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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.

AdVEGF-B(186) and AdVEGF-E are equally potent in inducing therapeutic angiogenesis in mouse myocardium and produce less side effects than AdVEGF-A(165).

Our results suggest that PDGF-C may play an important role in endothelium in normal and atherosclerotic arteries and in macrophages in lesions. PDGF-D was expressed in all types of lesions with the same intensity and thus differs from the expression of PDGF-C.

Efficient capillary enlargement leading to significant increases in tissue perfusion is needed to gain metabolic benefits after angiogenic gene therapy. However, the risk of systemic side effects can increase as the efficiency of angiogenic gene therapy is improved. Importantly, the unstable wall structure of the newly formed vessels seems not to compromise the metabolic benefits.

The Notch signaling pathway is essential for normal development due to its role in control of cell differentiation, proliferation and survival. It is also critically involved in tumorigenesis and cancer progression. A key enzyme in the activation of Notch signaling is the gamma-secretase protein complex and therefore, gamma-secretase inhibitors (GSIs)—originally developed for Alzheimer's disease—are now being evaluated in clinical trials for human malignancies. It is also clear that Notch plays an important role in angiogenesis driven by Vascular Endothelial Growth Factor A (VEGF-A)—a process instrumental for tumor growth and metastasis. The effect of GSIs on tumor vasculature has not been conclusively determined. Here we report that Compound X (CX), a GSI previously reported to potently inhibit Notch signaling in vitro and in vivo, promotes angiogenic sprouting in vitro and during developmental angiogenesis in mice. Furthermore, CX treatment suppresses tumor growth in a mouse model of renal carcinoma, leads to the formation of abnormal vessels and an increased tumor vascular density. Using a rabbit model of VEGF-A-driven angiogenesis in skeletal muscle, we demonstrate that CX treatment promotes abnormal blood vessel growth characterized by vessel occlusion, disrupted blood flow, and increased vascular leakage. Based on these findings, we propose a model for how GSIs and other Notch inhibitors disrupt tumor blood vessel perfusion, which might be useful for understanding this new class of anti-cancer agents.

Contrast pulse sequence ultrasound is an efficient and accurate noninvasive real-time perfusion imaging modality in small laboratory animals and also offers a means for the assessment of muscle perfusion in future clinical trials of therapeutic angiogenesis.

No abstract