Abstract
AbstractSphingosine 1-phosphate (S1P) is a bioactive sphingolipid generated through sphingosine kinase1 (SPK1)-mediated phosphorylation of sphingosine. We show here that injury-induced S1P upregulation increases corneal neovascularization through stimulating S1PR3, a cognate receptor. since this response was suppressed in S1PR3-knockout mice. Furthermore, Cayman10444, a selective S1PR3 inhibitor, reduced this response in WT mice. Such reductions in neovascularization were associated with reduced vascular endothelial growth factor A (VEGF-A) mRNA expression levels in WT TKE2 corneal epithelial cells and macrophages treated with CAY10444 as well as macrophages isolated from S1PR3 KO mice. S1P increased tube-like vessel formation in human vascular endothelial cells (HUVEC) and human retinal microvascular endothelial cells (HRMECs) cells expressing S1PR3. In S1PR3 KO mice, TGFβ1-induced increases in αSMA gene expression levels were suppressed relative to those in the WT counterparts. In S1PR3 deficient macrophages, VEGF-A expression levels were lower than in WT macrophages. Transforming growth factor β1(TGFβ1) upregulated SPK1 expression levels in ocular fibroblasts and TKE2 corneal epithelial cells. CAY10444 blocked S1P-induced increases in VEGF-A mRNA expression levels in TKE2 corneal epithelial cells. Endogenous S1P signaling upregulated VEGF-A and VE-cadherin mRNA expression levels in HUVEC. Unlike in TKE2 cells, SIS3 failed to block TGFβ1-induced VEGF-A upregulation in ocular fibroblasts. Taken together, these results indicate that injury-induced TGFβ1 upregulation increases S1P generation through increases in SPK1 activity. The rise in S1P formation stimulates the S1PR3-linked signaling pathway, which in turn increases VEGF-A expression levels and angiogenesis in mouse corneas.Injury-induced transforming growth factor β1 increases sphingosine 1-phosphate (S1P) generation by upregulating in sphingosine kinase 1 activity. The high levels of S1P formation stimulate the S1PR3-linked signaling pathway, which in turn increases vascular endothelial growth factor-A expression levels and angiogenesis in mouse corneas.
Highlights
Loss of corneal transparency following severe injury or infection may be due to maladaptive chronic neovascularization, inflammation and scarification
vascular endothelial growth factor A (VEGF-A) activation upregulates VE-cadherin expression [30, 31] and it is essential for inducing angiogenesis Using immunocytochemistry, the results shown in Fig. 11a reveal that inhibiting S1PR3 with CAY10444 suppressed VE-cadherin protein expression in human vascular endothelial cells (HUVEC) cultures (Fig. 11a)
We show here that activation by cauterization of the Sphingosine 1-phosphate (S1P)/ S1PRP3 signal pathway axis contributes to corneal stroma neovascularization since this response was larger in the WT mice than in the S1PR3 knockout counterpart
Summary
Loss of corneal transparency following severe injury or infection may be due to maladaptive chronic neovascularization, inflammation and scarification This undesirable outcome is induced by the activation of a host of different receptor-linked signaling pathways that are not self-limiting. The results show that crosstalk among cell signaling mediators in the pathways linked to VEGF and TGFβ cognate receptors with those in the S1P/S1PR3 cell signaling pathway axis modulate corneal injury-induced neovascularization in mice. The identity of such interactions provides additional targets whose modulation by selective agents may improve management of the corneal wound healing response induced by a penetrating injury to this tissue
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