G Protein Coupled Receptor Kinase 5 Role In The Development Of Angiogenesis Research

G protein-coupled receptors (GPCRs), the greatest family of transmembrane receptors, play a key role in transducing broad extracellular signals from diverse stimulations, including neuromediators, glycoproteinic hormones, sensory substances, as well as a large variety of small molecules, thus regulate a wide variety of biological processes. GPCRs have already become potential drug targets in various diseases. G protein-coupled receptor kinases (GRKs) are key regulatory kinases for GPCRs. Receptor phosphorylation by specific GRKs plays a key role in triggering rapid internalization and desensitization. There are7members in the GRK family (GRK1-7), which can be categorized into3subfamilies. Previous research mainly focused on the regulatory functions of GRK in differentiated cells and maturely developed organs. Roles of GRKs in the dynamic process of embryonic development were poorly defined.Angiogenesis which is subject to a complex control system with proangiogenesis and antiangiogenesis factors, is the most important stage of development processes. In adults, angiogenesis is tightly controlled by this "angiogenic balance", a physiological balance between the stimulatory and inhibitory signals for blood vessel growth. Once break up this balance, it will cause abnormal vessel growth and some diseases. GRK5ubiquitously distribute in multiple organs, highly expressed in heart and lung, it also expressed in endothelial cells and cardiac muscle cell, which play important roles in mediating a variety of physiological functions as well as pathological processes.In our research, we applied the animal model of mouse for the developmental angiogenesis study of GRK5,and depending on human umbilical vein endothelial cell phenotypes, we revealed de nove roles of GRK5in regulating angiogenesis during development stage. Our results showed that:1.During E8.5-E10.5,which angiogenesis is most active, GRK5deficiency leads to embryonic angiogenesis retarded. At E8.5, GRK5knockout (GRK5-/-) mice showed severe defects in angiogenic sprouting and vascular remodeling at mid-gestation. Only20%mice had grew inter-somitic vessels while80%WT mice seemed normal. At E9.5, although WT mice vasculature had matured to an elaborate and well organized system, GRK5knockout (GRK5-/-) mice retained a primitive vasculature in the head. The length of inter-somitic vessels and the width of primitive vessels are less than WT mice. These results revealed GRK5may involve in regulating embryonic angiogenesis.2. We further studied angiogenesis in mouse postnatal retina.We discovered that at P6, the area of blood vessel coverage, the branch points of blood vessels and the numbers of tip cells and filopodia, GRK5knockout (GRK5-/-) mice are reduced compared with WT mice. At P9, we assessed the perpendicular sprouting and neovascularization of the deeper retina and discovered that the area of blood vessel coverage is decreased in GRK5knockout (GRK5-/-) mice. It indicated that GRK5inhibited the neovascularization of postnatal retina.3. To further confirmed the role of GRK5in developmental angiogenesis, we interfered the expression of GRK5in the retina through intraocular injection of siRNA. Analysis of the coverage area of blood vessel in retina of GRK5-/-mice revealed significant decrease compared to the control animals, however, it seemed no apparent change in superficial vessels, which were already existed.4. Then we verified whether GRK5affect capillary-like tube formation of endothelial cells in vitro.HUVEC can spontaneously form a tubular capillary-like network on matrigel culture, however GRK5RNAi dramatically inhibited HUVEC tube formation, suggesting that GRK5regulated angiogenesis in vitro.Above all, our studies first discover GRK5as a key role in developmental angiogenesis through animal and cell models. It may promote blood vessel growth. On the basis of our findings, different drug effects on adults and fetals would be better elucidated. It would be helpful to prevent abnormal embryonic development. This phenotype also revealed that GRK5may participate in pathological angiogenesis. Moreover, in the therapy of some clinical diseases, such as diabetic retinopathy, heart disease, macular edema, rheumatoid arthritis, tumor and so on, GRK5may be a potential target to inhibit angiogenesis, further to provide a neoteric treatment.