A low-speed centrifugation concept leads to cell accumulation and vascularization of solid platelet-rich fibrin: an experimental study in vivo.

Platelet-rich fibrin (PRF) is generated from the patients' own venous blood by a single centrifugation step without the additional use of anticoagulants. Based on the previously described LSCC (low-speed centrifugation concept), our group showed that modification of the centrifugation setting, that is, reducing the relative centrifugal force (RCF) and mildly increasing the centrifugation time, resulted in modified solid and liquid PRF-matrices with increased number of platelets, leukocytes, and growth factors' concentrations. The aim of this study was to determine whether RCF reduction might also result in different tissue reactions toward the two PRF-based matrices, especially vascularization and cell distribution in vivo. Two centrifugation protocols (PRF-high [719 g] and PRF-medium [222 g]) were compared in a subcutaneous implantation model of SCID mice at 5 and 10 days. Histological and histomorphometrical analyses were performed to quantify lymphocyte, neutrophil, human macrophage, and monocyte populations. CD31 was used to detect newly formed vessels, while all human cells were detected by using human vimentin as a pan-cellular marker. The results demonstrated that PRF-high elicited a dense and stable fibrin structure and prevented cellular penetration of the host tissue. By contrast, PRF-medium was more porous, had a significantly higher in vivo vascularization rate, and included significantly more human cells, especially at day 10, compared to PRF-high. These findings highlight the possibility of modifying the structure and composition of PRF matrices and thus selectively altering their regenerative potential in vivo. Clinical studies now must evaluate the different PRF matrices for bone and soft-tissue regeneration to validate possible benefits using personalized preparation protocols.

Influence of concentration and preparation of platelet rich fibrin on human bone marrow mononuclear cells (in vitro).

Large bone defects have always been a big challenge. The use of bone marrow mononuclear cells (BMCs) combined with an osteoconductive scaffold has been proved a good alternative for the treatment of large bone defects. Another autologous source for tissue engineering is platelet rich fibrin (PRF). PRF is a blood concentrate system obtained through a one-step centrifugation. The generated 3D matrix of the PRF clot serves as a reservoir of growth factors. Those growth factors might support the regenerative response of BMC, and therefore the effect of PRF, centrifuged with either high medium (208 g) or low (60 g) relative centrifugation force (RCF) on BMCs was evaluated in vitro in the present study. The two PRF matrices obtained were initially characterized and compared to human serum. Significantly increased concentrations of insulin-like growth factor (IGF), soluble intercellular adhesion molecule-1 (sICAM1) and transforming growth factor (TGF)-ß were found in PRF compared to human serum whereas VEGF concentration was not significantly altered. A dose-response study revealed no further activation of BMC's metabolic activity, if concentration of both PRF matrices exceeded 10% (v/v). Effect of both PRF preparations [10%] on BMC was analyzed after 2, 7, and 14 days in comparison to human serum [10%]. Metabolic activity of BMC increased significantly in all groups on day 14. Furthermore, gene expression of matrix metalloproteinases (MMP)-2, -7, and -9 was significantly stimulated in BMC cultivated with the respective PRF matrices compared to human serum. Apoptotic activity of BMC incubated with PRF was not altered compared to BMC cultivated with serum. In conclusion, PRF could be used as a growth factor delivery system of autologous or allogeneic source with the capability of stimulating cells such as BMC.

Radiation Sensitization of Basal Cell and Head and Neck Squamous Cell Carcinoma by the Hedgehog Pathway Inhibitor Vismodegib.

Vismodegib, an inhibitor of the Hedgehog signaling pathway, is an approved drug for monotherapy in locally advanced or metastatic basal cell carcinoma (BCC). Data on combined modality treatment by vismodegib and radiation therapy, however, are rare. In the present study, we examined the radiation sensitizing effects of vismodegib by analyzing viability, cell cycle distribution, cell death, DNA damage repair and clonogenic survival in three-dimensional cultures of a BCC and a head and neck squamous cell carcinoma (HNSCC) cell line. We found that vismodegib decreases expression of the Hedgehog target genes glioma-associated oncogene homologue (GLI1) and the inhibitor of apoptosis protein (IAP) Survivin in a cell line- and irradiation-dependent manner, most pronounced in squamous cell carcinoma (SCC) cells. Furthermore, vismodegib significantly reduced proliferation in both cell lines, while additional irradiation only slightly further impacted on viability. Analyses of cell cycle distribution and cell death induction indicated a G1 arrest in BCC and a G2 arrest in HNSCC cells and an increased fraction of cells in SubG1 phase following combined treatment. Moreover, a significant rise in the number of phosphorylated histone-2AX/p53-binding protein 1 (γH2AX/53BP1) foci in vismodegib- and radiation-treated cells was associated with a significant radiosensitization of both cell lines. In summary, these findings indicate that inhibition of the Hedgehog signaling pathway may increase cellular radiation response in BCC and HNSCC cells.

Reduction of the relative centrifugal force influences cell number and growth factor release within injectable PRF-based matrices.

Platelet rich fibrin (PRF) is a blood concentrate system obtained by centrifugation of peripheral blood. First PRF matrices exhibited solid fibrin scaffold, more recently liquid PRF-based matrix was developed by reducing the relative centrifugation force and time. The aim of this study was to systematically evaluate the influence of RCF (relative centrifugal force) on cell types and growth factor release within injectable PRF- in the range of 60-966 g using consistent centrifugation time. Numbers of cells was analyzed using automated cell counting (platelets, leukocytes, neutrophils, lymphocytes and monocytes) and histomorphometrically (CD 61, CD- 45, CD-15+, CD-68+, CD-3+ and CD-20). ELISA was utilized to quantify the concentration of growth factors and cytokines including PDGF-BB, TGF-ß1, EGF, VEGF and MMP-9. Leukocytes, neutrophils, monocytes and lymphocytes had significantly higher total cell numbers using lower RCF. Whereas, platelets in the low and medium RCF ranges both demonstrated significantly higher values when compared to the high RCF group. Histomorphometrical analysis showed a significantly high number of CD61+, CD-45+ and CD-15+ cells in the low RCF group whereas CD-68+, CD-3+ and CD-20+ demonstrated no statistically significant differences between all groups. Total growth factor release of PDGF-BB, TGF-ß1 and EGF had similar values using low and medium RCF, which were both significantly higher than those in the high RCF group. VEGF and MMP-9 were significantly higher in the low RCF group compared to high RCF. These findings support the LSCC (low speed centrifugation concept), which confirms that improved PRF-based matrices may be generated through RCF reduction. The enhanced regenerative potential of PRF-based matrices makes them a potential source to serve as a natural drug delivery system. However, further pre-clinical and clinical studies are required to evaluate the regeneration capacity of this system.

Multinucleated giant cells in the implant bed of bone substitutes are foreign body giant cells-New insights into the material-mediated healing process.

In addition to macrophages, multinucleated giant cells (MNGCs) are involved in the tissue reaction to a variety of biomaterials. Especially in the case of bone substitute materials it has been assumed that the MNGCs are osteoclasts, based on the chemical and physical similarity of many materials to the calcified matrix and the bony environment in which they are used. However, many studies indicate that these cells belong to the cell line of the foreign body giant cells (FBGCs), which are of "inflammatory origin", although they have been shown to possess both a pro- and also anti-inflammatory phenotype. Moreover, no information is available about their role in the tissue reaction to bone substitute materials. The present study was conducted to analyze the origin of MNGCs in the implant beds of a synthetic and a xenogeneic bone substitute and focused on the application of immunohistochemical methods. Two antibodies against integrin molecules specific for osteoclasts (ß-3 integrin) or FBGCs (ß-2 integrin) were used to distinguish both giant cell types. The results of the present study indicate that the MNGCs induced by both kinds of bone substitutes are FBGCs, as they express only ß-2 integrin in contrast to the osteoclasts outside of the immediate implantation areas, which only demonstrate ß-3 integrin expression. These data give new insight into the tissue reaction to both xenogeneic and synthetic bone substitutes. Based on this new knowledge further research concerning the proteomic profile of the FBGCs especially based on the different physicochemical properties of bone substitutes is necessary. This may show that specific characteristics of bone substitutes may exhibit a substantial influence on the regeneration process via the expression of anti-inflammatory molecules by FBGCs. Based on this information it may be possible to formulate and choose bone substitutes that can guide the process of bone tissue regeneration on the molecular level. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1105-1111, 2017.

Analysis of the in vitro degradation and the in vivo tissue response to bi-layered 3D-printed scaffolds combining PLA and biphasic PLA/bioglass components - Guidance of the inflammatory response as basis for osteochondral regeneration.

The aim of the present study was the in vitro and in vivo analysis of a bi-layered 3D-printed scaffold combining a PLA layer and a biphasic PLA/bioglass G5 layer for regeneration of osteochondral defects in vivo Focus of the in vitro analysis was on the (molecular) weight loss and the morphological and mechanical variations after immersion in SBF. The in vivo study focused on analysis of the tissue reactions and differences in the implant bed vascularization using an established subcutaneous implantation model in CD-1 mice and established histological and histomorphometrical methods. Both scaffold parts kept their structural integrity, while changes in morphology were observed, especially for the PLA/G5 scaffold. Mechanical properties decreased with progressive degradation, while the PLA/G5 scaffolds presented higher compressive modulus than PLA scaffolds. The tissue reaction to PLA included low numbers of BMGCs and minimal vascularization of its implant beds, while the addition of G5 lead to higher numbers of BMGCs and a higher implant bed vascularization. Analysis revealed that the use of a bi-layered scaffold shows the ability to observe distinct in vivo response despite the physical proximity of PLA and PLA/G5 layers. Altogether, the results showed that the addition of G5 enables to reduce scaffold weight loss and to increase mechanical strength. Furthermore, the addition of G5 lead to a higher vascularization of the implant bed required as basis for bone tissue regeneration mediated by higher numbers of BMGCs, while within the PLA parts a significantly lower vascularization was found optimally for chondral regeneration. Thus, this data show that the analyzed bi-layered scaffold may serve as an ideal basis for the regeneration of osteochondral tissue defects. Additionally, the results show that it might be able to reduce the number of experimental animals required as it may be possible to analyze the tissue response to more than one implant in one experimental animal.

"Race for the Surface": Eukaryotic Cells Can Win.

With an aging population and the consequent increasing use of medical implants, managing the possible infections arising from implant surgery remains a global challenge. Here, we demonstrate for the first time that a precise nanotopology provides an effective intervention in bacterial cocolonization enabling the proliferation of eukaryotic cells on a substratum surface, preinfected by both live Gram-negative, Pseudomonas aeruginosa, and Gram-positive, Staphylococcus aureus, pathogenic bacteria. The topology of the model black silicon (bSi) substratum not only favors the proliferation of eukaryotic cells but is biocompatible, not triggering an inflammatory response in the host. The attachment behavior and development of filopodia when COS-7 fibroblast cells are placed in contact with the bSi surface are demonstrated in the dynamic study, which is based on the use of real-time sequential confocal imaging. Bactericidal nanotopology may enhance the prospect for further development of inherently responsive antibacterial nanomaterials for bionic applications such as prosthetics and implants.

Relative expression of α-smooth muscle actin and matrix metalloproteinases-2 in ameloblastoma of a black African sub-population.

BACKGROUND: Ameloblastoma although a benign odontogenic tumor, is locally invasive. The abundant presence of myofibroblasts (marked by α-smooth muscle actin [α-SMA]) in the stroma and expression of matrix metalloproteinase-2 (MMP-2) in the neoplastic or stromal cells have been linked with the tumor's ability for both local and distant spread. We aim to estimate the relative expression of α-SMA and MMP-2 in ameloblastoma from a black African subgroup to gauge their relative potential for enhancing local invasiveness and hence, their prospects as possible chemotherapeutic targets. MATERIALS AND METHODS: Twenty-five formalin-fixed paraffin-embedded blocks of ameloblastoma cases from Nigeria were prepared for antibody processing to α-SMA (Dako Monoclonal Mouse Anti-Human α-SMA antibody clone 1A4) and MMP-2 (Abcam Mouse Monoclonal Anti-MMP-2 antibody [CA-4001/CA719E3C] ab3158). The score for percentage positivity of the tumor cells and the score for staining intensities were then multiplied in order to generate an immunoreactive score. RESULTS: α-smooth muscle actin was only expressed in the fibrous connective tissues adjacent to the tumor islands while MMP-2 was expressed in the ameloblasts, stellate reticulum, and the connective tissues in varying proportions. All the variants analyzed expressed α-SMA mildly or moderately, except for the follicular variant that either did not express α-SMA or expressed it mildly. The highest number of strong immunoreactivity to MMP-2 in the ameloblast region was found in the plexiform variant. CONCLUSION: Chemotherapeutic targeting of both molecules may, therefore, be a vital step in the control of local ameloblastoma invasiveness.

Vismodegib hedgehog-signaling inhibition and treatment of basal cell carcinomas as well as keratocystic odontogenic tumors in Gorlin syndrome.

Vismodegib hedgehog signaling inhibition treatment has potential for reducing the burden of multiple skin basal cell carcinomas and jaw keratocystic odontogenic tumors. They are major criteria for the diagnosis of Gorlin syndrome, also called nevoid basal cell carcinoma syndrome. Clinical features of Gorlin syndrome are reported, and the relevance of hedgehog signaling pathway inhibition by oral vismodegib for maxillofacial surgeons is highlighted. In summary, progressed basal cell carcinoma lesions are virtually inoperable. Keratocystic odontogenic tumors have an aggressive behavior including rapid growth and extension into adjacent tissues. Interestingly, nearly complete regression of multiple Gorlin syndrome-associated keratocystic odontogenic tumors following treatment with vismodegib. Due to radio-hypersensitivity in Gorlin syndrome, avoidance of treatment by radiotherapy is strongly recommended for all affected individuals. Vismodegib can help in those instances where radiation is contra-indicated, or the lesions are inoperable. The effect of vismodegib on basal cell carcinomas was associated with a significant decrease in hedgehog-signaling and tumor proliferation. Vismodegib, a new and approved drug for the treatment of advanced basal cell carcinoma, is a specific oncogene inhibitor. It also seems to be effective for treatment of keratocystic odontogenic tumors and basal cell carcinomas in Gorlin syndrome, rendering the surgical resections less challenging.

PTCH-1 and MDM2 expression in ameloblastoma from a West African sub-population: implication for chemotherapeutics.

INTRODUCTION: Ameloblastoma is a slow growing, painless odontogenic swelling which can attain sizes that result in severe deformities of the craniofacial complex. It is the most commonly encountered odontogenic tumor in Nigeria. Surgical intervention is currently the method of treatment; however identification of altered molecular pathways may inform chemotherapeutic potential. The Protein Patched homolog 1 (PTCH-1) is overexpressed in ameloblastoma. Also, mutation in the MDM2 gene can reduce the tumor suppressor function of p53 and promote ameloblastoma growth. No study however has characterized the molecular profile of African cases of ameloblastoma with a view to developing chemotherapeutic alternatives. The objective was to characterize the PTCH-1 genetic profile of Ameloblastoma in Nigerian patients as a first step in investigating its potential for chemotherapeutic intervention. METHODS: Twenty-eight FFPE blocks of ameloblastoma cases from Nigerian patients were prepared for antibody processing to PTCH-1 (Polyclonal Anti-PTCH antibody ab39266) and MDM2 (Monoclonal Anti-MDM2 antibody (2A10) ab16895). Cytoplasmic brown staining was considered as positive for PTCH while nuclear staining was positive for MDM2. RESULTS: Moderate and strong expressions for PTCH in ameloblast and stellate reticulum were 78.6% and 60.7% respectively. Only 3 (10.7%) cases expressed MDM2. CONCLUSION: The importance of our study is that it supports, in theory, anti-PTCH/SHH chemotherapeutics for Nigerian ameloblastoma cases and also infers the possible additional use of anti-p53 agents.

Porcine Dermis and Pericardium-Based, Non-Cross-Linked Materials Induce Multinucleated Giant Cells After Their In Vivo Implantation: A Physiological Reaction?

The present study analyzed the tissue reaction to 2 novel porcine-derived collagen materials: pericardium versus dermis. By means of the subcutaneous implantation model in mice, the tissue reactions were investigated at 5 time points: 3, 10, 15, 30, and 60 days after implantation. Histologic, histochemical, immunhistologic, and histomorphometric analysis methodologies were applied. The dermis-derived material underwent an early degradation while inducing mononuclear cells together with some multinucleated giant cells and mild vascularization. The pericardium-derived membrane induced 2 different cellular tissue reactions. The compact surface induced mononuclear cells and multinucleated giant cells, and underwent a complete degradation until day 30. The spongy surface of the membrane induced mainly mononuclear cells, and served as a stable barrier membrane for up to 60 days. No transmembranous vascularization was observed within the spongy material surface layer. The present data demonstrate the diversity of the cellular tissue reaction toward collagen-based materials from different tissues. Furthermore, it became obvious that the presence of multinucleated giant cells was associated with the material breakdown/degradation and vascularization. Further clinical data are necessary to assess extent to which the presence of multinucleated giant cells observed here will influence the materials stability, integration, and, correspondingly, tissue regeneration within human tissue.

Porcine Dermis-Derived Collagen Membranes Induce Implantation Bed Vascularization Via Multinucleated Giant Cells: A Physiological Reaction?

In this study, the tissue reactions to 2 new porcine dermis-derived collagen membranes of different thickness were analyzed. The thicker material (Mucoderm) contained sporadically preexisting vessel skeletons and fatty islands. The thinner membrane (Collprotect) had a bilayered structure (porous and occlusive side) without any preexisting structures. These materials were implanted subcutaneously in mice to analyze the tissue reactions and potential transmembranous vascularization. Histological and histomorphometrical methodologies were performed at 4 time points (3, 10, 15, and 30 days). Both materials permitted stepwise connective tissue ingrowth into their central regions. In the Mucoderm matrix, newly built microvessels were found within the preexisting vessel and fatty island skeletons after 30 days. This vascularization was independent of the inflammation-related vascularization on both material surfaces. The Collprotect membrane underwent material disintegration by connective tissue strands in combination with vessels and multinucleated giant cells. The histomorphometric analyses revealed that the thickness of Mucoderm did not decrease significantly, while an initial significant decrease of membrane thickness in the case of Collprotect was found at day 15. The present results demonstrate that the 2 analyzed collagen membranes underwent a multinucleated giant cell-associated vascularization. Neither of the materials underwent transmembraneous vascularization. The microvessels were found within the preexisting vessel and fatty island skeletons. Additional long-term studies and clinical studies are necessary to determine how the observed foreign body giant cells affect tissue regeneration.

Advanced platelet-rich fibrin: a new concept for cell-based tissue engineering by means of inflammatory cells.

Choukroun's platelet-rich fibrin (PRF) is obtained from blood without adding anticoagulants. In this study, protocols for standard platelet-rich fibrin (S-PRF) (2700 rpm, 12 minutes) and advanced platelet-rich fibrin (A-PRF) (1500 rpm, 14 minutes) were compared to establish by histological cell detection and histomorphometrical measurement of cell distribution the effects of the centrifugal force (speed and time) on the distribution of cells relevant for wound healing and tissue regeneration. Immunohistochemistry for monocytes, T and B -lymphocytes, neutrophilic granulocytes, CD34-positive stem cells, and platelets was performed on clots produced from four different human donors. Platelets were detected throughout the clot in both groups, although in the A-PRF group, more platelets were found in the distal part, away from the buffy coat (BC). T- and B-lymphocytes, stem cells, and monocytes were detected in the surroundings of the BC in both groups. Decreasing the rpm while increasing the centrifugation time in the A-PRF group gave an enhanced presence of neutrophilic granulocytes in the distal part of the clot. In the S-PRF group, neutrophils were found mostly at the red blood cell (RBC)-BC interface. Neutrophilic granulocytes contribute to monocyte differentiation into macrophages. Accordingly, a higher presence of these cells might be able to influence the differentiation of host macrophages and macrophages within the clot after implantation. Thus, A-PRF might influence bone and soft tissue regeneration, especially through the presence of monocytes/macrophages and their growth factors. The relevance and feasibility of this tissue-engineering concept have to be proven through in vivo studies.

Scaffold vascularization in vivo driven by primary human osteoblasts in concert with host inflammatory cells.

Successful cell-based tissue engineering requires a rapid and thorough vascularization in order to ensure long-term implant survival and tissue integration. The vascularization of a scaffold is a complex process, and is modulated by the presence of transplanted cells, exogenous and endogenous signaling proteins, and the host tissue reaction, among other influencing factors. This paper presents evidence for the significance of pre-seeded osteoblasts for the in vivo vascularization of a biodegradable scaffold. Human osteoblasts, cultured on silk fibroin micronets in vitro, migrated throughout the interconnected pores of the scaffold and produced extensive bone matrix. When these constructs were implanted in SCID mice, a rapid and thorough vascularization of the scaffold by the host blood capillaries occurred. This profound response was not seen for the silk fibroin scaffold alone. Moreover, when the pre-cultivation time of human osteoblasts was reduced from 14 days to only 24 h, the significant effect these cells exerted on vascularization rate in vivo was still detectable. From these studies, we conclude that matrix and soluble factors produced by osteoblasts can serve to instruct host endothelial cells to migrate, proliferate, and initiate the process of scaffold vascularization. This finding represents a potential paradigm shift for the field of tissue engineering, especially in bone, as traditional strategies to enhance scaffold vascularization have focused on endovascular cells and regarded osteoblasts primarily as cell targets for mineralization. In addition, the migration of host macrophages and multinucleated giant cells into the scaffold was also found to influence the vascularization of the biomaterial. Therefore, the robust effect on scaffold vascularization seen by pre-culturing with osteoblasts appears to occur in concert with the pro-angiogenic stimuli arising from host immune cells.

Histological and histomorphometrical analysis of a silica matrix embedded nanocrystalline hydroxyapatite bone substitute using the subcutaneous implantation model in Wistar rats.

The clinical suitability of a bone substitute material is determined by the ability to induce a tissue reaction specific to its composition. The aim of this in vivo study was to analyze the tissue reaction to a silica matrix-embedded, nanocrystalline hydroxyapatite bone substitute.The subcutaneous implantation model in Wistar rats was chosen to assess the effect of silica degradation on the vascularization of the biomaterial and its biodegradation within a time period of 6 months. Already at day 10 after implantation, histomorphometrical analysis showed that the vascularization of the implantation bed reached its peak value compared to all other time points. Both vessel density and vascularization significantly decreased until day 90 after implantation. In this time period, the bone substitute underwent a significant degradation initiated by TRAP-positive and TRAP-negative multinucleated giant cells together with macrophages and lymphocytes. Although no specific tissue reaction could be related to the described silica degradation, the biomaterial was close to being fully degraded without a severe inflammatory response. These characteristics are advantageous for bone regeneration and remodeling processes.

A RYR1 mutation associated with recessive congenital myopathy and dominant malignant hyperthermia in Asian families.

In this study we present 3 families with malignant hyperthermia (MH), all of Indian subcontinent descent. One individual from each of these families was fully sequenced for RYR1 and presented with the non-synonymous change c.11315G>A/p.R3772Q. When present in the homozygous state c.11315*A is associated with myopathic symptoms.

Induction of macrophage chemotaxis by aortic extracts of the mgR Marfan mouse model and a GxxPG-containing fibrillin-1 fragment.

BACKGROUND: The primary cause of early death in untreated Marfan syndrome (MFS) patients is aortic dilatation and dissection. METHODS AND RESULTS: We investigated whether ascending aortic samples from the fibrillin-1-underexpressing mgR mouse model for MFS or a recombinant fibrillin-1 fragment containing an elastin-binding protein (EBP) recognition sequence can act as chemotactic stimuli for macrophages. Both the aortic extracts from the mgR/mgR mice and the fibrillin-1 fragment significantly increased macrophage chemotaxis compared with extracts from wild-type mice or buffer controls. The chemotactic response was significantly diminished by pretreatment of macrophages with lactose or with the elastin-derived peptide VGVAPG and by pretreatment of samples with a monoclonal antibody directed against an EBP recognition sequence. Mutation of the EBP recognition sequence in the fibrillin-1 fragment also abolished the chemotactic response. These results indicate the involvement of EBP in mediating the effects. Additionally, investigation of macrophages in aortic specimens of MFS patients demonstrated macrophage infiltration in the tunica media. CONCLUSIONS: Our findings demonstrate that aortic extracts from mgR/mgR mice can stimulate macrophage chemotaxis by interaction with EBP and show that a fibrillin-1 fragment possesses chemotactic stimulatory activity similar to that of elastin degradation peptides. They provide a plausible molecular mechanism for the inflammatory infiltrates observed in the mgR mouse model and suggest that inflammation may represent a component of the complex pathogenesis of MFS.

A fibrillin-1-fragment containing the elastin-binding-protein GxxPG consensus sequence upregulates matrix metalloproteinase-1: biochemical and computational analysis.

Mutations in the gene for fibrillin-1 cause Marfan syndrome (MFS), a common hereditary disorder of connective tissue. Recent findings suggest that proteolysis, increased matrix metalloproteinase activity, and fragmentation of fibrillin-rich microfibrils in tissues of persons with MFS contribute to the complex pathogenesis of this disorder. In this study we show that a fibrillin-1 fragment containing a EGFEPG sequence that conforms to a putative GxxPG elastin-binding protein (EBP) consensus sequence upregulates the expression and production of matrix metalloproteinase (MMP)-1 by up to ninefold in a cell culture system. A mutation of the GxxPG consensus sequence site abrogated the effects. This is the first demonstration of such an effect for ligands other than elastin fragments. Molecular dynamics analysis of oligopeptides with the wildtype and mutant sequence support our biochemical results by predicting significant alterations of structural characteristics such as the potential for forming a type VIII beta-turn that are thought to be important for binding to the EBP. These results suggest that fibrillin-1 fragments may regulate MMP-1 expression, and that the dysregulation of MMPs related to fragmentation of fibrillin might contribute to the development of MFS. Our Gene Ontology (GO) analysis of the human proteome shows that proteins with multiple GxxPG motifs are highly enriched for GO terms related to the extracellular matrix. Matrix proteins with multiple GxxPG sites include fibrillin-1, -2, and -3, elastin, fibronectin, laminin, and several tenascins and collagens. Some of these proteins have been associated with disorders involving alterations in MMP regulation, and the results of the present study suggest a potential mechanism for these observations.

Calcium-dependent self-association of the C-type lectin domain of versican.

Versican is a large (1-2 x 10(6) Da) chondroitin-sulfate proteoglycan that can form large aggregates by means of interaction with hyaluronan and also binds to a series of other extracellular matrix proteins, chemokines and cell-surface molecules. Versican is a multifunctional molecule with roles in cell adhesion, matrix assembly, cell migration and proliferation. Characterization of the binding interactions mediated by the various domains of versican is a first step towards understanding the functions of versican and interacting molecules in the extracellular matrix. In this study we investigated a recombinant construct corresponding to the C-type lectin domain of versican and demonstrated a calcium-dependent self-association of this region by blot overlay and plasmon surface resonance assays. Electron microscopy provided further evidence of the relevance of the binding reaction by demonstrating a mixture of monomers, dimers and complex aggregates of recombinant versican C-type lectin domain. This binding reaction could contribute to the ability of versican to organize formation of the proteoglycan extracellular matrix by inducing binding of individual versican molecules or by modulating binding reactions to other matrix components.

RGD-containing fibrillin-1 fragments upregulate matrix metalloproteinase expression in cell culture: a potential factor in the pathogenesis of the Marfan syndrome.

The Marfan syndrome (MFS), a relatively common autosomal dominant disorder of connective tissue, is caused by mutations in the gene for fibrillin-1 (FBN1). Fibrillin-1 is the main component of the 10- to 12-nm microfibrils that together with elastin form elastic fibers found in tissues such as the aortic media. Recently, FBN1 mutations have been shown to increase the susceptibility of fibrillin-1 to proteolysis in vitro, and other findings suggest that up-regulation of matrix metalloproteinases (MMP), as well as fragmentation of microfibrils, could play a role in the pathogenesis of MFS. In the present work, we have investigated the influence of fibrillin-1 fragments on the expression of MMP-1, MMP-2, and MMP-3 in a cell culture system. Cultured human dermal fibroblasts were incubated with several different recombinant fibrillin-1 fragments. The expression level of MMP-1, MMP-2, and MMP-3, was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and the concentration of the corresponding proteins was estimated by quantitative Western blotting. Our results establish that treatment of cultured human dermal fibroblasts with recombinant fibrillin-1 fragments containing the arginine-glycine-aspartic acid (RGD) integrin-binding motif of fibrillin-1 induces up-regulation of MMP-1 and MMP-3. A similar effect was seen upon stimulation with a synthetic RGD peptide. The expression of MMP-2 was not influenced by treatment. Our results suggest the possibility that fibrillin fragments could themselves have pathogenic effects by leading to up-regulation of MMPs, which in turn may be involved in the progressive breakdown of microfibrils thought to play a role in MFS.