Type IV collagen is a novel DEJ biomarker that is reduced by radiotherapy.

The dental basement membrane (BM) is composed of collagen types IV, VI, VII, and XVII, fibronectin, and laminin and plays an inductive role in epithelial-mesenchymal interactions during tooth development. The BM is degraded and removed during later-stage tooth morphogenesis; however, its original position defines the location of the dentin-enamel junction (DEJ) in mature teeth. We recently demonstrated that type VII collagen is a novel component of the inner enamel organic matrix layer contiguous with the DEJ. Since it is frequently co-expressed with and forms functional complexes with type VII collagen, we hypothesized that type IV collagen should also be localized to the DEJ in mature human teeth. To identify collagen IV, we first evaluated defect-free erupted teeth from various donors. To investigate a possible stabilizing role, we also evaluated extracted teeth exposed to high-dose radiotherapy--teeth that manifest post-radiotherapy DEJ instability. We now show that type IV collagen is a component within the morphological DEJ of posterior and anterior teeth from individuals aged 18 to 80 yr. Confocal microscopy revealed that immunostained type IV collagen was restricted to the 5- to 10-µm-wide optical DEJ, while collagenase treatment or previous in vivo tooth-level exposure to > 60 Gray irradiation severely reduced immunoreactivity. This assignment was confirmed by Western blotting with whole-tooth crown and enamel extracts. Without reduction, type IV collagen contained macromolecular α-chains of 225 and 250 kDa. Compositionally, our results identify type IV collagen as the first macromolecular biomarker of the morphological DEJ of mature teeth. Given its network structure and propensity to stabilize the dermal-epidermal junction, we propose that a collagen-IV-enriched DEJ may, in part, explain its well-known fracture toughness, crack propagation resistance, and stability. In contrast, loss of type IV collagen may represent a biochemical rationale for the DEJ instability observed following oral cancer radiotherapy.

Effects of pulsed and sinusoidal electromagnetic fields on MMP-2, MMP-9, collagen type IV and E-cadherin expression levels in the rat kidney: an immunohistochemical study.

OBJECTIVE: To investigate the role of extremely low frequency pulsed and sinusoidal electromagnetic fields on kidney tissues. STUDY DESIGN: Twenty-seven male Wistar albino rats were used. The rats were divided into 3 groups (n = 9): control group, sinusoidal electromagnetic field (SEMF) group, and pulsed electromagnetic field (PEMF) group. The SEMF and PEMF groups (pulse time 25 microsn, pulse frequency 50 Hz) were subjected to 1.5 mT, 50 Hz, exposure 6 hours a day, 5 days a week for 28 days in methacrylate boxes. Formalin-fixed, paraffin-embedded kidney tissue sections were stained with hematoxylin-eosin, Gomori and periodic acid-Schiff. In addition, matrix metalloproteinase-2 (MMP-2) and 9 (MMP-9), E-cadherin and collagen type IV expression levels were examined immunohistochemically. RESULTS: Thickening of glomerular basement membranes was evident in electromagnetic fields, especially in the SEMF group. In addition, expression levels of E-cadherin were decreased with electromagnetic field (EMF) exposure. The expression level of MMP-9 increased, and MMP-2 and collagen type IV expression levels were not altered with EMF exposure. CONCLUSION: Both EMFs changed the molecular component of the kidney adversely.

Tissue-engineered mucosa is a suitable model to quantify the acute biological effects of ionizing radiation.

The aim of this study was to evaluate the suitability of tissue-engineered mucosa (TEM) as a model for studying the acute effects of ionizing radiation (IR) on the oral mucosa. TEM and native non-keratinizing oral mucosa (NNOM) were exposed to a single dose of 16.5Gy and harvested at 1, 6, 24, 48, and 72h post-irradiation. DNA damage induced by IR was determined using p53 binding protein 1 (53BP1), and DNA repair was determined using Rad51. Various components of the epithelial layer, basement membrane, and underlying connective tissue were analyzed using immunohistochemistry. The expression of cytokines interleukin-1ß (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) was analyzed using an enzyme-linked immunosorbent assay. The expression of DNA damage protein 53BP1 and repair protein Rad51 were increased post-irradiation. The expression of keratin 19, vimentin, collage type IV, desmoglein 3, and integrins α6 and ß4 was altered post-irradiation. Proliferation significantly decreased at 24, 48, and 72h post-irradiation in both NNOM and TEM. IR increased the secretion of IL-1ß, whereas TGF-ß1 secretion was not altered. All observed IR-induced alterations in TEM were also observed in NNOM. Based on the similar response of TEM and NNOM to IR we consider our TEM construct a suitable model to quantify the acute biological effects of IR.

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.

Low-energy helium-neon laser induces melanocyte proliferation via interaction with type IV collagen: visible light as a therapeutic option for vitiligo.

BACKGROUND: The treatment of vitiligo remains a challenge for clinical dermatologists. We have previously shown that the helium-neon laser (He-Ne laser, 632.8 nm) is a therapeutic option for treatment of this depigmentary disorder. OBJECTIVES: Addressing the intricate interactions between melanocytes, the most important cellular component in the repigmentation scheme of vitiligo, and their innate extracellular matrix collagen type IV, the current study aimed to elucidate the effects of the He-Ne laser on melanocytes. METHODS: Cultured melanocytes were irradiated with the He-Ne laser. Relevant biological parameters including cell attachment, locomotion and growth were evaluated. In addition, the potentially involved molecular pathways were also determined. RESULTS: Our results show that in addition to suppressing mobility but increasing attachment to type IV collagen, the He-Ne laser stimulates melanocyte proliferation through enhanced alpha2beta1 integrin expression. The expression of phosphorylated cyclic-AMP response element binding protein (CREB), an important regulator of melanocyte growth, was also upregulated by He-Ne laser treatment. Using a specific mitochondrial uncoupling agent [carbonyl cyanide m-chlorophenyl-hydrazone (CCCP)], the proliferative effect of the He-Ne laser on melanocytes was abolished and suppression of melanocyte growth was noted. CONCLUSIONS: In summary, we have demonstrated that the He-Ne laser imparts a growth stimulatory effect on functional melanocytes via mitochondria-related pathways and proposed that other minor pathways including DNA damage may also be inflicted by laser treatment on irradiated cells. More importantly, we have completed the repigmentation scheme of vitiligo brought about by He-Ne laser light in vitro and provided a solid theoretical basis regarding how the He-Ne laser induces recovery of vitiligo in vivo.