMID: 35763380), show PCLS efficacy in human PH drug assessment. In this study, we used bovine and/or human PCLS to evaluate the hypoxia and inflammation induced responses in control PCLS and the effects of a new HDAC inhibitor (HDACi) and a bromodomain protein inhibitor (BRDi) on these responses and established PH phenotypes in hypertensive PCLS. Methods: PCLS from calves and/or humans with PH (PH-PCLS) and normal subjects (CO-PCLS) were used as ex vivo model system. PCLS viability was evaluated using a PrestoBlue assay and fluorescent-based LIVE/DEAD kit. CO-PCLS were exposed to hypoxia alone or combined with a pro-inflammatory cytokine IL-1β, with or without BRDi (JQ1) treatment. PH-PCLS were treated with HDACi (OKI-5) or BRDi (JQ1) or the combination of both. OKI-5 is a derivative of largazole, a novel, potent, and selective class Ι HDACi (US patent). Expression level of pro-inflammatory, pro-proliferative and pro-fibrotic genes were evaluated using RT-PCR and immunohistochemistry (IHC) staining. Additionally, transmission electron microscopy (TEM) analysis of PCLS was performed to determine the presence of extracellular vesicles (EVs) in native pulmonary arteries. Results: PCLS Maintain structure, activity, mixture of cells for at least 14 days ex vivo. H&E and IHC staining indicated that PCLS culture ex vivo recapitulates pulmonary vascular remodeling in PH by comparing to in vivo source tissue. Hypoxia (1.5% O2) exposure combined with IL-1β (not hypoxia alone) for 48 hours induced PH associated phenotypes such as inflammatory responses in CO-PCLS indicated by the increased expression of IL-6, which can be inhibited by BRDi (JQ1). PH featured proinflammatory genes (e.g, CCL2), pro-proliferative genes (e.g, Mki67) and pro-fibrotic genes (e.g, tenascin-C) were decreased by the treatment of combined BRDi (JQ1) and HDACi (OKI-5) in PH-PCLS. Additionally, TEM analysis of PCLS revealed the presence and potential role of EVs in fibroblast-macrophage interaction in native pulmonary artery adventitia. Conclusion: PCLS can maintain pulmonary vasculature viability for at least 14 days, and facilitate mechanistic investigation and drug testing, offering promising applications of PCLS in PH research.

Rationale: In pulmonary hypertension (PH), females generally exhibit better right ventricular (RV) function than males, despite having higher PH prevalence. While PH alters RV metabolism, the mechanisms of sex-specific RV protection and the metabolic effects on individual cell types remain unknown. Previous studies showed high intensity interval training (HIIT) exercise lowered RV systolic pressures (RVSP) and improved cardiac index (CI) in rat monocrotaline-induced mild PH. We hypothesized that HIIT would improve RV hemodynamics and restore RV metabolism in rat Sugen Hypoxia (SuHx) severe PH in a sexually dimorphic manner.Methods: 8-week-old Sprague Dawley rats (n=14 male, n=8 female) received Sugen (SU5416) 20mg/kg and were housed for 3 weeks in hypobaric hypoxia followed by 3 weeks normoxia. SuHx rats were randomized 1:1 to remain sedentary (SuHx-Sed) or undergo HIIT exercise (SuHx-HIIT) on a treadmill 5x weekly for 6 weeks. Control rats (n=6 male, n=4 female) were housed in normoxia for 6 weeks. Rats underwent critical speed testing at baseline and 6 weeks. Hemodynamics were obtained followed by collection of peripheral blood and heart tissues. Blood was fractionated into plasma, red blood cells (RBCs), and peripheral blood mononuclear cells (PBMCs). The RV was dissected and digested to isolate myocytes, macrophages, and endothelial cells. Metabolomic profiling was performed on all samples using Ultra-High Performance Liquid Chromatography-Mass Spectrometry.Results: At baseline, female rats demonstrated better exercise endurance than males, but this sex difference was abrogated in SuHx-Sed and SuHx-HIIT conditions. SuHx-Sed males developed more severe PH and had higher RV contractility than SuHx-Sed females. Female rats developed higher mPAP and RVESP in response to HIIT exercise. One male and one female rat in the SuHx-HIIT group died during exercise before the end of the study period. HIIT had no significant effects on RV contractility, RV/PA coupling, RV cardiac output or cardiac index on surviving rats. Statistically significant sexual dimorphisms in the metabolome were seen in both peripheral and RV cellular compartments, and changes in metabolites in response to SuHx and SuHX with HIIT were observed in a preliminary analysis of samples from 12 rats. Conclusions: HIIT exercise may be detrimental to RV hemodynamics in severe rat SuHx PH, in contrast to previous studies showing benefit in mild PH. Metabolomic analyses are ongoing to determine whether changes in the peripheral compartment correlate with changes in the RV cellular compartment, and whether metabolomic shifts in response to SuHx and SuHX with HIIT exhibit sexual dimorphisms.

Background: Pulmonary hypertension (PH) is a severe cardiopulmonary disorder characterized by high blood pressure in the pulmonary arteries (PAs), resulting in significant morbidity and reduced life expectancy. Disrupted cell-cell communication triggers inflammation and vascular remodeling, eventually leading to PH. Recently we have shown complement-containing sEVs from adventitial fibroblasts induce proinflammatory and metabolic reprogramming in macrophages. Most studies to date have focused on sEVs derived from cultured cells or body fluids, such as plasma, serum, urine, semen, and milk. More recent studies have revealed two distinct types of nanovesicles present in tissues: matrix-bound nanovesicles (MBVs) and liquid-phase nanovesicles, also known as sEVs. The roles of these nanovesicles in ECM remodeling and cell-cell communication, particularly within intact tissue in the context of PH, remain unexplored. Methods and Results: Lung tissue from young control bovines and two-week hypoxic bovine models, which develop severe PH in 2week of hypoxia, was inflated with agarose and sectioned using a vibratome to prepare precision-cut lung slices (PCLS). Distal PAs of similar size, along with adjacent airways, were excised, and the regions of interest (1-2 mm x 1-2 mm) were processed for transmission electron microscopy (TEM). Thin sections (∼70 nm) of resin-embedded tissue were then imaged by TEM. Our observations revealed two distinct types of sEVs in the tissue: one group, MBV, located in close proximity to extracellular matrices (ECM), such as collagen and elastin fibers, and another group as liquid phase nanovesicles / sEVs located in extracellular space. To quantify these vesicles, we isolated sEVs from dPA and found a significant increase in sEVs quantity in the PH dPA compared to control dPA. Additionally, pathways analysis proteomic data of the decellularized ECM from dPA showed enrichment of complement and coagulation cascades, suggesting that MBVs are closely associated with ECM proteins and may play a role in complement-mediated ECM remodeling. To confirm the proteomic findings and complement abundance in individual nanovesicles, immuno-TEM with C3 immunogold labeling of purified sEVs from dPA revealed a higher expression of complement C3 protein in PH samples than controls. Conclusions: This study identifies two distinct types of sEVs in pulmonary tissue, with a higher quantity of sEVs and complement C3 protein expression in PH. These findings also suggest that MBVs may contribute to complement-mediated ECM remodeling and macrophage activation toward a proinflammatory phenotype, potentially driving disease progression in pulmonary hypertension (Fig.1). Further exploration of these pathways could lead to new therapeutic strategies.

Pulmonary hypertension (PH) significantly impairs exercise capacity and the quality of life in patients, which is influenced by dysfunctions in multiple organ systems, including the right ventricle, lungs, and skeletal muscles. Recent research has identified metabolic reprogramming and mitochondrial dysfunction as contributing factors to reduced exercise tolerance in PH patients. In this study, we investigated the therapeutic potential of enhancing mitochondrial function through the activation of the mitochondrial deacetylase SIRT3, using SIRT3 activator Honokiol combined with the SIRT3 co-factor nicotinamide adenine dinucleotide (NAD), in a Sugen/Hypoxia-induced PH rat model. Our results show that Sugen/Hypoxia-induced PH significantly impairs RV, lung, and skeletal muscle function, leading to reduced exercise capacity. Treatment with Honokiol and NAD notably improved exercise endurance, primarily by restoring SIRT3 levels in skeletal muscles, reducing proteolysis and atrophy in the gastrocnemius, and enhancing mitochondrial complex I levels in the soleus. These effects were independent of changes in cardiopulmonary hemodynamics. We concluded that targeting skeletal muscle dysfunction may be a promising approach to improving exercise capacity and overall quality of life in PH patients.

BackgroundSchistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. Type 2 inflammation contributes to the development of Schistosoma-induced PH. Specifically, interstitial macrophages (IMs) derived from monocytes play a pivotal role by producing thrombospondin-1 (TSP-1), which in turn activates TGF-β, thereby driving the pathology of PH. Resident and recruited IM subpopulations have recently been identified. We hypothesized that in Schistosoma-PH, one IM subpopulation expresses monocyte recruitment factors, whereas recruited monocytes become a separate IM subpopulation that expresses TSP-1.MethodsMice were intraperitoneally sensitized and then intravenously challenged with S. mansoni eggs. Flow cytometry on lungs and blood was performed on wildtype and reporter mice to identify IM subpopulations and protein expression. Single-cell RNA sequencing (scRNAseq) was performed on flow-sorted IMs from unexposed and at day 1, 3 and 7 following Schistosoma exposure to complement flow cytometry based IM characterization and identify gene expression.ResultsFlow cytometry and scRNAseq both identified 3 IM subpopulations, characterized by CCR2, MHCII, and FOLR2 expression. Following Schistosoma exposure, the CCR2+ IM subpopulation expanded, suggestive of circulating monocyte recruitment. Schistosoma exposure caused increased monocyte-recruitment ligand CCL2 expression in the resident FOLR2+ IM subpopulation. In contrast, the vascular pathology-driving protein TSP-1 was greatest in the CCR2+ IM subpopulation.ConclusionSchistosoma-induced PH involves crosstalk between IM subpopulations, with increased expression of monocyte recruitment ligands by resident FOLR2+ IMs, and the recruitment of CCR2+ IMs which express TSP-1 that activates TGF-β and causes PH.

IntroductionHypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitial macrophages (IMs) play pivotal roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs’ response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development.MethodsWe conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia.ResultsOur analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a pro-remodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the pro-remodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways.DiscussionOur findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension.

Rationale: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by extensive pulmonary vascular remodeling caused by plexiform and obliterative lesions, media hypertrophy, inflammatory cell infiltration, and alterations of the adventitia. Objective: We sought to test the hypothesis that microscopic IPAH vascular lesions express unique molecular profiles, which collectively are different from control pulmonary arteries. Methods: We used digital spatial transcriptomics to profile the genomewide differential transcriptomic signature of key pathological lesions (plexiform, obliterative, intima+media hypertrophy, and adventitia) in IPAH lungs (n = 11) and compared these data with the intima1media hypertrophy and adventitia of control pulmonary artery (n = 5). Measurements and Main Results: We detected 8,273 transcripts in the IPAH lesions and control lung pulmonary arteries. Plexiform lesions and IPAH adventitia exhibited the greatest number of differentially expressed genes when compared with intima1media hypertrophy and obliterative lesions. Plexiform lesions in IPAH showed enrichment for 1) genes associated with transforming growth factor beta signaling and 2) mutated genes affecting the extracellular matrix and endothelial-mesenchymal transformation. Plexiform lesions and IPAH adventitia showed upregulation of genes involved in immune and IFN signaling, coagulation, and complement pathways. Cellular deconvolution indicated variability in the number of vascular and inflammatory cells between IPAH lesions, which underlies the differential transcript profiling. Conclusions: IPAH lesions express unique molecular transcript profiles enriched for pathways involving pathogenetic pathways, including genetic disease drivers, innate and acquired immunity, hypoxia sensing, and angiogenesis signaling. These data provide a rich molecular-structural framework in IPAH vascular lesions that inform novel biomarkers and therapeutic targets in this highly morbid disease.

Pulmonary hypertension (PH) is a chronic and progressive disease with significant morbidity and mortality. It is characterized by remodeled pulmonary vessels associated with perivascular and intravascular accumulation of inflammatory cells. Although there is compelling evidence that bone marrow-derived cells, such as macrophages and T cells, cluster in the vicinity of pulmonary vascular lesions in humans and contribute to PH development in different animal models, the role of dendritic cells in PH is less clear. Dendritic cells' involvement in PH is likely since they are responsible for coordinating innate and adaptive immune responses. We hypothesized that dendritic cells drive hypoxic PH. We demonstrate that a classical dendritic cell (cDC) subset (cDC2) is increased and activated in wild-type mouse lungs after hypoxia exposure. We observe significant protection after the depletion of cDCs in ZBTB46 DTR chimera mice before hypoxia exposure and after established hypoxic PH. In addition, we find that cDC depletion is associated with a reduced number of two macrophage subsets in the lung (FolR2+ MHCII+ CCR2+ and FolR2+ MHCII+ CCR2-). We found that depleting cDC2s, but not cDC1s, was protective against hypoxic PH. Finally, proof-of-concept studies in human lungs show increased perivascular cDC2s in patients with Idiopathic Pulmonary Arterial Hypertension (IPAH). Our data points to an essential role of cDCs, particularly cDC2s, in the pathophysiology of experimental PH.

Pulmonary hypertension (PH) is a heterogeneous and life-threatening cardiopulmonary disorder in which mitochondrial dysfunction is believed to drive pathogenesis, although the underlying mechanisms remain unclear. To determine if abnormal SIRT3 (sirtuin 3) activity is related to mitochondrial dysfunction in adventitial fibroblasts from patients with idiopathic pulmonary arterial hypertension (IPAH) and hypoxic PH calves (PH-Fibs) and whether SIRT3 could be a potential therapeutic target to improve mitochondrial function, SIRT3 concentrations in control fibroblasts, PH-Fibs, and lung tissues were determined using quantitative real-time PCR and western blot. SIRT3 deacetylase activity in cells and lung tissues was determined using western blot, immunohistochemistry staining, and immunoprecipitation. Glycolysis and mitochondrial function in fibroblasts were measured using respiratory analysis and fluorescence-lifetime imaging microscopy. The effects of restoring SIRT3 activity (by overexpression of SIRT3 with plasmid, activation SIRT3 with honokiol, and supplementation with the SIRT3 cofactor nicotinamide adenine dinucleotide [NAD+]) on mitochondrial protein acetylation, mitochondrial function, cell proliferation, and gene expression in PH-Fibs were also investigated. We found that SIRT3 concentrations were decreased in PH-Fibs and PH lung tissues, and its cofactor, NAD+, was also decreased in PH-Fibs. Increased acetylation in overall mitochondrial proteins and SIRT3-specific targets (MPC1 [mitochondrial pyruvate carrier 1] and MnSOD2 [mitochondrial superoxide dismutase]), as well as decreased MnSOD2 activity, was identified in PH-Fibs and PH lung tissues. Normalization of SIRT3 activity, by increasing its expression with plasmid or with honokiol and supplementation with its cofactor NAD+, reduced mitochondrial protein acetylation, improved mitochondrial function, inhibited proliferation, and induced apoptosis in PH-Fibs. Thus, our study demonstrated that restoration of SIRT3 activity in PH-Fibs can reduce mitochondrial protein acetylation and restore mitochondrial function and PH-Fib phenotype in PH.

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.

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.

This study analyzed microarray data of right ventricular (RV) tissue from rats exposed to pulmonary embolism to understand the initial dynamic transcriptional response to mechanical stress and compare it with experimental pulmonary hypertension (PH) models. The dataset included samples harvested from 55 rats at 11 different time points or RV locations. We performed principal component analysis (PCA) to explore clusters based on spatiotemporal gene expression. Relevant pathways were identified from fast gene set enrichment analysis using PCA coefficients. The RV transcriptomic signature was measured over several time points, ranging from hours to weeks after an acute increase in mechanical stress, and was found to be highly dependent on the severity of the initial insult. Pathways enriched in the RV outflow tracts of rats at 6 weeks after severe PE share many commonalities with experimental PH models, but the transcriptomic signature at the RV apex resembles control tissue. The severity of the initial pressure overload determines the trajectory of the transcriptomic response independent of the final afterload, but this depends on the location where the tissue is biopsied. Chronic RV pressure overload due to PH appears to progress toward similar transcriptomic endpoints.

Background and aim: Metabolic syndrome (MetS) is a complex disease of physiological imbalances interrelated to abnormal metabolic conditions, such as abdominal obesity, type II diabetes, dyslipidemia and hypertension. In the present pilot study, we investigated the nutraceutical bitter melon (Momordica charantia L) -intake induced transcriptome and metabolome changes and the converging metabolic signaling networks underpinning its inhibitory effects against MetS-associated risk factors. Experimental procedure: Metabolic effects of lyophilized bitter melon juice (BMJ) extract (oral gavage 200 mg/kg/body weight-daily for 40 days) intake were evaluated in diet-induced obese C57BL/6J male mice [fed-high fat diet (HFD), 60 kcal% fat]. Changes in a) serum levels of biochemical parameters, b) gene expression in the hepatic transcriptome (microarray analysis using Affymetrix Mouse Exon 1.0 ST arrays), and c) metabolite abundance levels in lipid-phase plasma [liquid chromatography mass spectrometry (LC-MS)-based metabolomics] after BMJ intervention were assessed. Results and conclusion: BMJ-mediated changes showed a positive trend towards enhanced glucose homeostasis, vitamin D metabolism and suppression of glycerophospholipid metabolism. In the liver, nuclear peroxisome proliferator-activated receptor (PPAR) and circadian rhythm signaling, as well as bile acid biosynthesis and glycogen metabolism targets were modulated by BMJ (p < 0.05). Thus, our indepth transcriptomics and metabolomics analysis suggests that BMJ-intake lowers susceptibility to the onset of high-fat diet associated MetS risk factors partly through modulation of PPAR signaling and its

Few studies have examined lung interstitial macrophage (IM) molecular phenotypes after being exposed to hypoxia in vivo at the single-cell level, even though macrophages contribute to hypoxic pulmonary hypertension (PH). We aimed to determine IM diversity and its association with hypoxia-induced PH. We hypothesized that integrating single-cell RNA sequencing (scRNAseq) and binary hierarchal clustering (BHC) could resolve IM heterogeneity under normal homeostatic conditions and changes induced by hypoxia exposure. Cx3cr1 GFP/+ reporter mice were exposed to normoxic conditions (∼21% [Formula: see text]) or exposed to 1 day ( D1) or 7 days ( D7) of hypoxia (∼10% [Formula: see text]). We used flow cytometry to isolate Cx3cr1 + IMs and the 10X Genomics platform for scRNAseq, Cell Ranger, Seurat, ClusterMap, monocle, ingenuity pathway analysis, and Fisher’s exact test ( q value < 0.05) for functional investigations. n = 374 (normoxia), n = 2,526 ( D1), and n = 1,211 ( D7) IMs were included in the analyses. We identified three normoxia-related cell types, five hypoxia-associated cell types that emerged at D1, and three that appeared at D7. We describe the existence of a putative resident trained innate IM, which is present in normoxia, transiently depleted at D1, and recovered after 7 days of sustained hypoxia. We also define a rare putative pathogenic population associated with transcripts implicated in PH development that emerges at D7. In closing, we describe the successful integration of BHC with scRNAseq to determine IM heterogeneity and its association with PH. These results shed light on how resident-trained innate IMs become more heterogeneous but ultimately accustomed to hypoxia.

Medical Profession was included under The Consumer Protection Act (CPA) in 1986 to provide a forum tosafeguard the rights of the customers and establish guidelines for the speedy redressal of their grievancesagainst unethical medical practices. This by far has been the most crucial legislations introduced in theframework of health professionals which over the years seems to have propelled them to practice in a guardedmanner. In recent times this also seems to be flanked by a spurt in Violence against the Doctors enormously.It is thereby imperative for all medical professionals to not only have a comprehensive knowledge of ethicsand laws related to health care and medicolegal issues but also its implications in their profession. In thisvery light the current study was conducted as an attempt to assess the perception of undergraduate medicalstudents towards medical ethics, CPA and its influence on Violence against Doctors.