Sustained Release Of Vascular Endothelial Growth Factor Research Articles

Development and Validation of Small‐diameter Vascular Tissue From a Decellularized Scaffold Coated With Heparin and Vascular Endothelial Growth Factor

To overcome shortcomings of current small-diameter vascular prostheses, we developed a novel allogenic vascular graft from a decellularized scaffold modified through heparin immobilization and vascular endothelial growth factor (VEGF) coating. The VEGF coating and release profiles were assayed by enzyme-linked immunosorbent assay, the biological activity of modified surface was validated by human umbilical vein endothelial cells seeding and proliferation for 10 days in vitro. In vivo, we implanted either a modified or a nonmodified scaffold as bilateral carotid allogenic graft in canines (n = 15). The morphological examination of decellularized scaffolds showed complete removal of cellular components while the extracellular matrix structure remained intact. After modification, the scaffolds possessed local sustained release of VEGF up to 20 days, on which the cells cultured showed significantly higher proliferation rate throughout the time after incubation compared with the cells cultured on nonmodified scaffolds (P < 0.0001). After 6 months of implantation, the luminal surfaces of modified scaffolds exhibited complete endothelium regeneration, however, only a few disorderly cells and thrombosis overlay the luminal surfaces of nonmodified scaffolds. Specifically, the modified scaffolds exhibited significantly smaller hyperplastic neointima area compared with the nonmodified, not only at midportion (0.56 +/- 0.07 vs. 2.04 +/- 0.12 mm(2), P < 0.0001), but also at anastomotic sites (1.76 +/- 0.12 vs. 3.67 +/- 0.20 mm(2), P < 0.0001). Moreover, modified scaffolds had a significantly higher patency rate than the nonmodified after 6 months of implantation (14/15 vs. 7/15, P = 0.005). Overall, this modified decellularized scaffold provides a promising direction for fabrication of small-diameter vascular grafts.

Bioactivation of collagen matrices through sustained VEGF release from PLGA microspheres

The success of any tissue engineering implant relies upon prompt vascularization of the cellular construct and, hence, on the ability of the scaffold to broadcast specific activation of host endothelium and guide vessel ingrowth. Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator, and if released in a controlled manner it may enhance and guide scaffold vascularization. Therefore, the aim of this work was to realize a scaffold with integrated depots able to release VEGF in a controlled rate and assess the ability of this scaffold to promote angiogenesis. VEGF-loaded poly(lactide-co-glycolide) (PLGA) microspheres were produced and included in a collagen scaffold. The release of VEGF from microspheres was tailored to be sustained over several weeks and occurred at a rate of approximately 0.6 ng/day per mg of microspheres. It was found that collagen scaffolds bioactivated with VEGF-loaded microspheres strongly enhanced endothelial cell activation and vascular sprouting both in vitro and in vivo as compared with a collagen scaffold bioactivated with free VEGF. This report demonstrates that by finely tuning VEGF release rate within a polymeric scaffold, sprouting of angiogenic vessels can be guided within the scaffolds interstices as well as broadcasted from the host tissues.

Controlled Angiogenic Factor Release from Alginate Gels

Therapeutic angiogenesis by localized delivery of angiogenic factors is a promising approach to treat patients with cardiovascular disease and to engineer large tissues. Vascular endothelial growth factor (VEGF) is the most common and biologically active form of the VEGF family, which acts as a mitogen to endothelial cells and is capable of specific binding to heparin. However, when VEGF is administered via bolus injection, it can be widely distributed, and its concentration is likely to be within the effective window for only a short time period due to rapid degradation. Delivery of angiogenic factors, using controlled drug delivery strategies, offers great potential to promote angiogenesis at a specific site while reducing the unwanted side effects that may occur with systemic delivery. We now report on the sustained release of VEGF from alginate gels, modified with a heparin-binding peptide. Briefly, a small peptide with the sequence of G5K(βA)FAKLAARLYRKA, which is known to specifically interact with heparin, was chemically conjugated to alginate, and the peptide-modified alginate formed gels after mixing with heparin and VEGF. The release rate of VEGF from the gels slowed in vitro for over 45 days, compared with release from non-modified alginate gels. This result suggests potential applications of alginate gels in promoting angiogenesis for therapeutic purposes, as well as for tissue engineering.

Vascular endothelial growth factor VEGF189 induces human neutrophil chemotaxis in extravascular tissue via an autocrine amplification mechanism

Vascular endothelial growth factor (VEGF) is a potent and specific endothelial cell mitogen involved in normal and pathological angiogenesis. Our group recently reported that, among the several VEGF isoforms, VEGF189 (V189) is selectively induced in decidual endometrial cells during the mid-late phase of the menstrual cycle, together with polymorphonuclear neutrophil (PMN) influx. We thus compared the effects of various VEGF isoforms on PMN migration in vitro, and the mechanisms involved. In transmigration and under-agarose assays, V189 was both chemotactic and chemokinetic for PMN, while VEGF165 (V165) was only chemokinetic. The chemokinetic effect of V189 for PMN was blocked by neutralizing anti-VEGF antibodies, but not by neutralizing anti-KDR antibodies, suggesting that the Flt-1 VEGF receptor that is expressed in PMN mediates these effects. Flow cytometric analysis of several adhesion molecules at the PMN surface showed that all VEGF isoforms slightly upregulated β1- and β2-integrins and PECAM, and downregulated L-selectin; all these molecules are activation markers. The involvement of β1-integrins was further supported by the ability of blocking antibodies to reduce VEGF-induced PMN migration. As human PMN can secrete several cytokines and growth factors, the selective secretion of VEGF isoforms was also further examined. RT-PCR analysis showed that V165 mRNA was more strongly expressed than V189 mRNA. Conversely, the major protein isoform secreted after optimal PMN degranulation was V189, which was located in both azurophilic and specific granules. PMN-derived VEGF can thus modulate PMN migration. This autocrine amplification mechanism would allow sustained VEGF release to occur at inflammatory sites, and may contribute to both normal and pathological angiogenesis.

Intravitreal Sustained Release of VEGF Causes Retinal Neovascularization in Rabbits and Breakdown of the Blood– Retinal Barrier in Rabbits and Primates

Vascular endothelial growth factor (VEGF) has been identified as a possible mediator of retinal neovascularization (NV), but it is not certain if VEGF alone is sufficient to cause retinal NV. We sought to investigate this issue by implanting ethylene-vinyl acetate copolymer pellets that slowly release VEGF into the vitreous cavity of rabbits and primates. Eyes were examined by indirect ophthalmoscopy, fundus photography, and fluorescein angiography and then animals were killed at various time points and immunocytochemical and ultrastructural evaluations were carried out.Seven days after implantation of a pellet containing 30 μg of human recombinant VEGF into the vitreous cavity of rabbits, retinal blood vessels became dilated and tortuous, and between days 14 and 21, retinal NV was noted in all eyes. Fluorescein angiography showed profuse leakage of dye from the anomalous vessels. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) showed positively staining nuclei in many of the endothelial cells of new blood vessels on the surface of the retina. Six eyes implanted with control pellets containing vehicle and two eyes implanted with pellets containing 30 μg of human serum albumin alone showed no retinal vascular abnormalities.Implantation of pellets containing 100 μg of VEGF into the vitreous cavity of primates resulted in iris NV and retinal vascular dilation and tortuosity very much like that seen in humans with ischemic retinopathies. Immunohistochemical staining for serum albumin showed widespread severe breakdown of the blood–retinal barrier (BRB). Histology showed dilated thin-walled retinal vessels, but unequivocal retinal NV could not be identified and staining for PCNA was negative.These findings indicate that sustained intravitreal release of VEGF causes widespread retinal vascular dilation and breakdown of the BRB. Retinal NV seems to require persistent high levels of VEGF at the retinal surface and can be achieved in rabbits providing a potentially useful model of retinal NV, but is difficult to achieve in primates. The extensive VEGF-induced disruption of the blood-retinal barrier suggests that VEGF antagonists may provide a new therapy for patients with ischemic retinopathies and macular edema.