Poulsen Ryan R, McClaskey Carolyn M, Rivkees Scott A, Wendler Christopher C
Section of Developmental Endocrinology and Biology, Yale Child Health Research Center, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
BMC Dev Biol. 2011 Jun 13;11:37. doi: 10.1186/1471-213X-11-37.
Sphingosine-1-phosophate (S1P) is a biologically active sphingolipid metabolite that influences cellular events including differentiation, proliferation, and migration. S1P acts through five distinct cell surface receptors designated S1P1-5R, with S1P1R having the highest expression level in the developing heart. S1P1R is critical for vascular maturation, with its loss leading to embryonic death by E14.5; however, its function during early cardiac development is not well known. Our previous studies demonstrated that altered S1P levels adversely affects atrioventricular (AV) canal development in vitro, with reduced levels leading to cell death and elevated levels inhibiting cell migration and endothelial to mesenchymal cell transformation (EMT).
We determined, by real-time PCR analysis, that S1P1R was expressed at least 10-fold higher than other S1P receptors in the developing heart. Immunohistochemical analysis revealed S1P1R protein expression in both endothelial and myocardial cells in the developing atrium and ventricle. Using AV canal cultures, we observed that treatment with either FTY720 (an S1P1,3,4,5R agonist) or KRP203 (an S1P1R-specific agonist) caused similar effects on AV canal cultures as S1P treatment, including induction of cell rounding, inhibition of cell migration, and inhibition of EMT. In vivo, morphological analysis of embryonic hearts at E10.5 revealed that S1P1R-/- hearts were malformed with reduced myocardial tissue. In addition to reduced myocardial tissue, E12.5 S1P1R-/- hearts had disrupted morphology of the heart wall and trabeculae, with thickened and disorganized outer compact layer and reduced fibronectin (FN) deposition compared to S1P1R+/+ littermates. The reduced myocardium was accompanied by a decrease in cell proliferation but not an increase in apoptosis.
These data indicate that S1P1R is the primary mediator of S1P action in AV canal cultures and that loss of S1P1R expression in vivo leads to malformed embryonic hearts, in part due to reduced fibronectin expression and reduced cell proliferation.
1-磷酸鞘氨醇（S1P）是一种生物活性鞘脂代谢产物，可影响包括分化、增殖和迁移在内的细胞活动。S1P通过五种不同的细胞表面受体发挥作用，分别命名为S1P1 - 5R，其中S1P1R在发育中的心脏中表达水平最高。S1P1R对血管成熟至关重要，其缺失会导致胚胎在E14.5时死亡；然而，其在心脏早期发育过程中的功能尚不清楚。我们之前的研究表明，体外S1P水平改变会对房室（AV）管发育产生不利影响，水平降低会导致细胞死亡，而水平升高则会抑制细胞迁移以及内皮细胞向间充质细胞转化（EMT）。
通过实时PCR分析，我们确定在发育中的心脏中，S1P1R的表达比其他S1P受体至少高10倍。免疫组织化学分析显示，S1P1R蛋白在发育中的心房和心室的内皮细胞和心肌细胞中均有表达。使用AV管培养物，我们观察到用FTY720（一种S1P1、3、4、5R激动剂）或KRP203（一种S1P1R特异性激动剂）处理对AV管培养物产生的影响与S1P处理相似，包括诱导细胞变圆、抑制细胞迁移和抑制EMT。在体内，对E10.5胚胎心脏的形态学分析显示，S1P1R基因敲除小鼠的心脏畸形，心肌组织减少。除了心肌组织减少外，E12.5的S1P1R基因敲除小鼠心脏的心脏壁和小梁形态紊乱，与S1P1R基因野生型同窝小鼠相比，外层致密层增厚且紊乱，纤连蛋白（FN）沉积减少。心肌减少伴随着细胞增殖的减少，但细胞凋亡没有增加。
这些数据表明，S1P1R是AV管培养物中S1P作用的主要介质，体内S1P1R表达缺失会导致胚胎心脏畸形，部分原因是纤连蛋白表达减少和细胞增殖减少。
