198 G. L. Semenza

Calf blood pressure (BP) was measured preoperatively and on postoperative day 14, at which time angiography was repeated (Fig. 1D), the animal was sacrificed, and adductor muscle tissue was excised and fixed. Compared to AdLacZ-treated rabbits, AdCA5-treated animals showed a significant improvement in the calf BP ratio (coiled:non-coiled limb) and a significant decrease in the time required for complete perfusion of the femoral circulation after contrast injection (Fig. 2).

Anti-CD31 immunohistochemistry revealed a significant increase in capillary:myocyte ratio in adductor muscle for AdCA5-treated animals (Fig. 3). More importantly, whereas the total number of arteries was not changed, AdCA5 increased the total luminal area of arteries > 100 µm in diameter, as determined by image analysis of sections of adductor muscle stained with anti-α-smooth muscle actin antibodies (Fig. 4). Thus, AdCA5 administration significantly stimulated the remodeling of pre-existing collateral vessels (arteriogenesis) (Fig. 5), which is the major determinant of blood flow following arterial occlusion.78 AdCA5 treatment induced increased expression of PDGF-B, PLGF, MCP-1, SDF-1, and VEGF mRNA in adductor muscle.78 Both MCP-1 and PLGF have been shown to promote arteriogenesis in previous studies.79,80

Taken together these two studies demonstrate that AdCA5 can induce both angiogenesis and arteriogenesis. In both cases, AdCA5 was shown to induce the expression of multiple angiogenic growth factors and cytokines. As a result, HIF-1α gene therapy may have advantages over gene therapy approaches that only increase the expression of a single angiogenic growth factor or cytokine — a strategy that has been unsuccessful in clinical trials. Further studies are required to determine whether AdCA5 administration may represent a novel treatment option for patients with extensive peripheral vascular disease who are not candidates for conventional therapies.

6. Control of Tumor Angiogenesis by HIF-1

Human colon cancer cells transfected with an expression vector encoding HIF-1α manifest a dramatic increase in tumor xenograft growth and angiogenesis, with significant increases in tumor vascular volume and vascular permeability demonstrated in vivo by magnetic resonance

A B

C D

Fig. 2. Representative pelvic and hindlimb angiograms of an AdCA5-treated rabbit. (A) Digital angiogram, day 0, before coiling. (B) Anterior-posterior digital spot image showing coils (arrows) in the left femoral artery and six needles (bracketed area) placed on the sites for adenoviral injection. (C) Digital subtraction angiogram, day 0, immediately after coiling. Arrowheads indicate location of coils. (D) Digital subtraction angiogram of an AdCA5-treated rabbit (day 14) showing collateral vessel development in the medial thigh. Arrowheads indicate location of the coils. (Reprinted from Ref. 78, copyright 2005 with permission from the European Society of Cardiology.)

imaging.81 Conversely, human gastric cancer cells transfected with an expression vector encoding a dominant negative form of HIF-1α (HIF- 1αDN) manifest a striking reduction in tumor growth, either as subcutaneous xenografts or after orthotopic transplantation into the gastric

A B

Fig. 4. Capillary morphometry of sections from left adductor muscles harvested on day 14. CD-31 staining of the adductor muscle injected with AdCA5 (A) and AdLacZ (B). Magnification: × 400. Scale bars: 100 µm. (C) Capillary/myocyte ratio on day 14 ( p < 0.05). (Reprinted from Ref. 78, copyright 2005 with permission from the European Society of Cardiology.)

hypoxia and by growth factor signal transduction. A critical hallmark of cancer cells is the acquisition of independence from external sources of growth factor due to the establishment of autocrine signaling pathways in which the tumor expresses both the growth factor and its receptor. HIF-1 appears to play an important role in these pathways because HIF-1 is both upstream and downstream of the receptors. A consequence of receptor (e.g. EGFR, IGF-R1) activation by ligand is an

Table 4. Effect of HIF-1a overexpression in human cancers.

Small molecule inhibitors of growth factor receptors or downstream signaling molecules block a major stimulus for HIF-1α synthesis.83 The resulting decrease in angiogenesis may lead to tumor hypoxia, which will induce HIF-1 activity via the reduced activity of the prolyl and asparaginyl hydroxylases that negatively regulate HIF-1, thus