CD248 activates TGF-β receptor I to promote vascular remodeling in pulmonary arterial hypertension

Abstract

I.BackgroundPulmonary arterial hypertension (PAH) is a debilitating cardiopulmonary disease characterized by progressive remodeling of the pulmonary vasculature. Pathologic transforming growth factor-{beta} (TGF-{beta}) signaling is an essential driver of vascular remodeling in PAH. While global inhibitors of TGF-{beta} exist, their clinical application is limited by systemic adverse effects. Therefore, a critically unmet need in PAH is to identify pulmonary vascular-specific regulators of the TGF-{beta} axis, which would selectively enhance clinical efficacy while minimizing adverse effects. As the clinical care of PAH largely promotes vasodilation, and only one FDA-approved agent targets vascular remodeling, this study aimed to identify selective, therapeutically targetable regulators of the TGF-{beta} axis in the PAH pulmonary vasculature. MethodsCD248 was identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics in human lungs. CD248 levels were assessed across human, rat, and mouse lung tissues using western blotting, RTqPCR, and/or immunofluorescence techniques. CD248-null (CD248-/-) mice were used to study the contribution of CD248 to hypoxia-sugen (H/S)-induced PAH. The mechanistic role of CD248 in PAH vascular remodeling and TGF-{beta} signaling was assessed by genetic (siRNA knockdown; overexpression) and pharmacologic (Ontuxizumab) manipulation of primary human pulmonary vascular cells. ResultsLC-MS/MS proteomics coupled with pathway enrichment analysis of human lung tissue identified CD248 as a putative mediator of vascular remodeling that is elevated in PAH lungs. CD248 was elevated in PAH pulmonary artery smooth muscle cells (PASMCs) across human, rat, and mouse lung tissue. CD248-/- mice were protected from H/S-induced elevations in right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary artery muscularization. CD248 knock-down reduced cell proliferation and migration of primary PAH PASMCs. CD248 was essential for phospho-activation of TGF-{beta} receptor I (T{beta}RI) at S165 and canonical phosphorylation of SMAD3 at S423/425. CD248 loss blunted TGF-{beta}-induced gene expression (FN1, Col11, -SMA) and activated expression of the vasoprotective matrix metalloprotease, MMP-8. Mechanistically, CD248 interacted with and enhanced de novo phosphorylation and stability of T{beta}RI, blocking its ubiquitin-mediated proteasomal degradation. Ontuxizumab promoted T{beta}RI instability and attenuated the production of FN1, Col11, and -SMA in primary PAH PASMCs. ConclusionsThis work identifies CD248 as a previously unrecognized co-activator of T{beta}RI in PAH. As CD248 is largely quiescent in most adult tissues yet pathologically upregulated in the PAH pulmonary vasculature, this study supports the potential of anti-CD248 therapy as a novel pulmonary vascular-specific alternative to systemic TGF-{beta} inhibition.