Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

Low transformation growth factor-β1 production and collagen synthesis correlate with the lack of hepatic periportal fibrosis development in undernourished mice infected with Schistosoma mansoni

Undernourished mice infected (UI) submitted to low and long-lasting infections by Schistosoma mansoni are unable to develop the hepatic periportal fibrosis that is equivalent to Symmers’ fibrosis in humans. In this report, the effects of the host’s nutritional status on parasite (worm load, egg viability and maturation) and host (growth curves, biology, collagen synthesis and characteristics of the immunological response) were studied and these are considered as interdependent factors influencing the amount and distribution of fibrous tissue in hepatic periovular granulomas and portal spaces. The nutritional status of the host influenced the low body weight and low parasite burden detected in UI mice as well as the number, viability and maturation of released eggs. The reduced oviposition and increased number of degenerated or dead eggs were associated with low protein synthesis detected in deficient hosts, which likely induced the observed decrease in transformation growth factor (TGF)-β1 and liver collagen. Despite the reduced number of mature eggs in UI mice, the activation of TGF-β1 and hepatic stellate cells occurred regardless of the unviability of most miracidia, due to stimulation by fibrogenic proteins and eggshell glycoproteins. However, changes in the repair mechanisms influenced by the nutritional status in deficient animals may account for the decreased liver collagen detected in the present study.

Early onset collagen VI myopathies: Genetic and clinical correlations.

OBJECTIVE: Mutations in the genes encoding the extracellular matrix protein collagen VI (ColVI) cause a spectrum of disorders with variable inheritance including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate phenotypes. We extensively characterized, at the clinical, cellular, and molecular levels, 49 patients with onset in the first 2 years of life to investigate genotype-phenotype correlations. METHODS: Patients were classified into 3 groups: early-severe (18%), moderate-progressive (53%), and mild (29%). ColVI secretion was analyzed in patient-derived skin fibroblasts. Chain-specific transcript levels were quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and mutation identification was performed by sequencing of complementary DNA. RESULTS: ColVI secretion was altered in all fibroblast cultures studied. We identified 56 mutations, mostly novel and private. Dominant de novo mutations were detected in 61% of the cases. Importantly, mutations causing premature termination codons (PTCs) or in-frame insertions strikingly destabilized the corresponding transcripts. Homozygous PTC-causing mutations in the triple helix domains led to the most severe phenotypes (ambulation never achieved), whereas dominant de novo in-frame exon skipping and glycine missense mutations were identified in patients of the moderate-progressive group (loss of ambulation). INTERPRETATION: This work emphasizes that the diagnosis of early onset ColVI myopathies is arduous and time-consuming, and demonstrates that quantitative RT-PCR is a helpful tool for the identification of some mutation-bearing genes. Moreover, the clinical classification proposed allowed genotype-phenotype relationships to be explored, and may be useful in the design of future clinical trials.

High-density collagen gel tubes as a matrix for primary human bladder smooth muscle cells.

Tissue-engineered grafts for the urinary tract are being investigated for the potential treatment of several urologic diseases. These grafts, predominantly tubular-shaped, usually require in vitro culture prior to implantation to allow cell engraftment on initially cell-free scaffolds. We have developed a method to produce tubular-shaped collagen scaffolds based on plastic compression. Our approach produces a ready cell-seeded graft that does not need further in vitro culture prior to implantation. The tubular collagen scaffolds were in particular investigated for their structural, mechanical and biological properties. The resulting construct showed an especially high collagen density, and was characterized by favorable mechanical properties assessed by axial extension and radial dilation. Young modulus in particular was greater than non-compressed collagen tubes. Seeding densities affected proliferation rate of primary human bladder smooth muscle cells. An optimal seeding density of 10(6) cells per construct resulted in a 25-fold increase in Alamar blue-based fluorescence after 2 wk in culture. These high-density collagen gel tubes, ready seeded with smooth muscle cells could be further seeded with urothelial cells, drastically shortening the production time of graft for urinary tract regeneration.

Production of a product similar to gelatin from chicken feet collagen

Chicken feet can be used as an alternative source of collagen for the development of new products. In this sense, the aim of this study was the production of a product similar to gelatin from collagen extracted from chicken feet and the evaluation of sensory quality. The products were produced in two distinct flavors, with grape flavor called GU and pineapple flavor called GA. Subsequently, we compared these formulations with gelatin of a trademark established in the market. We used in the verification of sensory acceptability of products a hedonic scale of 9 points and the availability of consuming the product by 30 untrained tasters. According to the results, all formulations showed good levels of acceptability, indicating the collagen from chicken feet as an alternative source of high quality in the production of gelatin.

Distribution of collagen types I, III, and IV in gastric tissue of marmosets (Callithrix spp., Callitrichidae: Primates)

Extracellular matrix (ECM) components such as fibrillar collagens play a fundamental role in wound repair and have also been studied in association with the gastric ulcer healing process in gastroenterology. Nevertheless, there have been no studies in the literature to date regarding the description and characterization of ECM components, neither in normal nor in injured gastric tissue of primate species. The objective of this study was to investigate the expression of gastric collagen types I, III, and IV in marmosets (Callithrix sp.). Histological specimens from the stomach of 6 Callithrix jacchus, 12 C. kuhli, and 12 C. geoffroyi were evaluated. The specimens were immunostained with anti-types I and III collagen polyclonal antibodies and anti-type IV collagen monoclonal antibody. Collagen types I and III were detected in the submucosa and lamina propria between the mucosal glands while collagen type IV was detected in the muscularis mucosae, muscular layers, blood vessels, and gastric mucosa between the mucosal glands. It is hoped that these findings can contribute to future studies on the gastric extracellular matrix components in primates and to comparative studies in the area of gastroenterology.

Collagen arrangement in hepatic granuloma in mice infected with Schistosoma mansoni: dependence on fiber radiation centers

The collagen structure of isolated and in situ liver granuloma from Swiss Webster mice infected with Schistosoma mansoni was sequentially and three-dimensionally analyzed during different times of infection (early acute, acute, transitional acute-chronic, and chronic phases) by laser scanning confocal microscopy and electron scanning variable vacuum microscopy. The initial granuloma structure is characterized by vascular collagen residues and by anchorage points (or fiber radiation centers), from where collagenous fibers are angularly shed and self-assembled. During the exudative-productive stage, the self-assembly of these fibers minimizes energy and mass through continuous tension and focal compression. The curvature or angles between collagen fibers probably depends on the fibroblastic or myofibroblastic organization of stress fibers. Gradually, the loose unstable lattice of the exudative-productive stage transforms into a highly packed and stable architecture as a result of progressive compactness. The three-dimensional architecture of granulomas provides increased tissue integrity, efficient distribution of soluble compounds and a haptotactic background to the cells.

Bone mineral density in young women of the city of São Paulo, Brazil: correlation with both collagen type I alpha 1 gene polymorphism and clinical aspects

Osteoporosis is a multifactorial disease with great impact on morbidity and mortality mainly in postmenopausal women. Although it is recognized that factors related to life-style and habits may influence bone mass formation leading to greater or lower bone mass, more than 85% of the variation in bone mineral density (BMD) is genetically determined. The collagen type I alpha 1 (COLIA1) gene is a possible risk factor for osteoporosis. We studied a population of 220 young women from the city of São Paulo, Brazil, with respect to BMD and its correlation with both COLIA1 genotype and clinical aspects. The distribution of COLIA1 genotype SS, Ss and ss in the population studied was 73.6, 24.1 and 2.3%, respectively. No association between these genotypes and femoral or lumbar spine BMD was detected. There was a positive association between lumbar spine BMD and weight (P<0.0001), height (P<0.0156), and body mass index (BMI) (P<0.0156), and a negative association with age at menarche (P<0.0026). There was also a positive association between femoral BMD and weight (P<0.0001), height (P<0.0001), and BMI (P<0.0001), and a negative correlation with family history for osteoporosis (P<0.041). There was no association between the presence of allele s and reduced BMD. We conclude that a family history of osteoporosis and age at menarche are factors that may influence bone mass in our population.

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37ºC and 43ºC (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.

Effect of an aqueous extract of Phaseolus vulgaris on the properties of tail tendon collagen of rats with streptozotocin-induced diabetes

Changes in the structural and functional properties of collagen caused by advanced glycation might be of importance for the etiology of late complications in diabetes. The present study was undertaken to investigate the influence of oral administration of aqueous pod extract (200 mg/kg body weight) of Phaseolus vulgaris, an indigenous plant used in Ayurvedic Medicine in India, on collagen content and characteristics in the tail tendon of streptozotocin-diabetic rats. In diabetic rats, collagen content (117.01 ± 6.84 mg/100 mg tissue) as well as its degree of cross-linking was increased, as shown by increased extent of glycation (21.70 ± 0.90 µg glucose/mg collagen), collagen-linked fluorescence (52.8 ± 3.0 AU/µmol hydroxyproline), shrinkage temperature (71.50 ± 2.50ºC) and decreased acid (1.878 ± 0.062 mg hydroxyproline/100 mg tissue) and pepsin solubility (1.77 ± 0.080 mg hydroxyproline/100 mg tissue). The alpha/ß ratio of acid- (1.69) and pepsin-soluble (2.00) collagen was significantly decreased in streptozotocin-diabetic rats. Administration of P. vulgaris for 45 days to streptozotocin-diabetic rats significantly reduced the accumulation and cross-linking of collagen. The effect of P. vulgaris was compared with that of glibenclamide, a reference drug administered to streptozotocin-diabetic rats at the dose of 600 µg/kg body weight for 45 days by gavage. The effects of P. vulgaris (collagen content, 64.18 ± 1.97; extent of glycation, 12.00 ± 0.53; collagen-linked fluorescence, 33.6 ± 1.9; shrinkage temperature, 57.0 ± 1.0; extent of cross-linking - acid-soluble collagen, 2.572 ± 0.080, and pepsin-soluble collagen, 2.28 ± 0.112) were comparable with those of glibenclamide (collagen content, 71.5 ± 2.04; extent of glycation, 13.00 ± 0.60; collagen-linked fluorescence, 38.9 ± 2.0; shrinkage temperature, 59.0 ± 1.5; extent of cross-linking - acid-soluble collagen, 2.463 ± 0.078, and pepsin-soluble collagen, 2.17 ± 0.104). In conclusion, administration of P. vulgaris pods had a positive influence on the content of collagen and its properties in streptozotocin-diabetic rats.

Serum laminin, type IV collagen and hyaluronan as fibrosis markers in non-alcoholic fatty liver disease

Hepatic fibrosis in patients with non-alcoholic fatty liver disease is associated with progression of the disease. In the present study, we analyzed the discriminative ability of serum laminin, type IV collagen and hyaluronan levels to predict the presence of fibrosis in these patients. In this preliminary report, we studied 30 overweight patients divided into two groups according to the absence (group I, N = 19) or presence (group II, N = 11) of fibrosis in a liver biopsy. Triglycerides, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltranspeptidade, hyaluronan (noncompetitive fluoroassay), type IV collagen, and laminin (ELISA) were determined. Group II presented significantly higher mean laminin, hyaluronan, type IV collagen, and aspartate aminotransferase values, which were due to the correlation between these parameters and the stage of fibrosis in the biopsy (Spearman's correlation coefficient, rS = 0.65, 0.62, 0.53, and 0.49, respectively). Analysis of the ROC curve showed that laminin values >282 ng/ml were those with the best diagnostic performance, with 87% accuracy. Association of laminin with type IV collagen showed improvement in the positive predictive value (100%), but with reduction in diagnostic sensitivity (64%). When compared with the criteria of Ratziu et al. [Gastroenterology (2000) 118: 1117-1123] for the diagnosis of septal fibrosis, laminin values presented a better diagnostic accuracy (83 vs 70%). Determination of extracellular matrix components in serum, especially of laminin, may identify patients with non-alcoholic fatty liver disease and fibrosis and these components may be used as indicators for liver biopsy in these patients.

Collagen content, but not the ratios of collagen type III/I mRNAs, differs among hypertensive, alcoholic, and idiopathic dilated cardiomyopathy

Cardiac interstitial fibrosis may contribute to ventricular dysfunction and the prognosis of patients with dilated cardiomyopathy. The objective of the present study was to determine if total myocardial collagen content and collagen type III/I (III/I ratio) mRNAs differ in hypertensive, alcoholic, and idiopathic dilated cardiomyopathy subjects. Echocardiography and exercise cardiopulmonary testing were performed in patients with idiopathic (N = 22), hypertensive (N = 12), and alcoholic (N = 11) dilated cardiomyopathy. Morphometric analysis of collagen was performed in fragments obtained by endomyocardial biopsy with picrosirius red staining. The collagen III/I ratio was determined by reverse transcription polymerase chain reaction. Samples of controls (N = 10) were obtained from autopsy. Echocardiographic variables and maximal oxygen uptake were not different among dilated cardiomyopathy groups. Collagen was higher in all dilated cardiomyopathy groups (idiopathic, hypertensive and alcoholic, 7.36 ± 1.09%) versus controls (1.12 ± 0.18%), P < 0.05. Collagen was lower in idiopathic dilated cardiomyopathy (4.97 ± 0.83%) than hypertensive (8.50 ± 1.11%) and alcoholic (10.77 ± 2.09%) samples (P < 0.005 for both). The collagen III/I ratio in all samples from dilated cardiomyopathy patients was higher compared to that in controls (0.29 ± 0.04, P < 0.05) but was the same in the samples from idiopathic (0.77 ± 0.07), hypertensive (0.75 ± 0.07), and alcoholic (0.81 ± 0.16) dilated cardiomyopathy groups. Because of the different physical properties of the types of collagen, the higher III/I ratio may contribute to progressive ventricular dilation and dysfunction in dilated cardiomyopathy patients.

Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

Supraorganized collagen enhances Schwann cell reactivity and organization in vitro

We investigated the reactivity and expression of basal lamina collagen by Schwann cells (SCs) cultivated on a supraorganized bovine-derived collagen substrate. SC cultures were obtained from sciatic nerves of neonatal Sprague-Dawley rats and seeded on 24-well culture plates containing collagen substrate. The homogeneity of the cultures was evaluated with an SC marker antibody (anti-S-100). After 1 week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, S-100 and p75NTR (pan neurotrophin receptor) and for scanning electron microscopy (SEM). Positive labeling with antibodies to the cited molecules was observed, indicating that the collagen substrate stimulates SC alignment and adhesion (collagen IV labeling - organized collagen substrate: 706.33 ± 370.86, non-organized collagen substrate: 744.00 ± 262.09; S-100 labeling - organized collagen: 3809.00 ± 120.28, non-organized collagen: 3026.00 ± 144.63, P < 0.05) and reactivity (p75NTR labeling - organized collagen: 2156.33 ± 561.78, non-organized collagen: 1424.00 ± 405.90, P < 0.05; means ± standard error of the mean in absorbance units). Cell alignment and adhesion to the substrate were confirmed by SEM analysis. The present results indicate that the collagen substrate with an aligned suprastructure, as seen by polarized light microscopy, provides an adequate scaffold for SCs, which in turn may increase the efficiency of the nerve regenerative process after in vivo repair.