TWIST1 Drives Smooth Muscle Cell Proliferation in Pulmonary Hypertension via Loss of GATA-6 and BMPR2.

Rationale: The bHLH (basic helix-loop-helix) transcription factor TWIST1 (Twist-related protein 1) controls cell proliferation and differentiation in tissue development and disease processes. Recently, endothelial TWIST1 has been linked to pulmonary hypertension (PH) and endothelial-to-mesenchymal transition, yet the role of TWIST1 in smooth muscle cells (SMCs) remains so far unclear.Objectives: To define the role of TWIST1 in SMCs in the pathogenesis of PH.Methods: SMC-specific TWIST1-deficient mice, SMC-specific TWIST1 silencing in rats, mass spectrometry, immunoprecipitation, and chromatin immunoprecipitation were used to delineate the role of SMC TWIST1 in PH.Measurements and Main Results: In pulmonary vessels from patients with PH and rodent PH models, TWIST1 expression was markedly increased and predominantly localized to SMCs. SMC-specific TWIST1 deficiency or silencing attenuated the development of PH and distal vessel muscularization in chronically hypoxic mice and in monocrotaline-treated rats. In vitro, TWIST1 inhibition or silencing prevented pulmonary artery SMC proliferation and migration. Mechanistically, the observed effects were mediated, at least in part, by TWIST1-dependent degradation of GATA-6 (GATA-binding protein 6). BMPR2 (bone morphogenetic protein receptor-2) was identified as a novel downstream target of GATA-6, which directly binds to its promoter. Inhibition of TWIST1 promoted the recruitment of GATA-6 to the BMPR2 promoter and restored BMPR2 functional expression.Conclusions: Our findings identify a key role for SMC TWIST1 in the pathogenesis of lung vascular remodeling and in PH that is partially mediated via reduced GATA-6-dependent BMPR2 expression. Inhibition of SMC TWIST1 may constitute a new therapeutic strategy for the treatment of PH.

Physiological exercise loading suppresses post-traumatic osteoarthritis progression via an increase in bone morphogenetic proteins expression in an experimental rat knee model.

OBJECTIVE: To evaluate the dose-response relationship of exercise loading in the cartilage-subchondral bone (SB) unit in surgically-induced post-traumatic osteoarthritis (PTOA) of the knee. DESIGN: Destabilized medial meniscus (DMM) surgery was performed on the right knee of 12-week-old male Wistar rats, and sham surgery was performed on the contralateral knee. Four weeks after the surgery, the animals were subjected to moderate (12 m/min) or intense (21 m/min) treadmill exercises for 30 min/day, 5 days/week for 4 weeks. PTOA development in articular cartilage and SB was examined using histological and immunohistochemical analyses, micro-computed tomography (micro-CT) analysis, and biomechanical testing at 8 weeks after surgery. Gremlin-1 was injected to determine the role of bone morphogenetic protein (BMP) signaling on PTOA development following moderate exercise. RESULTS: Moderate exercise increased BMP-2, BMP-4, BMP-6, BMP receptor 2, pSmad-5, and inhibitor of DNA binding protein-1 expression in the superficial zone chondrocytes and suppressed cartilage degeneration, osteophyte growth, SB damage, and osteoclast-mediated SB resorption. However, intense exercise had little effect on BMP expression and even caused progression of these osteoarthritis (OA) changes. Gremlin-1 injection following moderate exercise caused progression of the PTOA development down to the level of the non-exercise DMM-operated knee. CONCLUSIONS: Exercise regulated cartilage-SB PTOA development in DMM-operated knees in a dose-dependent manner. Our findings shed light on the important role of BMP expression in superficial zone chondrocytes in attenuation of PTOA development following physiological exercise loading. Further studies to support a mechanism by which BMPs would be beneficial in preventing PTOA progression are warranted.

[Bushen Tiaojing Recipe Regulated Expressions of BMPR I[/ALK6-Smads in Mouse Oocytes Cul- ture in vitro].

Objective To observe the effects of Bushen Tiaojing Recipe (BTR) on the counts of survival preantral follicles and the bone morphogenetic protein receptor II (BMPR II )/activin receptor- like kinase 6-drosophila mothers against decapentaplegic proteins (ALK6-Smads) signal pathway in oocytes cultured in vitro, and to study its mechanism for improving the quality of oocytes. Methods Prean- tral follicles were mechanically isolated from 65 female 12-day old healthy Kunming mice, which were inoculated by normal rats' serum (as the control group) , high, medium, low dose BTR containing serums (as Shen-supplementing groups) , high dose BTR containing serum + K02288 (as the inhibitor group) , respectively. All were cultured by common method in vitro. On the 6th day the counts of survival preantral follicles were compared between each Shen-supplementing group and the control group respectively. mR- NA expressions of BMPR II, ALK6, Smad1 , Smad5, and Smad8 were detected by Real-time fluorescence quantitative PCR. The protein expressions of indices mentioned above and phospho-Smadl/5/8 (p- Smadl/5/8) were detected by cellular immunofluorescence test. Results Compared with the control group, the quantity of survival preantral follicles increased in the high dose BTR containing serum group; mRNA expressions of BMPR II, ALK6, Smad5, and Smad8 were elevated, protein expressions of indi- ces mentioned above and p-Smadl/5/8 were increased in the 3 Shen-supplementing groups (P <0. 05) ; mRNA and protein expressions of Smad1 were increased in high and medium dose BTR containing serum groups (P<0.05). Compared with the high dose BTR containing serum group, protein expressions of Smad1/5/8 were reduced in the inhibitor group (P <0.05). Conclusion BTR could elevate the quantity of survival preantral follicles cultured in vitroand improve the quality of oocytes, which might be possibly as- sociated to regulating the BMPR II/ALK6-Smads signal pathway in oocytes.

Elafin Reverses Pulmonary Hypertension via Caveolin-1-Dependent Bone Morphogenetic Protein Signaling.

RATIONALE: Pulmonary arterial hypertension is characterized by endothelial dysfunction, impaired bone morphogenetic protein receptor 2 (BMPR2) signaling, and increased elastase activity. Synthetic elastase inhibitors reverse experimental pulmonary hypertension but cause hepatotoxicity in clinical studies. The endogenous elastase inhibitor elafin attenuates hypoxic pulmonary hypertension in mice, but its potential to improve endothelial function and BMPR2 signaling, and to reverse severe experimental pulmonary hypertension or vascular pathology in the human disease was unknown. OBJECTIVES: To assess elafin-mediated regression of pulmonary vascular pathology in rats and in lung explants from patients with pulmonary hypertension. To determine if elafin amplifies BMPR2 signaling in pulmonary artery endothelial cells and to elucidate the underlying mechanism. METHODS: Rats with pulmonary hypertension induced by vascular endothelial growth factor receptor blockade and hypoxia (Sugen/hypoxia) as well as lung organ cultures from patients with pulmonary hypertension were used to assess elafin-mediated reversibility of pulmonary vascular disease. Pulmonary arterial endothelial cells from patients and control subjects were used to determine the efficacy and mechanism of elafin-mediated BMPR2 signaling. MEASUREMENTS AND MAIN RESULTS: In Sugen/hypoxia rats, elafin reduced elastase activity and reversed pulmonary hypertension, judged by regression of right ventricular systolic pressure and hypertrophy and pulmonary artery occlusive changes. Elafin improved endothelial function by increasing apelin, a BMPR2 target. Elafin induced apoptosis in human pulmonary arterial smooth muscle cells and decreased neointimal lesions in lung organ culture. In normal and patient pulmonary artery endothelial cells, elafin promoted angiogenesis by increasing pSMAD-dependent and -independent BMPR2 signaling. This was linked mechanistically to augmented interaction of BMPR2 with caveolin-1 via elafin-mediated stabilization of endothelial surface caveolin-1. CONCLUSIONS: Elafin reverses obliterative changes in pulmonary arteries via elastase inhibition and caveolin-1-dependent amplification of BMPR2 signaling.

Sildenafil potentiates bone morphogenetic protein signaling in pulmonary arterial smooth muscle cells and in experimental pulmonary hypertension.

OBJECTIVE: Mutations in the bone morphogenetic protein type II receptor (BMPR-II) are responsible for the majority of cases of heritable pulmonary arterial hypertension (PAH), and BMPR-II deficiency contributes to idiopathic and experimental forms of PAH. Sildenafil, a potent type-5 nucleotide-dependent phosphodiesterase inhibitor, is an established treatment for PAH, but whether sildenafil affects bone morphogenetic protein (BMP) signaling in the pulmonary circulation remains unknown. METHODS AND RESULTS: Studies were undertaken in human pulmonary arterial smooth muscle cells (PASMCs) and in vivo in the monocrotaline rat model of PAH. In PASMCs, sildenafil enhanced BMP4-induced phosphorylation of Smad1/5, Smad nuclear localization, and Inhibitor of DNA binding protein 1 gene and protein expression. This effect was mimicked by 8-bromo-cyclic GMP. Pharmacological inhibition or small interfering RNA knockdown of cyclic GMP-dependent protein kinase I inhibited the effect of sildenafil on BMP signaling. In functional studies, we observed that sildenafil potentiated the antiproliferative effects of BMP4 on PASMC proliferation. Furthermore, sildenafil restored the antiproliferative response to BMP4 in PASMCs harboring mutations in BMPR-II. In the monocrotaline rat model of PAH, which is characterized by BMPR-II deficiency, sildenafil prevented the development of pulmonary hypertension and vascular remodeling, and partly restored Smad1/5 phosphorylation and Inhibitor of DNA binding protein 1 gene expression in vivo in monocrotaline exposed rat lungs. CONCLUSIONS: Sildenafil enhances canonical BMP signaling via cyclic GMP and cyclic GMP-dependent protein kinase I in vitro and in vivo, and partly restores deficient BMP signaling in BMPR-II mutant PASMCs. Our findings demonstrate a novel mechanism of action of sildenafil in the treatment of PAH and suggest that targeting BMP signaling may be beneficial in this disease.

Interactions of regenerative, inflammatory and biomechanical signals on bone morphogenetic protein-2 in periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Regeneration of periodontal tissues by EMD remains a major challenge because a number of modifying factors are as yet unknown. The effects of EMD seem to be mediated, at least in part, by bone morphogenetic protein-2 (BMP-2). This in vitro study was performed to examine whether the effects of EMD on BMP-2 activity are modulated by inflammatory and/or biomechanical signals. MATERIAL AND METHODS: Periodontal ligament cells were seeded on BioFlex(®) plates and exposed to EMD under normal, inflammatory or biomechanical loading conditions for 1 and 6 d. In order to mimic proinflammatory or biomechanical loading conditions in vitro, cells were stimulated with interleukin-1ß (IL-1ß), which is increased at inflamed periodontal sites, and cyclic tensile strain of various magnitudes, respectively. The synthesis of BMP-2, its receptors (BMPR-1A, BMPR-1B and BMPR-2) and its inhibitors (follistatin, matrix gla protein and noggin) were analyzed using real-time RT-PCR and ELISA. RESULTS: In EMD-treated cells, BMP-2 synthesis was increased significantly at 1 d. EMD also induced the expression of all BMP receptors, and of the BMP inhibitors follistatin and noggin. In general, IL-1ß and biomechanical loading neither down-regulated BMP-2 nor up-regulated BMP inhibitors in EMD-stimulated cells. However, IL-1ß and biomechanical loading, when applied for a longer time period, caused a down-regulation of EMD-induced BMP receptors. CONCLUSION: EMD induces not only BMP-2, but also its receptors and inhibitors, in PDL cells. IL-1ß and biomechanical forces may counteract the beneficial effects of EMD on BMP-2 activity via the down-regulation of BMP receptors.

Narrative review: the enigma of pulmonary arterial hypertension: new insights from genetic studies.

Pulmonary arterial hypertension (PAH) occurs as an idiopathic disease (formerly called primary pulmonary hypertension) and as a consequence of other illnesses. These illnesses include connective tissue diseases, portal hypertension, diet and stimulant drug use, HIV infection, and congenital heart disease. Inherited susceptibility to PAH occurs in families and is almost always due to mutations in genes of the TGF-beta family of receptors. The most common mutation leading to PAH is in bone morphogenetic protein receptor type 2 (BMPR2), originally discovered to be involved in bone healing. Mutations in BMPR2 have also been found in patients with idiopathic PAH, although the true prevalence of this susceptibility has not been determined. About 20% of individuals with a BMPR2 mutation develop symptomatic pulmonary hypertension. Evidence is growing that imbalanced activation of other TGF-beta receptors coupled with reduced activity of mutated BMPR2 increases the likelihood of development of PAH. Many signaling systems have been found to participate in PAH, including K channels, serotonin, angiopoietin, and cyclooxygenases. An interaction of these signaling systems with BMPR2 is a focus of research in PAH. Approaches to altering the imbalance of activation of BMPR2 and other TGF-beta receptors may yield future therapies for PAH.