Molecular mechanism of vascular endothelial/mural cell differentiation by receptor tyrosine kinases

在本研究中，我们检测了受体酪氨酸激酶在小鼠胚胎干细胞（ES）来源的VEGFR 2 ^+细胞分化为内皮细胞和壁细胞中的作用。（1）血管内皮生长因子受体3（VEGFR 3）在内皮细胞分化中的作用VEGFR 3是VEGF-C和VEGF-D的受体。为了研究VEGFR 3在内皮细胞分化中的作用，我们采用Supertransfer技术建立了表达VEGFR 3的ES细胞，然后通过体外分化和MACS制备了VEGFR 2 ^+细胞。这些细胞分化为内皮细胞，并在VEGF-C存在下表达淋巴管内皮标记物LYVE-1。VEGF-C刺激VEGFR 3以及VEGFR 3，而VEGF-C（C152 S）突变体仅刺激VEGFR 3。VEGF-C（C152 S）不能诱导内皮细胞分化。在用VEGF-C刺激后，VEGFR 2信号传导对于内皮分化是不可或缺的，并且VEGFR 3信号传导另外赋予内皮细胞淋巴管内皮样表型。（2）血管内皮/平滑肌分化中的FGF信号我们检测了FGF-2存在下VEGFR 2 ^+细胞的分化2天。45%的细胞分化为PECAM 1 ^+内皮细胞，20%的细胞分化为SMA^+壁细胞，35%的细胞两种标记物均为阴性。因此，FGF-2不指导细胞分化为一个特定的谱系。我们还研究了VEGF-A和FGF-2的共刺激作用。VEGF-A诱导内皮细胞分化，而VEGF-A/FGF-2共刺激诱导95%的内皮细胞和5%的壁细胞。尤其是附着在内上皮层上的壁细胞。我们发现细胞间的通讯是通过内源性PDGF-B/PDGFRβ信号转导介导的。

In the present study, we examined the roles of receptor tyrosine kinases in differentiation of mouse embryonic stem (ES) cell-derived VEGFR2^+ cells into endothelial cells and mural cells.(1)VEGFR3 signaling in endothelial differentiationVEGFR3 is a receptor for VEGF-C and VEGF-D. In order to examine the role of VEGFR3 in endothelial differentiation, we established ES cells expressing VEGFR3 by Supertransfection technique and then prepared VEGFR2^+ cells through in vitro differentiation and MACS. These cells differentiated into endothelial cells and expressed lymphatic endothelial marker LYVE-1 in the presence of VEGF-C. VEGF-C stimulates VEGFR3 as well as VEGFR3, whereas VEGF-C (C152S) mutant exclusively stimulates VEGFR3. VEGF-C (C152S) failed to induce endothelial differentiation. Upon stimulation with VEGF-C, VEGFR2 signaling is indispensable for endothelial differentiation, and VEGFR3 signaling additionally confers lymphatic endothelial-like phenotypes to endothelial cells.(2)FGF signaling in endothelial/smooth muscle differentiationWe examined the differentiation of VEGFR2^+ cells in the presence of FGF-2 for 2 days. 45% of the cells differentiated PECAM1^+ endothelial cells, 20% of them differentiated into SMA^+ mural cells, and 35% of them remained negative for both markers. FGF-2 thus does not direct cell differentiation to one specific lineage. We also examined the co-stimulatory effect of VEGF-A and FGF-2. Whereas VEGF-A caused exclusive endothelial differentiation, VEGF-A/FGF-2 co-stimulation induced 95% endothelial cells and 5% mural cells. Notably mural cells attached to the entdothelial sheets. We found that the cell-cell communication was mediated through endogenonus PDGF-B/PDGFRβ signaling.