Morphological and quantitative study of collagen fibers in healthy and diseased human gingival tissues.

OBJECTIVES: This study aimed to evaluate types I, III and IV collagen in healthy gingival tissue and to compare them to gingival tissues suffering from chronic gingivitis and chronic periodontitis. MATERIALS AND METHODS: Thirty-two man patients were selected. The patients belonged to three diagnostic categories: healthy gingiva (HG), chronic gingivitis (CG) and chronic periodontitis (CP), based on clinical and radiographical criteria. Gingival tissue samples were obtained from patients who underwent periodontal surgery procedures. Hematoxylin and Eosin (HE), Picrosirius red, indirect immunofluorescence by confocal microscopy and quantitative analyses were performed to identify the presence and location of types I, III and IV collagen. Statistical significance was verified using the Kruskal-Wallis test. RESULTS: Samples from HG group showed thick collagen fibers arranged in a parallel pattern. Samples from CG group showed dilated blood vessels; collagen fibers and inflammatory cells were found dispersed throughout the tissue. Samples from CP group showed the extracellular matrix severely damaged, disorganized collagen fibers and large amount of inflammatory cells. The HG group showed an apparent higher expression of type I collagen, when compared to tissues with CG and CP, however no statistical differences were detected (p=0.064). The types III and IV collagen fibers showed no difference in expression in tissues with gingivitis and periodontitis. CONCLUSIONS: Following the periodontal disease there was a morphological destruction of the extracellular matrix with lower expression of collagen, which led to a change in tissue architecture that might compromise its functional capacity. There were differences in type I collagen expression among healthy, chronic gingivitis and chronic periodontitis tissue samples.

Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome.

PURPOSE: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. METHODS: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c.3277C>T, a nonsense mutation, and c.3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. RESULTS: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. CONCLUSIONS: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

Collagen XVIII/endostatin is associated with the epithelial-mesenchymal transformation in the atrioventricular valves during cardiac development.

Type XVIII collagen is a multidomain protein that contains cleavable C-terminal NC1 and endostatin fragments, which have been shown to either induce or inhibit cell migration. Endostatin is being intensely studied because of its anti-angiogenic activity. Three variants of type XVIII collagen have been reported to be distributed in epithelial and endothelial basement membranes in a tissue-specific manner. The single gene encoding collagen XVIII is on chromosome 21 within the region associated with the congenital heart disease phenotype observed in Down's syndrome. In this study, we investigated the expression pattern of collagen XVIII in embryonic mouse hearts during formation of the atrioventricular (AV) valves. We found that collagen XVIII is localized not only in various basement membranes but is also highly expressed throughout the connective tissue core of the endocardial cushions and forming AV valve leaflets. It was closely associated with the epithelial-mesenchymal transformation of endothelial cells into mesenchymal cushion tissue cells and was localized around these cells as they migrated into the cardiac jelly to form the initial connective tissue elements of the valve leaflets. However, after embryonic day 17.5 collagen XVIII expression decreased rapidly in the connective tissue and thereafter remained detectable only in the basement membranes of the endothelial layer covering the leaflets. The staining pattern observed within the AV endocardial cushions suggests that collagen XVIII may have a role in cardiac valve morphogenesis. These results may help us to better understand normal heart development and the aberrant mechanisms that cause cardiac malformations in Down's syndrome.

Collagen XVIII and fibronectin involvement in bullous scleroderma.

BACKGROUND: Endostatin, an anti-angiogenic C-terminal fragment of collagen XVIII, has been recently reported to play a role in scleroderma pathogenesis, but collagen XVIII immunohistochemistry in scleroderma skin has still not been performed. Bullous scleroderma, a rare form of scleroderma, may have altered angiogenic and lymphangiogenic characteristics. OBJECTIVE: Our aim is to report a rare case of bullous scleroderma, studying the presence of fibronectin and collagens type I, III and XVIII in sclerodermic skin. METHODS: We describe the progression of bullous scleroderma in a 67-year-old patient since the first symptoms. Histological and immunohistochemical aspects of skin biopsies are compared to normal skin from a patient without scleroderma and are correlated with the pathogenesis of the disease. Indirect immunofluorescence measured by laser confocal microscopy allows quantitative determination of fibronectin and collagens type I, III and XVIII. RESULTS AND CONCLUSIONS: Dermo-epidermal cleavage, fibrosis and inflammation are the main histological findings. The dermal distribution and amounts of collagens and in the scleroderma patient are similar to normal skin. Conversely, both fibronectin and collagen XVIII are increased in scleroderma skin, suggesting their involvement in the pathogenesis of bullous scleroderma.

Structurally altered basement membranes and hydrocephalus in a type XVIII collagen deficient mouse line.

Type XVIII collagen/endostatin is known to be crucial for the eye, as witnessed by severe eye defects in Knobloch syndrome patients with mutations in this collagen and in Col18a1(-/-) mice. We show here that in a specific C57BL background, 20% of the Col18a1(-/-) mice developed hydrocephalus, and dilation of the brain ventricles was observed by MRI in all of the mutant mice. Significant broadening was observed in the epithelial basement membrane (BM) of the choroid plexuses (CP), its width being 86.4+/-10.52 nm, compared with 61.4+/-6.05 nm in wild-type mice. The CP epithelial cell morphology was balloon-shaped rather than cuboidal, and the microvilli of the apical surface of the CP epithelium contained more vacuoles in the null mice than in the wild-type, as also did the CP epithelial cells, which is suggestive of alterations in cerebrospinal fluid production. Analysis of BMs elsewhere in the body revealed a broadened epidermal BM in the Col18a1(-/-) mice, but this did not result in any apparent functional deficiencies. Moreover, markedly broadened BMs were found in the atrioventricular valves of the heart and in the kidney tubules, whereas the glomerular mesangial matrix of the kidneys was expanded in the mutant mice and serum creatinine levels were elevated, indicating alterations in kidney filtration capacity. We thus suggest that type XVIII collagen is a structurally important constituent of BMs, and that its absence can result in a variety of phenotypic alterations.