Daniel Hernandez-Saavedra

Altered metabolism in pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs) contributes to the pathology of pulmonary hypertension (PH), but changes in substrate uptake and how substrates are utilized have not been fully characterized. We hypothesized stable isotope metabolomics would identify increased glucose, glutamine and fatty acid uptake and utilization in human PASMCs and PAECs from PH versus control specimens, and that TGF-β treatment would phenocopy these metabolic changes. We used ¹³C-labeled glucose, glutamine or a long-chain fatty acid mixture added to cell culture media, and mass spectrometry-based metabolomics to detect and quantify ¹³C-labeled metabolites. We found PH PASMCs had increased glucose uptake and utilization by glycolysis and the pentose shunt, but no changes in glutamine or fatty acid uptake or utilization. Diseased PAECs had increased proximate glycolysis pathway intermediates, less pentose shunt flux, increased anaplerosis from glutamine, and decreased fatty acid β-oxidation. TGF-β treatment increased glycolysis in PASMCs, but did not recapitulate the PAEC disease phenotype. In TGF-β-treated PASMCs, glucose, glutamine and fatty acids all contributed carbons to the TCA cycle. In conclusion, PASMCs and PAECs collected from PH subjects have significant changes in metabolite uptake and utilization, partially recapitulated by TGF-β treatment.

Aims: Transforming growth factor-β (TGF-β) signalling is required for chronic hypoxia-induced pulmonary hypertension (PH). The activation of TGF-β by thrombospondin-1 (TSP-1) contributes to the pathogenesis of hypoxia-induced PH. However, neither the cellular source of pathologic TSP-1 nor the downstream signalling pathway that link activated TGF-β to PH have been determined. In this study, we hypothesized that circulating monocytes, which are recruited to become interstitial macrophages (IMs), are the major source of TSP-1 in hypoxia-exposed mice, and TSP-1 activates TGF-β with increased Rho-kinase signalling, causing vasoconstriction. Methods and results: Flow cytometry revealed that a specific subset of IMs is the major source of pathologic TSP-1 in hypoxia. Intravenous depletion and parabiosis experiments demonstrated that these cells are circulating prior to recruitment into the interstitium. Rho-kinase-mediated vasoconstriction was a major downstream target of active TGF-β. Thbs1 deficient bone marrow (BM) protected against hypoxic-PH by blocking TGF-β activation and Rho-kinase-mediated vasoconstriction. Conclusion: In hypoxia-challenged mice, BM derived and circulating monocytes are recruited to become IMs which express TSP-1, resulting in TGF-β activation and Rho-kinase-mediated vasoconstriction.

Background: Inflammation underlies many forms of pulmonary hypertension ( PH ), including that resulting from Schistosoma infection, a major cause of PH worldwide. Schistosomiasis‐associated PH is proximately triggered by embolization of parasite eggs into the lungs, resulting in localized type 2 inflammation. However, the role of CD 4 ⁺ T cells in this disease is not well defined. Methods and Results: We used a mouse model of schistosomiasis‐associated PH , induced by intraperitoneal egg sensitization followed by intravenous egg challenge, with outcomes including right ventricle systolic pressure measured by cardiac catheterization, and cell density and phenotype assessed by flow cytometry. We identified that embolization of Schistosoma eggs into lungs of egg‐sensitized mice increased the perivascular density of T‐helper 2 (Th2) CD 4 ⁺ T cells by recruitment of cells from the circulation and triggered type 2 inflammation. Parabiosis confirmed that egg embolization is required for localized type 2 immunity. We found Th2 CD 4 ⁺ T cells were necessary for Schistosoma ‐induced PH , given that deletion of CD 4 ⁺ T cells or inhibiting their Th2 function protected against type 2 inflammation and PH following Schistosoma exposure. We also observed that adoptive transfer of Schistosoma ‐sensitized CD 4 ⁺ Th2 cells was sufficient to drive type 2 inflammation and PH . Conclusions: Th2 CD 4 ⁺ T cells are a necessary and sufficient component for the type 2 inflammation‐induced PH following Schistosoma exposure.

Schistosomiasis is a leading cause of pulmonary hypertension (PH) worldwide. Recent studies reveal that the type‐2 immune cytokines IL‐4 and IL‐13, as well as consequent activation of TGF‐β, are key factors in the pathogenesis of Schistosoma‐PH. Paclitaxel has been reported to act as an adjuvant for Th2 inflammation while downregulating TGF‐β activation. Moreover, paclitaxel blocks PH in monocrotaline and SU5416‐hypoxia models. We hypothesized that paclitaxel would augment Th2 inflammation while blocking TGF‐β activation and PH after schistosomiasis exposure. Wild‐type mice (C57BL6/J; 6/group) were intraperitoneally (IP) sensitized and then intravenously (IV) challenged with Schistosoma mansoni eggs. One day after IV egg challenge, the mice were treated with a single IP dose of 25 mg/kg paclitaxel or vehicle. Right ventricular (RV) catheterization was performed and granuloma volumes and vascular remodeling were quantified. Lung cytokines were quantified by ELISA and reverse transcription polymerase chain reaction, and the quantity of active TGF‐β was determined using a cell reporter line. We also investigated hypoxia‐induced PH. Paclitaxel treatment significantly protected mice from Schistosoma‐PH, with decreased RV systolic pressure (P = 0.005) and pulmonary vascular media thickness. Inflammation was significantly suppressed, contrary to our hypothesis, with decreased IL‐4 and IL‐13 levels, smaller granulomas, and less active TGF‐β following paclitaxel treatment. There was no change in IFN‐γ or FoxO1 or FoxO3 expression. Paclitaxel did not suppress chronic hypoxia‐induced PH, which is also TGF‐β‐driven but independent of type‐2 immunity. Paclitaxel protects against Schistosoma‐induced PH in mice, although by blocking proximate Th2 inflammation rather than suppressing distal TGF‐β activation.

Optimal right ventricular (RV) function in pulmonary hypertension (PH) requires structural and functional coupling between the RV cardiomyocyte and its adjacent capillary network. Prior investigations have indicated that RV vascular rarefaction occurs in PH, which could contribute to RV failure by reduced delivery of oxygen or other metabolic substrates. However, it has not been determined if rarefaction results from relative underproliferation in the setting of tissue hypertrophy or from actual loss of vessels. It is also unknown if rarefaction results in inadequate substrate delivery to the RV tissue. In the present study, PH was induced in rats by SU5416-hypoxia-normoxia exposure. The vasculature in the RV free wall was assessed using stereology. Steady-state metabolomics of the RV tissue was performed by mass spectrometry. Complementary studies were performed in hypoxia-exposed mice and rats. Rats with severe PH had evidence of RV failure by decreased cardiac output and systemic hypotension. By stereology, there was significant RV hypertrophy and increased total vascular length in the RV free wall in close proportion, with evidence of vessel proliferation but no evidence of endothelial cell apoptosis. There was a modest increase in the radius of tissue served per vessel, with decreased arterial delivery of metabolic substrates. Metabolomics revealed major metabolic alterations and metabolic reprogramming; however, metabolic substrate delivery was functionally preserved, without evidence of either tissue hypoxia or depletion of key metabolic substrates. Hypoxia-treated rats and mice had similar but milder alterations. There is significant homeostatic vascular adaptation in the right ventricle of rodents with PH.

When evaluating the role of redox-regulating signaling in pulmonary vascular diseases, it is intriguing to consider the modulation of key antioxidant enzymes like superoxide dismutase (SOD) because SOD isoforms are regulated by redox reactions, and, in turn, modulate downstream redox sensitive processes. The emerging field of redox biology is built upon understanding the regulation and consequences of tightly controlled and specific reduction–oxidation reactions that are critical for diverse cellular processes including cell signaling. Of relevance, both the site of production of specific reactive oxygen and nitrogen species and the site of the antioxidant defenses are highly compartmentalized within the cell. For example, superoxide is generated during oxidative phosphorylation in the mitochondria as well as by a number of enzymatic sources within the cytosol and at the cell membrane. In the pulmonary circulation, these sources include the mitochondrial electron transport chain, NADPH oxidases (NOX1–4, Duox1,2), nitric oxide synthases, and xanthine oxidase; this important topic has been thoroughly reviewed recently [1]. In parallel with these different cellular sites of superoxide production, the three SOD isoforms are also specifically localized to the cytosol (SOD1), mitochondria (SOD2) or extracellular compartment (SOD3). This chapter focuses on the role of redox mechanisms regulating SOD2 and SOD3, with an emphasis on these processes in the setting of 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 inSchistosoma-exposed mice. FollowingSchistosomaexposure, 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.

Tobacco smoke (TS) causes chronic obstructive pulmonary disease, including chronic bronchitis, emphysema, and asthma. Rtp801, an inhibitor of mechanistic target of rapamycin, is induced by oxidative stress triggered by TS. Its up-regulation drives lung susceptibility to TS injury by enhancing inflammation and alveolar destruction. We postulated that Rtp801 is not only increased by reactive oxygen species (ROS) in TS but also instrumental in creating a feedforward process leading to amplification of endogenous ROS generation. We used cigarette smoke extract (CSE) to model the effect of TS in wild-type (Wt) and knockout (KO-Rtp801) mouse lung fibroblasts (MLF). The production of superoxide anion in KO-Rtp801 MLF was lower than that in Rtp801 Wt cells after CSE treatment, and it was inhibited in Wt MLF by silencing nicotinamide adenine dinucleotide phosphate oxidase–4 (Nox4) expression with small interfering Nox4 RNA. We observed a cytoplasmic location of ROS formation by real-time redox changes using reduction-oxidation–sensitive green fluorescent protein profluorescent probes. Both the superoxide production and the increase in the cytoplasmic redox were inhibited by apocynin. Reduction in the activity of Sod and decreases in the expression of Sod2 and Gpx1 genes were associated with Rtp801 CSE induction. The ROS produced by Nox4 in conjunction with the decrease in cellular antioxidant enzymatic defenses may account for the observed cytoplasmic redox changes and cellular damage caused by TS.