Immunolocalization of collagen types I and III in the arterial wall of the rat

Summary Collagen types I and III were located by immunofluorescence procedures in the aorta and coronary arteries of the rat. Type I collagen was most prevalent in the adventitia of the aorta with only small amounts present in the intima and media. Type III collagen appeared to be a significant component in the media of the aorta and also in the adventitia of both blood vessels. The intima and media of the coronary arteries did not stain strongly for either type I or III collagen. Neither staining procedure was altered with preincubation of the sections with hyaluronidase or chondroitinase ABC. These studies indicate that type III collagen is a major component of the adventitia which has previously not been recognized by immunohistochemical techniques, possibly due to masking of collagen staining with glycosaminoglycans.     

Expression of collagen type I,II, X and Ki-67 in osteochondroma compared to human growth plate cartilage

In order to characterize the consequences for the process of endochondral ossification we performed an immunohistochemical study and compared the expression of collagen type I, II and X as markers of cartilage differentiation and Ki-67 as a marker of cell proliferation in solitary (7-26 years, n=9) and multiple (11-42 years, n=6) osteochondromas with their expression in human fetal and postnatal growth plates. In fetal and young postnatal controls, we found a thin superficial layer of articular cartilage that stained positive for collagen type I while collagen II was expressed in the rest of the cartilage and collagen type X was restricted to the hypertrophic zone. Osteochondromas from children showed lobular collagen type II-positive areas surrounded by collagen type I. In adults, the separation of collagen type I- and type II-positive areas was more blurred, or the cartilaginous cap was missing. Collagen type X was detected in a pericellular distribution pattern within hypertrophic zones but also deeper between bone trabecula. The proliferative activity of osteochondromas from children younger than 14 years of age was comparable to postnatal growth plates, whereas in cartilage from individuals older than 14 years of age, we could not detect significant proliferative activity.     

Immunochemical and mechanical characterization of cartilage subtypes in rabbit.

Cartilage is categorized into three general subgroups, hyaline, elastic, and fibrocartilage, based primarily on morphologic criteria and secondarily on collagen (Types I and II) and elastin content. To more precisely define the different cartilage subtypes, rabbit cartilage isolated from joint, nose, auricle, epiglottis, and meniscus was characterized by immunohistochemical (IHC) localization of elastin and of collagen Types I, II, V, VI, and X, by biochemical analysis of total glycosaminoglycan (GAG) content, and by biomechanical indentation assay. Toluidine blue staining and safranin-O staining were used for morphological assessment of the cartilage subtypes. IHC staining of the cartilage samples showed a characteristic pattern of staining for the collagen antibodies that varied in both location and intensity. Auricular cartilage is discriminated from other subtypes by interterritorial elastin staining and no staining for Type VI collagen. Epiglottal cartilage is characterized by positive elastin staining and intense staining for Type VI collagen. The unique pattern for nasal cartilage is intense staining for Type V collagen and collagen X, whereas articular cartilage is negative for elastin (interterritorially) and only weakly positive for collagen Types V and VI. Meniscal cartilage shows the greatest intensity of staining for Type I collagen, weak staining for collagens V and VI, and no staining with antibody to collagen Type X. Matching cartilage samples were categorized by total GAG content, which showed increasing total GAG content from elastic cartilage (auricle, epiglottis) to fibrocartilage (meniscus) to hyaline cartilage (nose, knee joint). Analysis of aggregate modulus showed nasal and auricular cartilage to have the greatest stiffness, epiglottal and meniscal tissue the lowest, and articular cartilage intermediate. This study illustrates the differences and identifies unique characteristics of the different cartilage subtypes in rabbits. The results provide a baseline of data for generating and evaluating engineered repair cartilage tissue synthesized in vitro or for post-implantation analysis.     

Collagen-related gene and protein expression changes in the lung in response to chronic hypoxia

Collagen accumulation likely contributes to increased vascular and airway impedance in hypoxia-induced pulmonary hypertension (HPH). Collagen exists in multiple subtypes and can accumulate via increased synthesis or decreased degradation. To better understand the individual contributions of fibrillar (FB) and basement membrane (BM) collagen, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) to pulmonary vascular and airway remodeling in HPH, we investigated the temporal changes in gene and protein expression in the lungs of mice exposed to hypoxia for 0, 3, 6, 10 and 15 days. The earliest and largest change in gene expression was of type I FB collagen, which was significantly increased over control levels at 6, 10 and 15 days of hypoxia (p  <  0.05). Type III FB and type IV BM collagen were increased at 10 and 15 days of hypoxia (p <  0.05); MMP and TIMP gene expression levels were typically higher but sometimes lower than control levels at various time points. Collagen protein content was increased in whole lungs as early as 6 days of hypoxia and increased monotonically with longer exposures. However, neither qualitative nor semi-quantitative analysis of immunohistochemistry demonstrated accumulation of type I FB collagen in compartments of the lung other than large airways, suggesting that other collagen subtypes may be important contributors to collagen protein accumulation. These results provide insight into the patterns of gene and protein expression relevant to collagen accumulation in the lung in response to chronic hypoxia, through which we can develop a better understanding of the time course of changes in matrix biology and biomechanics that occur in HPH.     

Assembly of collagen into microribbons: effects of pH and electrolytes

Collagen represents the major structural protein of the extracellular matrix. Elucidating the mechanism of its assembly is important for understanding many cell biological and medical processes as well as for tissue engineering and biotechnological approaches. In this work, conditions for the self-assembly of collagen type I molecules on a supporting surface were characterized. By applying hydrodynamic flow, collagen assembled into ultrathin ( approximately 3 nm) highly anisotropic ribbon-like structures coating the entire support. We call these novel collagen structures microribbons. High-resolution atomic force microscopy topographs show that subunits of these microribbons are built by fibrillar structures. The smallest units of these fibrillar structures have cross-sections of approximately 3 x 5nm, consistent with current models of collagen microfibril formation. By varying the pH and electrolyte of the buffer solution during the self-assembly process, the microfibril density and contacts formed within this network could be controlled. Under certain electrolyte compositions the microribbons and microfibers display the characteristic D-periodicity of approximately 65 nm observed for much thicker collagen fibrils. In addition to providing insight into the mechanism of collagen assembly, the ultraflat collagen matrices may also offer novel ways to bio-functionalize surfaces.     

Upregulation of HSP47 and collagen type III in the dermal fibrotic disease, keloid

Keloid is a dermal fibrotic disease characterized by excessive accumulation of mainly type I collagen in extracellular matrix of the dermis. We have studied the expression levels of collagen types I and III, and its molecular chaperone HSP47 in keloid lesions and surrounding unaffected skin using Northern and Western blotting and immunohistochemical analyses. Collagen types I and III mRNA levels were found to be upregulated 20-fold in keloid tissues, contradicting previous reports of nearly normal type III collagen levels in this disease. HSP47 expression in keloid lesions was also highly upregulated; eightfold at mRNA level and more than 16-fold at the protein level. Strong upregulation of these three proteins in keloid was confirmed by immunohistochemical staining. These results suggest that accumulation of both type I and type III collagen is important for the development of keloid lesions, and that HSP47 plays a role in the rapid and extensive synthesis of collagen in keloid tissues.     

Type I collagen inhibits hydroxyl radical-induced apoptosis

The extracellular matrix (ECM) plays an important role in cell differentiation and apoptosis. Collagen is the major component of ECM. Here, an ESR signal of the hydroxyl radicals (.OH) generated via Fe2+ -mediated Fenton reaction was found to be significantly inhibited by type I collagen. Further study showed that type I collagen also inhibited cell apoptosis induced by.OH, as evidenced by morphological criteria (DAPI and annexin V staining) and quantitive assays for apoptotic cells (MTT and flow-cytometric assay for subG1 cells). By addition of type I collagen in HeLa cells, the lipid peroxidation caused by.OH was inhibited and the cellular GSH was protected. In comparison with type I collagen, BSA and the denatured collagen, gelatin, lacked such antioxidative and antiapoptotic effects. Together, the results suggest that type I collagen can uniquely prevent.OH-mediated apoptosis by scavenging free radicals.     

Mutations in the COL5A1 gene are causal in the Ehlers-Danlos syndromes I and II.

The Ehlers-Danlos syndrome (EDS) is a heterogeneous connective-tissue disorder of which at least nine subtypes are recognized. Considerable clinical overlap exists between the EDS I and II subtypes, suggesting that both are allelic disorders. Recent evidence based on linkage and transgenic mice studies suggest that collagen V is causally involved in human EDS. Collagen V forms heterotypic fibrils with collagen I in many tissues and plays an important role in collagen I fibrillogenesis. We have identified a mutation in COL5A1, the gene encoding the pro(alpha)1(V) collagen chain, segregating with EDS I in a four-generation family. The mutation causes the substitution of the most 5' cysteine residue by a serine within a highly conserved sequence of the pro(alpha)1(V) C-propeptide domain and causes reduction of collagen V by preventing incorporation of the mutant pro(alpha)1(V) chains in the collagen V trimers. In addition, we have detected splicing defects in the COL5A1 gene in a patient with EDS I and in a family with EDS II. These findings confirm the causal role of collagen V in at least a subgroup of EDS I, prove that EDS I and II are allelic conditions, and represent a, so far, unique example of a human collagen disorder caused by substitution of a highly conserved cysteine residue in the C-propeptide domain of a fibrillar collagen.     

The discoidin domain receptor DDR2 is a receptor for type X collagen.

During endochondral ossification, collagen X is deposited in the hypertrophic zone of the growth plate. Our previous results have shown that collagen X is capable of interacting directly with chondrocytes, primarily via integrin alpha2beta1. In this study, we determined whether collagen X could also interact with the non-integrin collagen receptors, discoidin domain receptors (DDRs), DDR1 or DDR2. The widely expressed DDRs are receptor tyrosine kinases that are activated by a number of different collagen types. Collagen X was found to be a much better ligand for DDR2 than for DDR1. Collagen X bound to the DDR2 extracellular domain with high affinity and stimulated DDR2 autophosphorylation, the first step in transmembrane signalling. Expression of DDR2 in the epiphyseal plate was confirmed by RT-PCR and immunohistochemistry. The spatial expression of DDR2 in the hypertrophic zone of the growth plate is consistent with a physiological interaction of DDR2 with collagen X. Surprisingly, the discoidin domain of DDR2, which fully contains the binding sites for the fibrillar collagens I and II, was not sufficient for collagen X binding. The nature of the DDR2 binding site(s) within collagen X was further analysed. In addition to a collagenous domain, collagen X contains a C-terminal NC1 domain. DDR2 was found to recognise the triple-helical region of collagen X as well as the NC1 domain. Binding to the collagenous region was dependent on the triple-helical conformation. DDR2 autophosphorylation was induced by the collagen X triple-helical region but not the NC1 domain, indicating that the triple-helical region of collagen X contains a specific DDR2 binding site that is capable of receptor activation. Our study is the first to describe a non-fibrillar collagen ligand for DDR2 and will form the basis for further studies into the biological function of collagen X during endochondral ossification.     

Rheology of heterotypic collagen networks

Collagen fibrils are the main structural element of connective tissues. In many tissues, these fibrils contain two fibrillar collagens (types I and V) in a ratio that changes during tissue development, regeneration, and various diseases. Here we investigate the influence of collagen composition on the structure and rheology of networks of purified collagen I and V, combining fluorescence and atomic force microscopy, turbidimetry, and rheometry. We demonstrate that the network stiffness strongly decreases with increasing collagen V content, even though the network structure does not substantially change. We compare the rheological data with theoretical models for rigid polymers and find that the elasticity is dominated by nonaffine deformations. There is no analytical theory describing this regime, hampering a quantitative interpretation of the influence of collagen V. Our findings are relevant for understanding molecular origins of tissue biomechanics and for guiding rational design of collagenous biomaterials for biomedical applications.     

Influence of serum on adult and fetal dermal fibroblast migration adhesion, and collagen expression

Summary The wound healing response to injury can be affected by many factors such as cell migration and extracellular matrix elaboration. The objective of this study was to examine the serum- and age-dependent effects on cell migration, adhesion, and collagen expression by skin fibroblasts. Dermal fibroblasts were isolated and plated with and without serum for up to 7 d. Cell migration was determined by quantitative image analysis, adhesion was quantified using a centrifugation assay, and collagen expression was assessed by PCR and immunohistochemical staining. Both adult and fetal fibroblasts migrated significantly faster in serum-containing medium compared to serum-free medium. There was no significant difference in migration between the two cell types in either serum-containing or serum-free medium. There was no significant difference in adhesion in the presence of serum, although there was a greater faction of adherent fetal skin fibroblasts than adult fibroblasts in serum-free medium. Moreover, the adherent fraction of fetal fibroblasts in serum-free medium was not significantly different from that in serum-containing medium, suggesting that fetal skin fibroblasts possess serum-independent adhesion properties. Collagen mRNA expression was significantly up-regulated in serum-free compared to serum-containing medium for both cell types. With respect to collagen immunohistochemistry, both dermal fibroblast populations exhibited greater type I collagen compared to type III collagen staining. Quantitative assessment of collagen staining indicated significantly enhanced type I collagen secretion in the presence of serum by fetal skin fibroblasts. These findings suggest that intrinsic cellular characteristics may govern the observed differences in adult and fetal wound healing.     

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage

Type X collagen is a short chain, non-fibrilforming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992 a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic α1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, α1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.     

Immunohistochemical localization of type II and type I collagens in articular cartilage of the femoral head of dexamethasone-treated rats

The immunohistochemical localization of type II and type I collagens was examined in the articular cartilage of the femoral head of growing rats injected systemically with 5 mg kg−1 dexamethasone for 2 weeks every other day. The intensities of immunostaining for type II collagen, measured by microphotometry, was highest in the flattened cell layer and high in the hypertrophic cell layer, moderate in the proliferative cell and transitional cell layers and low in the superficial layer. After dexamethasone administration, the intensities decreased markedly in the flattened cell layer and slightly in the hypertrophic cell layer, although the decreases in other layers were negligible. The staining intensities for type I collagen were highest in the flattened cell layer, low in the superficial and transitional cell layers and very low in the proliferative and hypertrophic cell layers. After dexamethasone administration, the intensities increased markedly in the flattened cell layer and slightly in the superficial and proliferative cell layers, but did not change in the transitional and hypertrophic cell layers. Thus, dexamethasone administration caused a decrease in type II collagen and an increase in type I collagen in the matrix of the surface portion of articular cartilage. The composition of isoforms of collagen in the matrix changed after the steroid administration. The results strongly suggest that the shift in collagen composition from type II to type I predominance is a cause of the degeneration of the articular cartilage after glucocorticoid administration. This revised version was published online in November 2006 with corrections to the Cover Date.     

Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis

Collagen monomers, oligomers, and fibrillar structures were isolated from chick tendons at various stages of development and studied by rotary shadowing. Monomers of Type I collagen, solubilized in 0.15 M NaCl solutions, were mostly present as collagen, pN-collagen, and pC-collagen with few procollagen molecules. They did not form polymers, nor were they associated with a carrier. Dimers of fibrillar collagen molecules were arranged in a 4-D stagger, suggesting that this was the preferred molecular interaction for the initiation of collagen fibrillogenesis. Type XII collagen molecules were mostly free, but some were attached by their central globular domain to one end of free fibrillar collagen molecules. Tenascin and Type VI collagen were also identified. The fibril populations consisted of collagen and beaded structures. These fibrils consisted of beads (globular domains) about 23 nm in diameter, separated by a period about 27 nm in length. Beads were linked by filamentous structures. These beaded fibrils probably represent the microfibrils of elastin.     

Immunological identification of types I and III collagen in bovine lens epithelium and its anterior lens capsule

To define the molecular structure of bovine lens epithelium and its anterior lens capsule, we investigated the composition of lens capsule basement membrane proteins. Immunofluorescence and immunogold techniques were used to demonstrate the presence of type I and type III collagen in the lens capsule and in primary explant epithelial cultures grown on protein-binding membranes. Immunofluorescence staining with specific antibodies indicated that type I and type III collagen were constituents of lens basement membrane. We observed that deposition of type III collagen was more than type I collagen. The synthesis of fibrillar collagen by lens epithelium and its deposition in the lens capsule was established by localization of fibrillar collagen by transmission immunoelectron microscopy. These results demonstrate for the first time that normal lens epithelium synthesize fibrillar collagen which is an intrinsic component of the anterior lens capsule basement membrane.     

4-Thiaproline reduces heart lipid peroxidation and collagen accumulation in the diabetic db/db mouse

Summary Collagen accumulation is a main pathological feature of diabetic cardiomyopathy. The underlying mechanisms seem to be increased cross linking by reactive carbonyles. The purpose of the study was to decrease the collagen content of total ventricular tissue by the oral administration of thiaproline, which could reduce collagen due to its functions as a proline analogue, blocking collagen production and as a free oxygen radical scavenger, blocking reactive carbonyles and oxygen species and subsequently collagen cross linking.Thiaproline was administered to genetically diabetic db/db mice and compared to untreated animals. Total ventricular collagen as expressed by hydroxyproline was significantly lower in the treated group (means 0.23 micromoles/10 tissue in the treated vs 0.35 micromoles/100 mg tissue in the untreated group, p < 0.001). Significantly more collagen could be eluted in the treated group (p < 0.001) and carboxymethyllysine was significantly reduced in the treated group (p < 0.001). Di-tyrosine and glycemic control did not differ between the groups. Glutathione was significantly increased in the TP treated experimental group (p < 0.001) and lipid peroxidation products were significantly decreased (means 0.221 absorbance in the treated group versus 0.321 absorbance in the untreated diabetic group) correlating with total ventricular collagen content (r = 0.87, p < 0.01).We conclude that thiaproline reduced total ventricular collagen content by inhibiting collagen cross linking as reflected by increased solubility of collagen and expressed by higher elution quantity of collagen. Thiaproline, and/or its metabolites induced increase of heart glutathione which may well have been scavenging reactive carbonyles derived from lipid peroxidation and advanced stage nonenzymatic glycosylation as shown by decreased total ventricular carboxy-methyllysine and lipid peroxidation products paralleling reduced heart collagen content.It remains to be shown that the successful reduction of heart collagen by thiaproline is paralleled by improved functional properties.     

The leech product saratin is a potent inhibitor of platelet integrin alpha2beta1 and von Willebrand factor binding to collagen

Subendothelial collagen plays an important role, via both direct and indirect mechanisms, in the initiation of thrombus formation at sites of vascular injury. Collagen binds plasma von Willebrand factor, which mediates platelet recruitment to collagen under high shear. Subsequently, the direct binding of the platelet receptors glycoprotein VI and alpha2beta1 to collagen is critical for platelet activation and stable adhesion. Leeches, have evolved a number of inhibitors directed towards platelet-collagen interactions so as to prevent hemostasis in the host during hematophagy. In this article, we describe the molecular mechanisms underlying the ability of the leech product saratin to inhibit platelet binding to collagen. In the presence of inhibitors of ADP and thromboxane A2, both saratin and 6F1, a blocking alpha2beta1 mAb, abrogated platelet adhesion to fibrillar and soluble collagen. Additionally, saratin eliminated alpha2beta1-dependent platelet adhesion to soluble collagen in the presence of an Src kinase inhibitor. Moreover, saratin prevented platelet-rich plasma adhesion to fibrillar collagen, a process dependent upon both alpha2beta1 and von Willebrand factor binding to collagen. Furthermore, saratin specifically inhibited the binding of the alpha2 integrin subunit I domain to collagen, and prevented platelet adhesion to collagen under flow to the same extent as observed in the presence of a combination of mAbs to glycoprotein Ib and alpha2beta1. These results demonstrate that saratin interferes with integrin alpha2beta1 binding to collagen in addition to inhibiting von Willebrand factor-collagen binding, presumably by binding to an overlapping epitope on collagen. This has significant implications for the use of saratin as a tool to inhibit platelet-collagen interactions.     

肺腺癌细胞中EGFR蛋白的表达与细胞内胶原化的相关性研究

目的探讨表皮生长因子受体（epidermal growth factor receptor,EGFR）在肺腺癌细胞中的表达及与细胞发生胶原化的相关性。方法从胸水中提取肺腺癌细胞为研究对象,以32例良性胸水中的增生上皮细胞、炎性细胞为对照,采用免疫细胞化学方法检测细胞中EGFR、E钙粘素蛋白、Vimentin、TTF-1和胶原蛋白亚型I的表达。Masson染色方法检测胶原纤维表达。结果78例胸水标本中,EGFR在肺腺癌细胞中的阳性率为79．5％,胶原蛋白亚型I为32．1％,Masson染色的阳性率为70．5％,明显高于对照组且EGFR和Masson染色的阳性表达结果的相关性具有统计学意义（P〈0．01）。结论EG—FR在肺腺癌细胞中阳性表达,可能与细胞内基质胶原蛋白形成有关。     

Collagen colocalizes with a protein containing a decorin-specific peptide in the tissues of the ascidian Styela plicata

Decorin is an extracellular matrix dermatan sulfate/chondroitin sulfate proteoglycan found in a variety of vertebrate species. In the extracellular matrix of mammals, decorin interacts with fibrillar collagen and regulates its morphology. We report here the occurrence and distribution of collagen type I and the peptide, CEASGIGPEVPDDRD, which is present in the human decorin proteoglycan, in the extracellular matrix of different tissues of the primitive invertebrate chordate Styela plicata. The content of collagen was estimated by hydroxyproline, and its distribution in the tissues by histochemistry. Collagen was detected biochemically in intestine, heart, pharynx and mantle, occurring in higher amounts in the heart, followed by pharynx, mantle and intestine. Histochemical analysis with Sirius red indicates that collagen is present in the extracellular matrix of intestine and pharynx. Further ultrastructural immuno-gold assays using polyclonal antibodies raised against the decorin-specific peptide CEASGIGPEVPDDRD and collagen type I showed a co-localization of these molecules. These data suggest the occurrence of a protein containing a decorin-like peptide sequence, which may be interacting with fibrillar collagen in this primitive chordate.     

Changes in Muscularis Propria of Anterior Vaginal Wall in Women with Pelvic Organ Prolapse

The objective of this study was to evaluate the morphological and immunohistochemical alterations of tissue removed from the upper third of anterior vaginal wall in a sample group of the female population presenting homogenous risk factors associated with pelvic organ prolapse (POP). The case study consisted of 14 patients with POP and there were 10 patients in the control group. Patient selection was carried on the basis of specific criteria and all of the patients involved in the study presented one or more of the recognized POP risk factors. Samples were taken from POP patients during vaginal plastic surgery following colpohysterectomy, and from control patients during closure of the posterior fornix following hysterectomy. Samples were processed for histological and immunohistochemical analyses for Collagen I and Collagen III, α-Smooth Muscle Actin (α-SMA), Platelet-Derived-Growth-Factor (PDGF), matrix metalloproteinase 3 (MMP3), tissue inhibitors metalloproteinase 1 (TIMP1), Caspase3. Immunofluorescence analyses for Collagen I and III and PDGF were also carried out. In prolapsed specimens our results show a disorganization of smooth muscle cells that appeared to have been displaced by an increased collagen III deposition resulting in rearrangement of the muscularis propria architecture. These findings suggest that the increase in the expression of collagen fibers in muscularis could probably be due to a phenotypic switch resulting in the dedifferentiation of smooth muscle cells into myofibroblasts. These alterations could be responsible for the compromising of the dynamic functionality of the pelvic floor.Key words: Pelvic organ prolapse, immunohistochemistry, smooth muscle cells, collagen, PDGF