Jianyun Yan

Resident c-kit + cells in the heart are not cardiac stem cells

Identifying a bona fide population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. Previously, cardiac c-kit+ cells were reported to be CSCs with a potential to become myocardial, endothelial and smooth muscle cells in vitro and after cardiac injury. Here we provide further insights into the nature of cardiac c-kit+ cells. By targeting the c-kit locus with multiple reporter genes in mice, we find that c-kit expression rarely co-localizes with the expression of the cardiac progenitor and myogenic marker Nkx2.5, or that of the myocardial marker, cardiac troponin T (cTnT). Instead, c-kit predominantly labels a cardiac endothelial cell population in developing and adult hearts. After acute cardiac injury, c-kit+ cells retain their endothelial identity and do not become myogenic progenitors or cardiomyocytes. Thus, our work strongly suggests that c-kit+ cells in the murine heart are endothelial cells and not CSCs.

Adriamycin-induced autophagic cardiomyocyte death plays a pathogenic role in a rat model of heart failure

BACKGROUND The mechanisms underlying heart failure induced by adriamycin are very complicated and still unclear. The aim of this study was to investigate whether autophagy was involved in the progression of heart failure induced by adriamycin, so that we can develop a novel treatment strategy for heart failure. METHODS 3-methyladenine (3MA), a specific inhibitor on autophagy was used in a heart failure model of rats induced by adriamycin. Neonatal cardiomyocytes were isolated from Sprague-Dawley rat hearts and randomly divided into controls, an adriamycin-treated group, and a 3MA plus adriamycin-treated group. We then examined the morphology, expression of beclin 1 gene, mitochondrial permeability transition (MPT), and Na+-K+ ATPase activity in vivo. We also assessed cell viability, mitochondrial membrane potential changes and counted autophagic vacuoles in cultured cardiomyocytes. In addition, we analyzed the expression of autophagy associated gene, beclin 1 using RT-PCR and Western blotting in an animal model. RESULTS 3MA significantly improved cardiac function and reduced mitochondrial injury. Furthermore, adriamycin induced the formation of autophagic vacuoles, and 3MA strongly downregulated the expression of beclin 1 in adriamycin-induced failing heart and inhibited the formation of autophagic vacuoles. CONCLUSION Autophagic cardiomyocyte death plays an important role in the pathogenesis of heart failure in rats induced by adriamycin. Mitochondrial injury may be involved in the progression of heart failure caused by adriamycin via the autophagy pathway.

Decorin GAG Synthesis and TGF-β Signaling Mediate Ox-LDL–Induced Mineralization of Human Vascular Smooth Muscle Cells

Objective—Decorin and oxidized low-density lipoprotein (Ox-LDL) independently induce osteogenic differentiation of vascular smooth muscle cells (VSMCs). We aimed to determine whether decorin glycosaminoglycan (GAG) chain synthesis contributes to Ox-LDL–induced differentiation and calcification of human VSMCs in vitro. Methods and Results—Human VSMCs treated with Ox-LDL to induce oxidative stress showed increased alkaline phosphatase (ALP) activity, accelerated mineralization, and a difference in both decorin GAG chain biosynthesis and CS/DS structure compared with untreated controls. Ox-LDL increased mRNA abundance of both xylosyltransferase (XT)-I, the key enzyme responsible for GAG chain biosynthesis and Msx2, a marker of osteogenic differentiation. Furthermore, downregulation of XT-I expression using small interfering RNA blocked Ox-LDL–induced VSMC mineralization. Adenoviral-mediated overexpression of decorin, but not a mutated unglycanated form, accelerated mineralization of VSMCs, suggesting GAG chain addition on decorin is crucial for the process of differentiation. The decorin-induced VSMC osteogenic differentiation involved activation of the transforming growth factor (TGF)-&bgr; pathway, because it was attenuated by blocking of TGF-&bgr; receptor signaling and because decorin overexpression potentiated phosphorylation of the downstream signaling molecule smad2. Conclusion—These studies provide direct evidence that oxidative stress–mediated decorin GAG chain synthesis triggers TGF-&bgr; signaling and mineralization of VSMCs in vitro.

Myocardial Tbx20 regulates early atrioventricular canal formation and endocardial epithelial-mesenchymal transition via Bmp2

During early embryogenesis, the formation of the cardiac atrioventricular canal (AVC) facilitates the transition of the heart from a linear tube into a chambered organ. However, the genetic pathways underlying this developmental process are poorly understood. The T-box transcription factor Tbx20 is expressed predominantly in the AVC of early heart tube. It was shown that Tbx20 activates Nmyc1 and suppresses Tbx2 expression to promote proliferation and specification of the atrial and ventricular chambers, yet it is not known if Tbx20 is involved in early AVC development. Here, we report that mice lacking Tbx20 in the AVC myocardium fail to form the AVC constriction, and the endocardial epithelial-mesenchymal transition (EMT) is severely perturbed. Tbx20 maintains expression of a variety of genes, including Bmp2, Tbx3 and Hand1 in the AVC myocardium. Intriguingly, we found Bmp2 downstream genes involved in the EMT initiation are also downregulated. In addition, re-expression of Bmp2 in the AVC myocardium substantially rescues the EMT defects resulting from the lack of Tbx20, suggesting Bmp2 is one of the key downstream targets of Tbx20 in AVC development. Our data support a complex signaling network with Tbx20 suppressing Tbx2 in the AVC myocardium but also indirectly promoting Tbx2 expression through Bmp2. The spatiotemporal expression of Tbx2 in the AVC appears to be balanced between these two opposing signals. Overall, our study provides genetic evidence that Tbx20 has essential roles in regulating AVC development that coordinate early cardiac chamber formation.

HGF/c-Met signalling promotes Notch3 activation and human vascular smooth muscle cell osteogenic differentiation in vitro

Objectives Vascular calcification is a major clinical problem and elucidating the underlying mechanism is important to improve the prognosis of patients with cardiovascular disease. We aimed to elucidate the role and mechanism of action of Hepatocyte Growth Factor (HGF)/c-Met signalling in vascular calcification and establish whether blocking this pathway could prevent mineralisation of vascular smooth muscle cells (VSMCs) in vitro. Methods and results We demonstrate increased HGF secretion and c-Met up-regulation and phosphorylation during VSMC osteogenic differentiation. Adenoviral-mediated over-expression of HGF (AdHGF) in VSMCs accelerated mineralisation, shown by alizarin red staining, and significantly increased 45Calcium incorporation (1.96 ± 0.54-fold [P < 0.05]) and alkaline phosphatase (ALP) activity (3.01 ± 0.8-fold [P < 0.05]) compared to controls. AdHGF also significantly elevated mRNA expression of bone-related proteins, Runx2, osteocalcin, BMP2 and osterix in VSMCs. AdHGF-accelerated mineralisation correlated with increased Akt phosphorylation, nuclear translocation of Notch3 intracellular domain (N3IC) and up-regulation of the Notch3 target protein, HES1. In contrast, adenoviral-mediated over-expression of the HGF antagonist, NK4, markedly attenuated VSMC mineralisation, and reduced c-Met phosphorylation, Akt activation and HES1 protein expression compared to AdHGF-treated cells. Furthermore, the Notch inhibitor, DAPT, attenuated N3IC nuclear translocation and AdHGF-induced mineralisation. Conclusion We demonstrate HGF induces VSMC osteogenic differentiation via c-Met/Akt/Notch3 signalling, highlighting these pathways as potential targets for intervention of vascular calcification.

Smad4 Regulates Ureteral Smooth Muscle Cell Differentiation during Mouse Embryogenesis

Proper formation of ureteral smooth muscle cells (SMCs) during embryogenesis is essential for ureter peristalsis that propels urine from the kidney to the bladder in mammals. Currently the molecular factors that regulate differentiation of ureteral mesenchymal cells into SMCs are incompletely understood. A recent study has reported that Smad4 deficiency reduces the number of ureteral SMCs. However, its precise role in the ureteral smooth muscle development remains largely unknown. Here, we used Tbx18:Cre knock-in mouse line to delete Smad4 to examine its requirement in the development of ureteral mesenchyme and SMC differentiation. We found that mice with specific deletion of Smad4 in Tbx18-expressing ureteral mesenchyme exhibited hydroureter and hydronephrosis at embryonic day (E) 16.5, and the mutant mesenchymal cells failed to differentiate into SMCs with increased apoptosis and decreased proliferation. Molecular markers for SMCs including alpha smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) were absent in the mutant ureters. Moreover, disruption of Smad4 significantly reduced the expression of genes, including Sox9, Tbx18 and Myocardin associated with SMC differentiation. These findings suggest that Smad4 is essential for initiating the SMC differentiation program during ureter development.

TLR4/NF-κB/Ceramide signaling contributes to Ox-LDL-induced calcification of human vascular smooth muscle cells

&NA; Vascular calcification is a major feature of advanced atherosclerosis and highly associated with cardiovascular diseases. Oxidized low density lipoprotein (Ox‐LDL) has been recognized as a critical risk factor for atherosclerosis and osteogenic differentiation of vascular smooth muscle cells (VSMCs). Previous studies have demonstrated that toll like receptor 4 (TLR4) is highly expressed in atherosclerotic lesions and participates in the progression of atherosclerosis. However, the role of TLR4 in vascular calcification remains unknown. In this study, we investigated whether TLR4 modulates vascular calcification induced by Ox‐LDL. TLR4 expression was up‐regulated in cultured human VSMCs treated with Ox‐LDL. Knockdown of TLR4 by small interfering RNA (siRNA) significantly reduced Ox‐LDL‐induced calcification, detected by alizarin red staining and calcium content assay. TLR4 siRNA also decreased the mRNA expression of bone‐related proteins including Msx2, osterix, BMP2 and KLF4, but increased the expression of VSMC contractile proteins including SMA and SM22&agr; in VSMCs. In addition, Ox‐LDL stimulated nuclear translocation of nuclear factor kappa B (NK‐&kgr;B) p65. These effects of Ox‐LDL on VSMCs were reversed by TLR4 siRNA. Furthermore, NK‐&kgr;B inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated Ox‐LDL‐induced VSMC calcification, which was rescued by C2‐ceramide treatment. In conclusion, these findings suggest that TLR4 regulates VSMC calcification induced by Ox‐LDL through activation of NK‐&kgr;B, highlighting the critical role of TLR4/NK‐&kgr;B signaling in vascular calcification.

Generation of a tamoxifen inducible Tnnt2MerCreMer knock‐in mouse model for cardiac studies

Tnnt2, encoding thin‐filament sarcomeric protein cardiac troponin T, plays critical roles in heart development and function in mammals. To develop an inducible genetic deletion strategy in myocardial cells, we generated a new Tnnt2:MerCreMer (Tnnt2MerCreMer/+) knock‐in mouse. Rosa26 reporter lines were used to examine the specificity and efficiency of the inducible Cre recombinase. We found that Cre was specifically and robustly expressed in the cardiomyocytes at embryonic and adult stages following tamoxifen induction. The knock‐in allele on Tnnt2 locus does not impact cardiac function. These results suggest that this new Tnnt2MerCreMer/+ mouse could be applied towards the temporal genetic deletion of genes of interests in cardiomyocytes with Cre‐LoxP technology. The Tnnt2MerCreMer/+ mouse model also provides a useful tool to trace myocardial lineage during development and repair after cardiac injury. genesis 53:377–386, 2015.

WISP1 overexpression promotes proliferation and migration of human vascular smooth muscle cells via AKT signaling pathway.

Proliferation and migration of vascular smooth muscle cells (VSMCs) play crucial roles in the development of vascular restenosis. Our previous study showed that CCN4, namely Wnt1 inducible signaling pathway protein 1 (WISP1), significantly promotes proliferation and migration of rat VSMCs, but its mechanism remains unclear. This study aims to investigate whether and how WISP1 stimulates proliferation and migration of human VSMCs. Western blot analysis showed that FBS treatment increased WISP1 protein levels in human VSMCs in a dose-dependent manner. Overexpression of WISP1 using adenovirus encoding WISP1 (AD-WISP1) significantly increased proliferation rate of human VSMCs by 2.98-fold compared with empty virus (EV)-transfected cells, shown by EdU incorporation assay. Additionally, Scratch-induced wound healing assay revealed that adenovirus-mediated overexpression of WISP1 significantly increased cell migration compared with EV-transfected cells from 6h (4.56±1.14% vs. 11.23±2.25%, P<0.05) to 48h (25.25±5.51% vs. 97.54±13.12%, P<0.01) after injury. Transwell Migration Assay confirmed that WISP1 overexpression significantly promoted human VSMC migration by 2.25-fold compared with EV. Furthermore, WISP1 overexpression stimulated Akt signaling activation in human VSMCs. Blockage of Akt signaling by Akt inhibitor AZD5363 or PI3K inhibitor LY294002, led to an inhibitory effect of WISP1-induced proliferation and migration in human VSMCs. Moreover, we found that WISP1 overexpression stimulated GSK3α/β phosphorylation, and increased expression of cyclin D1 and MMP9 in human VSMCs, and this effect was abolished by AZD5363. Collectively, we demonstrated that Akt signaling pathway mediates WISP1-induced migration and proliferation of human VSMCs, suggesting that WISP1 may act as a novel potential therapeutic target for vascular restenosis.

Curcumin attenuates osteogenic differentiation and calcification of rat vascular smooth muscle cells.

Vascular calcification has been considered as a biological process resembling bone formation involving osteogenic differentiation. It is a major risk factor for cardiovascular morbidity and mortality. Previous studies have shown the protective effects of curcumin on cardiovascular diseases. However, whether curcumin has effects on osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) has not been reported. In the present study, we used an in vitro model of VSMC calcification to investigate the role of curcumin in the progression of rat VSMC calcification. Curcumin treatment significantly reduced calcification of VSMCs in a dose-dependent manner, detected by alizarin red staining and calcium content assay. Similarly, ALP activity and expression of bone-related molecules including Runx2, BMP2, and Osterix were also decreased in VSMCs treated with curcumin. In addition, flow cytometry analysis and caspase-3 activity assay revealed that curcumin treatment significantly suppressed apoptosis of VSMCs, which plays an important role during vascular calcification. Furthermore, we found that pro-apoptotic molecules including p-JNK and Bax were up-regulated in VSMCs treated with calcifying medium, but they were reduced in VSMCs after curcumin treatment. However, curcumin treatment has no effect on expression of NF-κB p65. Taken together, these findings suggest that curcumin attenuates apoptosis and calcification of VSMCs, presumably via inhibition of JNK/Bax signaling pathway.