Basement Membrane Proteins Collagen Type Research Articles

ANTI-COLLAGEN TYPE IV ANTIBODIES AND THE DEVELOPMENT OF MICROVASCULAR COMPLICATIONS IN DIABETIC PATIENTS WITH ARTERIAL HYPERTENSION

S U M M A R Y Background and Aims: Thickening of basement membrane in capillaries and small vessels is a well-known finding and important in the progression of diabetic microangiopathy. Patients with diabetes mellitus and arterial hypertension are at higher risk of vascular disease. Material and methods: To monitor the metabolism of the basement membrane protein collagen type IV (CIV) in type 2 diabetes mellitus (T2DM), serum levels of antibodies to CIV (ACIV) IgG, IgM and IgA were measured using an ELISA method in 93 patients with type 2 diabetes mellitus and arterial hypertension (AH) (mean age 61,4±11,3 years, diabetes duration 9,88±3,12 years; hypertension duration 9,28±4,98). These values were compared to serum antibodies to CIV in 42 age and sex matched controls. Diabetics were divided in two groups according to presenceGroup 1 (n=67) or absenceGroup 2 (n=26) of microangiopathy. Results: Patients with T2DM and AH showed statistically significant higher levels of ACIV IgG in comparison to healthy controls (0.30±0.12 vs. 0.21±0.08) (p=0.0001). Group 1 showed significantly hihger levels of ACIV IgG than Group 2 (0.32±0.13vs. 0.24±0.08) (p=0.009) and healthy controls (0.32±0.13vs. 0.21±0.08) (p=0.0001). ACIV IgG are statistically significant higher in diabetics with retinopathy than this without (0.33±0.10 vs. 0.26±0.13) (ð=0.04). ACIV IgG correlates with diabetes duration (r=0.49); (p=0.0004), retinopathy (r=0.20); (p=0.05) and BMI (r=-0.24); (p=0.05). Serum ACIV IgM and IgA levels in patients with T2DM and AH were lower than these in controls, but the differences are not statistically significant. Conclusion: Our study showed a relationship between elevation of serum levels of ACIV IgG in diabetics and development of microangiopathy.

Hydrogel encapsulation environments functionalized with extracellular matrix interactions increase islet insulin secretion

The individual and synergistic effects of extracellular matrix interactions on isolated islet function in culture were investigated within a three-dimensional poly(ethylene glycol) (PEG) hydrogel encapsulation environment. First, we observed similar glucose-stimulated insulin secretion from unencapsulated murine islets and islets photoencapsulated in PEG gels. Then islets were encapsulated in gels containing the basement membrane proteins collagen type IV and laminin, individually and in combination, at a total protein concentration of 100μg/ml, and islet insulin secretion in response to high glucose was measured over time. Specific laminin interactions were investigated via islet encapsulation with adhesive peptide sequences found in laminin as well as via functional blocking of cell surface receptors known to bind laminin. Over 32days, islet interactions with collagen type IV and laminin localized within the three-dimensional extracellular environment contributed to two-fold and four-fold increases in insulin secretion, respectively, relative to islets encapsulated without matrix proteins. Hydrogel compositions containing both matrix proteins and > 75% laminin further increased islet insulin secretion to approximately six-fold that of islets encapsulated in the absence of matrix proteins. Encapsulation with the peptide sequence IKVAV resulted in increased islet insulin secretion, but not to the extent observed in the presence of whole laminin. Increased insulin secretion in the presence of laminin was eliminated when islets were exposed to functionally blocking anti-α 6 integrin antibody prior to islet encapsulation with laminin. Our results demonstrate the potential of specific matrix interactions within an islet encapsulation microenvironment to promote encapsulated islet function.

Serum antibodies to collagen type IV and development of diabetic vascular complications in children with type 1 (insulin-dependent) diabetes mellitus: A longitudinal study

Thickening of basement membrane in capillaries and small vessels is a well-known finding and important in the progression of diabetic microangiopathy. To monitor the metabolism of the basement membrane protein collagen type IV (CIV) in diabetes mellitus, serum levels of IgG, IgM and IgA to CIV were measured using an ELISA method in 28 children with Type 1 diabetes mellitus over a period of 6 years. These values were compared to serum antibodies to CIV in 24 age- and sex-matched controls. At the end of the study, 11 children had diabetic microangiopathy. IgG to CIV was associated with age ( r=.33, P=.026), diabetes duration ( r=.32, P=.021), HbA1c ( r=.31, P=.019), microalbuminuria ( r=.32, P=.022) and anti-AGE antibodies ( r=.47, P=.0007). IgM to CIV correlated with age ( r=.46, P=.001), diabetes duration ( r=.45, P=.001), HbA1c ( r=.26, P=.038) and anti-AGE antibodies ( r=.26, P=.038) and IgA to CIV with triglycerides ( r=.29, P=.038) and anti-AGE antibodies ( r=.44, P=.0025). We suggest that serum levels of IgG to CIV can be used as a marker for the development of diabetic microalbuminuria.

Detection of serum collagen type IV in children with type 1 (insulin-dependent) diabetes mellitus--a longitudinal study.

The basement membrane is a major focus of scientific interest because of its role in a variety of diseases. In diabetes mellitus, a thickening of the capillary basement membrane results in microangiopathic lesions. To monitor the metabolism of the basement membrane protein collagen type IV (CIV) in diabetes mellitus, serum levels of CIV were measured using an enzyme-linked immunosorbent assay (ELISA) method in 28 children with type 1 diabetes mellitus over a period of 6 years. These values were compared to serum CIV levels in 24 age and sex matched controls. In the first 3 years, serum CIV levels were normal. In the 4th year, 1 patient and in years 5 and 6, 4 patients had increased CIV serum levels. At the end of the investigation, 3 children had developed retinopathy, 6 microalbuminuria, and 2 both microalbuminuria and retinopathy. Only those patients with microalbuminuria had increased CIV serum levels. In conclusion, we suggest that CIV serum levels can be used as a marker for the development of diabetic microalbuminuria.

Arachidonic Acid Activates Mitogen-activated Protein (MAP) Kinase-activated Protein Kinase 2 and Mediates Adhesion of a Human Breast Carcinoma Cell Line to Collagen Type IV through a p38 MAP Kinase-dependent Pathway

Adhesion of metastatic human mammary carcinoma MDA-MB-435 cells to the basement membrane protein collagen type IV can be activated by treatment with arachidonic acid. We initially observed that this arachidonic acid-mediated adhesion was inhibited by the tyrosine kinase inhibitor genistein. Therefore, we examined the role of the mitogen-activated protein (MAP) kinase family tyrosine phosphorylation-regulated pathways in arachidonic acid-stimulated cell adhesion. Arachidonic acid stimulated the phosphorylation of p38, the activation of MAP kinase-activated protein kinase 2 (MAPKAPK2, a downstream substrate of p38), and the phosphorylation of heat shock protein 27 (a downstream substrate of MAP kinase-activated protein kinase 2). Treatment with the p38 inhibitor PD169316 completely and specifically inhibited arachidonic acid-mediated cell adhesion to collagen type IV. p38 activity was specifically associated with arachidonic acid-stimulated adhesion; this was demonstrated by the observation that 12-O-tetradecanoylphorbol 13-acetate-activated cell adhesion was not blocked by inhibiting p38 activity. Extracellular signal-regulated protein kinases (ERKs) 1 and 2 were also activated by arachidonic acid; however, cell adhesion to collagen type IV was not highly sensitive to PD98059, an inhibitor of MAP kinase kinase/ERK kinase 1 (MEK1) that blocks activation of the ERKs. c-Jun NH(2)-terminal kinase was not activated by arachidonic acid treatment of these cells. Together, these data suggest a novel role for p38 MAP kinase in regulating adhesion of breast cancer cells to collagen type IV.

Transforming growth factor-β1 stimulates the synthesis of basement membrane proteins laminin, collagen type IV and entactin in rat liver sinusoidal endothelial cells

Background/Aims: It is suggested that during fibrogenesis as well as during carcinogenesis of the liver, the hepatic microvascular phenotype is transformed fromsinusoids - which lack a basement membrane - into continuous capillaries which rest on a basement membrane. As transforming growth factor (TGF)-β1 seems to be the most effective mediator in the stimulation of matrix protein synthesis, we were interested in the modulation of basement membrane proteins collagen type IV, laminin, and entactin expression by TGF-β1 in liver sinusoidal endothelial cells (SECs), especially since a stimulation of the synthesis of collagen type IV but not of entactin and laminin by TGF-β1 has been demonstrated in a fibrosarcoma cell line.Methods: The synthesis of the basement membrane (BM) proteins entactin, laminin, and collagen type IV and of the extracellular matrix (ECM) proteins tenascin and fibronectin with or without TGF-β1 - stimulation was analyzed by immunostaining, immunoprecipitation of endogenously labeled proteins and Northern blot analysis of total RNA extracted from freshly isolated or cultured SECs from rat or guinea pig livers. Furthermore, SECs were isolated from acutely and chronically CCl4-damaged rat livers and were analyzed for matrix protein expression.Results: SECs were adherent 24 h after isolation and formed confluent monolayers on day 4 of primary culture. Specific immunoprecipitates and specific transcripts for the BM proteins entactin, laminin, and collagen type IV and for ECM proteins tenascin and fibronectin were detectable in freshly isolated or cultured SECs. The synthesis of all tested BM proteins and ECM proteins was stimulated at least 3-fold by TGF-β1. In SECs isolated after CCl4-induced acute and chronic liver damage, increased levels of matrix protein transcripts were detectable.Conclusions: The stimulation of the synthesis of all BM-proteins by TGF-β1 in vitro and the accumulation of ECM transcripts in SECs isolated from CCl4-treated livers, suggests that SECs are involved in the formation of a basement membrane during the“capillarization” of the sinusoids during liver disease.

P53 protein expression in human breast cancer: an immunohistochemical study including correlation with steroid receptor status, proliferation indices, collagen type IV, laminin, C-erbB-2 oncoprotein and Cathepsin D

In a study of 83 breast cancer patients, tumour p53 protein expression was studied by inimunohistochemistry using the monoclonal antibody DO-7. Its relationship with several prognostic variables such as: steroid receptors, cerbB-2 oncogene, Cathepsin D (CD), proliferative indices Ki-67 and PCNA, as well as with expression of the basement membrane proteins collagen type IV and laminin was investigated. p53 protein expression was detected in 51.8% of tumours (43 of 83). Expression was independent of c-erbB-2 overexpression, proliferating index as measured by Ki-67, CD expression and steroid receptor status. Also, p53 accumulation was not correlated with any other conventional clinicopathological parameter considered in this study. A significant positive correlation was found between p53 protein expression and collagen type IV expression (P < 0.05), and PCNA-LI (P < 0.05). These data suggest that p53 protein could play an important role in the expansion of breast cancer and in proliferative activity, as determined by PCNA-LI. The results do not resolve whether detectable p53 protein expression represents a random product in tumours with high collagen type IV expression or whether it is an important feature of the malignant phenotype in the expansion of breast cancer.

Mouse polymorphonuclear granulocyte binding to extracellular matrix molecules involves beta 1 integrins.

The mechanism of adhesion of purified mouse polymorphonuclear granulocytes (PMN) to extracellular matrix proteins characteristic of basement membranes and the interstitium has been investigated and compared with the adhesion of a mouse progranulocytic cell line, 32DC13, and a mouse monocytic cell line, WEHI 78/24. All three cell types bound specifically to fibronectin and vitronectin to different degrees under different cellular activation states. 32DC13 bound to fibronectin and vitronectin strongly, and this binding increased upon cellular activation with phorbol 12-myristate-13-acetate (PMA) but not with formyl-Met-Leu-Phe. Only 32DC13 showed significant binding to laminin-1. By contrast, WEHI 78/24 and PMN bound only fibronectin and vitronectin; this binding was weak and was altered only marginally upon activation with PMA. In the case of WEHI 78/24, a slight increase in adhesion both to fibronectin and to vitronectin was observed after cellular activation with PMA, while PMN adhesion to both substrates was slightly reduced. The mechanism of binding to fibronectin and vitronectin was similar in the three cell types. The integrin alpha5 beta1 mediated fibronectin adhesion, demonstrating for the first time the existence of a functionally active beta1 integrin on mouse PMN. Vitronectin binding was mediated by alpha(v) beta3, as demonstrated by the ability of alpha(v)-specific cyclic L-Arg-L-Gly-L-Asp-D-Phe-L-Val (RGDfV) peptide (EMD66203), and anti-beta3 antibody to inhibit cell adhesion. 32DC13 adhesion to laminin-1 was via the alpha6 beta1 integrin. None of the three cell types tested bound to the basement membrane proteins collagen type IV and perlecan, or to the interstitial stromal constituents tenascin, collagen types I, V and VI. Interestingly, perlecan and collagen type IV were found to repel all three cell types. The relative inability of PMN, WEHI 78/24, and 32DC13 to bind to extracellular matrix proteins characteristic of basement membranes and their ability to bind inflammatory markers of the interstitium is discussed with respect to leukocyte extravasation processes.

The extracellular matrix in pigmented skin lesions: an immunohistochemical study.

In recent years the interaction between tumour cells and the surrounding extracellular matrix in the process of tumour development, invasion and metastasis has been a focus of interest. We studied frozen sections of nine naevocellular naevi (junctional, compound and intradermal), 40 dysplastic naevi, six pagetoid in situ melanomas and 12 superficial spreading melanomas in order to determine the expression of: the basement membrane proteins collagen type IV and laminin, the interstitial collagen types I, III and VI, and fibronectin and tenascin. An indirect immunoperoxidase technique was used. In the various stages of melanocytic tumour progression we observed: 1 loss of type IV collagen and laminin within dermal melanocytic cell nests; 2 de novo expression of basement membrane type IV collagen and increased expression of the interstitial collagen types I, III and VI, as well as tenascin and fibronectin in the dermal stroma surrounding dysplastic naevus cells and melanoma cells; 3 presence of extracellular matrix components in close association with intra-epidermally located invading atypical melanocytes. These data demonstrate the complex alterations of the composition of the extracellular matrix from bland naevi through lesions with progressive atypia to invasive melanoma. The changes described result in a molecular environment which melanocytes with an altered adhesion molecule profile are able to invade.

High‐affinity binding of the basement membrane protein collagen type IV to the crystalline virulence surface protein array of Aeromonas salmonicida

The surface of the fish pathogen Aeromonas salmonicida is covered by a paracrystalline array (the A-layer) which is a virulence factor for the organism. Quantification of the ability of A. salmonicida cells to bind collagen types I and IV in a 125I-radiolabelled liquid-phase assay showed that A-layer-positive cells bound high levels of collagen type IV, but significantly lower levels of collagen type I. Collagen type IV binding was confirmed using non-radiolabelled enzyme-linked immunosorbent assays. 125I-Collagen type IV binding was rapid, specific, saturable, high affinity, and essentially irreversible by unlabelled collagen type IV. The A-layer was responsible for collagen type IV binding because binding was inactivated by selective removal of the A-layer at pH 2.2, and neither isogenic A-layer-deficient A. salmonicida mutants nor strains of Aeromonas hydrophila possessing a morphologically similar paracrystalline array bound this basement membrane protein.

High-affinity binding of the basement membrane proteins collagen type IV and laminin to the gastric pathogen Helicobacter pylori

The ability of 16 isolates of the human gastroduodenal pathogen Helicobacter pylori to bind 125I-radiolabelled tissue proteins was quantitated by liquid-phase assay. While capable of binding generally low levels of collagen types I and II, vitronectin, and fibronectin (average binding, 8%; highest binding, 23%), the various H. pylori isolates were good binders of the basement membrane proteins collagen type IV and laminin (average binding, 27%; highest binding, 60%). Campylobacter species tested bound lower levels of collagen type IV and laminin (average binding, 12%; highest binding, 17%). Trypsin and proteinase K treatment of H. pylori cells markedly reduced the binding of collagen type IV and laminin, as did heat treatment, suggesting that the binding of basement membrane proteins is mediated by bacterial surface proteins. Binding of both basement membrane proteins was rapid and saturable. 125I-collagen type IV binding to H. pylori 915 was inhibited by preincubation with unlabelled collagen type IV but was not inhibited by laminin or a number of other proteins. Once bound, radiolabelled collagen type IV but was not displaced by an excess of unlabelled collagen type IV, indicating that the binding interaction was of high affinity. Binding of laminin was partially reversible, and analysis in a solid-phase nonradiolabel assay showed that the interaction was of high affinity, with a Kd of 7.9 nM. This interaction was affected by salt, indicating the presence of a hydrophobic component in the ability of H. pylori to bind laminin.