9.3 Results

Extensive vitreous neovascularization was noted in oxygen-induced mice, in which the number of endothelial nuclei were 22 ± 3.5 (Fig. 9.1a). While the control mice showed lesser neovascularization, and we detected only a single endothelial cell nuclei each in 4 control sections (Fig. 9.1b; P < 0.01).

Fig. 9.1 HE strain, a: Normal control; b: Oxygen-induced group, which shows the endothelial cell entering into the internal limiting membrane

Positive brown signals in endothelial nuclei in the neovascularized region from IH and ISH were detected in the retina of all oxygen-induced group (Figs. 9.2a and 9.3a) and few in control group (Figs. 9.2b and 9.3b). The intensity of labeling recorded by Image-pro Plus software system were 0.214 ± 0.005 for IH and 0.209 ± 0.007 for ISH, while that in the control were 0.081 ± 0.007 for IH (P < 0.01) and 0.077 ± 0.005 for ISH (P < 0.01).

Fig. 9.3 ISH, a: control group, in which few expression of Smad 4 shown; b: Oxygen-induced group, marked expression of Smad 4

9.4 Discussion

TGF-β is a multifunctional cytokine with an array of biological effects such as cell growth, differentiation, and immunomodulation (Wakefield and Roberts 2002). In the experiment we found that expression of TGF-β1 and Smad-4 were increased in the retinal neovascular from oxygen-induced mice. But the roles of TGF-β1 and Smad 4 in the formation of neovascular are not clear.

Members of the transforming growth factor family (TGFb1, TGFb2, and TGFb3) are multifunctional proteins that regulate cell growth, differentiation, migration, and extracellular matrix production and also play important roles in embryonic development, wound healing, immune responses, and vascular development(Blobe et al. 2000; Behzadian et al. 2001). VEGF, on the other hand, shown to be play a critical role in the development of CNV and other retinal neovascularization disorders. There are some studies on the relationship between TGF-β and VEGF, which

showed all three isoforms of TGF-β enhanced secretion of VEGF significantly. Increase in the mRNA levels of VEGF by TGF-β and blocking of its enhanced secretion by actinomycin-D, an inhibitor of transcription, suggested that TGF-β induced VEGF expression predominantly by transcriptional activation (Nagineni et al. 2003).

Current concept of TGFβ-Smad signaling pathway, which transfer the stimulating signal from outside into the affected cells by binding of the active form of TGF-β, to TGF-β receptor type II that initiates phosphorylation of TGF-β1 receptor type I, which is followed by the phosphorylation of receptor regulated Smad-2 and 3 proteins. These phosphorylated Smad proteins bind to costimulatory Smad protein (Smad-4) and are translocated to the nucleus for transmission of transcriptional signals (Mehra and Wrana 2000; Yamashita et al. 1997).

But on the contrary to the above findings, TGF-β has been shown to inhibit vascular tumor growth (Dong et al. 1996), in vitro studies have shown that TGF-β can inhibit proliferation of vascular endothelial cells and smooth muscle cells (Orlidge and D’Amore 1987). And also VEGF over expression in photoreceptors did not develop CNV in the transgenic mice. Therefore, the alternative hypothesis is that angiogenesis is stimulated by VEGF and other angiogenic factors released by the inflammatory cells and in cases where inflammatory reactions not observed, TGF-β does not induce angiogenesis. But in our experiment, we did not notice the inflammatory reaction, and therefore, we suggest that it may be related to the upregulated TGF-β (Zhao and Overbeek 2001).

In summary, our present study demonstrates the significant increase of TGF-β1 and Smad-4, which may play an important role in regulation of ocular vascular development. Further investigations are required to find out how TGF-β1 and Smad 4 signaling pathways act at molecular levels in regulation of vascular development. Understanding of the mechanism of TGF-β and Smadmediated formation of neovascularization will provide new ideas to prevent or treat ocular neovascularization.

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