The Role Of Notch Signaling In Choroidal Neovascularization

BACKGROUNDOcular angiogenesis is the common pathological change and clinical feature in several ocular diseases such as age related macular degeneration (AMD), proliferateive diabetic retinopathy (PDR), retinal vein occlusion (RVO) and retinopathy of premature (ROP), with the accompany by a series of incidents including exudates, bleeding and fibrosis, which cause serious disruption in the structure and function of the eye and lead to the various extents of vision loss. It is a group of factors that commit to the disruption of retina and choroid, and correspondently, excessive reactivity of the tissue repair represents the histopathological changes of epiretinal and subretinal neovascularization.Among various ocular angiogenesis, choroidal neovascularization (CNV) is the growth of new blood vessels between Bruch's membrane and retinal epithelium (RPE), or between neural retina and RPE, or bwtween RPE and choroidal capillary plexus, by the migration and proliferation of choroidal capillary endothelial cells from the rupture of Bruch's membrane. Multifactors including degeneration, heredity, inflammation, tumor and trauma could cause the damage in RPE-Bruch's membrane-choriodal capillary complex, which consequently induce the complication of CNV. AMD associated CNV is the major reason of blindness among the elderly. It is often observed in the macular area, thus causing the major loss of the central vision. To date, the specific mechanism of CNV is still unclear. However, several types of cells, growth factors and extracellular matrix (ECM) and signaling pathways constitute a microenvironment for CNV development, where the interaction of cells, growth factors and signaling pathways plays a significant role in the regulation the growth of CNV. Therefore, in-depth studies on the mechanisms of CNV may offer new candidates for its therapeutic application. The current concept of anti-VEGF has been approved by FDA, and verified its effectiveness in the inhibition of new vessels growth and the improvement of vision. However, due to the physiological function of VEGF including maintenance of vascular permeability and neural protection, extensive observation are required on the safety and curative stability with long term and repeated usage of anti-VEGF drugs. More notably, in the microenvironment of CNV, VEGF is absolutely not the single growth factor which is involved in the complicated angiogenic process, thus studies on other key factors in this condition and their therapeutic potentials could be the new target in the CNV clinical treatment.Notch signaling pathway is highly conserved pathway that widely expresses in all tissues in the development of vertebrate and invertebrate. Recent studies have identified the expressions of several ligands and receptors of Notch signaling pathway in vascular system, which play a critical role in the regulation of physiological and pathological angiogenesis during the embryonic and postnatal stage. It is notable that in the studies that loss of even single allele of gene (haploinsufficiency) can cause embryonic lethality due to the vascular abnormity, only two genes have been confirmed with this effect: VEGF and Notch signaling ligand Delta like 4 (Dll4), indicating the equivalent importance of Notch signaling with VEGF in the development of the vascular system. But, the regulatory modulation of Notch signaling is distinct from VEGF. Notch signaling pathway functions in neighboring cells via the binding of Notch ligands and receptors, whereas VEGF is secreted and exert a remote effect to the distant cells, in the control of the activation and function of EC, mural cells and other types of cell. In recent years, it has been unveiled and emphasized that Notch signaling pathway is vital in the development of retinal vasculature and some ocular angiogenesis, but involvement of Notch signaling in the development of CNV and its correspondent therapeutic potential remain in investigation.AIMSTo investigate the contribution of Notch signaling in the maintenance of ocular and systemic vascular homeostasis and to elucidate the regulatory role of Notch signaling in the development of CNV as well as the effects of Notch signaling on the key cells in ocular angiogenesis and its underlying mechanisms, with a further study is to investigate the possibility of its therapeutic potential in the treatment of CNVMETHODS1. RBP-Jflox/WT, Mx-Cre and ROSA mice were mated to get Mx-Cre-RBP-Jflox/WT, RBP-Jflox/flox and Mx-Cre-RBP-Jflox-ROSA mice. Four weeks after birth, the above mice were underwent injection with 500μg polyI-C (five times in total) to get the final RBP-J knockout (KO) mice and control (CON) mice. The activity of Cre in endothelium was observed by X-gal staining;2. By using the histopathological techniques, the vascular changes in the eyes and other organs after the knockout of RBP-J/Notch signaling were observed, as well as the vessel growth in the subcutaneous Matrigel gel;3. RBP-J KO chimeric mice and CON chimeric mice were developed by transplanting bone marrow cells from RBP-J KO and CON mice to adult C57BL/6J mice and comfirmed by flow cytometry one month later. CNV were induced in experimental mice (RBP-J KO and CON mice, RBP-J KO chimeric and CON chimeric mice) by laser injury of Bruch's membrane and the severity of CNV was observed by and fundus fluorescence angiogram (FFA) and histopathological techniques.4. The expressions and regulatory functions of Notch signaling molecules were studied by using mice retinal flatmount, in vitro cultures of the aortic endothelial cells, liver sinusoidal endothelial cells and bone marrow derived endothelial progenitor cells from KO mice, primary culture of human RPE cells and rhesus monkey choroid–retinal endothelial cell line RF/6A.5. The effect of activation of Notch signaling was observed by ?-secretase inhibitor GSI and exogenous recombinant Dll1 treatment on the RF/6A and RPE cells.6. To observe the effect of activation of Notch signaling in vivo, neonatal C57BL/6J mice and C57BL/6J mice with laser induced CNV were respectively treated with subcutaneous and intravenous injection of murine Dll1-RGD. Retnial flatmount and RPE-choroidal-scleral complex flatmount were used in this part.RESULT1. Mx-Cre-RBP-Jflox/WT and Mx-Cre-RBP-Jflox/flox mice were obtained by mating RBP-Jflox/WT and Mx-Cre;Mx-Cre-RBP-Jflox-ROSA was obtained by mating Mx-Cre-RBP-Jflox and Mx-Cre-ROSA. Mx-Cre-RBP-Jflox/WT, Mx-Cre-RBP-Jflox/flox and Mx-Cre-RBP-Jflox-ROSA mice were undergone 500μg poly:I-C injection (5 times in total). The skin and retinal vasculatures of Mx-Cre-RBP-Jflox-ROSA exhibited blue staining by X-gal, indicating that the Cre expression in mice vescular system. The expression of Cre simutanously knocked out RBP-J gene by DNA recombination, which finaly resulted in production of RBP-J (-/-) mice named Knockout mice (KO) and RBP-J (+/-) named control mice (CON). Through the observation on the ocular and systemic vascular system of KO mice and CON mice, we found the lack of RBP-J/Notch signaling caused spontaneously angiogenesis in the iris, cronea, retina, liver and lung. Five days after Matrigel subcutanous implantation in KO and CON mice, a large mount of neovessels grew into the gel of KO mice with extensive bleeding. In addition, in the vitro studies, we found the proliferation of arotic endothelial cells was greatly enhanced and the sprouting of liver sinus endothelial cells was increased. The expression of VEGFR2 was upregulated, while the expression of VEGFR1 was downregulated. And also, cell cycle regulatory protein p21WAF1/CIP1 was downregulated in the transcriptional level.2. When Notch signaling was inhibited, laser induced CNV on KO mice was more severe than that in CON mice. FFA exhibited that the leakage and the incidence of CNV were enchanced in KO mice. The area and thickness of CNV in KO mice were increased statistically than those in KO mice. In vitro studies, inhibition of Notch signaling ?-secretase inhibitor GSI reduced the proliferation and migration of RPE; While GSI promoted the proliferation but weakened the tube formation of RF/6A.3. Chimeric mice were obtained by successfully transplanted bone marrow from KO and CON mice to wild type C57BL/6J mice. Laser induced CNV were set up in the chemiric mice one month after bone marrow transplantation. The severity of CNV in the KO chimeric mice was enhanced. Bone marrow derived cells from KO and CON mice were stained by Dio dye and transplanted in to the wild type C57BL/6J mice. Cells with green fluorenscein were observed in the laser induced CNV area, and expression of CXCR4 was detected on these cells. There was no significant change in the number of EPC in the peripheral blood; however, the mature endothelial cells were increased in the circulation. In the study of the vitro culture of EPC, EPC from KO mice exhibited less responsiveness to the chemotaxis to SDF-1, which was possibly caused by the downregulation of SDF-1 receptor CXCR4.4. On the retinal vasculatures of P5 and P15 neonatal C57BL/6J mice, the activation of Notch signaling was respectively expressed in the tip cells and the vascular branches. And Notch signaling was also activated in the CNV area of C57BL/6J mice. After subcutaneously injection of Dll1-RGD to the neonatal mice, the tip cells and branches were decreased compared with the control group. After intravenously injection of Dll1-RGD in the C57BL/6J mice with laser induced CNV model, the area of CNV was markedly reduced in the comparesion with the control group. In vitro study, after the Dll1 treatment, the invasion capacity of RF/6A was weakened, but its tube formation was enhanced. For RPE cells, Dll1 treatment caused the reduction of cell migration.CONCLUSIONIn this study, we identified that Notch signaling plays an important regulatory role in CNV. Lack of Notch signaling exacerbates the CNV formation, wherease enhancement of Notch signaling alleviates the CNV formation. In adult, Notch signaling contributes to the maintainance of ocular and systemic vascular system. When Notch signaling is inhibited, the EC's proliferation and specification to tip cells could be enhanced, which is possibly due to the transcriptional regulation of VEGFR2, VEGFR1 and p21 by Notch signaling.BMCs also participate in the CNV formation, of which EPC is considered as a dominating type in the growth of CNV. The comparision on the number of EPCs and ECs suggests that Notch may modulate the differentiation of EPC to EC. In addition, the reduced responsiveness of EPC to SDF-1 caused by lack of Notch signaling is possibly due to the downregulated expression of CXCR4. Notch signaling could also regulate the biological function of RPE cell including cell proliferation and migration, which finally commit to the progression of CNV. However, it is notated that, as the mice model we used is systemic gene knockout, whether Notch signaling regulates other types of cells in the CNV formation is still in investigation.Based on the observation that lack of RBP-J/Notch signaling exacerbates the CNV formation, we adopted a new fusion protein called Dll1-RGD to specifically activate the Notch signaling in vascular endothelial cells. Systemic treatment of Dll1-RGD could inhibite the formation of mice retinal vasculatrures and the growth of CNV, indicating that activation of Notch signaling might have a therapeutic potential for the treatment of CNV. But more work including the toxic effect and its associated molecular mechanism are needed in future studies.