Effects of high power-pulsed Nd:YAG laser irradiation on the release of transforming growth factor-beta (TGF-ß) and vascular endothelial growth factor (VEGF) from human gingival fibroblasts.

The purpose of this study was to investigate the effect of different high-power energy settings of a neodymium:yttrium-aluminum-garnet (Nd:YAG) laser (1064 nm) on cell viability of human gingival fibroblasts (GFs) and release of transforming growth factor-beta (TGF-ß) and vascular endothelial growth factor (VEGF) on these cells. GFs were isolated from human gingival connective tissues during the crown lengthening procedure. GFs were irradiated with different laser parameters as follows: group 1: 1 W (100 mJ, 10 Hz) 10 seconds; group 2: 1.5 W (150 mJ, 10 Hz) 10 seconds; group 3: 2 W (200 mJ, 10 Hz) 10 seconds; group 4: 1 W (100 mJ, 10 Hz) 20 seconds; group 5: 1.5 W (150 mJ, 10 Hz) 20 seconds; and group 6: 2 W (200 mJ, 10 Hz) 20 seconds. Cell viability/cell proliferation was analyzed with XTT (tetrazolium salt, cell proliferation kit) staining. The release levels of TGF-ß and VEGF were analyzed by the enzyme-linked immunosorbent assay. No significant differences were observed in the different laser irradiation groups compared to the control group in terms of cell viability (p > 0.05). The release of TGF-ß was not affected by different laser irradiation settings (p > 0.05). Only group 6 promoted significantly higher VEGF release from GFs in 24 hours compared to the control group (p ˂ 0.05). These findings suggest that high-power Nd:YAG laser is probably safe but has a very limited effect for wound healing.

HIF-1-dependent stromal adaptation to ischemia mediates in vivo tumor radiation resistance.

PURPOSE: Hypoxia-inducible factor 1 (HIF-1) promotes cancer cell survival and tumor progression. The specific role played by HIF-1 and tumor-stromal interactions toward determining tumor resistance to radiation treatment remains undefined. We applied a multimodality preclinical imaging platform to mechanistically characterize tumor response to radiation, with a focus on HIF-1-dependent resistance pathways. METHODS: C6 glioma and HN5 human squamous carcinoma cells were stably transfected with a dual HIF-1 signaling reporter construct (dxHRE-tk/eGFP-cmvRed2XPRT). Reporter cells were serially interrogated in vitro before and after irradiation as monolayer and multicellular spheroid cultures and as subcutaneous xenografts in nu/nu mice. RESULTS: In vitro, single-dose irradiation of C6 and HN5 reporter cells modestly impacted HIF-1 signaling in normoxic monolayers and inhibited HIF-1 signaling in maturing spheroids. In contrast, irradiation of C6 or HN5 reporter xenografts with 8 Gy in vivo elicited marked upregulation of HIF-1 signaling and downstream proangiogenic signaling at 48 hours which preceded recovery of tumor growth. In situ ultrasound imaging and dynamic contrast-enhanced (DCE) MRI indicated that HIF-1 signaling followed acute disruption of stromal vascular function. High-resolution positron emission tomography and dual-contrast DCE-MRI of immobilized dorsal skin window tumors confirmed postradiotherapy HIF-1 signaling to spatiotemporally coincide with impaired stromal vascular function. Targeted disruption of HIF-1 signaling established this pathway to be a determinant of tumor radioresistance. CONCLUSIONS: Our results illustrate that tumor radioresistance is mediated by a capacity to compensate for stromal vascular disruption through HIF-1-dependent proangiogenic signaling and that clinically relevant vascular imaging techniques can spatially define mechanisms associated with tumor irradiation.

Inhibition of transforming growth factor-beta, hypoxia-inducible factor-1alpha and vascular endothelial growth factor reduced late rectal injury induced by irradiation.

Tumor hypoxia and angiogenesis associated with malignant progression have been studied widely. The efficacy of angiogenesis inhibition combined with radiotherapy has been demonstrated in cancer treatment. Here, we studied the effect of hypoxia and angiogenesis inhibition on radiation-induced late rectal injury. The rectum of C57BL/6N mice was irradiated locally with a single dose of 25 Gy. Radiation-induced histological changes were examined at 90 days after irradiation by hematoxylin-eosin (H.E.) staining and azan staining. Pimonidazole was administered and its distribution was assayed by immunohistochemistry staining. Expression of transforming growth factor beta1 (TGF-beta1), hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) was assessed on the fibrotic region using real-time PCR and immunohistochemistry. In addition, the effects of TGF-beta, VEGF and HIF-1alpha on radiation-induced injury were investigated by the administration of neutralizing antibody of TGF-beta, antibody of VEGF or YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole) which was developed as an agent for inhibiting HIF-1 expression after irradiation respectively. Fibrosis and uptake of pimonidazole were found 90 days after irradiation. The expression of TGF-beta1, HIF-1alpha and VEGF significantly increased with the formation of fibrosis induced by irradiation compared with unirradiated controls. In addition, treatment of neutralizing antibody of TGF-beta, antibody of VEGF or YC-1 reduced the development of radiation-induced injury. Our results suggested that radiation-induced hypoxia may play an important role in late rectal injury. Although the inhibition of HIF-1alpha and VEGF reduced the radiation induced late injury, the precise mechanism is still unclear.

Vascular endothelial growth factor expression predicts outcome after primary radiotherapy for head and neck squamous cell cancer.

AIMS: To establish whether the expression of vascular endothelial growth factors (VEGFs) predicts prognosis in patients treated with primary radiotherapy for cancers of the upper aerodigestive tract. MATERIALS AND METHODS: A retrospective analysis was undertaken of VEGF and VEGF-D expression in tumour tissue in pre-treatment biopsies from 27 patients who had been treated with primary radiotherapy for stage II-IV squamous head and neck carcinomas. Serial sections (4 microm) were cut from formalin-fixed, paraffin-embedded specimens and stained with monoclonal antibodies using standard immunoperoxidase methods. Two independent investigators assessed the staining intensity in a randomised, blind manner. Both negative and positive controls (placenta and/or tonsil) were included in the staining procedure. All patients were followed for a minimum of 5 years, or until death. Local control and overall survival were taken as end points for the comparative analysis between patients whose tumours expressed low levels and those that expressed high levels of the two growth factors. Comparisons were made using the Log-rank test with Kaplan-Meier actuarial survival analysis. RESULTS: In patients with tumours expressing low levels of VEGF, 5-year local control was seen in 75% compared with 18% for those with high levels; overall survival was 75 and 23%, respectively. For those with low levels of VEGF-D, 5-year local control was 64% compared with 17% for those with high levels; overall survival was 58 and 20%, respectively. CONCLUSION: Our results suggest that the expression of endothelial growth factors in squamous head and neck cancers may predict outcome after radiotherapy.