Search

Introduction: In pulmonary hypertension (PH), pulmonary arterial remodeling is often accompanied by perivascular inflammation. The inflammation is characterized by the accumulation of activated macrophages and lymphocytes within the adventitial stroma, which is comprised primarily of fibroblasts. The well-known ability of fibroblasts to secrete interleukins and chemokines has previously been implicated as contributing to this tissue-specific inflammation in PH vessels. We were interested if pulmonary fibroblasts from PH arteries contribute to microenvironmental changes that could activate and polarize T-cells in PH., Methods: We used single-cell RNA sequencing of intact bovine distal pulmonary arteries (dPAs) from PH and control animals and flow cytometry, mRNA expression analysis, and respirometry analysis of blood-derived bovine/human T-cells exposed to conditioned media obtained from pulmonary fibroblasts of PH/control animals and IPAH/control patients (CM-(h)PH Fibs vs CM-(h)CO Fibs)., Results: Single-cell RNA sequencing of intact bovine dPAs from PH and control animals revealed a pro-inflammatory phenotype of CD4+ T-cells and simultaneous absence of regulatory T-cells (FoxP3+ Tregs). By exposing T-cells to CM-(h)PH Fibs we stimulated their proinflammatory differentiation documented by increased IFNγ and decreased IL4, IL10, and TGFβ mRNA and protein expression. Interestingly, we demonstrated a reduction in the number of suppressive T-cell subsets, i.e., human/bovine Tregs and bovine γδ T-cells treated with CM-(h)PH-Fibs. We also noted inhibition of anti-inflammatory cytokine expression (IL10, TGFβ, IL4). Pro-inflammatory polarization of bovine T-cells exposed to CM-PH Fibs correlated with metabolic shift to glycolysis and lactate production with increased prooxidant intracellular status as well as increased proliferation of T-cells. To determine whether metabolic reprogramming of PH-Fibs was directly contributing to the effects of PH-Fibs conditioned media on T-cell polarization, we treated PH-Fibs with the HDAC inhibitor SAHA, which was previously shown to normalize metabolic status and examined the effects of the conditioned media. We observed significant suppression of inflammatory polarization associated with decreased T-cell proliferation and recovery of mitochondrial energy metabolism., Conclusion: This study demonstrates how the pulmonary fibroblast-derived microenvironment can activate and differentiate T-cells to trigger local inflammation, which is part of the vascular wall remodeling process in PH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Plecitá-Hlavatá, Brázdová, Křivonosková, Hu, Phang, Tauber, Li, Zhang, Hoetzenecker, Crnkovic, Kwapiszewska and Stenmark.)

Hypoxia-inducible factor (HIF) has received much attention as a potential pulmonary hypertension (PH) treatment target because inhibition of HIF reduces the severity of established PH in rodent models. However, the limitations of small-animal models of PH in predicting the therapeutic effects of pharmacologic interventions in humans PH are well known. Therefore, we sought to interrogate the role of HIFs in driving the activated phenotype of PH cells from human and bovine vessels. We first established that pulmonary arteries (PAs) from human and bovine PH lungs exhibit markedly increased expression of direct HIF target genes ( CA9, GLUT1 , and NDRG1 ), as well as cytokines/chemokines ( CCL2, CSF2, CXCL12 , and IL6 ), growth factors ( FGF1, FGF2, PDGFb , and TGFA ), and apoptosis-resistance genes ( BCL2, BCL2L1 , and BIRC5 ). The expression of the genes found in the intact PAs was determined in endothelial cells, smooth muscle cells, and fibroblasts cultured from the PAs. The data showed that human and bovine pulmonary vascular fibroblasts from patients or animals with PH (termed PH-Fibs) were the cell type that exhibited the highest level and the most significant increases in the expression of cytokines/chemokines and growth factors. In addition, we found that human, but not bovine, PH-Fibs exhibit consistent misregulation of HIFα protein stability, reduced HIF1α protein hydroxylation, and increased expression of HIF target genes even in cells grown under normoxic conditions. However, whereas HIF inhibition reduced the expression of direct HIF target genes, it had no impact on other "persistently activated" genes. Thus, our study indicated that HIF inhibition alone is not sufficient to reverse the persistently activated phenotype of human and bovine PH-Fibs.

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.

Metabolic reprogramming is considered important in the pathogenesis of the occlusive vasculopathy observed in pulmonary hypertension (PH). However, the mechanisms that link reprogrammed metabolism to aberrant expression of genes, which modulate functional phenotypes of cells in PH, remain enigmatic. Herein, we demonstrate that, in mice, hypoxia-induced PH was prevented by glucose-6-phosphate dehydrogenase deficiency (G6PD Def ), and further show that established severe PH in Cyp2c44 -/- mice was attenuated by knockdown with G6PD shRNA or by G6PD inhibition with an inhibitor (N-ethyl-N'-[(3β,5α)-17-oxoandrostan-3-yl]urea, NEOU). Mechanistically, G6PD Def , knockdown and inhibition in lungs: 1 ) reduced hypoxia-induced changes in cytoplasmic and mitochondrial metabolism, 2 ) increased expression of Tet methylcytosine dioxygenase 2 ( Tet2 ) gene, and 3 ) upregulated expression of the coding genes and long noncoding (lnc) RNA Pint , which inhibits cell growth, by hypomethylating the promoter flanking region downstream of the transcription start site. These results suggest functional TET2 is required for G6PD inhibition to increase gene expression and to reverse hypoxia-induced PH in mice. Furthermore, the inhibitor of G6PD activity (NEOU) decreased metabolic reprogramming, upregulated TET2 and lncPINT , and inhibited growth of control and diseased smooth muscle cells isolated from pulmonary arteries of normal individuals and idiopathic-PAH patients, respectively. Collectively, these findings demonstrate a previously unrecognized function for G6PD as a regulator of DNA methylation. These findings further suggest that G6PD acts as a link between reprogrammed metabolism and aberrant gene regulation and plays a crucial role in regulating the phenotype of cells implicated in the pathogenesis of PH, a debilitating disorder with a high mortality rate.