Collagen XII interacts with avian tenascin-X through its NC3 domain

Large oligomeric proteins often contain several binding sites for different molecules and can therefore induce formation of larger protein complexes. Collagen XII, a multidomain protein with a small collagenous region, interacts with fibrillar collagens through its C-terminal region. However, no interactions to other extracellular proteins have been identified involving the non-collagenous N-terminal NC3 domain. To further elucidate the components of protein complexes present close to collagen fibrils, different extracellular matrix proteins were tested for interaction in a solid phase assay. Binding to the NC3 domain of collagen XII was found for the avian homologue of tenascin-X that in humans is linked to Ehlers-Danlos disease. The binding was further characterized by surface plasmon resonance spectroscopy and supported by immunohistochemical co-localization in chick and mouse tissue. On the ultrastructural level, detection of collagen XII and tenascin-X by immunogold labeling confirmed this finding.

Expression of phosphoproteins and amelotin in teeth

The organic material of our teeth consists of collagens and a number of calcium-binding phosphoproteins. Six of these phosphoproteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins), namely osteopontin, bone sialoprotein, dentin matrix protein (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE) and enamelin. We prepared a cDNA library from rat incisors in order to identify the genes involved in tooth formation. The library was screened by subtractive hybridization with two probes; one specific for teeth, the other for bone. We found that the vast majority of the clones from our library were expressed at similar levels in bone and teeth, demonstrating the close relationship of the two tissues. Only 7% of all the clones were expressed in a tooth-specific fashion. These included clones for the enamel proteins; amelotin, amelogenin, ameloblastin and enamelin; for the dentin proteins DSPP and DMP1; and for the intermediate filament protein cytokeratin 13. Several typical bone proteins, including collagen I, osteocalcin, alkaline phosphatase and FATSO, were also expressed at significantly higher levels in teeth than in bone, probably due to the extreme growth rate of rat incisors. The amino acid sequence of rat amelotin showed 62% identity with the sequence from humans. It was expressed considerably later than the other enamel proteins, suggesting that amelotin may serve a function different from those of amelogenin, ameloblastin and enamelin.

Collagen response and glycoprotein VI function decline progressively as canine platelets age in vivo

Clinical and experimental observations suggest that platelet function deteriorates quickly with cell age. However, efforts to define age-dependent alterations have detected only modest biochemical changes occurring late in the cell life span. In this report, we demonstrate two significant alterations of the collagen response occurring during in vivo aging of canine platelets: a progressive increase in the EC50 for collagen types I, III and V and the emergence of a population of aged platelets which are refractory to collagen. Experiments with convulxin, a specific agonist for the collagen receptor glycoprotein VI (GPVI), also demonstrate an age-dependent decline in activation and the appearance of a non-reactive, aged population as observed with native collagens. Our studies indicate that canine platelets have two distinct binding levels for FITC-labeled convulxin and that the higher binding level disappears upon cell aging. During these studies one dog (#428) was identified whose platelets not only failed to demonstrate an age-dependent decrease in convulxin reactivity but also maintained a high convulxin-binding ability throughout their otherwise normal life span. Transfusion of biotinylated platelets from control dogs into dog #428 showed that the expected changes in collagen response and GPVI function did not occur in the transfused platelets. These observations demonstrate that the canine platelet response towards collagen is strongly dependent upon cell-age and suggest that this functional decline is at least partly due to an extrinsic-mediated alteration, possibly proteolytic, of GPVI.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Platelet collagen receptors

Collagens are important platelet activators in the vascular subendothelium and vessel wall. Since the regulation of platelet activation is a key step in distinguishing normal haemostasis from pathological thrombosis, collagen interactions with platelets are important targets for pharmacological control. Platelets have two major receptors for collagens, the integrin alpha2beta1, with a major role in adhesion and platelet anchoring and the Ig superfamily member, GPVI, principally responsible for signalling and platelet activation. In addition, GPIb-V-IX, can be considered as an indirect collagen receptor acting via von Willebrand factor as bridging molecule and is essential for platelet interactions with collagen at high shear rates. There is some evidence for additional receptors, which may regulate the response to individual collagen types. This review discusses how these receptors work separately with specific agonists and proposes possible mechanisms for how they work together to regulate platelet activation by collagen, which remains controversial and poorly understood.

Platelet collagen receptors: a new target for inhibition?

Collagen is a major component of extracellular matrix and a wide variety of types exist. Cells recognise collagen in different ways depending on sequence and structure. They can recognise predominantly primary sequence, they may require triple-helical structure or they can require fibrillar structures. Since collagens are major constituents of the subendothelium that determine the thrombogenicity of the injured or pathological vessel wall, a major role is induction of platelet activation and aggregation as the start of repair processes. Platelets have at least two direct and one indirect (via von Willebrand factor) receptors for collagen, and collagen has specific recognition motifs for these receptors. These receptors and recognition motifs are under intensive investigation in the search for possible methods to control platelet activation in vivo. A wide range of proteins has been identified and, in part, characterised from both haematophageous insects and invertebrates but also from snake venoms that inhibit platelet activation by collagen or induce platelet activation via collagen receptors on platelets. These will provide model systems to test the effect of inhibition of specific collagen-platelet receptor interactions for both effectiveness as well as for side effects and should provide assay systems for the development of small molecule inhibitors. Since platelet inhibitors for long-term prophylaxis of cardiovascular diseases are still in clinical trials there are many unanswered questions about long-term effects both positive and negative. The major problem which still has to be definitively solved about these alternative approaches to inhibition of platelet activation is whether they will show advantages in terms of dose-response curves while offering decreased risks of bleeding problems. Preliminary studies would seem to suggest that this is indeed the case.

Sensitivity of osteoblasts, fibroblasts, bone marrow cells, and dendritic cells to 5-aminolevulinic acid based photodynamic therapy

INTRODUCTION: Photodynamic therapy with 5-aminolevulinic acid (5-ALA-PDT) exerts cell type specific effects on target cells. Since chondrocytes were found to be more resistant than osteoblasts to 5-ALA-PDT, the pre-treatment of osteochondral grafts with 5-ALA-PDT may represent a means to devitalize the osseous portion while maintaining functional cartilage. The present study was designed to determine the effects of 5-ALA-PDT in vitro on cell populations residing in skeletal tissues. METHODS: Osteoblasts, fibroblasts, bone marrow cells, and dendritic cells were incubated with 0.5 mM 5-ALA for 4 h. Protoporphyrin IX (PpIX) accumulation and after exposure to light cellular functions were assessed for up to 6 days. RESULTS: Accumulation of PpIX reached a plateau at 0.5 mM in osteoblasts, fibroblasts, and dendritic cells, and at 2.0 mM in bone marrow cells. At 0.5 mM 5-ALA, similar responses to illumination were observed in all cells with a survival rate of less than 12% at a light dose of 20 J/cm(2). The function of osteoblasts (proliferation, levels of mRNA encoding collagen type I, alkaline phosphatase activity) and fibroblasts (proliferation, levels of mRNAs encoding collagens type I and III) was not affected, when the cells were treated with 5-ALA and light doses of < or =10 J/cm(2). Paralleling the reduction of viable cells after 5-ALA-PDT, the capacity of dendritic cells to stimulate T cells in a mixed leukocyte reaction decreased to 4+/-2% at 20 J/cm(2). CONCLUSION: The investigated cell types were sensitive to 5-ALA-PDT and the residual cell debris did not elicit an allogenic response. These findings, together with the resistance of chondrocytes to 5-ALA-PDT, encourage the further investigation of this protocol in the pretreatment of osteochondral allografts.

Mechanical signals regulating extracellular matrix gene expression in fibroblasts

Mechanical forces are essential for connective tissue homeostasis. The extracellular matrix (ECM) plays a key role in the transmission of forces generated by the organism (e.g. muscle contraction) and externally applied (e.g. gravity). The expression of specific ECM proteins such as collagens and tenascin-C, as well as of matrix metalloproteinases, involved in their turnover, is influenced by mechanical stimuli. The precise mechanisms by which mechanical strains are translated into chemical signals and lead to differential gene expression are however not fully understood. Cell-matrix adhesion sites are good candidates for hosting a "mechanosensory switch", as they transmit forces from the ECM to the cytoskeleton and vice versa by physically linking the cytoskeleton to the ECM. Integrins, transmembrane proteins located to these adhesion sites, have been shown to trigger a set of internal signaling cascades after mechanical stimulation. We have shown that the expression level of tenascin-C directly correlates with externally applied mechanical stress, as well as with RhoA/RhoA-dependent kinase-mediated cytoskeletal tension. Presumably other genes are regulated in a similar manner. The changes in ECM composition and mechanical properties derived from mechanical stress are relevant in medical intervention after ligament and tendon injury.

Tenocytes of chronic rotator cuff tendon tears can be stimulated by platelet-released growth factors

BACKGROUND Bone-to-tendon healing after rotator cuff repairs is mainly impaired by poor tissue quality. The tenocytes of chronic rotator cuff tendon tears are not able to synthesize normal fibrocartilaginous extracellular matrix (ECM). We hypothesized that in the presence of platelet-released growth factors (PRGF), tenocytes from chronically retracted rotator cuff tendons proliferate and synthesize the appropriate ECM proteins. MATERIALS AND METHODS Tenocytes from 8 patients with chronic rotator cuff tears were cultured for 4 weeks in 2 different media: standard medium (Iscove's Modified Dulbecco's Media + 10% fetal calf serum + 1% nonessential amino acids + 0.5 μg/mL ascorbic acid) and media with an additional 10% PRGF. Cell proliferation was assessed at 7, 14, 21, and 28 days. Messenger (m)RNA levels of collagens I, II, and X, decorin, biglycan, and aggrecan were analyzed using real time reverse-transcription polymerase chain reaction. Immunocytochemistry was also performed. RESULTS The proliferation rate of tenocytes was significantly higher at all time points when cultured with PRGF. At 21 days, the mRNA levels for collagens I, II, and X, decorin, aggrecan, and biglycan were significantly higher in the PRGF group. The mRNA data were confirmed at protein level by immunocytochemistry. CONCLUSIONS PRGFs enhance tenocyte proliferation in vitro and promote synthesis of ECM to levels similar to those found with insertion of the normal human rotator cuffs. CLINICAL RELEVANCE Biologic augmentation of repaired rotator cuffs with PRGF may enhance the properties of the repair tissue. However, further studies are needed to determine if application of PRGF remains safe and effective in long-term clinical studies. LEVEL OF EVIDENCE Basic Science Study, Cell Biology.

Glomerular and renal vascular structural changes in alpha8 integrin-deficient mice

Integrins are matrix receptors that regulate cell-matrix interactions during development and in adult tissue. In the adult kidney, the alpha8 chain is specifically expressed in glomerular mesangial cells and vascular smooth muscle cells. alpha8-deficient (alpha8-/-) mice demonstrate reductions in renal mass, which can range from complete renal agenesis to the development of kidneys that are only slightly smaller than wild-type kidneys. No histologic abnormalities of these kidneys have been described. However, considering the prominent expression of alpha8 in glomeruli and renal vessels, it seemed unlikely that the kidneys of alpha8-/- mice would be completely normal. Therefore, the renal phenotype of adult alpha8-/- mice was investigated, for assessment of more subtle morphologic alterations in kidney tissue. alpha8-/- mice displayed a significant reduction in nephron number and an increase in glomerular volume, compared with wild-type control animals. Albuminuria was not different in wild-type and alpha8-/- mice. Quantitative morphologic analyses revealed that the glomeruli of alpha8-/- mice were hypercellular, with an increased number of mesangial cells, compared with wild-type mice. Mesangial matrix deposition (as demonstrated for collagen IV and the alpha8 ligand fibronectin) was expanded in alpha8-/- mice, compared with wild-type mice. Collagens I and III, which are not normally present in glomeruli, were detected in the glomeruli of alpha8-/- mice. Staining for other glomerular integrins demonstrated an increased abundance of the collagen receptor alpha2 integrin in alpha8-/- mice. The glomerular capillary length density was significantly greater in alpha8-/- mice than in wild-type mice. Cortical arterial vessel walls were not altered in alpha8-/- mice, but the capillaries of the peritubular network were widened. Despite the strong mesangial and vascular expression of alpha8, glomerular and renal vascular alterations in alpha8-/- mice were relatively mild. Only aged alpha8-/- mice demonstrated increased glomerular capillary widening, compared with control animals. The results suggest that the lack of alpha8 can be largely compensated for, at least in younger alpha8-/- mice. It is not yet clear whether the occurrence of collagens that are not normally present in glomeruli and the increased abundance of the collagen receptor alpha2 contribute to maintaining the glomerular structure in alpha8-/- mice. The compensatory mechanisms involved will be the subject of future research.

Collagen XII: Protecting bone and muscle integrity by organizing collagen fibrils.

Collagen XII, largest member of the fibril-associated collagens with interrupted triple helix (FACIT) family, assembles from three identical α-chains encoded by the COL12A1 gene. The molecule consists of three threadlike N-terminal noncollagenous NC3 domains, joined by disulfide bonds and a short interrupted collagen triple helix toward the C-terminus. Splice variants differ considerably in size and properties: "small" collagen XIIB (220 kDa subunit) is similar to collagen XIV, whereas collagen XIIA (350 kDa) has a much larger NC3 domain carrying glycosaminoglycan chains. Collagen XII binds to collagen I-containing fibrils via its collagenous domain, whereas its large noncollagenous arms interact with other matrix proteins such as tenascin-X. In dense connective tissues and bone, collagen XII is thought to regulate organization and mechanical properties of collagen fibril bundles. Accordingly, recent findings show that collagen XII mutations cause Ehlers-Danlos/myopathy overlap syndrome associated with skeletal abnormalities and muscle weakness in mice and humans.

How best to preserve and reveal the structural intricacies of cartilaginous tissue.

No single processing technique is capable of optimally preserving each and all of the structural entities of cartilaginous tissue. Hence, the choice of methodology must necessarily be governed by the nature of the component that is targeted for analysis, for example, fibrillar collagens or proteoglycans within the extracellular matrix, or the chondrocytes themselves. This article affords an insight into the pitfalls that are to be encountered when implementing the available techniques and how best to circumvent them. Adult articular cartilage is taken as a representative pars pro toto of the different bodily types. In mammals, this layer of tissue is a component of the synovial joints, wherein it fulfills crucial and diverse biomechanical functions. The biomechanical functions of articular cartilage have their structural and molecular correlates. During the natural course of postnatal development and after the onset of pathological disease processes, such as osteoarthritis, the tissue undergoes structural changes which are intimately reflected in biomechanical modulations. The fine structural intricacies that subserve the changes in tissue function can be accurately assessed only if they are faithfully preserved at the molecular level. For this reason, a careful consideration of the tissue-processing technique is indispensable. Since, as aforementioned, no single methodological tool is capable of optimally preserving all constituents, the approach must be pre-selected with a targeted structure in view. Guidance in this choice is offered.