Philip M. Bauer

Pulmonary hypertension (PH) is a progressive and fatal disease with no cure. Vascular remodeling in PH involves intraluminal growth of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Cell growth in these lesions is quasi-neoplastic, with evidence of monoclonality, apoptosis resistance and cancer-like metabolic derangements. Herein we tested the effect of human interferon alpha 2b (IFNα), a pleiotropic cytokine and anti-cancer therapeutic, on the development and progression of PH in the rat SU5416/hypoxia (SUH) model and mouse hypoxia model of the disease. In both models IFNα attenuated the development of PH and reversed established PH as assessed by measuring right ventricular systolic pressure and right ventricular hypertrophy. The effect of IFNα was dependent on the type I interferon receptor (IFNAR) since mice lacking a subunit of the IFNAR were not protected by IFNα. Morphometric analysis of pulmonary aterioles from hypoxic mice or SUH rats showed that IFNα inhibited pulmonary vascular remodeling in both models and that IFNα reversed remodeling in SUH rats with established disease. Immunohistochemical staining revealed that IFNα decreased the number of PCNA and Tunel positive cells in the wall of pulmonary arterioles. In vitro, IFNα inhibited proliferation of human pulmonary artery smooth muscle cells and as well as human pulmonary artery endothelial cell proliferation and apoptosis. Together these findings demonstrate that IFNα reverses established experimental PH and provide a rationale for further exploration of the use of IFNα and other immunotherpies in PH.

Rationale:: Recent studies demonstrate a role for toll-like receptor 4 (TLR4) in the pathogenesis of pulmonary hypertension (PH); however, the cell types involved in mediating the effects of TLR4 remain unknown. Objectives:: The objective of this study was to determine the contribution of TLR4 expressed on nonparenchymal cells to the pathogenesis of PH. Methods and Results:: TLR4 bone marrow chimeric mice revealed an equal contribution of TLR4 on nonparenchymal and parenchymal cells in the pathogenesis of PH as determined by measuring right ventricular (RV) systolic pressure and RV hypertrophy. However, the deletion of TLR4 from myeloid lineage cells had no effect on the development of PH because we found no difference in RV systolic pressure or RV hypertrophy in wild-type versus LysM-TLR4 ^−/− mice. To explore the potential role of platelet TLR4 in the pathogenesis of PH, platelet-specific TLR4 ^−/− mice were generated (PF4-TLR4 ^−/− mice). TLR4 ^−/− platelets from either global TLR4 ^−/− or PF4-TLR4 ^−/− mice were functional but failed to respond to lipopolysaccharide, demonstrating a lack of TLR4. PF4-TLR4 ^−/− mice demonstrated significant protection from hypoxia-induced PH, including attenuated increases in RV systolic pressure and RV hypertrophy, decreased platelet activation, and less pulmonary vascular remodeling. The deletion of TLR4 from platelets attenuated serotonin release after chronic hypoxia, and lipopolysaccharide-stimulated platelets released serotonin and promoted pulmonary artery smooth muscle cell proliferation in a serotonin-dependent manner. Conclusions:: Our data demonstrate that TLR4 on platelets contributes to the pathogenesis of PH and further highlights the role of platelets in PH.

Survival rates for patients with pulmonary hypertension (PH) remain low, and our understanding of the mechanisms involved are incomplete. Here we show in a mouse model of chronic hypoxia (CH)-induced PH that the nuclear protein and damage-associate molecular pattern molecule (DAMP) high mobility group box 1 (HMGB1) contributes to PH via a Toll-like receptor 4 (TLR4)-dependent mechanism. We demonstrate extranuclear HMGB1 in pulmonary vascular lesions and increased serum HMGB1 in patients with idiopathic pulmonary arterial hypertension. The increase in circulating HMGB1 correlated with mean pulmonary artery pressure. In mice, we similarly detected the translocation and release of HMGB1 after exposure to CH. HMGB1-neutralizing antibody attenuated the development of CH-induced PH, as assessed by measurement of right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling and endothelial activation and inflammation. Genetic deletion of the pattern recognition receptor TLR4, but not the receptor for advanced glycation end products, likewise attenuated CH-induced PH. Finally, daily treatment of mice with recombinant human HMGB1 exacerbated CH-induced PH in wild-type (WT) but not Tlr4(-/-) mice. These data demonstrate that HMGB1-mediated activation of TLR4 promotes experimental PH and identify HMGB1 and/or TLR4 as potential therapeutic targets for the treatment of PH.

Caveolin-1 (Cav-1)−/− mice develop mild pulmonary hypertension as they age. In this study, we sought to determine the effect of chronic hypoxia, an established model of pulmonary hypertension, on young Cav-1−/− mice with no measurable signs of pulmonary hypertension. Exposure of Cav-1−/− mice to chronic hypoxia resulted in an initial rise in right ventricular (RV) systolic pressure (RVSP) similar to wild-type (WT) mice. By three weeks RVSP decreased in the Cav-1−/− mice, whereas it was maintained in WT mice. The drop in RVSP in Cav-1−/− mice was accompanied by decreased cardiac output, increased RV hypertrophy, RV interstitial fibrosis, decreased RV sarco(endo)plasmic reticulum Ca²⁺-ATPase 2a mRNA and decreased RV function compared with WT mice. Importantly, minimal differences were noted in pulmonary vascular remodeling between WT and Cav-1−/− mice, and left ventricular function was normal in hypoxic Cav-1−/− mice. Mechanistically, increased endothelial nitric oxide synthase uncoupling and increased tyrosine nitration of protein kinase G were detected in the RV of Cav-1−/− mice. These hemodynamic, histological, and molecular changes were prevented in Cav-1−/− mice expressing an endothelial-specific Cav-1 transgene or by nitric oxide synthase inhibition. These data suggest that, in Cav-1−/− mice, increased oxidative/nitrosative stress due to endothelial nitric oxide synthase uncoupling modifies the response of the RV to pressure overload, accelerating the deterioration of RV function.

Evidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension. Immunofluorescent staining revealed significant C3d deposition in lung sections from IPAH patients and C57Bl6/J wild-type mice exposed to three weeks of chronic hypoxia to induce pulmonary hypertension. Right ventricular systolic pressure and right ventricular hypertrophy were increased in hypoxic vs. normoxic wild-type mice, which were attenuated in C3−/− hypoxic mice. Likewise, pulmonary vascular remodeling was attenuated in the C3−/− mice compared to wild-type mice as determined by the number of muscularized peripheral arterioles and morphometric analysis of vessel wall thickness. The loss of C3 attenuated the increase in interleukin-6 and intracellular adhesion molecule-1 expression in response to chronic hypoxia, but not endothelin-1 levels. In wild-type mice, but not C3−/− mice, chronic hypoxia led to platelet activation as assessed by bleeding time, and flow cytometry of platelets to determine cell surface P-selectin expression. In addition, tissue factor expression and fibrin deposition were increased in the lungs of WT mice in response to chronic hypoxia. These pro-thrombotic effects of hypoxia were abrogated in C3−/− mice. Herein, we provide compelling genetic evidence that the complement system plays a pathophysiologic role in the development of PAH in mice, promoting pulmonary vascular remodeling and a pro-thrombotic phenotype. In addition we demonstrate C3d deposition in IPAH patients suggesting that complement activation plays a role in the development of PAH in humans.

Objective— Heme oxygenase-1 (HO-1), via its enzymatic degradation products, exhibits cell and tissue protective effects in models of vascular injury and disease. The migration of vascular smooth muscle cells (VSMC) from the medial to the intimal layer of blood vessels plays an integral role in the development of a neointima in these models. Despite this, there are no studies addressing the effect of increased HO-1 expression on VSMC migration. Results and Methods— The effects of increased HO-1 expression, as well as biliverdin, bilirubin, and carbon monoxide (CO), were studied in in vitro models of VSMC migration. Induction of HO-1 or CO, but not biliverdin or bilirubin, inhibited VSMC migration. This effect was mediated by the inhibition of Nox1 as determined by a range of approaches, including detection of intracellular superoxide, nicotinamide adenine dinucleotide phosphate oxidase activity measurements, and siRNA experiments. Furthermore, CO decreased platelet-derived growth factor-stimulated, redox-sensitive signaling pathways. Conclusion— Herein, we demonstrate that increased HO-1 expression and CO decreases platelet-derived growth factor-stimulated VSMC migration via inhibition of Nox1 enzymatic activity. These studies reveal a novel mechanism by which HO-1 and CO may mediate their beneficial effects in arterial inflammation and injury.

Rationale: Fatty acid nitroalkenes are endogenously generated electrophilic byproducts of nitric oxide and nitrite-dependent oxidative inflammatory reactions. Existing evidence indicates nitroalkenes support posttranslational protein modifications and transcriptional activation that promote the resolution of inflammation. Objective: The aim of this study was to assess whether in vivo administration of a synthetic nitroalkene could elicit antiinflammatory actions in vivo using a murine model of vascular injury. Methods and Results: The in vivo administration (21 days) of nitro-oleic acid (OA-NO2) inhibited neointimal hyperplasia after wire injury of the femoral artery in a murine model (OA-NO2 treatment resulted in reduced intimal area and intima to media ratio versus vehicle- or oleic acid (OA)-treated animals, P<0.0001). Increased heme oxygenase (HO)-1 expression accounted for much of the vascular protection induced by OA-NO2 in both cultured aortic smooth muscle cells and in vivo. Inhibition of HO by Sn(IV)-protoporphyrin or HO-1 small interfering RNA reversed OA-NO2-induced inhibition of platelet-derived growth factor-stimulated rat aortic smooth muscle cell migration. The upregulation of HO-1 expression also accounted for the antistenotic actions of OA-NO2 in vivo, because inhibition of neointimal hyperplasia following femoral artery injury was abolished in HO-1(-/-) mice (OA-NO2-treated wild-type versus HO-1(-/-) mice, P=0.016). Conclusions: In summary, electrophilic nitro-fatty acids induce salutary gene expression and cell functional responses that are manifested by a clinically significant outcome, inhibition of neointimal hyperplasia induced by arterial injury. (Circ Res. 2009;105:965-972.)