AimsCalcific aortic valve disease (CAVD) is a progressive heart disease that is particularly prevalent in elderly patients. The current treatment of CAVD is surgical valve replacement, but this is not a permanent solution, and it is very challenging for elderly patients. Thus, a pharmacological intervention for CAVD may be beneficial. In this study, we intended to rescue aortic valve (AV) calcification through inhibition of TGFβ1 and SMAD3 signaling pathways.Methods and ResultsThe klotho gene, which was discovered as an aging-suppressor gene, has been observed to play a crucial role in AV calcification. The klotho knockout (Kl–/–) mice have shorter life span (8–12 weeks) and develop severe AV calcification. Here, we showed that increased TGFβ1 and TGFβ-dependent SMAD3 signaling were associated with AV calcification in Kl–/– mice. Next, we generated Tgfb1- and Smad3-haploinsufficient Kl–/– mice to determine the contribution of TGFβ1 and SMAD3 to the AV calcification in Kl–/– mice. The histological and morphometric evaluation suggested a significant reduction of AV calcification in Kl–/–; Tgfb1± mice compared to Kl–/– mice. Smad3 heterozygous deletion was observed to be more potent in reducing AV calcification in Kl–/– mice compared to the Kl–/–; Tgfb1± mice. We observed significant inhibition of Tgfb1, Pai1, Bmp2, Alk2, Spp1, and Runx2 mRNA expression in Kl–/–; Tgfb1± and Kl–/–; Smad3± mice compared to Kl–/– mice. Western blot analysis confirmed that the inhibition of TGFβ canonical and non-canonical signaling pathways were associated with the rescue of AV calcification of both Kl–/–; Tgfb1± and Kl–/–; Smad3± mice.ConclusionOverall, inhibition of the TGFβ1-dependent SMAD3 signaling pathway significantly blocks the development of AV calcification in Kl–/– mice. This information is useful in understanding the signaling mechanisms involved in CAVD.

In the bone marrow (BM) microenvironment, where breast cancer (BC)-disseminated tumor cells (DTCs) can remain dormant for decades, NG2+/Nestin+ mesenchymal stem cells (MSCs) promote hematopoietic stem cell quiescence. Here we reveal that periarteriolar BM-resident NG2+/Nestin+ MSCs can also instruct BC DTCs to enter dormancy. NG2+/Nestin+ MSCs produce transforming growth factor (TGF)-β2 and bone morphogenetic protein (BMP)7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase, results in p27 induction. Genetic depletion of MSCs or conditional knockout of TGF-β2 in MSCs using an NG2-CreER driver led to bone metastatic outgrowth of otherwise dormant p27+/Ki67− DTCs. Also, patients with estrogen receptor-positive BC without systemic recurrence displayed higher frequency of TGF-β2 and BMP7 detection in the BM. Our results provide direct proof that hematopoietic stem cell dormancy niches control BC DTC dormancy and suggest that aging or extrinsic factors that affect the NG2+/Nestin+ MSC niche homeostasis may result in a break from dormancy and BC bone relapse. Aguirre-Ghiso and colleagues report that bone-marrow-resident mesenchymal stem cells promote cancer dormancy through TGF-β family members.

Among the three transforming growth factor beta (TGFβ) ligands, TGFβ2 is essential for heart development and is produced by multiple cell types, including myocardium. Heterozygous mutations in TGFB2 in patients of connective tissue disorders result in congenital heart defects and adult valve malformations, including mitral valve prolapse (MVP) with or without regurgitation. Tgfb2 germline knockout fetuses exhibit multiple cardiac defects but the role of myocardial-TGFβ2 in heart development is yet to be elucidated. Here, myocardial Tgfb2 conditional knockout (CKO) embryos were generated by crossing Tgfb2flox mice with Tgfb2+/−; cTntCre mice. Tgfb2flox/− embryos were normal, viable. Cell fate mapping was done using dual-fluorescent mT/mG+/− mice. Cre-mediated Tgfb2 deletion was assessed by genomic PCR. RNAscope in situ hybridization was used to detect the loss of myocardial Tgfb2 expression. Histological, morphometric, immunohistochemical, and in situ hybridization analyses of CKOs and littermate controls at different stages of heart development (E12.5–E18.5) were used to determine the role of myocardium-derived TGFβ2 in atrioventricular (AV) cushion remodeling and myocardial development. CKOs exhibit a thin ventricular myocardium, AV cushion remodeling defects and developed incomplete AV septation defects. The loss of myocardial Tgfb2 resulted in impaired cushion maturation and dysregulated cell death. Phosphorylated SMAD2, a surrogate for TGFβ signaling, was “paradoxically” increased in both AV cushion mesenchyme and ventricular myocardium in the CKOs. Our results indicate that TGFβ2 produced by cardiomyocytes acting as cells autonomously on myocardium and via paracrine signaling on AV cushions are required for heart development.

In the bone marrow (BM) microenvironment, NG2 + /Nestin + mesenchymal stem cells (MSCs) promote hematopoietic stem cell (HSC) quiescence 1,2 . Importantly, the BM can also harbour disseminated tumour cells (DTCs) from multiple cancers, which, like HSCs, can remain dormant 3 . The BM signals are so growth-restrictive that dormant BM DTCs can persist for years to decades only to awaken and fuel lethal metastasis 3–10 . The mechanisms and niche components regulating DTC dormancy remain largely unknown. Here, we reveal that periarteriolar BM-resident NG2 + /Nestin + MSCs can instruct breast cancer (BC) DTCs to enter dormancy. NG2 + /Nestin + MSCs produce TGFβ2 and BMP7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase result in p27-CDK inhibitor induction. Importantly, genetic depletion of the NG2 + /Nestin + MSCs or conditional knock-out of TGFβ2 in the NG2 + /Nestin + MSCs led to awakening and bone metastatic expansion of otherwise dormant p27 + /Ki67 − DTCs. Our results provide a direct proof that HSC dormancy niches control BC DTC dormancy. Given that aged NG2 + /Nestin + MSCs can lose homeostatic control of HSC dormancy, our results suggest that aging or extrinsic factors that affect the NG2 + /Nestin + MSC niche may result in a break from dormancy and BC bone relapse.

Transforming growth factor beta3 (TGFB3) gene mutations in patients of arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD1) and Loeys-Dietz syndrome-5 (LDS5)/Rienhoff syndrome are associated with cardiomyopathy, cardiac arrhythmia, cardiac fibrosis, cleft palate, aortic aneurysms, and valvular heart disease. Although the developing heart of embryos express Tgfb3, its overarching role remains unclear in cardiovascular development and disease. We used histological, immunohistochemical, and molecular analyses of Tgfb3−/− fetuses and compared them to wildtype littermate controls. The cardiovascular phenotypes were diverse with approximately two thirds of the Tgfb3−/− fetuses having one or more cardiovascular malformations, including abnormal ventricular myocardium (particularly of the right ventricle), outflow tract septal and alignment defects, abnormal aortic and pulmonary trunk walls, and thickening of semilunar and/or atrioventricular valves. Ventricular septal defects (VSD) including the perimembranous VSDs were observed in Tgfb3−/− fetuses with myocardial defects often accompanied by the muscular type VSD. In vitro studies using TGFβ3-deficient fibroblasts in 3-D collagen lattice formation assays indicated that TGFβ3 was required for collagen matrix reorganization. Biochemical studies indicated the ‘paradoxically’ increased activation of canonical (SMAD-dependent) and noncanonical (MAP kinase-dependent) pathways. TGFβ3 is required for cardiovascular development to maintain a balance of canonical and noncanonical TGFβ signaling pathways.