Human PDGF adenoviral particles

Therapeutic angiogenesis through delivery of vascular growth factors is an attractive strategy for treating debilitating occlusive vascular diseases, but clinical trials to date have failed to demonstrate efficacy. One challenge is that the master regulator of angiogenesis, vascular endothelial growth factor (VEGF), induces dysfunctional vessels if expressed outside of a narrow dosing window. Here, researchers tested the hypothesis that co-delivery of platelet-derived growth factor-BB (PDGF-BB), which recruits pericytes, can induce normal angiogenesis in skeletal muscle regardless of VEGF levels. Co-expression of VEGF and PDGF-BB encoded by different vectors in either different cells or in the same cell only partially corrected aberrant angiogenesis. In stark contrast, co-expression of both factors at fixed relative levels in each cell via a single bicistronic vector resulted in robust, uniform, normal angiogenesis, even when VEGF expression levels were high and heterogeneous. Notably, single-vector expression effectively promoted collateral artery growth, vascular remodeling, increased blood flow, and reduced tissue damage in an ischemic hindlimb model. Furthermore, these results were confirmed in a clinically applicable gene therapy approach delivered by adenovirus-mediated bicistronic vector. Thus, coordinated expression of VEGF and PDGF-BB by a single vector is a novel strategy to harness the power of VEGF to induce safe and effective angiogenesis.

Here, the researchers generated four different adenoviral vectors expressing the same cassette constructs as the retroviral vectors used to generate CD8, V, P, and VIP myoblast populations described above, and named them Ad-CD8, Ad-VEGF, Ad-PDGF, and Ad-VIP, respectively. After in vitro infection of HEK293 cells, the supernatants were tested by ELISA to confirm that the recombinant adenoviruses efficiently produced the growth factors. Each virus was injected into the auricular muscle of SCID mice. Intravascular lectin staining 2 weeks later showed that neither the Ad-CD8 nor the Ad-PDGF vectors affected the pre-existing vascular network (Figure 1A, B), while Ad-VEGF caused the widespread appearance of abnormal hemangioma-like structures that were of varying sizes and irregular shapes (Figure 1C), similar to the structures induced by myoblasts expressing VEGF alone. In contrast, PDGF-BB expression coordinated by the Ad-VIP bicistronic vector prevented all abnormal angiogenesis and induced only the growth of homogeneous normal capillaries (Figure 1D), consistent with a myoblast-mediated gene delivery system.

These experiments were confirmed in limb muscles by injecting viral vectors into the tibialis anterior muscle. Immunostaining of frozen tissue sections showed that the Ad-PDGF vector did not induce any angiogenesis above that of the control Ad-CD8 vector (Figure 1E, F). The Ad-VEGF vector induced abnormal vasculature (Figure 1G). In contrast, Ad-VIP vector injection resulted in efficient growth of mature, pericyte-covered capillaries (Figure 1H) and prevented the appearance of abnormal, smooth muscle-covered hemangioma-like structures. These studies indicate that gene therapy via adenoviral delivery of bicistronic vectors encoding VEGF and PDGF-BB to leg muscles, the target of ischemia, results in normal angiogenesis.

Figure 1. Adenoviral delivery of a single bicistronic vector leads to coordinated expression of VEGF and PDGF-BB and prevents aberrant angiogenesis. (Banfi A, et al., 2012)