Vascular Endothelial Growth Factor-A and Platelet-Derived Growth Factor-B Combination Gene Therapy Prolongs Angiogenic Effects via Recruitment of Interstitial Mononuclear Cells and Paracrine Effects Rather Than Improved Pericyte Coverage of Angiogenic Vessels

Abstract

Vessel stabilisation and the inhibition of side-effects such as tissue edema are essential in angiogenic gene therapy. Thus, combination gene transfers (GT) stimulating both endothelial cell and pericyte proliferation have become of interest. However, there is currently little data to support combination GT in large animal models. In this study we evaluated the potential advantages of such a strategy by combining the transfer of adenoviral (Ad) vascular endothelial growth factor A (VEGF-A) and platelet derived growth factor B (PDGF-B) into rabbit hindlimb skeletal muscle. AdLacZ alone or in combination with AdVEGF-A were used as controls. Contrast-enhanced ultrasound, modified Miles assay and immunohistology were used to quantify perfusion, vascular permeability and capillary size, respectively. Confocal microscopy was utilized in the assessment of pericyte-coverage. The transfer of AdPDGF-B alone and in combination with AdVEGF-A induced prominent proliferation of α-sma-, CD31-, RAM11-, HAM56-, and VEGF- positive cells. Although, pericyte recruitment to angiogenic vessels was not improved, combination GT induced a longer-lasting increase in perfusion in both intact and ischemic muscles than AdVEGF-A GT alone. In conclusion, intramuscular delivery of AdVEGF-A and AdPDGF-B, combined, resulted in a prolonged angiogenic response. However, the effects were most likely mediated via paracrine mechanisms rather than an increase in vascular pericyte coverage.

Long-term VEGF-A expression promotes aberrant angiogenesis and fibrosis in skeletal muscle

“…Enhanced extracellular matrix is proangiogenic, but fibrosis may affect flexibility and contractility of skeletal muscles. VEGF-A has been shown to recruit mononuclear cells in many studies via VEGFR-1 and neuropilin-1 stimulation [22][23][24] and can prolong angiogenesis via paracrine mechanims. We also observed that long-term VEGF-A expression recruits macrophages, which can promote endogenous expression of VEGF-A and lead to further enhanced angiogenesis.…”

“…Intramuscular gene transfer was done in the hindlimb semimembranosus muscle as described. 3,22 Briefly, rabbits were anesthetized with medetomidine (Domitor 0.3 mg kg À1 , Orion, Espoo, Finland) and ketamine (Ketalar 20 mg kg À1 , Pfizer, Espoo, Finland) and the total dose of 1Â10 11 virus particles in 1 ml of 0.9% NaCl was divided into 10 100 ml injections. To induce local ischemia in the target semimembranosus muscle, ligation of the profound femoral artery was performed as described 3 and gene transfer in ischemic muscles was done on the same day as the ischemia operation.…”

“…3,22 Evans Blue dye (30 mg kg À1 ) binding to plasma proteins was injected 30 min before euthanasia via the ear vein. Muscle samples were taken from both transduced muscles and contralateral intact muscles and weighted.…”

Add Some Fat to Vascular Progenitor Cell Therapy

“…A very recent study combining PDGF and VEGF gene therapy in an ischemic setting showed very similar data corroborating the crucial and interacting role of these 2 factors. 18 The combination of these 2 factors resulted in longer-lasting improvements in perfusion of ischemic hindlimbs, even though no improved pericyte coverage was detected. However, we have to keep in mind that VEGF has also been reported to counteract vessel maturation if recombinant VEGF and PDGF are coadministered in neovascularization models in vivo.…”

Formation of Stable Vascular Networks in Engineered Tissues

“…Transient vegf gene expression in vivo results in potent angiogenic sprouting, however, the resulting vasculature appears to be immature and leaky, similar to the results of VEGF protein delivery. (Rissanen et al, 2003) When vegf gene is co-administrated with genes encoding angiogenic maturation factors, such as pdgf-b (Hao et al, 2004a;Korpisalo et al, 2008;Kupatt et al, 2010) and ang-1 Siddiqui et al, 2003;Su et al, 2009), the vasculature is longer-lasting and less permeable,with increased perfusion, which is also similar to the results of dual protein growth factor delivery. Other genes delivered for stable vascular networks formation includes ang-1 (Shyu et al, 1998), pdgf-b (Shea et al, 1999, fgf-2 with pdgf-b (de Paula et al, 2009;Hao et al, 2004b), inf-β (Dickson et al, 2007, and neutrophin-3 (Cristofaro et al, 2010).…”