Isolation, characterization, and localization of cardiac collagen type VI. Associations with other extracellular matrix components.

We have isolated and characterized collagen type VI from murine, canine, and nonhuman primate hearts. In the three species studied, collagen type I was the major collagenous component of the cardiac interstitium (80% of total collagen), whereas collagen type VI represented approximately 5% of total collagen. To define the exact distribution of collagen type VI and its possible interactions with other components of the cardiac extracellular matrix, collagen types I, III, IV, and VI, laminin, and fibronectin were localized in the rat myocardium by immunohistochemistry, using monospecific antibodies. In the rat myocardium, collagen type VI was prevalent in the media and adventitia of muscular arteries, in fine connective tissue septa, in the area surrounding capillaries, and in the delicate endomysium in proximity to myocardial cells. When compared with the immunohistochemical localization of collagen types I, III, and IV, laminin, and fibronectin, the continuity and hierarchical organization of the cardiac extracellular matrix became apparent. The matrix forms a continuous network extending from the pericardium to the endocardium. Furthermore, there is an arborescent hierarchy in the system such that collagen type I is more prevalent in the wider septa, collagen type III being more obvious in medium-sized branches, and fibronectin and collagen type VI prevailing in the terminal (pericellular) aspects of the network. In this pericellular location, fibronectin and collagen type VI, by means of specific interactions, may act as anchor components linking the myocardial cell basement membranes not only to the extracellular matrix but also to the cardiac interstitial cells. This continuity, organization, and coupling of the cardiac extracellular matrix appears well suited to integrate and distribute the physical stress generated by the continuous contraction and relaxation of the myocardium.     

Myocardial glycoproteins in diabetes: type VI collagen is a major PAS-reactive extracellular matrix protein.

An investigation of myocardial glycoproteins was undertaken to elucidate the molecules responsible for the periodic acid-Schiff (PAS) reactivity of the increased extracellular matrix of diabetic cardiomyopathy. Perfusion with radiolabeled mannose indicated an enhanced formation of matrix components in the diabetic compared to the normal rat heart. Electrophoretic separation of radiolabeled extracts demonstrated the presence of glycoproteins with Mr values of 205, 142 and 90 kDa which could be separated by Bio-Gel A-5 m filtration. Fractionation of non-perfused hearts resulted in the isolation of only the 205 and 142 kDa components, which were shown by amino acid analyses and collagenase digestion to belong to the collagen family of proteins and by immunoblotting to represent type VI collagen. The carbohydrate content of these rat myocardial type VI collagen subunits, determined from monosaccharide analyses, was 11 and 12%, respectively, and N-glycanase digestion of the 142 kDa chain resulted in a decrease in size of approximately 14 kDa, indicating the presence of asparagine-linked units. Examination of normal and diabetic rat heart sections indicated that the latter contained abundant PAS-positive strands and nodules which corresponded to the distribution of anti type VI collagen reactivity. Moreover, immunoblots showed higher levels of Type VI collagen in diabetic than in normal heart extracts. Type VI collagen therefore appears to represent a major glycoprotein of myocardial extracellular matrix and to be implicated in diabetic cardiomyopathy.     

Digoxin-like immunoreactivity: occurrence in three molecular forms and partial characterization

Digoxin-like immunoreactivity (DLI) has been found in serum from subjects with a variety of physiological and pathological conditions, including uremia, liver disease, pregnancy, and the neonatal period. The physicochemical nature of this material is still not known. Using gel filtration chromatography, extraction with methylene chloride, and treatment with beta-glucuronidase, we determined that serum DLI exists in three forms: protein-bound, glucuronidated, and free, with respective mol wt of 5000, 400, and 230. In serum, DLI exists in the protein-bound and free forms, while in urine, DLI is found in the glucuronidated and free states.     

Modulation of extracellular matrix components during dimethylnitrosamine-induced cirrhosis

Liver fibrosis was induced in rats after administration of dimethylnitrosamine (DMN) intraperitoneally three times a week for 3 weeks. Incomplete septa appeared after 7 days and evidence of nodulation of the parenchyma was observed after 21 days. Both distribution of extracellular matrix components (collagen type I, type III and type IV, laminin, fibronectin, heparan sulphate proteoglycan) and the distribution of desmin as a marker of lipocytes (Ito cells) and of iso-alpha-smooth muscle actin were studied with immunoperoxidase. Changes in the distribution of extracellular matrix components outlined both the formation of septa and the development of nodules with changes in the sinusoidal pattern evoking aspects of capillarization. The number of desmin-positive cells increased in DMN-treated animals, showing a prominent reaction in the fibrous septa. In the normal liver, lipocytes were positive for laminin and negative for actin, but septal and juxta-septal lipocytes were positive for both antigens, suggesting the presence of transitional cells with mixed immunoreactivity. This was confirmed by ultrastructural studies which showed typical intraseptal myofibroblasts and other elements exhibiting the structural features of both myofibroblasts and lipocytes.     

Bovine prolactin-related protein-I is anchored to the extracellular matrix through interactions with type IV collagen

The bovine placenta produces an array of proteins structurally similar to pituitary prolactin (PRL). At least ten genes of the bovine placental PRL family, including bovine placental lactogen (bPL) and ten bovine PRL-related protein-I to -X (bPRP-I to -X), encode hormones/cytokines predicted to be involved in the establishment and maintenance of pregnancy. Targets and biological roles for most members of the bovine PRL family have yet to be specified. This study focused on three members of bovine PRL family, bPL, bPRP-I, and bPRP-VI. An alkaline phosphatase (AP) tagging strategy was used to monitor interactions of the ligands with their targets. AP-bPRP-I and AP-bPRP-VI specifically bound to tissue sections of the bovine placentome. AP-bPRP-I and AP-bPRP-VI binding within the placentome mimicked the distribution of the extracellular matrix (ECM). Consequently, AP fusion protein binding to individual ECM components (heparin, laminin, fibronectin, type I collagen, and type IV collagen) was evaluated. AP-bPRP-I specifically bound to type IV collagen, but not to the other ECM components. AP-bPRP-VI exhibited weak interactions with ECM components, while AP-bPL and AP did not show significant binding to any of the ECM components. Binding of AP-bPRP-I to type IV collagen was concentration-dependent, influenced by salt concentrations, and specific to the N-terminal cross-linking domain (7S) of type IV collagen but not its triple-helical domain. The interaction of bPRP-I with type IV collagen suggests that bPRP-I accumulates in the ECM where it likely acts on cells traversing the bovine placentome.     

Synovial extracellular matrix II. Specific incorporation of immunoglobulin into the cell-free matrix of pannus.

To determine whether immune complex-like material is incorporated into the extracellular matrix (ECM) of proliferated RA synovium, cell-free matrices were isolated from pannus removed at joint replacement surgery, and were subjected to differential extraction. When the IgG and albumin concentrations in the ECM extracts were compared to those in simultaneously obtained synovial fluids, the IgG was found to be enriched 8.8-fold. Approximately 95% of the IgG was extractable with 6M Guanidine-HCl and 8 M Urea-B-ME. Further extraction with collagenase and low-pH buffers did not result in any additional recovery of IgG. Matrix-associated IgG demonstrated a restricted mobility on IEF with a pI of 4.8. The extracellular matrix of RA pannus is enriched in an acidic IgG species. Incorporation of IgG appears to be secondary to non-covalent interactions and may represent an additional reservoir of immune complex material in the rheumatoid joint.     

Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin.

Regeneration of the dermis does not occur spontaneously in the adult mammal. The epidermis is regenerated spontaneously provided there is a dermal substrate over which it can migrate. Certain highly porous, crosslinked collagen-glycosaminoglycan copolymers have induced partial morphogenesis of skin when seeded with dermal and epidermal cells and then grafted on standard, full-thickness skin wounds in the adult guinea pig. A mature epidermis and a nearly physiological dermis, which lacked hair follicles but was demonstrably different from scar, were regenerated over areas as large as 16 cm2. These chemical analogs of extracellular matrices were morphogenetically active provided that the average pore diameter ranged between 20 and 125 microns, the resistance to degradation by collagenase exceeded a critical limit, and the density of autologous dermal and epidermal cells inoculated therein was greater than 5 x 10(4) cells per cm2 of wound area. Unseeded copolymers with physical structures that were within these limits delayed the onset of wound contraction by about 10 days but did not eventually prevent it. Seeded copolymers not only delayed contraction but eventually arrested and reversed it while new skin was being regenerated. The data identify a model extracellular matrix that acts as if it were an insoluble growth factor with narrowly specified physiochemical structure, functioning as a transient basal lamina during morphogenesis of skin.     

Embryonic neural retina collagen: in vitro synthesis of high molecular weight forms of type II plus a new genetic type.

Chicken neural retina cells from 6- to 7-day embryos were labeled with [(3)H]proline for 24 and 72 hr and the collagenous proteins of the medium were analyzed. Ninety percent of the collagenous proteins eluted from DEAE-cellulose columns as a peak near the middle of the gradient. Upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, this material consisted predominantly of a slowly migrating procollagen, a smaller amount of an intermediate migrating form, and some alpha-chain material. Limited enzymatic digestion of this DEAE peak material plus mapping of its CNBr peptides identified this material as a type II precursor. The remaining 10% of the collagenous proteins eluted from DEAE-cellulose at the end of the gradient. Electrophoresis of this fraction showed four major bands. Two migrated near the type II precursors, a third migrated somewhat more slowly, and the fourth was near beta-chain dimers. In addition, there were two minor bands. Limited pepsin digestion of this DEAE peak material produced two bands: one migrated slightly more slowly than alpha1, and the second, considerably more slowly. The CNBr peptide pattern of this material appears to be different from any previously described. Thus, neural retina cells in culture synthesize at least two genetically distinct classes of collagenous proteins. One represents precursors to type II. The second is composed of multiple, very high molecular weight forms which may represent precursors (procollagens) of a new genetic type(s) of collagen.     

Immunolocalization of extracellular matrix components and integrins during mouse liver development

Intrahepatic biliary cell differentiation takes place in periportal hepatoblasts under the influence of the subjacent connective tissue, the mechanism of which is still unclear. This study was undertaken to analyze the immunolocalization of extracellular matrix components and their cellular receptors during mouse liver development, with special attention given to biliary differentiation and vascular development. In young fetal mouse liver, primitive structures of sinusoids were developed between hepatic cords associated with hematopoietic cells demonstrated by immunohistochemistry of basal laminar components, the alpha6 integrin subunit, and PECAM-1. Portal veins and hepatic veins showed different staining intensities of alpha2, alpha3, and alpha6 integrin subunits from early stages of development. Anti-beta4 integrin subunit antibodies reacted with portal veins, but not with hepatic veins after perinatal stages. Their different phenotypes may be related to the preferential differentiation of periportal bile ducts. In intrahepatic bile duct development, periportal hepatoblasts adjacent to the connective tissue were immunostained for each basal laminar component on the basal side at almost the same time; alpha3, alpha5, alpha6, and beta4 integrin subunits were immunohistochemically detectable later than the basal laminar components. These staining patterns of intrahepatic bile duct cells clearly differed from those of extrahepatic bile duct cells from the beginning of their development, suggesting that these ducts are of different origins. In conclusion, the vascular structures, including sinusoids, portal veins, and hepatic veins, develop from early stages of liver development, and the extracellular matrix components may play important roles in biliary differentiation and vascular development. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).     

Mutant fibrillin 1 from tight skin mice increases extracellular matrix incorporation of microfibril-associated glycoprotein 2 and type I collagen

OBJECTIVE: Skin fibrosis in the TSK mouse, a model of skin fibrosis seen in systemic sclerosis (SSc), is caused by a large in-frame duplication in the Fbn1 gene, tsk-Fbn1. We investigated whether tsk-Fbn1 might cause dermal fibrosis by affecting Fbn1 and associated extracellular matrices. We also studied whether deposition of microfibril-associated glycoprotein 2 (MAGP-2), a protein that is associated with fibrillin 1, was altered in the skin of patients with SSc. METHODS: An in vitro model of the TSK mouse was created by conditionally expressing tsk-Fbn1 in mouse embryonic fibroblasts (MEFs). Cell cultures were examined by immunofluorescence and Western and Northern blotting to determine the effect of tsk-Fbn1 on the structure, expression, and deposition of fibrillin 1 (Fbn-1), type I collagen, and MAGP-2. The skin of TSK mice and SSc patients was analyzed by immunohistochemistry for MAGP-2 expression. RESULTS: Expression of tsk-Fbn1 in cultured MEF cells altered the morphology of Fbn-1 fibers and increased the deposition of type I collagen into the extracellular matrix (ECM) without concomitantly changing messenger RNA expression, secretion, or processing of type I procollagen. Moreover, MEF cells expressing tsk-Fbn1 showed increased MAGP-2 matrix. MAGP-2 was increased in the dermis of TSK mice. Fibrotic SSc skin also showed higher levels of MAGP-2 in the dermis than nonfibrotic SSc skin and normal skin. CONCLUSION: Tsk-Fbn1 altered ECM organization and caused fibrosis by affecting the deposition of MAGP-2 or other Fbn-1-associated proteins. Alterations in microfibril structure or deposition might contribute to fibrosis in SSc.     

Type XII collagen: distinct extracellular matrix component discovered by cDNA cloning.

We have screened a cDNA library constructed from tendon fibroblast mRNA for the presence of collagenous coding sequences. Nucleotide sequence analysis of one isolated clone, pMG377, reveals that the clone encodes a polypeptide that is homologous to, yet distinctly different from, type IX short-chain collagen polypeptides. The structure of the conceptual translation product of the cDNA is also different from that of all other collagen types. Therefore, we have given the type IX-like collagen chain encoded by pMG377 the designation alpha 1(XII). Ribonuclease protection assays with single-stranded cRNA probes demonstrate that alpha 1(XII) mRNA is present in several tissues such as calvaria, tendon, and sternal cartilage of 17-day-old chicken embryo and in cornea from 6-day-old embryos. Using pMG377 as the hybridization probe, we isolated a fragment of the corresponding gene from a chicken genomic library. Partial nucleotide sequence analysis of the genomic clone DG12 shows that the exon/intron structure of the alpha 1(XII) collagen gene appears to be homologous to that of the alpha 1(IX) and alpha 2(IX) collagen genes. Our data demonstrate that types IX and XII collagen are two homologous members of a family of unique collagenous proteins that show tissue-specific patterns of expression. Based on their structure and the properties of their genes, we conclude that this family of collagens is distinctly different from that of fibrillar collagens.     

Architecture and the extracellular matrix: the still unappreciated components of the adipose tissue

Fibrosis is usually characterized by the modification of both the amount and composition of a wide panel of extracellular matrix (ECM) proteins. In the liver, pancreas, kidney and lung the accumulation of fibrosis disrupts cellular processes and appears detrimental for organ function. This review highlights the available evidence supporting an important ECM remodelling in adipose tissue (AT) and, in particular, during the development of obesity. The modifications and occurrence of new adipose ECM components leads to an abnormal accumulation of fibrosis in this tissue. This phenomenon was well described in rodent models and evidence is beginning to emerge in humans; however, the origin and potential impact of these depots in AT biology are unclear. Two animal models with disruptions in ECM components (secreted proteins acidic in nature rich in cysteine null mice and ob/ob collagen VI null mice) suggest that fibrosis limits adipocyte hypertrophy and may cause the metabolic disorders associated with obesity. Over‐expression of Hypoxia‐inducible factor 1 leading to an increase in collagen expression suggests a role for hypoxia in fibrosis development. We conclude this review with possible hypotheses regarding the cellular and molecular contributors of fibrosis initiation.     

Immunolocalisation of type VI collagen in the intervertebral disc.

Type VI collagen has been isolated from many connective tissues, including the intervertebral disc. Distribution of this collagen, however, varies considerably within different tissues. In adult mammalian nasal and articular cartilage it is localised preferentially in the matrix immediately surrounding the cell. Intervertebral discs from various species and of various ages were studied and a similar pericellular localisation was found. When antisera to type VI collagen were used staining was seen around the cells of all sections of intervertebral disc, being particularly prominent in the nucleus pulposus. Staining on or around the cells was also seen in the adjacent cartilaginous end plate and bone.     

Increased rat myocardial type VI collagen in diabetes mellitus and hypertension

Summary Diabetic cardiomyopathy, a condition characterized by the accumulation of carbohydrate-containing material surrounding the myocardial small blood vessels, has been studied in alloxan-diabetic normotensive and hypertensive rats. Immunochemical techniques were used to monitor several extracellular matrix constituents present in extracts of cardiac tissue, namely types I, IV and VI collagen, laminin and fibronectin, as well as myosin. These studies have indicated that after induction of diabetes, type VI collagen but none of the other matrix components studied, was significantly increased (from 2.29±0.04 mg/g in normal to 2.85±0.18 mg/g in diabetic ventricles, p<0.01). Hypertension, whether induced by the clipping of one renal artery or genetically determined (spontaneously hypertensive rats), resulted in a similar elevation in type VI collagen (2.71±0.12 mg/g, p<0.005 compared to normal rats). In the presence of diabetes plus hypertension the effect was not additive, the type VI collagen level being 2.93±0.15 (p<0.001 compared to normal rats). Basement membrane collagen (type IV) in the myocardium appeared to be unaffected by diabetes or hypertension and the myosin contents of the hearts of the four experimental groups were similar. Quantitative determinations indicate that compared to type IV collagen, laminin or fibronectin, type VI collagen represents the major periodic acid-Schiff-reactive extracellular constituent of the rat ventricle. Its preferential increase in the heart in diabetes may provide insight into the molecular mechanisms of the diabetic microvascular disease.     

Regulation of matrix metallo-proteinase expression by extracellular matrix components in cultured hepatic stellate cells

Hepatic stellate cells (HSC) changed their morphology and function including production of matrix metalloproteinases (MMPs) in response to extracellular matrix (ECM) component used as a substratum in culture. We examined in this study the regulatory role of ECM component on expression of MMPs and tissue inhibitor of metalloproteinase (TIMP) in rat HSCs cultured on polystyrene, type I collagen-coated surface, type I collagen gel, or Matrigel, respectively. When cultured on type I collagen gel, HSCs showed the asteroid cell shape and MMP-1 activity, as detected by in situ zymography. Expression of MMP-1 protein and mRNA were examined by using immunofluorescence staining and RT-PCR analysis in HSCs cultured on type I collagen gel. Active form of MMP-2 was detected by gelatin zymography in the conditioned medium of HSCs cultured on type I collagen gel, whereas it was not detected when HSCs were cultured on polystyrene, type I collagen-coated surface, or Matrigel. Increased MMP-2 mRNA was detected by RT-PCR in HSCs cultured on type I collagen gel. Increased MT1-MMP proteins were shown to localize on the cell membrane by using immunofluorescence staining in HSCs cultured on type I collagen gel. Elevated expression of membrane-type matrix metallproteinase-1 (MT1-MMP) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was detected by RT-PCR in HSCs cultured on type I collagen-coated surface or type I collagen gel. These results indicate that expression of MMPs and TIMP-2 is regulated by ECM components in cultured HSCs, suggesting an important role of HSCs in the remodeling of liver tissue.     

Ascorbate regulation of collagen biosynthesis in Ehlers-Danlos syndrome, type VI

We studied two unrelated individuals with Ehlers-Danlos syndrome type VI, which is characterized by congenital hypotonia, lax joints, severe kyphoscoliosis, friable skin, and hemorrhagic hypotrophic scars. The diagnosis was confirmed by decreased hydroxylysine residues in dermal collagen and decreased collagen lysyl hydroxylase activities in their cultured skin fibroblasts. Despite the diminished hydroxylysine residues in dermal collagen from the probands, we found no differences in hydroxylysyl residues of collagen synthesized by fibroblasts in culture. When patient 1 was given oral sodium ascorbate (5 g/d) for 3 weeks, ascorbate concentrations increased two-fold in plasma and 300-fold in urine. Urinary excretion of hydroxylysine and hydroxyproline increased during ascorbate administration. After a 1-year interval, bleeding time, wound healing, and muscle strength improved. Ascorbate supplementation (50 micrograms/mL) to confluent fibroblasts cultured from the two patients and controls increased hydroxyprolyl and hydroxylysyl residues of fibroblasts four to seven and three to four-fold respectively. Total protein associated with the cell layer increased 14% to 32% without concomitant change in cellular DNA. Total soluble collagenous material recovered from culture media increased 61% to 103% with ascorbate supplementation. These studies demonstrate that ascorbate improves the clinical status of patients with impaired collagen lysyl hydroxylase activity by enhancing lysyl and prolyl hydroxylation and total collagen production.