Endothelial progenitor cells: past, state of the art, and future

Recent evidences suggest that endothelial progenitor cells (EPCs) derived from bone marrow (BM) contribute to de novo vessel formation in adults occurring as physiological and pathological responses. Emerging preclinical trials have shown that EPCs home to sites of neovascularization after ischemic events in limb and myocardium. On the basis of these aspects, EPCs are expected to develop as a key strategy of therapeutic applications for the ischemic organs. Such clinical requirements of EPCs will tentatively accelerate the translational research aiming at the devices to acquire the optimized quality and quantity of EPCs. In this review, we attempt to discuss about biological features of EPCs and speculate on the clinical potential of EPCs for therapeutic neovascularization.     

Aquaporin 1 Expression in Retinal Neovascularization in a Mouse Model of Retinopathy of Prematurity

Abstract

Recently, it was found that Aquaporin 1 (AQP1) is expressed strongly in proliferating microvessels, but the role of AQP1 in retinal neovascularization remains unknown. Here, we report the distribution of AQP1 expression during neovascularization of the retina in a mouse model of retinopathy of prematurity. AQP1 was expressed in all of the samples examined in P15 mouse and P17 mouse, including experimental and control groups. Immunostaining results showed that AQP1 is located in microvessel endothelia in retinas with proliferative retinopathy and prominently in the outer retina. Expression of AQP1 was significantly increased in experimental animals at P17, compared with control mice. No significant difference was seen in the levels of AQP1 on P12 or P15, compared with control mice. These results suggest that AQP1 may play an important role in retinal neovascularization.     

In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to αvβ3 integrin localization

Summary This study deals with the role of the mechanical properties of matrices in in vitro angiogenesis. The ability of rigid fibrinogen matrices with fibrin gels to promote capillarylike structures was compared. The role of the mechanical properties of the fibrin gels was assessed by varying concentration of the fibrin gels. When the concentration of fibrin gels was decreased from 2 mg/ml to 0.5 mg/ml, the capillarylike network increased. On rigid fibrinogen matrices, capillarylike structures were not formed. The extent of the capillarylike network formed on fibrin gels having the lowest concentration depended on the number of cells seeded. The dynamic analysis of capillarylike network formation permitted a direct visualization of a progressive stretching of the 0.5 mg/ml fibrin gels. This stretching was not observed when fibrin concentration increases. This analysis shows that 10 h after seeding, a prearrangement of cells into ringlike structures was observed. These ringlike structures grew in size. Between 16 and 24 h after seeding, the capillarylike structures were formed at the junction of two ringlike structures. Analysis of the α_vβ₃ integrin localization demonstrates that cell adhesion to fibrinogen is mediated through the α_vβ₃ integrin localized into adhesion plaques. Conversely, cell adhesion to fibrin shows a diffuse and dot-contact distribution. We suggest that the balance of the stresses between the tractions exerted by the cells and the resistance of the fibrin gels triggers an angiogenic signal into the intracellular compartment. This signal could be associated with modification in the α_vβ₃ integrin distribution.     

CC chemokine receptor-3 as new target for age-related macular degeneration

CC chemokine receptor-3 (CCR3) is involved in angiogenic processes. Recently, CCR3 was accounted to participate in choroidal neovascularization (CNV) and CCR3 targeting was reported to be superior to standard antivascular endothelial growth factor-A (VEGF-A) administration when tested in an artificially induced CNV in animals. As human CCR3 studies are lacking in age-related macular degeneration (AMD) patients we sought to determine if CCR3 has any association with inflammatory processes that occur in CNV. A total of 176 subjects were included on the basis of inclusion criteria. Real time PCR was used to analyze the single nucleotide polymorphism in CCR3 of AMD (115) and normal controls (n = 61). Genotype frequency was adjusted for possible confounders like cigarette smoking, alcohol, meat consumption and other risk factors. Chi-square test was used for analysis of polymorphism. The genotype distribution of CCR3 (rs3091250) polymorphism was significantly different in AMD patients in the Indian population. GT (heterozygous) and TT (homozygous) at the rs3091250 SNP increased risk of AMD as compared to the GG genotypes (OR = 4.8, CI 95% = 2.2–10.8 and OR = 4.1, CI 95% = 1.6–10.1 respectively). Subgroup analysis of AMD patients in wet and dry revealed no significant differences. There was no significant difference for rs3091312 in AMD and control group. A significant association between AMD and CCR3 (rs3091250) polymorphism localized on chromosome 3p21.3 was detected. The results suggest the possible contribution of rs3091250, a new predisposing allele in AMD.     

内皮前体细胞的动员与调节

胚胎发生时期,内皮前体细胞(endothelial progenitor cells,EPCs)参与了原始血管形成的最初过程(血管发生)。已有的证据显示,分化为内皮细胞(endothelial cells,Ecs)的前体也存在于成人中,正常情况下,EPCs停留在成人的骨髓,但是,可以通过细胞因子或血管生成因子信号被动员到循环血,迁移到生理或病理条件下的新血管形成位点,并原位分化成内皮细胞,快速和及时地修复损伤的血管。自源的EPCs原住动员或移植是治疗性血管再生的一个潜在、有效的方法,因此,探究EPCs从骨髓的动员和调节,对血管再生以及修复器官功能具有重要的意义。     

Topical dimethyl sulfoxide inhibits corneal neovascularization and stimulates corneal repair in rabbits following acid burn

Neovascularization of the cornea is characterized by the growth of blood vessels caused by imbalances between angiogenic and anti-angiogenic factors. We investigated whether the expression of Vascular endothelial growth factor (VEGF), Vascular endothelial growth factor receptor (VEGF), Vascular endothelial growth inhibitor (VEGI) receptors, as well as topical drug treatments, participate in regulating corneal neovascularization after corneal damage and remodeling. We used 72 mature male New Zealand rabbits. Corneal burns were induced by hydrofluoric acid under general anesthesia. The rabbits then were treated with indomethacin or dimethyl sulfoxide (DMSO). The animals were euthanized on days 2, 7 and 14 after injury. Each cornea was fixed with 10% neutral formalin. On days 2, 7 and 14, VEGF, flk1/KDR and flt1/fms were strongly expressed in the epithelial, stromal and inflammatory cells, but not in the corneal endothelial cells. On day 7, newly formed blood vessels were observed growing toward the center of the cornea. In the control, indomethacin treated, DMSO–treated, and indomethacin + DMSO–treated animals, VEGI, VEGF, and the receptors, flk1/KDR, flt1/fms and flt4, were expressed at different densities in the neovascular regions. This was particularly evident in the indomethacin- and indomethacin + DMSO–treated groups on days 7 and 14, compared to day 2. Treatment with VEGF and DMSO stimulated repair of corneal damage. We suggest that VEGI in the endothelial cells of neovascularized cornea may act as a signaling protein that promotes balance between cell proliferation and apoptosis. Topical administration of DMSO inhibited corneal neovascularization more effectively than indomethacin.     

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro–computed tomography (µCT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and µCT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, µCT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7^th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7^th and 10^th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.     

Brivanib,a multitargeted small-molecule tyrosine kinase inhibitor,suppresses laser-induced CNV in a mouse model of neovascular AMD

In age-related macular degeneration (AMD), choroidal neovascularization (CNV), a major pathologic feature of neovascular AMD (nAMD), affects 10% of patients, potentially causing serious complications, including vision loss. Vascular endothelial growth factor receptor 2 (VEGFR2) and fibroblast growth factor receptor 1 (FGFR1) contribute to the pathogenesis of CNV. Brivanib is an oral selective dual receptor tyrosine kinase (RTK) inhibitor of FGFRs and VEGFRs, especially VEGFR2 and FGFR1. In this study, brivanib inhibited zebrafish embryonic angiogenesis without impairing neurodevelopment. In a mouse CNV model, brivanib intravitreal injection blocked phosphorylation of FGFR1 and VEGFR2 and reduced CNV leakage, area, and formation without causing intraocular toxicity. Moreover, brivanib oral gavage reduced CNV leakage and area. Accordingly, brivanib remained at high concentrations (above 14,000 ng/ml) in retinal/choroidal/scleral tissues following intravitreal injection. Similarly, brivanib remained at high concentrations (over 10,000 ng/ml) in retinal/choroidal/scleral tissues following oral gavage. Finally, in vitro cell experiments demonstrated that brivanib inhibited the proliferation, migration and tube formation of microvascular endothelial cells. In conclusion, our study suggested that brivanib treatment could be a novel therapeutic strategy for nAMD.