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

PI3K, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells

We recently reported that vasa vasorum expansion occurs in the pulmonary artery (PA) adventitia of chronically hypoxic animals and that extracellular ATP is a pro-angiogenic factor for isolated vasa vasorum endothelial cells (VVEC). However, the sources of extracellular ATP in the PA vascular wall, as well as the molecular mechanisms underlying its release, remain elusive. Studies were undertaken to explore whether VVEC release ATP in response to hypoxia and to determine signaling pathways involved in this process. We found that hypoxia (1–3% O2) resulted in time- and O2-dependent ATP release from VVEC. Preincubation with the inhibitors of vesicular transport (monensin, brefeldin A, and N-ethylmaleimide) significantly decreased ATP accumulation in the VVEC conditioned media, suggesting that hypoxia-induced ATP release occurs through vesicular exocytosis. Additionally, both hypoxia and exogenously added ATP resulted in the activation of PI3K and accumulation of GTP-bound RhoA in a time-dependent manner. Pharmacological inhibition of PI3K and ROCK or knockout of RhoA by small interfering RNA significantly abolished hypoxia-induced ATP release from VVEC. Moreover, RhoA and ROCK play a critical role in ATP-induced increases in VVEC DNA synthesis, migration, and tube formation, indicating a functional contribution of PI3K, Rho, and ROCK to both the autocrine mechanism of ATP release and ATP-mediated angiogenic activation of VVEC. Taken together, our findings provide novel evidence for the signaling mechanisms that link hypoxia-induced increases in extracellular ATP and vasa vasorum expansion

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

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-1β 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

Adventitial Fibroblasts Induce a Distinct Proinflammatory/Profibrotic Macrophage Phenotype in Pulmonary Hypertension

Macrophage accumulation is not only a characteristic hallmark but also a critical component of pulmonary artery (PA) remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Utilizing multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, as well as primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive Pas (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL4/IL13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation while complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, while deficiency in C/EBPβ or HIF1 attenuated fibroblast driven macrophage activation. These findings challenge the current paradigm of IL4/IL13-STAT6 mediated alternative macrophage activation as the sole driver of vascular remodeling in PH and uncover a crosstalk between adventitial fibroblasts and macrophages in which paracrine IL6 activated STAT3, HIF1, and C/EBPβ signaling is critical for macrophage activation and polarization. Thus, targeting IL6 signaling in macrophages by completely inhibiting C/EBPβ, HIF1a or partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL6 and absent IL4/IL13 signaling

TGF-β activation by bone marrow-derived thrombospondin-1 causes Schistosoma- and hypoxia-induced pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an obstructive disease of the precapillary pulmonary arteries. Schistosomiasis-associated PAH shares altered vascular TGF-β signalling with idiopathic, heritable and autoimmune-associated etiologies; moreover, TGF-β blockade can prevent experimental pulmonary hypertension (PH) in pre-clinical models. TGF-β is regulated at the level of activation, but how TGF-β is activated in this disease is unknown. Here we show TGF-β activation by thrombospondin-1 (TSP-1) is both required and sufficient for the development of PH in Schistosoma-exposed mice. Following Schistosoma exposure, TSP-1 levels in the lung increase, via recruitment of circulating monocytes, while TSP-1 inhibition or knockout bone marrow prevents TGF-β activation and protects against PH development. TSP-1 blockade also prevents the PH in a second model, chronic hypoxia. Lastly, the plasma concentration of TSP-1 is significantly increased in subjects with scleroderma following PAH development. Targeting TSP-1-dependent activation of TGF-β could thus be a therapeutic approach in TGF-β-dependent vascular diseases