Eiko Kohbayashi

Inhibition of smooth muscle cell migration by the p21 cyclin-dependent kinase inhibitor (Cip1)

In vascular smooth muscle cells (SMCs), proliferation and migration contribute to lesion formation after arterial injury. In the cell cycle, several cyclin-dependent kinases (cdks) inhibitors are implicated in the regulating of cyclin-cdk activity such as p21Cip1, p16Ink4 and p27Kip1. Although Cip1 inhibits SMC proliferation, its effects on SMC migration are unknown. To test the hypothesis that Cip1 inhibits SMCs migration and proliferation, we transfected the Cip1 gene into a strain of rabbit aortic SMCs (SM3 cells). Both the spreading and the attachment of Cip1-transfected SM3 cells to extracellular matrices (ECMs) were inhibited compared to that of vector-transfected cells. In the modified Boydens chamber assay the effect of fibronectin on the migratory activity of Cip1-transfected SM3 cells was significantly less than that of vector transfected cells in response to PDGF-BB. These data suggested that Cip1 inhibited both the migration and proliferation of SMC.

Adrenal androgen dehydroepiandrosterone sulfate inhibits vascular remodeling following arterial injury

Recent epidemiologic studies have suggested that serum dehydroepiandrosterone sulfate (DHEAS) levels have a significant inverse correlation with the incidence of cardiovascular diseases. However, direct evidence for the association with DHEAS and vascular disorders has not yet been explored. DHEAS significantly reduced neointima formation 28 days after surgery without altering other serum metabolite levels in a rabbit carotid balloon injury model. Immunohistochemical analyses revealed the reduction of proliferating cell nuclear antigen (PCNA) index and increase of TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) index, expressing differentiated vascular smooth muscle cell (VSMC) markers in the media 7 days after surgery. In vitro, DHEAS exhibited inhibitory effects on VSMC proliferation and migration activities, inducing G1 cell cycle arrest with upregulation of one of the cyclin dependent kinase (CDK) inhibitors p16(INK4a) and apoptosis with activating peroxisome proliferator-activated receptor (PPAR)-alpha in VSMCs. DHEAS inhibits vascular remodeling reducing neointima formation after vascular injury via its effects on VSMC phenotypic modulation, functions and apoptosis upregulating p16(INK4a)/activating PPARalpha. DHEAS may play a pathophysiological role for vascular remodeling in cardiovascular disease.

Cardiac Adipose-Derived Stem Cells Exhibit High Differentiation Potential to Cardiovascular Cells in C57BL/6 Mice

Adipose‐derived stem cells (AdSCs) have recently been shown to differentiate into cardiovascular lineage cells. However, little is known about the fat tissue origin‐dependent differences in AdSC function and differentiation potential. AdSC‐rich cells were isolated from subcutaneous, visceral, cardiac (CA), and subscapular adipose tissue from mice and their characteristics analyzed. After four different AdSC types were cultured with specific differentiation medium, immunocytochemical analysis was performed for the assessment of differentiation into cardiovascular cells. We then examined the in vitro differentiation capacity and therapeutic potential of AdSCs in ischemic myocardium using a mouse myocardial infarction model. The cell density and proliferation activity of CA‐derived AdSCs were significantly increased compared with the other adipose tissue‐derived AdSCs. Immunocytochemistry showed that CA‐derived AdSCs had the highest appearance rates of markers for endothelial cells, vascular smooth muscle cells, and cardiomyocytes among the AdSCs. Systemic transfusion of CA‐derived AdSCs exhibited the highest cardiac functional recovery after myocardial infarction and the high frequency of the recruitment to ischemic myocardium. Moreover, long‐term follow‐up of the recruited CA‐derived AdSCs frequently expressed cardiovascular cell markers compared with the other adipose tissue‐derived AdSCs. Cardiac adipose tissue could be an ideal source for isolation of therapeutically effective AdSCs for cardiac regeneration in ischemic heart diseases.

Implantation-Competent Blastocyst-Like Structures from Mouse Pluripotent Stem Cells

Soon after fertilization, the few totipotent cells of mammalian embryos diverge to form a structure called the blastocyst (BC). Although numerous types of cells, including germ cells and extended pluripotency stem cells, have been generated from pluripotent stem cells (PSCs) in-vitro, generating functional BCs only from PSCs has not yet been reported. Here we describe induced self-organizing 3D BC-like structures (iBCs) generated from mouse PSC culture in-vitro. Resembling natural BCs, iBCs have a blastocoel-like cavity and were formed with outer cells that are positive for trophectoderm lineage markers and with inner cells that are positive for pluripotency markers. iBCs transplanted to pseudopregnant mice uteruses implanted, induced decidualization, and exhibited growth and development before resorption, demonstrating that iBCs are implantation-competent. iBC production required the transcription factor Prdm14 and iBC precursor intermediates concomitantly activate the MERVL totipotency related cleavage stage reporter. Thus, our system may contribute to understanding molecular mechanisms underpinning totipotency, embryogenesis, and implantation. HIGHLIGHTS -Pluripotent cells self-organize blastocyst-like structures in defined conditions. -Structures have several extraembryonic and embryonic characteristics of blastocysts. -Structures can implant in the uterus and grow before resorption. -Totipotency is implicated concomitantly at loci that originate induced blastocysts.