The effect of dehydroglyasperin C on UVB-mediated MMPs expression in human HaCaT cells.

BACKGROUND: The ultraviolet B (UVB) from solar radiation increases the generation of reactive oxygen species (ROS), which mediate the production of matrix metalloproteinases (MMPs), and acts mainly on the epidermis layer of the skin. This study was aimed at assessing the anti-photoaging effects of dehydroglyasperin C isolated from Glycyrrhiza uralensis Fisch on MMPs levels in HaCaT human keratinocytes and to elucidate the underlying mechanism. METHODS: The cell viability was measured by MTT assay. Expression, phosphorylation and enzymatic activity of the protein were examined using ELISA, Western blot or gelatin zymography. Intracellular ROS measurement was evaluated by fluorescent ELISA and 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) assay. RESULTS: In the present study, we found that dehydroglyasperin C markedly inhibited UVB-mediated expression of collagenase (MMP-1) and gelatinase (MMP-9) by inhibiting ROS generation. Dehydroglyasperin C treatment also decreased the UVB irradiation-mediated activation of mitogen-activated protein kinase (MAPK), c-Jun phosphorylation, and c-Fos expression. In addition, the down-regulation of UVB-induced c-Jun phosphorylation caused by dehydroglyasperin C treatment was more than the down-regulation of c-Fos expression in the HaCaT human keratinocytes. CONCLUSION: Our results indicated that dehydroglyasperin C may function as a potential anti-photoaging agent by inhibiting UVB-mediated MMPs expression via suppression of MAPK and AP-1 signaling.

Differing energy densities with laser 670nm InGaP controls inflammation and collagen reorganization in burns.

PURPOSE: This study compared different energy densities of laser on second degrees burns in rats aiming to determine the most effective dosimetry in stimulation of the healing process. METHODS: Burns were induced in the dorsal skin of 54 animals divided into three groups (n: 18): 1-without treatment; 2-irradiated lesions by the Indium Gallium Phosphide (InGaP) 670nm (4.93J/cm2) laser; 3-irradiated lesions by the InGaP-670nm (9.86J/cm2) laser. Samples were collected on the 2, 10 and 18 days after injury for structural, morphometry, biochemical analysis and Western blotting. RESULTS: The energy densities examined were effective in significantly increasing the total number of fibroblasts and blood vessels and reduce the number of inflammatory cells particularly in irradiated lesions with 9.86J/cm2. This same energy density significantly increased the amount of GAGs (Glycosaminoglycans), decreased the TGF-ß1 (Transforming Growth Factor ß1) and increased the VEGF (Vascular and Endothelial Growth Factor) during the experimental period. This energy density also significantly increased the Collagen type I and decreased Collagen type III and the active isoform of metalloproteinase 9 (MMP-9). CONCLUSIONS: The energy density of 9.86J/cm2 was more effective in promoting cellular responses related to neoangiogenesis, decreasing inflammation and collagen fibers reorganization.

Low level laser therapy modulates viability, alkaline phosphatase and matrix metalloproteinase-2 activities of osteoblasts.

Low level laser therapy (LLLT) has been shown to stimulate bone cell metabolism but their impact on the matrix metalloproteinase (MMP) expression and activity is little explored. This study evaluated the influence of LLLT at two different wavelengths, red and infrared, on MC3T3-E1 preosteoblast viability, alkaline phosphatase (ALP) and MMP-2 and -9 activities. To accomplish this, MC3T3-E1 cells were irradiated with a punctual application of either red (660nm; InGaAIP active medium) or infrared (780nm; GaAlAs active medium) lasers both at a potency of 20mW, energy dose of 0.08 or 0.16J, and energy density of 1.9J/cm2 or 3.8J/cm2, respectively. The control group received no irradiation. Cellular viability, ALP and MMP-2 and -9 activities were assessed by MTT assay, enzymatic activity and zymography, respectively, at 24, 48 and 72h. The treatment of cells with both red and infrared lasers significantly increased the cellular viability compared to the non-irradiated control group at 24 and 48h. The ALP activity was also up modulated in infrared groups at 24 and 72h, depending on the energy densities. In addition, the irradiation with red laser at the energy density of 1.9J/cm2 promoted an enhancement of MMP-2 activity at 48 and 72h. However, no differences were observed for the MMP-9 activity. In conclusion, when used at these specific parameters, LLL modulates both preosteoblast viability and differentiation highlighted by the increased ALP and MMP-2 activities induced by irradiation.

Photobiomodulation therapy on collagen type I and III, vascular endothelial growth factor, and metalloproteinase in experimentally induced tendinopathy in aged rats.

This study investigates the effect of photobiomodulation therapy (PBMT) on collagen type I and III, matrix metalloproteinase (MMP), and vascular endothelial growth factor (VEGF) in experimentally induced tendinopathy in female aged rats. Tendinopathy was induced by the Achilles tendoncollagenase peritendinous. Forty-two Wistar rats (Norvegicus albinus) were used; groups consisted of 36 aged animals (18 months old; mean body weight, 517.7 ± 27.54 g) and 6 adult animals (12 weeks old; mean body weight, 266± 19.30 g). The animals were divided into three groups: control, aged tendinopathy, and aged tendinopathy PBMT; the aged groups were subdivided based on time to euthanasia: 7, 14, and 21 days. PBMT involved a gallium-arsenide-aluminum laser (Theralaser, DMC®) with active medium operating at wavelength 830 ± 10 nm, 50 mW power, 0.028 cm2 laser beam, 107 J/cm2 energy density, 1.8 W/cm2 power density, and an energy of 3 J per point. The laser was applied by direct contact with the left Achilles tendon during 60 s per point at a frequency of three times per week, until the euthanasia date (7, 14, and 21 days). VEGF, MMP-3, and MMP-9 were analyzed by immunohistochemistry, and collagen type I and III by Sirius red. PBMT increased the deposition of collagen type I and III in a gradual manner, with significant differences relative to the group aged tendonitis (p < 0.001), and in relation to VEGF (p < 0.001); decreased expression of MMP-3 and 9 were observed in group aged tendinopathy (p < 0.001). PBMT, therefore, increased the production of collagen type I and III, downregulated the expression of MMP-3 and MMP-9, and upregulated that of VEGF, with age and age-induced hormonal deficiency.

Biological effect of an alternating electric field on cell proliferation and synergistic antimitotic effect in combination with ionizing radiation.

Alternating electric fields at an intermediate frequency (100~300 kHz), referred to as tumour-treating fields (TTF), are believed to interrupt the process of mitosis via apoptosis and to act as an inhibitor of cell proliferation. Although the existence of an antimitotic effect of TTF is widely known, the proposed apoptotic mechanism of TTF on cell function and the efficacy of TTF are controversial issues among medical experts. To resolve these controversial issues, a better understanding of the underlying molecular mechanisms of TTF on cell function and the differences between the effects of TTF alone and in combination with other treatment techniques is essential. Here, we report experimental evidence of TTF-induced apoptosis and the synergistic antimitotic effect of TTF in combination with ionizing radiation (IR). For these experiments, two human Glioblastoma multiforme (GBM) cells (U373 and U87) were treated either with TTF alone or with TTF followed by ionizing radiation (IR). Cell apoptosis, DNA damage, and mitotic abnormalities were quantified after the application of TTF, and their percentages were markedly increased when TTF was combined with IR. Our experimental results also suggested that TTF combined with IR synergistically suppressed both cell migration and invasion, based on the inhibition of MMP-9 and vimentin.

The influence of sun exposure on the DNA methylation status of MMP9, miR-137, KRT14 and KRT19 genes in human skin.

BACKGROUND: Studies have shown that a variety of environmental factors and habits are associated with epigenetic changes. In addition, various genes are also found to respond to UV radiation. OBJECTIVES: The aim of this study was to investigate the sun exposure influence on the DNA methylation profile on the matrix metalloprotease-9 (MMP9), microRNA 137 (miR-137), cytokeratin 14 (KRT14) and 19 (KRT19) genes of skin cells of subjects with no history of skin diseases. METHODS: Skin biopsies (5mm) were obtained using a punch technique on sun-exposed (outer forearm) and sun-protected areas (inner arm) from 30 corpses from the Brazilian Service of Death Investigation. Skin types were ranked according to Fitzpatrick's criteria. Genomic DNA was extracted and a DNA methylation analysis was performed using Methylation Specific PCR (MSP) or Methylation-Sensitive Restriction Enzymes (MSRE) of sun-exposed and sun-protected skin areas. RESULTS: No differences were found among the areas (p>0.05; McNemar), with the partially methylated condition found to be a common event in skin for both MMP9 and miR-137 genes and the methylated condition for both KRT14 and KRT19 genes. Additional analysis showed no differences in the methylation status when age, gender and skin type were considered, however, the methylation status of miR-137 gene seems to be gender-related. CONCLUSIONS: We conclude that sun exposure does not induce changes in the DNA methylation status in MMP9, miR-137, KRT14 and KRT19 genes.

Effect of pulsed electromagnetic field on MMP-9 and TIMP-1 levels in chondrosarcoma cells stimulated with IL-1ß.

Chondrosarcoma, the second most common type of bone malignancy, is characterized by distant metastasis and local invasion. Previous studies have shown that treatment by pulsed electromagnetic field (PEMF) has beneficial effects on various cancer cells. In this study, we investigated the effects of PEMF applied for 3 and 7 days on the matrix metalloproteinase (MMP) levels in chondrosarcoma SW1353 cells stimulated with two different doses of IL-1ß. SW1353 cells were treated with (0.5 and 5 ng/ml) IL-1ß and PEMF exposure was applied either 3 or 7 days. MMP-9 and TIMP-1 levels were measured in conditioned media by enzyme-linked immunosorbent assay. The results were relative to protein levels. Statistical analyses were performed using one-way analysis of variance (ANOVA). P<0.05 was considered significant. PEMF treatment significantly decreased MMP-9 protein levels in human chondrosarcoma cells stimulated with 0.5 ng/ml IL-1ß at day 7, whereas it did not show any effect on cells stimulated with 5 ng/ml IL-1ß. There was no significant change in TIMP-1 protein levels either by IL-1ß stimulation or by PEMF treatment. The results of this study showed that PEMF treatment suppressed IL-1ß-mediated upregulation of MMP-9 protein levels in a dual effect manner. This finding may offer new perspectives in the therapy of bone cancer.

Irradiation alters MMP-2/TIMP-2 system and collagen type IV degradation in brain.

PURPOSE: Blood-brain barrier (BBB) disruption is one of the major consequences of radiation-induced normal tissue injury in the central nervous system. We examined the effects of whole-brain irradiation on matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in the brain. METHODS AND MATERIALS: Animals received either whole-brain irradiation (a single dose of 10 Gy γ-rays or a fractionated dose of 40 Gy γ-rays, total) or sham-irradiation and were maintained for 4, 8, and 24 h following irradiation. mRNA expression levels of MMPs and TIMPs in the brain were analyzed by real-time reverse transcriptase-polymerase chain reaction (PCR). The functional activity of MMPs was measured by in situ zymography, and degradation of ECM was visualized by collagen type IV immunofluorescent staining. RESULTS: A significant increase in mRNA expression levels of MMP-2, MMP-9, and TIMP-1 was observed in irradiated brains compared to that in sham-irradiated controls. In situ zymography revealed a strong gelatinolytic activity in the brain 24 h postirradiation, and the enhanced gelatinolytic activity mediated by irradiation was significantly attenuated in the presence of anti-MMP-2 antibody. A significant reduction in collagen type IV immunoreactivity was also detected in the brain at 24 h after irradiation. In contrast, the levels of collagen type IV were not significantly changed at 4 and 8 h after irradiation compared with the sham-irradiated controls. CONCLUSIONS: The present study demonstrates for the first time that radiation induces an imbalance between MMP-2 and TIMP-2 levels and suggests that degradation of collagen type IV, a major ECM component of BBB basement membrane, may have a role in the pathogenesis of brain injury.

Saponins from the roots of Platycodon grandiflorum suppress ultraviolet A-induced matrix metalloproteinase-1 expression via MAPKs and NF-κB/AP-1-dependent signaling in HaCaT cells.

Saponins from the roots of Platycodon grandiflorum (CKS) have been shown to exhibit many pharmacological activities, including anti-cancer and anti-inflammatory activities and antioxidant effects. However, anti-skin photoaging effects of CKS have not yet been reported. In this study, we investigated the protective effects of CKS against UVA damage on immortalized human keratinocytes (HaCaT). We then explored the inhibitory effects of CKS on UVA-induced MMP-1 and investigated the molecular mechanism underlying those effects. CKS increased the cell viability and inhibited reactive oxygen species (ROS) production in HaCaT cells exposed to UVA irradiation. Pre-treatment of HaCaT cells with CKS inhibited UVA-induced production of MMP-1 and MMP-9. In addition, CKS decreased UVA-induced expression of the inflammatory cytokines IL-1ß and IL-6. Western blot analysis further revealed that CKS markedly suppressed the enhancement of collagen degradation in UVA-exposed HaCaT cells. CKS also suppressed UVA-induced activation of NF-κB or c-Jun and c-Fos, and the phosphorylation of MAPKs, which are upstream modulators of NF-κB and AP-1.

Preoperative radiotherapy and extracellular matrix remodeling in rectal mucosa and tumour matrix metalloproteinases and plasminogen components.

BACKGROUND. Preoperative radiotherapy reduces recurrence but increases postoperative morbidity. The aim of this study was to explore the effect of radiotherapy in rectal mucosa and rectal tumour extracellular matrix (ECM) by studying enzymes and growth factors involved in ECM remodeling. MATERIALS AND METHODS. Twenty patients with short-term preoperative radiotherapy and 12 control patients without radiotherapy were studied. Biopsies from rectal mucosa and tumour were collected prior to radiotherapy and at surgery. Tissue MMP-1, -2, -9, TIMP-1, uPA, PAI-1, TGF-beta1 and calprotectin were determined by ELISA. Biopsies from irradiated and non-irradiated peritoneal areas were also analysed. RESULTS. Radiotherapy increased the tissue levels of MMP-2 and PAI-1 in both the rectal mucosa and tumours while calprotectin and uPA showed an increase only in the mucosa after irradiation. The increase of calprotectin was due to an influx of inflammatory cells as revealed by immunohistochemistry. Prior to irradiation, the tumour tissues had increased levels of MMP-1, -2, -9, total TGF-beta1, uPA, PAI-1 and calprotectin compared to mucosa, while TIMP-1 and the active TGF-beta1 fraction showed no statistical difference. CONCLUSIONS. This study indicates a radiation-induced effect on selected ECM remodeling proteases. This reaction may be responsible for early and late morbidity. Interference of this response might reduce these consequences.

Increased motility and invasiveness in tumor cells that survive 10 Gy irradiation.

Radiotherapy is an important noninvasive treatment for many types of cancer. However, it has been reported that the proliferative, invasive, and metastatic capacities of tumor cells can be increased in the repopulated tumors that survive radiotherapy. We have previously established a radiation-surviving cell model for the human non-small cell lung cancer cell line H1299 by harvesting relic cells 14 days after irradiation (IR cells). Here, we report that cell invasion, cell migration, and cell adhesion are enhanced in these surviving cancer cells. The mRNA expression levels of matrix metalloproteinases (MMPs), including mmp1, mmp2, and mmp9, were upregulated in IR cells compared with parental cells. A gelatin zymogram, wound healing assay, and invasion assay showed increased MMP activity, cell motility, and invasiveness in IR cells, respectively. Moreover, IR cells adhered more tightly to collagen-coated dishes than parental cells. Consistently, paxillin, phosphorylated FAK, integrin beta1, and vinculin were strongly localized at focal adhesions in IR cells, as visualized by immunofluorescence. In this report, we identify molecules responsible for the malignant properties of tumor cells that survive irradiation. These molecules may be important therapeutic targets for the control of repopulated tumors after radiotherapy.

[Effect of irradiation on matrix metalloproteinases, vascular endothelial growth factor and microvessel density of mice bearing Lewis lung cancer].

OBJECTIVE: To investigate the effect of irradiation on matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF) and microvessel density (MVD), which influence on tumor growth and metastases of mice bearing Lewis lung cancer (LLC). METHODS: C57BL/6 mice bearing LLC were randomized into two groups to receive radiation and no-radiation, respectively. When tumor reached about 190 mm3 in volume on the fourteenth day after inoculation, the radiation group was irradiated to the primary tumor (6MV-X, 5 Gy/day, d14, 16, 18, 20). On the eighth day after irradiation, animals were killed to examine primary tumors, normal tissue near tumors. MMP-2, MMP-9, VEGF and MVD were checked by immunohistochemical means. RESULTS: Immunohistochemical examination demonstrated an increased expression of MMP2, MMP9, VEGF and MVD in the tumor tissue of the radiation group (P<0.05). Additionally, the expression levels of MMP2 and MMP9 in the tumor tissue were higher than those in the normal tissue near tumor (P<0.05). CONCLUSION: This study showed that irradiation might increased MMP2, MMP9, VEGF and MVD expression in the tumor tissue of mice bearing Lewis lung cancer. Our finding supports a potential way to overcome tumor metastasis induced by radiation if MMP inhibitor and anti-vascularization are combined to irradiation clinically.

Peroxidized cholesterol-induced matrix metalloproteinase-9 activation and its suppression by dietary beta-carotene in photoaging of hairless mouse skin.

The activation of matrix metalloproteinase (MMP)-9 leading to the formation of wrinkle and sagging of skin is an essential step in the skin photoaging on exposure to ultraviolet A (UVA). This study attempted to elucidate the role of peroxidized cholesterol including cholesterol hydroperoxides (Chol-OOHs), primary products of lipid peroxidation in biomembranes, in MMP-9 activation and the effect of dietary beta-carotene in MMP-9 activation. Hairless mice were subjected to periodic UVA irradiation for 8 weeks. The amount of peroxidized cholesterol detected as total hydroxycholesterol in the skin was increased significantly by the exposure. The activity and protein level of MMP-9 were elevated with wrinkling and sagging formation. MMP-9 activity was also enhanced by the intracutaneous injection of Chol-OOHs into the mouse skin. Adding beta-carotene to the diet of the mice during the period of irradiation suppressed the activity and expression of MMP-9 as well as the wrinkling and sagging formation. The amount of cholesterol 5alpha-hydroperoxide, a singlet molecular oxygen oxygenation-specific peroxidized cholesterol, was significantly lowered by the addition of beta-carotene to the diet. These results strongly suggest that Chol-OOHs formed on exposure to UVA contribute to the expression of MMP-9, resulting in photoaging. Dietary beta-carotene prevents the expression of MMP-9, at least partly, by inhibiting photodynamic action involved in the formation of Chol-OOHs.

Vitamin D protects keratinocytes from deleterious effects of ionizing radiation.

BACKGROUND: Radiotherapy can induce severe skin responses that may limit the clinically acceptable radiation dose. The responses include erythema, dry and moist desquamation, erosions and dermal-epidermal blister formation. These effects reflect injury to, and reproductive failure of, epidermal cells and may also be due to dysregulation of the tissue remodelling process caused by excessive proteolytic activity. Calcitriol, the hormonally active vitamin D metabolite, protects keratinocytes from programmed cell death induced by various noxious stimuli. OBJECTIVE: To examine whether calcitriol protects proliferating keratinocytes from the damage inflicted by ionizing radiation under conditions similar to those employed during radiotherapy. METHODS: Autonomously proliferating HaCaT keratinocytes, used as a model for basal layer keratinocytes, were irradiated using a linear accelerator. Cell death was monitored by vital staining, executioner caspase activation, lactic dehydrogenase release and colony formation assay. Induction of matrix metalloproteinase-9 was assessed by gelatinase activity assay and mRNA determination. Levels of specific proteins were determined by immunoblotting. RESULTS: Treatment with calcitriol inhibited both caspase-dependent and -independent programmed cell death occurring within 48 h of irradiation and increased the colony formation capacity of irradiated cells. These effects may be attributable to inhibition of the c-Jun NH(2)-terminal kinase cascade and to upregulation of the truncated antiapoptotic isoform of p63. Treatment with the hormone also attenuated radiation-induced increase in matrix metalloproteinase-9 protein and mRNA levels. CONCLUSIONS: The results of this study suggest that active vitamin D derivatives may attenuate cell death and excessive proteolytic activity in the epidermis due to exposure to ionizing radiation in the course of radiotherapy.

Matrix metalloproteinase-9 inhibition down-regulates radiation-induced nuclear factor-kappa B activity leading to apoptosis in breast tumors.

PURPOSE: Novel strategies are needed to prevent the high mortality rates of several types of cancer. These high rates stem from tumor resistance to radiation therapy, which is thought to result from the induction of matrix metalloproteinases (MMP) and plasminogen activators. In the present study, we show that the modulation of MMP-9 expression, using adenoviral-mediated transfer of the antisense MMP-9 gene (MMP-9 adenoviral construct, Ad-MMP-9), affects breast cancer sensitivity to radiation. EXPERIMENTAL DESIGN: In the present study, we used antisense Ad-MMP-9 to down-regulate the expression of MMP-9 in MDA MB 231 breast cancer cell lines in vitro before irradiation and subsequently incubated cells in hypoxic condition. In vivo studies were done with orthotopic breast tumors, and radiosensitivity was evaluated both in vitro and in vivo. RESULTS: Ad-MMP-9 infection resulted in down-regulation of radiation-induced levels of hypoxia-inducible factor 1 alpha and MMP-9 under hypoxic conditions in MDA MB 231 breast cancer cells. In addition, Ad-MMP-9, in combination with radiation, decreased levels of the transcription factors nuclear factor-kappaB and activator protein 1, both of which contribute to the radioresistance of breast tumors. Finally, the triggering of the Fas-Fas ligand apoptotic cascade, which resulted in the cleavage of PARP-1 and caspase-10, caspase-3, and caspase-7, signifies the efficiency of combined treatment of Ad-MMP-9 and radiation. Treatment with Ad-MMP-9 plus radiation completely regressed tumor growth in orthotopic breast cancer model. CONCLUSIONS: In summary, integrating gene therapy (adenovirus-mediated inhibition of MMP-9) with radiotherapy could have a synergistic effect, thereby improving the survival of patients with breast cancer.

Modulation of matrix metalloproteinases by ultraviolet radiation in the canine cornea.

PURPOSE: To determine whether ultraviolet (UV) radiation can modulate expression and regulation of matrix metalloproteinases (MMP) in the canine cornea and to examine the expression of MMPs in canine chronic superficial keratitis (CSK). METHODS: Immunohistochemistry for MMP-2 and MMP-9 was performed on samples of CSK. In vitro, canine corneal epithelial cell (CEC) and stromal cell cultures were exposed to UV-irradiation. Following 2, 8 or 24 h, cells were harvested. MMP expression was examined by zymography, and RT-PCR was used to examine expression of Slug and Snail. CEC cultures treated with an EGFR inhibitor or a p38 inhibitor were UV-exposed and harvested 24 h later to examine expression of MMPs, Slug and Snail. RESULTS: Canine CSK had increased immunopositivity for both MMP-2 and MMP-9 compared to normal canine corneas. In vitro, CEC and stromal cell cultures exposed to UV showed generally increased expression of MMP-2, -9, Slug, and Snail; this response was dose and time dependent. Inhibition of the EGFR pathway did not prevent increased expression of MMP-2, -9, Slug or Snail in UV-exposed CEC; however, p38 inhibition did attenuate UV induction. CONCLUSIONS: We have found increased expression of MMPs in clinical samples of CSK compared to normal corneas. In addition, we have shown that there is a temporal association and dose dependency between UV exposure and production of MMPs, Slug, and Snail. These findings suggest that overexpression of MMPs due to UV-exposure may be linked to changes in the cornea that allow an influx of inflammatory cells and vascularization.

Visible light induces matrix metalloproteinase-9 expression in rat eye.

Up-regulation of matrix metalloproteinase-9 (MMP-9, gelatinase B) in the nervous system has been demonstrated when excitotoxicity-induced tissue remodeling and neuronal death occurs. Induction of MMP-9 by a natural stimulus has not been observed yet. Using RT-PCR and gelatin-zymography we demonstrated MMP-9 induction at transcriptional and protein levels in different structures of the rat eye following over-stimulation with white light. MMP-9 elevation occurred in the retina without reduction in photoreceptor number or major anatomical reorganization. A transient decrease in electroretinogram b-wave indicated the functional recovery. Retrobulbar injection of a broad-spectrum MMP-inhibitor GM6001, slowed the recovery rate of b-wave amplitude. Even room-light applied to dark-adapted awake animals induced MMP-9 increase in the retina, which suggests a role for MMP-9 in physiological functional plasticity of the nervous system, such as light adaptation. This is the first demonstration of MMP-9 induction by a sensory stimulus.

Effects of two conventional preoperative radiation schedules on anastomotic healing in the rat colon.

BACKGROUND: Preoperative radiotherapy (RT) is an increasingly popular form of adjunct therapy for rectal cancer; however, little is known about its effects on matrix metalloproteinase (MMP) expression in colonic anastomotic healing. METHODS: Wistar rats were irradiated to a total dose of 25 or 40 Gy. Four days after the end of RT, an end-to-end colorectal anastomosis was performed. Animals were sacrificed at 1, 3, and 7 days after the anastomosis. A control group was studied similarly, but was not irradiated. RESULTS: No significant differences were found in peritonitis rate and anastomotic complications. The average bursting pressure and breaking strength were only reduced significantly in the rats irradiated with 40 Gy. However, the concentration and the content of hydroxyproline in anastomotic tissues were unchanged. In irradiated rats, MMP-2 and MMP-9 were significantly increased at 40 Gy, but not at 25 Gy. On the other hand, 25-Gy irradiation induced a smaller increase in the levels of the tissue inhibitors of metalloproteinase-1 compared with the controls. CONCLUSION: Anastomotic strength is adversely affected by high-dose fractionated preoperative RT. In contrast, preoperative RT at 25 Gy in five fractions over 5 days is safe with regard to the maintenance of wound strength in colorectal anastomosis.

Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization.

Mast cells accumulate in tissues undergoing angiogenesis during tumor growth, wound healing, and tissue repair. Mast cells can secrete angiogenic factors such as vascular endothelial growth factor (VEGF). Ionizing irradiation has also been shown to have angiogenic potential in malignant and nonmalignant diseases. We observed that low-dose irradiation fosters mast cell-dependent vascular regeneration in a limb ischemia model. Irradiation promoted VEGF production by mast cells in a matrix metalloproteinase-9 (MMP-9)-dependent manner. Irradiation, through MMP-9 up-regulated by VEGF in stromal and endothelial cells, induced the release of Kit-ligand (KitL). Irradiation-induced VEGF promoted migration of mast cells from the bone marrow to the ischemic site. Irradiation-mediated release of KitL and VEGF was impaired in MMP-9-deficient mice, resulting in a reduced number of tissue mast cells and delayed vessel formation in the ischemic limb. Irradiation-induced vasculogenesis was abrogated in mice deficient in mast cells (steel mutant, Sl/Sl(d) mice) and in mice in which the VEGF pathway was blocked. Irradiation did not induce progenitor mobilization in Sl/Sl(d) mice. We conclude that increased recruitment and activation of mast cells following irradiation alters the ischemic microenvironment and promotes vascular regeneration in an ischemia model. These data show a novel mechanism of neovascularization and suggest that low-dose irradiation may be used for therapeutic angiogenesis to augment vasculogenesis in ischemic tissues.

Radiation induced MMP expression from rectal cancer is short lived but contributes to in vitro invasion.

AIMS: Matrix metalloproteinase (MMP) activity is increased after radiation. The aims of this study were to assess the time course of this increase and its effects on malignant cell invasion. METHODS: Colorectal cancer (HCT 116, LoVo, C 170 HM 2, CaCO-2), fibroblast (46-BR.IGI, CCD-18 Co) and fibrosarcoma (HT1080) cell lines were irradiated at 4 gray (4 Gy) and matrix metalloproteinase gene and protein expression examined over a 96 h period by real time polymerase chain reaction and gelatin zymography. Invasion was assessed on Matrigel. Human rectal tumour MMP expression was compared before and after long course radiotherapy. RESULTS: Radiation increased MMP gene expression of tumour cell lines, and resulted in increased MMP protein activity in the HT1080 line. HT1080 and HCT 116 in monoculture and LoVo in co-culture were more invasive after radiation at 48 h in vitro, but long course radiotherapy did not result in a consistent increase in MMP expression from human rectal tumour biopsies. CONCLUSIONS: Radiation results in increased MMP expression for a limited time period. This results in an early increase in cell line invasion. Further clinical research is required to clarify if MMP inhibition given perioperatively following radiotherapy decreases local recurrence rates.