The role of SMAD3 in pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a rare but fatal disease, characterized by elevated pulmonary vascular resistance due to remodelling of the pulmonary arteries. Remodelling occurs due to increased smooth muscle and endothelial cell proliferation as well as smooth muscle cell hyperplasia. The transforming growth factor ß receptor (TGF-ß receptor) superfamily is known to play an important role in the disease development of pulmonary hypertension. Its ligand TGF-ß, which is an important factor regulating cell proliferation, migration, apoptosis and immunosuppression, is elevated in PAH patients, and both, clinical and experimental data indicate a profound role for TGF-ß in the development of PAH. The major postreceptor mediators of the canonical TGF-ß induced signalling pathway are the SMAD proteins (SMAD: portmanteau from its homologs SMA (for small body size) and MAD (mothers against decapentaplegic) in C. elegans and Drosophila, respectively). In this proposal we aim to elucidate the role of SMAD3 signalling in lung vascular remodelling and the development of PAH based on the notion that i) SMAD3 is downregulated in experimental models and clinical samples of PH, and ii) loss of SMAD3 may promote cell proliferation and differentiation/hypertrophy of smooth muscle cells. Specifically, we hypothesize that decreased expression of SMAD3 leads to enhanced remodelling in vitro and in vivo via stabilization and/or disinhibition of myocardin-related transcription factor (MRTF). SMAD3 has been recently shown to inhibit MRTF, which is a key inducer of the serum response factor (SRF). SRF controls not only the expression of immediate early genes involved in proliferation (c-fos, Erg1) of many cell types, but is also a major driver of SM-actin (SMA) expression and smooth muscle cell differentiation. Conversely, inhibition or deficiency of MRTF may lead to decreased proliferation and cell hyperplasia and thus, reduced vascular remodelling. In addition, loss of SMAD3 may promote cell proliferation by MRTF-independent pathways as previously shown in tumour cells. We therefore propose to investigate the regulation and role of SMAD3 in hypertrophic and proliferative responses of human pulmonary arterial endothelial cells (PAEC) and SMCs (PASMCs) in vitro, and its underlying mechanisms, and test for the effects of inhibition of SMAD3 or its downstream signalling pathway on the development of pulmonary hypertension and vascular remodelling in experimental animal models of PH in vivo. The results of the proposed research are expected to provide important innovative insights into the molecular and cellular mechanisms underlying vascular remodelling in PH disease, to identify novel targets for future therapeutic interventions, and to establish cell-penetrating fusion peptides as novel therapeutic approach for the targeted inhibition of protein-protein interactions in lung vascular disease.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease, characterized by increased vascular resistance due to decreased vessel diameter. The decreased vessel diameter occurs due to increased pulmonary arterial smooth muscle and endothelial cell proliferation, as well as smooth muscle cell hyperplasia. The family of transforming growth factor receptors (TGF-? receptors) is known for its important role in the development of pulmonary hypertension. Their ligand TGF-?, which regulates proliferation, migration, apoptosis and immunosuppression, is elevated in PAH patients and clinical as well as preclinical data indicate its important role in the pathogenesis of PAH. The main downstream signaling mediators of TGF-? are the SMAD proteins. In the course of this project we could show that SMAD3 was downregulated in lungs of PAH patients and in pulmonary arteries of three independent PAH animal models. TGF-? treatment replicated the loss of SMAD3 in human pulmonary artery smooth muscle (huPASMCs) and endothelial (huPAECs) cells. SMAD3 silencing increased proliferation and migration in huPASMCs, huPAECs and hypertrophy in case of huPASMCs, all factors contributing to thickening of the vascular wall and therefore leading to pulmonary hypertension. In addition we found that decreased SMAD3 expression led to a disinhibition of the myocardin related transcription factor (MRTF), activating serum- response factor (SRF). SRF not only regulates the expression of so called early genes (c-fos, Erg1), participating in proliferation processes, but foremost also regulates the smooth muscle actin (SMA) expression and smooth muscle cell differentiation and therefore further participates in vessel wall thickening. Application of a MRTF inhibitor in a PAH animal model could reverse the vascular remodeling and decreased the pulmonary pressure in the treated animals proving our in vitro generated knowledge also in vivo. We can conclude that loss of SMAD3 presents a novel pathomechanism in PAH that promotes vascular cell proliferation and - via MRTF disinhibition - hypertrophy of huPASMC, thereby reconciling the parallel induction of a synthetic and contractile huPASMC phenotype. In summary we can state that with the help of this project we could gain important insight in the molecular and cellular mechanism, which participate in the vascular remodeling in pulmonary hypertension, and therefore could identify MRTF as a new target for future therapeutic interventions.