Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart

Abstract

Vascular endothelial growth factor (VEGF) plays important roles in physiological and pathological angiogenesis. Recent studies have demonstrated that direct injection of VEGF protein, plasmid DNA, or an adenoviral vector encoding the VEGF gene into ischemic myocardium or limb can induce collateral blood vessel formation and improve perfusion of the ischemic areas. However, these approaches have limitations ranging from a short-lasting effect to angioma formation. In this study, we investigated the feasibility of using adeno-associated viral (AAV) vectors to deliver VEGF genes to mouse myocardium. A cytomegalovirus promoter was used to drive genes for a human VEGF isoform, VEGF(165), and LacZ. A mouse myocardial ischemic model was generated by ligation of the anterior descending coronary artery. Approximately 10(11) copies of the AAV-VEGF vector mixed with 10(10) copies of AAV-LacZ were injected to one site of normal myocardium and a total of 10(11) copies of AAV-VEGF were injected to multiple sites of myocardium around the ischemic region. LacZ gene expression was observed up to 3 months after the vector inoculation. After AAV-VEGF inoculation, neoangiogenesis was observed in the ischemic heart model but not in normal heart tissue. An inflammatory-cell infiltration was not observed in the AAV-VEGF- and AAV-LacZ-inoculated hearts, and angioma-like structure was not observed. These results indicated that injection of the AAV vector directly to myocardium could mediate efficient gene transfer and transgene expression and that VEGF gene delivered by AAV vector can induce angiogenesis in ischemic myocardium.

Figure 1

AAV-LacZ infection of cultured mouse cardiomyocytes and adult mouse heart. (Left) Infected cardiomyocytes. (Middle) X-Gal staining of a whole heart. (Right) H&E staining of the myocardium. Note the lack of any inflammatory changes surrounding the cells expressing LacZ.

Figure 2

Photomicrographs of normal and ischemic hearts inoculated with AAV-VEGF (Normal/VEGF and Ischemic/VEGF) and ischemic heart not injected with AAV vector (Ischemic/control). Arrows, infarcted regions; arrowheads, small blood vessels. (A) Area around the infarcted regions. (Upper) H&E staining. (Lower) vWF staining. An increase in vessel formation is observed in the Ischemic/VEGF hearts. An inset in the micrograph of the vWF-stained ischemic/VEGF heart presents an enlarged area to show blood vessels more clearly. (B) Area around scar tissues formed by needle injections. H&E and vWF stains of cardiac myocardium of both the Ischemic/control and Ischemic/VEGF mice. Note the new blood vessel formation around the scar caused by the needles in the AAV-VEGF-injected heart but not in the control without injection of AAV.

Figure 3

Densities of small blood vessel. Bars: Ischemic/VEGF, ischemic heart injected with AAV-VEGF; Ischemic/PBS, ischemic heart injected with PBS; Normal/VEGF, normal heart injected with AAV-VEGF/AAV-LacZ. The mean is shown above each bar; error bars are the SEM. Differences between the ischemic/VEGF and ischemic /PBS groups and between the ischemic/VEGF and normal/VEGF groups are statistically significant (P < 0.01).