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Objective: To investigate the role of endothelial miR-126 in zebrafish vascular development. Methods and Results: Two homologs of miR-126, miR-126a (namely miR-126 in previous literature) and miR-126b, with only 1 nucleotide difference in their mature sequences, were identified in zebrafish genome. In vitro analysis showed that both precursors could sufficiently produce mature functional miRNAs. Expression analyses by Northern blot and quantitative RT-PCR showed that both miR-126s accumulated significantly 12 hours after fertilization and were specifically expressed in endothelial cells of zebrafish. Inhibition of miR-126a or miR-126b with specific morpholinos caused cranial hemorrhage, and simultaneous inhibition of both miR-126s resulted in a pronounced hemorrhage in higher percentage of embryos. Bioinformatics prediction showed that the targets of miR-126a/b partially overlapped but essentially differed. p21-activated kinase1 (pak1) was identified as a novel target of miR-126a/b, and pak1 3 untranslated region was differently regulated by these 2 miRNAs. Quantitative RT-PCR, in situ hybridization, and Western blot analyses showed that the level of pak1 was reduced when miR-126a/b were overexpressed. Notably, pak1 expression in endothelial cells was increased when miR-126a/b were knocked down. Furthermore, overexpression of the active form of human pak1 caused cranial hemorrhage, and knockdown pak1 effectively rescued the hemorrhage caused by inhibiting miR-126a/b. Key Words: miR-126 Ⅲ pak1 Ⅲ vascular development Ⅲ zebrafish T he vascular system is critical for the maintenance of blood flow to provide the organism with nutrition and oxygen. Endothelial cells play a central role in the organization and homeostasis of the functional vascular vessels. Their lineage commitment, proliferation, migration, and assembling are required for embryonic development and organogenesis. 1 MicroRNAs (miRNAs) are a set of small noncoding 18-to 22-nucleotide RNAs that regulate gene expression at the posttranscriptional level. 2 Emerging evidence showed that miRNAs are involved in development and various processes of pathogenesis. 3 To date, several miRNAs have been reported to play important roles in vascular development and diseases. 4 Among these miRNAs, miR-126 is an endothelial cell-enriched miRNA that regulates vascular development and angiogenesis by affecting vascular integrity and angiogenic signal pathways. [5][6][7] Zebrafish is an excellent model in vascular biology, because this organism is quickly developed and is transparent in the early days of development. Various transgenic lines 8 -10 are available to track the endothelial cells and hematopoietic cells, which greatly facilitated the investigation. Interestingly, many genes in this organism are duplicated, 11 and gene Original received May 25, 2010; revision received November 23, 2010; accepted November 29, 2010. In October 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.9 ...

BackgroundCardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule–mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line‐to‐line variation. Additionally, hurdles including line‐specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation.Methods and ResultsWe used the H9–human cardiac troponin T–eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer‐based and growth factor–free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53‐dependent apoptosis of human pluripotent stem cells during high‐density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production.ConclusionsWe have demonstrated that mammalian target of rapamycin exerts a stage‐specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional cardiomyocytes for disease modeling and in vitro drug screening.