莪术醇对斑马鱼的血管生成活性及作用机制研究

目的:初步研究莪术醇促进血管生成的活性及作用机制,为临床用药提供依据.方法:采用斑马鱼生物模式,观察莪术醇对斑马鱼胚胎体节间血管生长、成鱼剪尾后血管再生和仔鱼切尾后组织再生情况.采用相对荧光定量PCR法检测仔鱼切尾后体内血管内皮生长因子(VEGFA)及受体(VEGFR2)基因的表达.结果:莪术醇能使斑马鱼胚胎体节间血管侧生出新的血管,使成鱼剪尾后血管再生加快,使仔鱼切尾后尾部再生加快.且莪术醇能提高仔鱼体内VEGFA及VEGFR2基因的表达量,促进仔鱼切尾后的尾部再生.结论:莪术醇能促进斑马鱼的血管生成,提高仔鱼切尾后体内VEGFA及VEGFR2基因的表达量,促进尾部再生.     

Looking at the blood-brain barrier: Molecular anatomy and possible investigation approaches

The blood-brain barrier (BBB) is a dynamic and complex interface between blood and the central nervous system that strictly controls the exchanges between the blood and brain compartments, therefore playing a key role in brain homeostasis and providing protection against many toxic compounds and pathogens. In this review, the unique properties of brain microvascular endothelial cells and intercellular junctions are examined. The specific interactions between endothelial cells and basement membrane as well as neighboring perivascular pericytes, glial cells and neurons, which altogether constitute the neurovascular unit and play an essential role in both health and function of the central nervous system, are also explored. Some relevant pathways across the endothelium, as well as mechanisms involved in the regulation of BBB permeability, and the emerging role of the BBB as a signaling interface are addressed as well. Furthermore, we summarize some of the experimental approaches that can be used to monitor BBB properties and function in a variety of conditions and have allowed recent advances in BBB knowledge. Elucidation of the molecular anatomy and dynamics of the BBB is an essential step for the development of new strategies directed to maintain or restore BBB integrity and barrier function and ultimately preserve the delicate interstitial brain environment.     

Pharmacophore based 3D-QSAR study of VEGFR-2 inhibitors

The growth and metastasis of solid tumors are dependent on angiogenesis. The vascular endothelial growth factor (VEGF) is of particular interest since it is essential for angiogenesis. The development of novel inhibitors of VEGF receptor type 2 (VEGFR-2) is important. Quantitative structure–activity relationship (QSAR) studies were performed to understand the structural factors affecting inhibitory potency of 4-aryl-5-cyano-2-aminopyrimidines. Pharmacophore models indicate that the importance of steric and hydrogen bond acceptor groups. The best-fitted pharmacophore-based alignment was used for comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Both CoMFA (q ² = 0.62, r ² = 0.87, and r ² _predictive = 0.7) and CoMSIA (q ² = 0.54, r ² = 0.86, and r ² _predictive = 0.61) gave reasonable results. Factors such as steric bulkiness, electrostatic effect, and hydrogen bond acceptor were found to be important for the inhibitory activity. It is suggested that negatively charged, bulky H-bond accepting groups around the piperazine nitrogen would enhance inhibition against VEGFR-2.     

Inhibition of VEGF receptor 2 increased cell death of dentate hilar neurons after traumatic brain injury

Post-traumatic epilepsy, partly due to the loss of hilar neurons of the hippocampus, is a frequent long-term consequence of traumatic brain injury (TBI). We and others found that the levels of vascular endothelial growth factor (VEGF) that can act as a neuroprotectant increase after TBI. Here we tested whether VEGF and its receptor VEGFR2 are involved in mediating the death or survival of hilar neurons after injury. We demonstrated that VEGFR2 is expressed by most, if not all, hilar neurons and that these neurons are dying in large numbers as indicated by Fluoro-Jade B histology after fluid percussion TBI. To directly test the involvement of VEGFR2 and VEGF in the injury-induced apoptotic death of hilar neurons, we delivered SU5416, an inhibitor to VEGFR2, or recombinant VEGF into the ipsilateral cerebral ventricle of injured animals. We found that blocking VEGFR2 by SU5416 significantly increased the number of apoptotic (TUNEL-positive) cells in the hilus. Infusion of VEGF, however, failed to reduce the number of TUNEL-positive cells. Our results suggest that VEGFR2 is involved in mediating death or survival of hilar neurons after injury but delivering additional exogenous VEGF does not provide further protection from TBI-induced death of hilar neurons.     

Inhibition of experimental choroidal neovascularization in mice by anti-VEGFA/VEGFR2 or non-specific siRNA

Choroidal neovascularization (CNV) is one of the severe pathological consequences of the end-stage of age-related macular degeneration (AMD). Several lines of evidence implicate increased levels of vascular endothelial growth factor (VEGF) in the retinas of AMD patients. Current available agents for the inhibition of VEGF protein such as bevacizumab show significant promise for the treatment of exudative AMD. However, this compound still has limited efficacy and requires multiple administrations; thus, it is associated with a variety of ocular complications, including endophthalmitis and retinal detachment. In this study, we used anti-VEGFA/VEGFR2 or non-specific siRNA and evaluated their suppression of laser-induced choroidal neovascularization (CNV) in mice. Male adult C57BL/6J mice were used in the study. The mice were subjected to laser rupture of Bruch’s membrane to induce CNV and then randomized to five groups with six mice per group. The five groups were blank control, vehicle control (5% glucose solution, GS), VEGFA.siRNA, VEGFR2.siRNA, and non-specific siRNA. Two days after laser photocoagulation, each group, with the exception of the blank control group, received 1 μl of the appropriate agent by intravitreal injection to both eyes. Seven days later, after taking fundus photography and fundus fluorescein angiography (FFA), the mice were killed for tissue sampling. Six eyes from three mice in each group were used for choroidal flatmounts to examine CNV. Six eyes from three mice in each group were subjected to RNA extraction for VEGF mRNA quantification by qRT-PCR. Retinal tissue from 2 mice without laser treatment was harvested as the assay reference. The incidence of burns which showed fluorescein leakage was 80.0% in blank control, 75.0% in GS, 55.0% in VEGFA.siRNA, 40.0% in VEGFR2.siRNA and 30.0% in non-specific siRNA group. The flatmounted specimens showed that the retinal pigment epithelium (RPE) was visualized as a uniform hexagonal array in nonlasered areas. On day 7 after laser burn, well-defined isolectin-B4 labeled CNV networks were shown within the burn spots of the 2 control groups. The inhibitory effects of the 3 siRNAs on CNV formation were statistically significant as compared to the 2 control groups. Compared to the control groups, the ocular expression of VEGF mRNA was decreased significantly in the 3 siRNA treated groups. The areas of CNV correlated with the expression of VEGF mRNA. Anti-VEGFA/VEGFR2 or non-specific siRNA can inhibit CNV and attenuate VEGF mRNA expression in a laser-induced mouse model of CNV. We conclude that the siRNAs may be an option for treating patients with exudative AMD, and more studies are needed to test the possible side-effects of the treatment.     

Green tea catechin, epigallocatechin-3-gallate, inhibits vascular endothelial growth factor angiogenic signaling by disrupting the formation of a receptor complex

A potential mechanism by which green tea may prevent cancer development is through the inhibition of angiogenesis. We have shown previously that the green tea catechin, epigallocatechin gallate (EGCG), inhibits endothelial cell tube formation through the inhibition of vascular endothelial growth factor (VEGF)-induced Akt activation and vascular endothelial (VE)-cadherin phosphorylation. Furthermore, EGCG can suppress oxidant-induced production of the proangiogenic cytokine interleukin (IL)-8. To further elucidate the antiangiogenic mechanisms of EGCG, we investigated its regulation of other molecular processes in VEGF-induced signaling in human umbilical vein endothelial cells (HUVECs). We show that EGCG at physiological doses (0.5-10 microM) markedly inhibits the formation of a vascular endothelial growth factor receptor 2 complex formed upon the binding of its ligand VEGF. This disruption results in a significant and dose-dependent decrease in PI3-kinase activity. Electrophoretic mobility shift assay revealed that EGCG decreased the PI3 kinase-dependent activation and DNA-binding ability of NF-kappaB, likely acting through decreasing phosphorylation and degradation of IkappaB. VEGF-induced IL-8 production at the mRNA (real time RT-PCR) and protein levels (ELISA) are also suppressed with EGCG. These results suggest a novel mechanism for green tea's anticancer effects where EGCG can abrogate VEGF signaling by interfering with the formation of a receptor complex, resulting in attenuated mitogenic and angiogenic signaling.     

Pseudocowpox virus encodes a homolog of vascular endothelial growth factor

We have identified a gene encoding a homolog of vascular endothelial growth factor (VEGF) in the Pseudocowpox virus (PCPV) genome. The predicted protein shows 27% amino acid identity to human VEGF-A. It also shows 41 and 61% amino acid identity to VEGFs encoded by orf virus (ORFV) strains NZ2 and NZ7, respectively. Assays of the expressed VEGF-like protein of PCPV (PCPV(VR634)VEGF) demonstrated that PCPV(VR634)VEGF is mitogenic for endothelial cells and is capable of inducing vascular permeability. PCPV(VR634)VEGF bound VEGF receptor-2 (VEGFR-2) but did not bind VEGFR-1 or VEGFR-3. These results indicate that PCPV(VR634)VEGF is a biologically active member of the VEGF family which shares with the ORFV-encoded VEGFs a receptor binding profile that differs from those of all cellular members of the VEGF family. It seems likely that the biological activities of PCPV(VR634)VEGF contribute to the proliferative and highly vascularized nature of PCPV lesions.     

Angiogenesis and lymphangiogenesis and expression of lymphangiogenic factors in the atherosclerotic intima of human coronary arteries

Little information regarding the development of lymphangiogenesis in coronary atherosclerosis is available. We immunohistochemically investigated the correlation among intimal neovascularization (CD34 for angiogenesis and lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1] and podoplanin for lymphangiogenesis), the expression of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGF-D), and the progression of atherosclerosis using 169 sections of human coronary arteries from 23 autopsy cases. The more the atherosclerosis advanced, the more often the neointimas contained newly formed blood vessels ( P < .0001). Vascular endothelial growth factor-C was expressed mostly in foamy macrophages and in some smooth muscle cells, whereas VEGF-D was abundantly expressed in both. The number of VEGF-C-expressing cells, but not that of VEGF-D-expressing cells, was increased as the lesion advanced and the number of intimal blood vessels increased ( P < .01). Lymphatic vessels were rare in the atherosclerotic intima (LYVE-1 vs CD34 = 13 vs 3955 vessels) compared with the number seen in the adventitia (LYVE-1 vs CD34 = 360 vs 6921 vessels). The current study suggests that VEGF-C, but not VEGF-D, may contribute to plaque progression and be a regulator for angiogenesis rather than lymphangiogenesis in coronary atherosclerotic intimas. Imbalance of angiogenesis and lymphangiogenesis may be a factor contributing to sustained inflammatory reaction during human coronary atherogenesis.