Angiogenic Gene Therapy for Experimental Critical Limb Ischemia

Abstract

Abstract-Recent studies suggest the possible therapeutic effect of intramuscular vascular endothelial growth factor (VEGF) gene transfer in individuals with critical limb ischemia. Little information, however, is available regarding (1) the required expression level of VEGF for therapeutic effect, (2) the related expression of endogenous angiogenic factors, including fibroblast growth factor-2 (FGF-2), and (3) the related adverse effects due to overexpression of VEGF.To address these issues, we tested effects of overexpression of VEGF165 using recombinant Sendai virus (SeV), as directly compared with FGF-2 gene transfer. Intramuscular injection of SeV strongly boosted FGF-2, resulting in significant therapeutic effects for limb salvage with increased blood perfusion associated with enhanced endogenous VEGF expression in murine models of critical limb ischemia. In contrast, VEGF165 overexpression, 5-times higher than that of baseline on day 1, also strongly evoked endogenous VEGF in muscles, resulting in an accelerated limb amputation without recovery of blood perfusion. Interestingly, viable skeletal muscles of either VEGF165-or FGF-2-treated ischemic limbs showed similar platelet-endothelial cell adhesion molecule-1-positive vessel densities. Maturation of newly formed vessels suggested by smooth muscle cell actin-positive cell lining, however, was significantly disturbed in muscles with VEGF. Further, therapeutic effects of FGF-2 were completely diminished by anti-VEGF neutralizing antibody in vivo, thus indicating that endogenous VEGF does contribute to the effect of FGF-2. These results suggest that VEGF is necessary, but should be delicately regulated to lower expression to treat ischemic limb. The therapeutic effect of FGF-2, associated with the harmonized angiogenic effects seen with endogenous VEGF, provides important insights into therapeutic angiogenesis.

Generation of a recombinant Sendai virus that is selectively activated and lyses human tumor cells expressing matrix metalloproteinases

“…4,5 Sendai virus (SeV), also a member of the Paramyxoviridae, infects most mammalian cells and directs highlevel expression not only in cultured cells but also in experimental animals of the genes within its genome that have been exploited for therapeutic use and vaccination. [6][7][8][9][10] A strong potential of NDV as a vaccine vector was also demonstrated. 11 SeV and most of the other mononegaviruses replicate independent of cellular nuclear functions and do not have a DNA phase in their lifecycle, and therefore are not considered to present a high risk of cell transformation by integration of the viral genetic information into the cellular genome.…”

Blockage of Src by Specific siRNA as a Novel Therapeutic Strategy to Prevent Destructive Repair in Steroid-Associated Osteonecrosis in Rabbits

“…(12) It is not clear whether locally delivered therapeutic agents can improve the vascularization of ischemic animal models (13,14) or inhibit bone resorption in SAON. (15) It has been shown that histopathologically proven destructive repair in SAON is characterized by continuous marrow edema closely coupled with persistent bone resorption.…”

Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene

“…tissue necrosis) in old animals 8,11 and in animals with compromised immune system. 9,12 Diminished angiogenesis in these animal models corresponded with decreased expression of VEGF. 8,9,13 Interestingly, substituting VEGF by exogenously delivered protein or gene has not shown therapeutic benefit in either aged mice 11 or in balb/c mice, 12 unlike in other mouse models.…”

“…9,12 Diminished angiogenesis in these animal models corresponded with decreased expression of VEGF. 8,9,13 Interestingly, substituting VEGF by exogenously delivered protein or gene has not shown therapeutic benefit in either aged mice 11 or in balb/c mice, 12 unlike in other mouse models. 9 Similarly, VEGF refractory, severe hindlimb ischemia was also reported in mice deficient of endothelial nitric oxide synthase (ecNOS-KO).…”