Zeini Miriam, Hang Calvin T, Lehrer-Graiwer Joshua, Dao Tiffany, Zhou Bin, Chang Ching-Pin
Division of Cardiovascular Medicine, Department of Medicine, Stanford University, Stanford, CA 94305, USA.
Development. 2009 Oct;136(19):3335-45. doi: 10.1242/dev.037903. Epub 2009 Aug 26.
Formation of the coronary vasculature requires reciprocal signaling between endothelial, epicardially derived smooth muscle and underlying myocardial cells. Our studies show that calcineurin-NFAT signaling functions in endothelial cells within specific time windows to regulate coronary vessel development. Mouse embryos exposed to cyclosporin A (CsA), which inhibits calcineurin phosphatase activity, failed to develop normal coronary vasculature. To determine the cellular site at which calcineurin functions for coronary angiogenesis, we deleted calcineurin in endothelial, epicardial and myocardial cells. Disruption of calcineurin-NFAT signaling in endothelial cells resulted in the failure of coronary angiogenesis, recapitulating the coronary phenotype observed in CsA-treated embryos. By contrast, deletion of calcineurin in either epicardial or myocardial cells had no effect on coronary vasculature during early embryogenesis. To define the temporal requirement for NFAT signaling, we treated developing embryos with CsA at overlapping windows from E9.5 to E12.5 and examined coronary development at E12.5. These experiments demonstrated that calcineurin-NFAT signaling functions between E10.5 and E11.5 to regulate coronary angiogenesis. Consistent with these in vivo observations, endothelial cells exposed to CsA within specific time windows in tissue culture were unable to form tubular structures and their cellular responses to VEGF-A were blunted. Thus, our studies demonstrate specific temporal and spatial requirements of NFAT signaling for coronary vessel angiogenesis. These requirements are distinct from the roles of NFAT signaling in the angiogenesis of peripheral somatic vessels, providing an example of the environmental influence of different vascular beds on the in vivo endothelial responses to angiogenic stimuli.
冠状动脉血管系统的形成需要内皮细胞、心外膜来源的平滑肌细胞和下方的心肌细胞之间的相互信号传导。我们的研究表明，钙调神经磷酸酶 - 活化T细胞核因子（calcineurin-NFAT）信号在特定时间窗口内的内皮细胞中发挥作用，以调节冠状动脉的发育。暴露于环孢素A（CsA）（抑制钙调神经磷酸酶活性）的小鼠胚胎无法发育出正常的冠状动脉血管系统。为了确定钙调神经磷酸酶在冠状动脉血管生成中发挥作用的细胞位点，我们在内皮细胞、心外膜细胞和心肌细胞中删除了钙调神经磷酸酶。内皮细胞中钙调神经磷酸酶 - NFAT信号的破坏导致冠状动脉血管生成失败，重现了在CsA处理的胚胎中观察到的冠状动脉表型。相比之下，在心外膜或心肌细胞中删除钙调神经磷酸酶在早期胚胎发育过程中对冠状动脉血管系统没有影响。为了确定NFAT信号的时间需求，我们在E9.5至E12.5的重叠窗口期间用CsA处理发育中的胚胎，并在E12.5检查冠状动脉发育。这些实验表明，钙调神经磷酸酶 - NFAT信号在E10.5和E11.5之间发挥作用以调节冠状动脉血管生成。与这些体内观察结果一致，在组织培养中特定时间窗口内暴露于CsA的内皮细胞无法形成管状结构，并且它们对VEGF-A的细胞反应减弱。因此，我们的研究证明了NFAT信号对冠状动脉血管生成的特定时间和空间需求。这些需求与NFAT信号在外周体血管血管生成中的作用不同，提供了不同血管床的环境影响对体内内皮细胞对血管生成刺激反应的一个例子。
