Effects of brachytherapy on intimal hyperplasia in arteriovenous fistulas in a porcine model.

PURPOSE: The hypotheses of this investigation were that endovascular radiation would reduce intimal hyperplasia in arteriovenous fistulas (AVFs) and that this reduction would be associated with decreased expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-A, and tumor necrosis factor (TNF)-alpha. MATERIALS AND METHODS: Bilateral end jugular vein-to-side carotid artery fistulas were constructed in pigs. At 48 hours, one AVF was randomly selected for endovascular radiation with (192) Iridium. The contralateral fistula received no radiation and served as a control. Animals in group 1 (n = 7) received 14 Gy of radiation at a depth of 2 mm and tissue was harvested at 29 days; animals in group 2 received 7 Gy of radiation at a depth of 2 mm and tissue was harvested at 29 days (n = 8); and animals in group 3 received 7 Gy of radiation at a depth of 2 mm and tissue was harvested at 56 days (n = 8). The area and maximum thickness of intimal hyperplasia were then measured blindly. Immunohistochemical results for VEGF, PDGF-A, and TNFalpha were obtained and analyzed blindly by assigning a score of 0-3, with 0 indicating no staining and 3 indicating maximum staining. RESULTS: Irradiation with 14 Gy caused severe fibrosis in the media of the vein, with thrombosis of three of seven AVFs. Compared with the control group, the group that underwent irradiation with 7 Gy had significantly reduced intimal area at 56 days (9.9 mm(2) +/- 4.9 vs 2.1 mm(2) +/- 1.1; P =.001). This reduction correlated with significant reduction in the expression of VEGF (score of 2.2 +/- 0.1 vs 1.2 +/- 0.2; P =.001) and TNFalpha (1.3 +/- 0.1 vs 0.9 +/- 0.1; P =.04). CONCLUSION: Fourteen grays is an excessive radiation dose for veins, causing medial fibrosis and thrombosis of the AVF. Irradiation with 7 Gy effectively inhibited the formation of intimal hyperplasia in AVF. This inhibition correlated with decreased expression of VEGF and TNFalpha.

In vivo optical imaging of expression of vascular endothelial growth factor following laser incision in skin.

BACKGROUND AND OBJECTIVE: Laser-tissue interaction studies have focused on laser-induced secondary effects on tissue and the postmortem histological analysis of laser wounds. In this study we addressed wound healing and possible impairment of wound healing due to collateral tissue damage by in vivo imaging of gene expression. STUDY DESIGN/MATERIALS AND METHODS: We used a transgenic mouse model containing a VEGF promoter driving a GFP reporter gene to image VEGF expression in vivo. Twenty-two mice received two full thickness incisions in the dorsal skin: one with the Free Electron Laser (lambda = 6.45 microm, 52.9 mJ/mm(2)) and one with a scalpel. Mice were imaged for GFP expression at 3 days, 1, 2, 3, and 4 weeks. Confocal microscopic imaging was performed at 2 weeks. RESULTS: Peak GFP expression was seen at 2-3 weeks and was localized in fibroblasts. FEL lesions showed more total GFP expression than scalpel lesions but this was only statistically significant (P < 0.05) at 2 and 4 weeks. The full-width half-max (FWHM) of the GFP expression was always larger for the FEL lesion compared to the scalpel lesion but was only statistically significant (P < 0.05) at 2 and 3 weeks. At 2 weeks the extent of the GFP expression in the laser lesion was on average 55 microm beyond that seen in the scalpel lesion but correlated with the number of laser passes. CONCLUSIONS: Feasibility of using transgenic mice carrying photoactive reporter genes for studying cellular process of laser-inflicted wound repair in a noninvasive, in vivo manner was shown. GFP expression mediated by the VEGF promoter in fibroblast showed minimal impairment of wound healing due to the laser.

Comparison between light induced and chemically induced oxidation of rhVEGF.

PURPOSE: The primary objective of this study was to compare the effects of light-and chemical-induced oxidation of recombinant human vascular endothelial growth factor (rhVEGF) and the impact of these reactions on protein formulation. METHODS: A liquid formulation of rhVEGF was exposed to fluorescent light (2 x 10(4) lux for up to 4 weeks), hydrogen peroxide (H2O2), or t-butythydroperoxide (t-BHP) to induce oxidation of rhVEGF. All samples were then treated by tryptic digest and analyzed by reversed phase HPLC to determine the extent of oxidation. Chemically treated samples were also examined by near-UV and far-UV circular dichroism spectroscopy to determine the effect of oxidation on the structure of the protein. RESULTS: Exposure to light for 2 weeks resulted in 8 to 40% oxidation of all 6 methionine residues of rhVEGF (Met3 > Met18 > Met55 > Met78.81 > Met94). This amount of oxidation did not affect the binding activity of rhVEGF to its kinase domain receptor (KDR). Light exposure for 4 weeks increased metsulfoxide formation at Met3 and Met18 by an additional 16%, but did not affect the other residues. This oxidation decreased the receptor binding capacity to 73%. possibly due to the role of Met 18in receptor binding. Chemical oxidation of rhVEGF resulted in a greater extent of oxidation at all 6 methionines. Complete oxidation of Met3, Met18 and Met55 was observed after treatment with H2O2, while these residues underwent 40 to 60% oxidation after treatment with t-BHP. The receptor binding capacity was significantly reduced to 25% and 55% after treatment with H2O2 and t-BHP, respectively. After chemical oxidation, no changes in the secondary or tertiary structure were observed by far-UV and near-UV CD spectroscopy, respectively. CONCLUSIONS: Methionine residues with exposed surface areas greater than 65 A2 and sulfur surface areas greater than 16 A2 were most susceptible to oxidation. Chemical oxidation resulted in higher metsulfoxide formation and decreased binding activity of the protein to KDR than light-induced oxidation. The reduction in KDR binding was not caused by measurable conformational changes in the protein. Photooxidation was dependent on the amount of energy imparted to the protein, while the ability of t-BHP or H2O2 to react with methionine was governed by solvent accessibility of the methionine residues and steric limitations of the oxidizing agent. Significant chemical oxidation occurred on sulfurs with minimum surface areas of 16 A2, while increased photooxidation occurred as a function of increasing surface areas of solvent exposed sulfur atoms. Such differences in the extent of oxidation should be considered during protein formulation since it may help predict potential oxidation problems.

Association of preoperative radiation effect with tumor angiogenesis and vascular endothelial growth factor in oral squamous cell carcinoma.

This study examined the relationship between tumor angiogenesis and the radiation-induced response, evaluated based on pathological changes, in oral squamous cell carcinoma patients treated with preoperative radiation therapy. Forty-one cases of squamous cell carcinoma treated with preoperative radiation therapy were investigated. Tumor angiogenesis was assessed by scoring the intratumor microvessel density (IMVD). Expression of vascular endothelial growth factor (VEGF) was also evaluated before and after preoperative radiotherapy. There was no correlation between IMVD in the specimens before therapy and the pathological response to radiation therapy. However, radiation therapy decreased IMVD in the specimens after therapy. A significant association was observed between VEGF expression and resistance to radiation therapy: only 4 of the 21 patients whose tumors exhibited a high level (2 + or 3 + ) of VEGF staining experienced a major (3 + or 4 + ) pathological response to radiation therapy. Furthermore, an increasing level of VEGF expression after radiation therapy was observed in non-effective (0 to 2 + ) response cases. These results suggest that VEGF expression and the induction of this protein are related to radiosensitivity and could be used to predict the effects of preoperative radiation therapy on oral squamous cell carcinoma.

Induction by carbon-ion irradiation of the expression of vascular endothelial growth factor in lung carcinoma cells.

PURPOSE: To investigate the induction by carbon- ion irradiation of vascular endothelial growth factor (VEGF) mRNA and protein. MATERIALS AND METHODS: RERF-LC-AI lung squamous carcinoma cells were irradiated with carbon ions of either 13.3, 50 or 90keV/microm. Colony formation was used to determine cell survival. VEGF mRNA and protein of the irradiated cells were quantified by Northern blot analysis and ELISA assay, respectively. Genistein, Src tyrosine kinase inhibitor and H7, protein kinase C inhibitor, were used to inhibit VEGF mRNA expression. RESULTS: The relative biological effectiveness (RBE) of carbon ions (13.3, 50 and 90keV/microm) was 1.10, 1.97 and 2.30, respectively, in terms of D10 values. Single doses of 15 Gy with either X-rays or carbon ions significantly induced VEGF mRNA expression at 16-24h after irradiation with a maximum induction of 2.81-fold. A significant increase was also observed in VEGF protein levels, detected in culture supernatant 24h after irradiation with 50 and 90keV/microm carbon ions. Neither mRNA nor protein induction showed a dependence on LET. The induction of VEGF mRNA by carbon-ion irradiation was completely inhibited by pretreating cells with genistein and H7, indicating that Src tyrosine kinase and protein kinase C on cell surface membranes is involved in the induction. CONCLUSION: Irradiation of lung carcinoma cells with carbon ions induced VEGF mRNA expression and increased protein levels. The induction was dose-dependent. Radiation-induced DNA damage and/or its repair may not be a prerequisite for the induction of VEGF mRNA.