Timing and targeting of cell-based VEGF165 gene expression in ischemic tissue.

BACKGROUND: Therapeutic angiogenesis has become a key technology in experimental and clinical medicine. Only few data are available on the effects of timing and targeting of therapeutic proteins after cell-based gene transfer. This work investigates such effects after temporary expression of vascular endothelial growth factor 165 (VEGF(165)), the most commonly used angiogenic protein for therapeutic purposes. METHODS: We established a cell-based gene-transfer model using fibroblasts to temporarily produce VEGF(165). Cells were implanted into 40 rats. Protein expression and angiogenic effects were measured by PCR, immunohistology, and microangiography. To determine an improvement for survival of ischemically challenged tissue, cells were implanted in an ischemic flap model at different locations and time points. RESULTS: After implantation of modified cells, a temporary increase was found in the target tissue for VEGF(165), endothelial cell counts, and capillary network formations. Four wk later, histological alterations in the target tissue area were not different from controls. Implantation of modified cells into flap plus wound margin 1 wk before surgery showed significant improvement of tissue survival demonstrated by planimetric measurements and blood vessels counting in the target tissue. CONCLUSION: In our model, temporary expression of VEGF(165) induces therapeutically relevant angiogenesis and improves blood supply only if applied 1 wk before ischemia. It is essential to include the surrounding area for induction of angiogenesis in this model. In contrast, the angiogenic effects are not effective in the target area and its surrounding tissue, if therapeutic gene expression is started during onset of ischemia or 2 wk before ischemia in this model.

Identification of a 94-bp GC-rich element in the smooth muscle myosin heavy-chain promoter controlling vascular smooth muscle cell-specific gene expression.

The previously described rabbit 2.3-kilobase smooth muscle myosin heavy-chain (SMHCwt) promoter targets gene expression in transgenic animals to vascular smooth muscle cells (SMCs), including coronary arteries. Therefore, SMHCwt is thought to provide a promising tool for human gene therapy. In the present study, we examined tissue specificity and expression levels of wild-type and mutated SMHC promoters within the system of high-capacity adenoviral (hcAd) vectors. SMHCwt and a series of SMHC promoter deletion mutants, a triple promoter as well as a cytomegalovirus-SMHC hybrid promoter driving the enhanced green fluorescence protein (EGFP) reporter gene were transiently transfected into aortic SMCs. Fluorescence intensity was measured by flow cytometric analysis. Consecutively, hcAd vectors were constructed with the SMHCwt and the mutant promoter with the highest fluorescence activity. Levels of EGFP expression were determined after transduction of SMCs derived from human coronary arteries. For analysis of tissue specificity, embryonic stem (ES) cell-derived SMCs (ESdSMHCs) and cardiomyocytes (ESdCMs) were used. In comparison with SMHCwt, only the SMHCdel94 mutant lacking a 94-bp GC-rich element revealed a 1.5-fold increased fluorescence activity. Transduction of primary SMCs of human coronary arteries with hcAd vectors confirmed an increased EGFP expression driven by the SMHCdel94 promoter. In ES-cell-derived embryoid bodies, SMHCwt was exclusively active in transduced ESdSMCs. In contrast, expression of SMHCdel94 was also found in ESdCMs and other nontarget cells of the embryoid body. The tissue-specific rabbit SMHCwt promoter seems to be suitable for adenoviral gene transfer in SMCs of human coronary arteries and deletion of a 94-bp negative cis-acting GC-rich element results in loss of specificity.