Evaluating the Efficacy of a Thermoresponsive Hydrogel for Delivering Anti-Collagen Antibodies to Reduce Posttraumatic Scarring in Orthopedic Tissues.

Excessive posttraumatic scarring in orthopedic tissues, such as joint capsules, ligaments, tendons, muscles, and peripheral nerves, presents a significant medical problem, resulting in pain, restricted joint mobility, and impaired musculoskeletal function. Current treatments for excessive scarring are often ineffective and require the surgical removal of fibrotic tissue, which can aggravate the problem. The primary component of orthopedic scars is collagen I-rich fibrils. Our research team has developed a monoclonal anti-collagen antibody (ACA) that alleviates posttraumatic scarring by inhibiting collagen fibril formation. We previously established the safety and efficacy of ACA in a rabbit-based arthrofibrosis model. In this study, we evaluate the utility of a well-characterized thermoresponsive hydrogel (THG) as a delivery vehicle for ACA to injury sites. Crucial components of the hydrogel included N-isopropylacrylamide, poly(ethylene glycol) diacrylate, and hyaluronic acid. Our investigation focused on in vitro ACA release kinetics, stability, and activity. Additionally, we examined the antigen-binding characteristics of ACA post-release from the THG in an in vivo context. Our preliminary findings suggest that the THG construct exhibits promise as a delivery platform for antibody-based therapeutics to reduce excessive scarring in orthopedic tissues.

Corneal Wound Healing in the Presence of Antifibrotic Antibody Targeting Collagen Fibrillogenesis: A Pilot Study.

Highly organized collagen fibrils interlacing with proteoglycans form the crucial architecture of the cornea and facilitate its transparency. Corneal scarring from accidental injury, surgery, or infection alters this highly organized tissue, causing severe consequences, including blindness. There are no pharmacological or surgical methods to effectively and safely treat excessive corneal scarring. Thus, we tested the anticorneal scarring utility of a rationally designed anticollagen antibody (ACA) whose antifibrotic effects have already been demonstrated in nonocular models. Utilizing a rabbit model with an incisional corneal wound, we analyzed ACA's effects on forming collagen and proteoglycan-rich extracellular matrices in scar neotissue. We used microscopic and spectroscopic techniques to quantify these components and measure crucial parameters characterizing the structure and organization of collagen fibrils. Moreover, we analyzed the spatial distribution of collagen and proteoglycans in normal and healing corneas. Our study demonstrated significant changes in the quality and quantity of the analyzed molecules synthesized in scar neotissue. It showed that these changes extend beyond incision margins. It also showed ACA's positive impact on some crucial parameters defining proper cornea structure. This pilot study provides a stepping stone for future tests of therapeutic approaches that target corneal extracellular scar matrix assembly.

Scar formation in the presence of mitomycin C and the anti-fibrotic antibody in a rabbit model of glaucoma microsurgery: A pilot study.

Purpose: This study aimed to evaluate the utility of a rationally engineered antibody that directly blocks collagen fibrillogenesis to reduce scar tissue formation associated with subconjunctival glaucoma surgery. Material and methods: Fourteen eyes of 7 adult rabbits underwent glaucoma filtering surgery using XEN 45 Gel Stent. The rabbits' eyes were divided randomly into three treatment groups: (i) treated with the antibody, (ii) treated with mitomycin C, and (iii) treated with the antibody and mitomycin C. Following surgeries, the intraocular pressure and bleb appearance were evaluated in vivo. The rabbits were sacrificed 8 weeks after the surgery, and their eyes were harvested and processed for tissue analysis. Subsequently, tissue samples were analyzed microscopically for fibrotic tissue and cellular markers of inflammation. Moreover, the collagen-rich fibrotic tissue formed around the stents was analyzed using quantitative histology and infrared spectroscopy. The outcomes of this study were analyzed using the ANOVA test. Results: This study demonstrated no significant differences in intraocular pressure, bleb appearance, or presence of complications such as bleb leak among the treatment groups. In contrast, we observed significant differences among the subpopulations of collagen fibrils formed within scar neo-tissue. Based on the spectroscopic analyses, we determined that the relative content of mature collagen cross-links in the antibody-treated group was significantly reduced compared to other groups. Conclusions: Direct blocking of collagen fibrillogenesis with the anti-collagen antibody offers potentially beneficial effects that may reduce the negative impact of the subconjunctival scarring associated with glaucoma filtering surgery.

Extracellular Targets to Reduce Excessive Scarring in Response to Tissue Injury.

Excessive scar formation is a hallmark of localized and systemic fibrotic disorders. Despite extensive studies to define valid anti-fibrotic targets and develop effective therapeutics, progressive fibrosis remains a significant medical problem. Regardless of the injury type or location of wounded tissue, excessive production and accumulation of collagen-rich extracellular matrix is the common denominator of all fibrotic disorders. A long-standing dogma was that anti-fibrotic approaches should focus on overall intracellular processes that drive fibrotic scarring. Because of the poor outcomes of these approaches, scientific efforts now focus on regulating the extracellular components of fibrotic tissues. Crucial extracellular players include cellular receptors of matrix components, macromolecules that form the matrix architecture, auxiliary proteins that facilitate the formation of stiff scar tissue, matricellular proteins, and extracellular vesicles that modulate matrix homeostasis. This review summarizes studies targeting the extracellular aspects of fibrotic tissue synthesis, presents the rationale for these studies, and discusses the progress and limitations of current extracellular approaches to limit fibrotic healing.

Profibrotic behavior of fibroblasts derived from patients that develop posttraumatic shoulder stiffness.

Background and Aims: Arthrofibrosis is a severe scarring condition characterized by joint stiffness and pain. Fundamental to developing arthrofibrotic scars is the accelerated production of procollagen I, a precursor of collagen I molecules that form fibrotic deposits in affected joints. The procollagen I production mechanism comprises numerous elements, including enzymes, protein chaperones, and growth factors. This study aimed to elucidate the differences in the production of vital elements of this mechanism in surgical patients who developed significant posttraumatic arthrofibrosis and those who did not. Methods: We studied a group of patients who underwent shoulder arthroscopic repair of the rotator cuff. Utilizing fibroblasts isolated from the patients' rotator intervals, we analyzed their responses to profibrotic stimulation with transforming growth factor ß1 (TGFß1). We compared TGFß1-dependent changes in the production of procollagen I. We studied auxiliary proteins, prolyl 4-hydroxylase (P4H), and heat shock protein 47 (HSP47), that control procollagen stability and folding. A group of other proteins involved in excessive scar formation, including connective tissue growth factor (CTGF), α smooth muscle actin (αSMA), and fibronectin, was also analyzed. Results: We observed robust TGFß1-dependent increases in the production of CTGF, HSP47, αSMA, procollagen I, and fibronectin in fibroblasts from both groups of patients. In contrast, TGFß1-dependent P4H production increased only in the stiff-shoulder-derived fibroblasts. Conclusion: Results suggest P4H may serve as an element of a mechanism that modulates the fibrotic response after rotator cuff injury.

Circulating inflammatory cytokines alter transcriptional activity within fibrotic tissue of Dupuytren's disease patients.

Dupuytren's disease is a benign fibroproliferative disorder of the hand that results in disabling digital contractures that impair function and diminish the quality of life. The incidence of this disease has been correlated with chronic inflammatory states, but any direct association between inflammatory cytokines and Dupuytren's disease is not known. We hypothesized that advanced fibroproliferation is associated with increased levels of circulating inflammatory cytokines. Blood and fibrotic cord tissue were collected preoperatively from patients with severe contracture and control patients. Blood plasma concentrations of known inflammatory cytokines were evaluated using a multiplex immunoassay. Proteins from the cord tissue were analyzed by RNA sequencing and immunohistochemistry. Moreover, collagen-rich cords were analyzed using Fourier-transform infrared spectroscopy. The results indicate that patients exhibited significantly elevated circulating inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-2, and IL-12p70, as compared with controls. Similarly, IL-4 and IL-13 were detected significantly more frequently in Dupuytren's disease as compared with control. RNA sequencing revealed 5311 differentially expressed genes and distinct clustering between diseased and control samples. In addition to increased expression of genes associated with fibroproliferation, we also observed upregulation of transcripts activated by inflammatory cytokines, including prolactin inducible protein and keratin intermediate filaments. IL-2, but not TNF-α, was detected in fibrotic cord tissue by immunohistochemistry. Finally, spectroscopic assays revealed a significant reduction of the collagen content and alterations of collagen cross-linking within the Dupuytren's disease tissues. In total, our results illustrate that patients with severe Dupuytren's disease exhibit substantially elevated circulating inflammatory cytokines that may drive fibroproliferation. Clinical Significance: The results from this study establish the basis for a specific cytokine profile that may be useful for diagnostic testing and therapeutic intervention in Dupuytren's disease.

Mechanisms of reducing joint stiffness by blocking collagen fibrillogenesis in a rabbit model of posttraumatic arthrofibrosis.

Posttraumatic fibrotic scarring is a significant medical problem that alters the proper functioning of injured tissues. Current methods to reduce posttraumatic fibrosis rely on anti-inflammatory and anti-proliferative agents with broad intracellular targets. As a result, their use is not fully effective and may cause unwanted side effects. Our group previously demonstrated that extracellular collagen fibrillogenesis is a valid and specific target to reduce collagen-rich scar buildup. Our previous studies showed that a rationally designed antibody that binds the C-terminal telopeptide of the α2(I) chain involved in the aggregation of collagen molecules limits fibril assembly in vitro and reduces scar formation in vivo. Here, we have utilized a clinically relevant arthrofibrosis model to study the broad mechanisms of the anti-scarring activity of this antibody. Moreover, we analyzed the effects of targeting collagen fibril formation on the quality of healed joint tissues, including the posterior capsule, patellar tendon, and subchondral bone. Our results show that blocking collagen fibrillogenesis not only reduces collagen content in the scar, but also accelerates the remodeling of healing tissues and changes the collagen fibrils' cross-linking. In total, this study demonstrated that targeting collagen fibrillogenesis to limit arthrofibrosis affects neither the quality of healing of the joint tissues nor disturbs vital tissues and organs.

Three Decades of Research on Recombinant Collagens: Reinventing the Wheel or Developing New Biomedical Products?

Collagens provide the building blocks for diverse tissues and organs. Furthermore, these proteins act as signaling molecules that control cell behavior during organ development, growth, and repair. Their long half-life, mechanical strength, ability to assemble into fibrils and networks, biocompatibility, and abundance from readily available discarded animal tissues make collagens an attractive material in biomedicine, drug and food industries, and cosmetic products. About three decades ago, pioneering experiments led to recombinant human collagens' expression, thereby initiating studies on the potential use of these proteins as substitutes for the animal-derived collagens. Since then, scientists have utilized various systems to produce native-like recombinant collagens and their fragments. They also tested these collagens as materials to repair tissues, deliver drugs, and serve as therapeutics. Although many tests demonstrated that recombinant collagens perform as well as their native counterparts, the recombinant collagen technology has not yet been adopted by the biomedical, pharmaceutical, or food industry. This paper highlights recent technologies to produce and utilize recombinant collagens, and it contemplates their prospects and limitations.

Collagen-rich deposit formation in the sciatic nerve after injury and surgical repair: A study of collagen-producing cells in a rabbit model.

INTRODUCTION: Posttraumatic scarring of peripheral nerves produces unwanted adhesions that block axonal growth. In the context of surgical nerve repair, the organization of the scar tissue adjacent to conduits used to span the gap between the stumps of transected nerves is poorly understood. The goal of this study was to elucidate the patterns of distribution of collagen-rich scar tissue and analyze the spatial organization of cells that produce fibrotic deposits around and within the conduit's lumen. METHODS: Employing a rabbit model of sciatic nerve transection injury, we studied the formation of collagen-rich scar tissue both inside and outside conduits used to bridge the injury sites. Utilizing quantitative immunohistology and Fourier-transform infrared spectroscopy methods, we measured cellular and structural elements present in the extraneural and the intraneural scar of the proximal and distal nerve fragments. RESULTS: Analysis of cells producing collagen-rich deposits revealed that alpha-smooth muscle actin-positive myofibroblasts were only present in the margins of the stumps. In contrast, heat shock protein 47-positive fibroblasts actively producing collagenous proteins were abundant within the entire scar tissue. The most prominent site of transected sciatic nerves with the highest number of cells actively producing collagen-rich scar was the proximal stump. CONCLUSION: Our findings suggest the proximal region of the injury site plays a prominent role in pro-fibrotic processes associated with the formation of collagen-rich deposits. Moreover, they show that the role of canonical myofibroblasts in peripheral nerve regeneration is limited to wound contracture and that a distinct population of fibroblastic cells produce the collagenous proteins that form scar tissue. As scarring after nerve injury remains a clinical problem with poor outcomes due to incomplete nerve recovery, further elucidation of the cellular and spatial aspects of neural fibrosis will lead to more targeted treatments in the clinical setting.

Collagen Structure-Function Mapping Informs Applications for Regenerative Medicine.

Type I collagen, the predominant protein of vertebrates, assembles into fibrils that orchestrate the form and function of bone, tendon, skin, and other tissues. Collagen plays roles in hemostasis, wound healing, angiogenesis, and biomineralization, and its dysfunction contributes to fibrosis, atherosclerosis, cancer metastasis, and brittle bone disease. To elucidate the type I collagen structure-function relationship, we constructed a type I collagen fibril interactome, including its functional sites and disease-associated mutations. When projected onto an X-ray diffraction model of the native collagen microfibril, data revealed a matrix interaction domain that assumes structural roles including collagen assembly, crosslinking, proteoglycan (PG) binding, and mineralization, and the cell interaction domain supporting dynamic aspects of collagen biology such as hemostasis, tissue remodeling, and cell adhesion. Our type III collagen interactome corroborates this model. We propose that in quiescent tissues, the fibril projects a structural face; however, tissue injury releases blood into the collagenous stroma, triggering exposure of the fibrils' cell and ligand binding sites crucial for tissue remodeling and regeneration. Applications of our research include discovery of anti-fibrotic antibodies and elucidating their interactions with collagen, and using insights from our angiogenesis studies and collagen structure-function model to inform the design of super-angiogenic collagens and collagen mimetics.

The impact of cholesterol deposits on the fibrillar architecture of the Achilles tendon in a rabbit model of hypercholesterolemia.

BACKGROUND: Increased tendon pain and tendon damage is a significant complication related to hyperlipidemia. Unlike the well-established pathogenesis associated with increased serum concentrations of total cholesterol, triglycerides, and low-density lipoprotein in atherosclerotic cardiovascular disease, the role of hyperlipidemia in promoting tendon damage remains controversial and requires mechanistic clarity. METHODS: In this study, we analyzed the consequences of hypercholesterolemia on the integrity of the collagen-based architecture of the Achilles tendon. The Achilles tendons from rabbits fed with normal-cholesterol (nCH) and high-cholesterol (hCH) diets were analyzed. We studied the morphology of tendons, distribution of lipids within their collagen-rich milieu, the relative amounts of fibrillar collagen I and collagen III, and selected biomechanical parameters of the tendons at the macroscale and the nanoscale. RESULTS: Histological assays of hCH tendons and tenosynovium demonstrated hypercellular areas with increased numbers of macrophages infiltrating the tendon structure as compared to the nCH tendons. While Oil Red staining revealed lipid-rich deposits in the hCH tendons, hybridization of tendon tissue with the collagen hybridizing peptide (CHP) demonstrated damage to the collagen fibers. Fourier-transform infrared (FTIR) spectra showed the presence of distinct peaks consistent with the presence of cholesterol ester. Additionally, the hCH tendons displayed regions of poor collagen content that overlapped with lipid-rich regions. The hCH tendons had a substantial fourfold increase in the collage III to collagen I ratio as compared to the nCH tendons. Tendons from the hCH rabbits showed poor biomechanical characteristics in comparison with control. The biomechanical changes were evident at the macrolevel and the nanolevel of tendon structure. CONCLUSIONS: Our findings support the hypothesis that hypercholesterolemia coincides with the weakening of the tendons. It is likely that the intimate contact between collagen fibrils and cholesterol deposits contributes to the weakening of the fibrillar structure of the tendons.

Naproxen impairs load-induced bone formation, reduces bone toughness, and diminishes woven bone formation following stress fracture in mice.

Debilitating stress fractures are surprisingly common in physically active individuals, including athletes, military recruits, and dancers. These individuals are overrepresented in the 30 million daily users of non-steroidal anti-inflammatory drugs (NSAIDs). We hypothesized that regular use of NSAIDs would predispose habitually loaded bones to stress fracture and delay the repair of these injuries. In this project, we used repetitive axial forelimb compression in mice as a model to test these hypotheses. First, adult mice were subjected to six bouts of forelimb compression over a period of two weeks, with aspirin, naproxen, or vehicle continuously administered through drinking water. Naproxen-treated mice had diminished load-induced bone formation as well as a significant loss in toughness in non-loaded bone, which were not observed in aspirin-treated mice. Furthermore, there were no differences in RANKL/OPG ratio or cortical bone parameters. Picrosirius red staining and second harmonic generation imaging revealed that alterations in bone collagen fibril size and organization were driving the loss of toughness in naproxen-treated mice. Separately, adult mice were subjected to an ulnar stress fracture generated by a single bout of fatigue loading, with NSAIDs provided 24 h before injury. Both aspirin-treated and naproxen-treated mice had normal forelimb use in the week after injury, whereas control mice favored the injured forelimb until day 7. However, woven bone volume was only significantly impaired by naproxen. Both NSAIDs were found to significantly inhibit Ptgs2 and Ngf expression following stress fracture, but only naproxen significantly affected serum PGE2 concentration. Overall, our results suggest that naproxen, but not aspirin, may increase the risk of stress fracture and extend the healing time of these injuries, warranting further clinical evaluation for patients at risk for fatigue injuries.

Thrombospondin-1 Is a Major Activator of TGF-ß Signaling in Recessive Dystrophic Epidermolysis Bullosa Fibroblasts.

Mutations in the gene encoding collagen VII cause the devastating blistering disease recessive dystrophic epidermolysis bullosa (RDEB). RDEB is characterized by severe skin fragility and nonhealing wounds aggravated by scarring and fibrosis. We previously showed that TSP1 is increased in RDEB fibroblasts. Because transforming growth factor-ß (TGF-ß) signaling is also increased in RDEB, and TSP1 is known to activate TGF-ß, we investigated the role of TSP1 in TGF-ß signaling in RDEB patient cells. Knockdown of TSP1 reduced phosphorylation of smad3 (a downstream target of TGF-ß signaling) in RDEB primary fibroblasts, whereas overexpression of collagen VII reduced phosphorylation of smad3. Furthermore, inhibition of TSP1 binding to the LAP/TGF-ß complex decreased fibrosis in engineered extracellular matrix formed by RDEB fibroblasts, as evaluated by picrosirius red staining and analyses of birefringent collagen fibrillar deposits. We show that collagen VII binds TSP1, which could potentially limit TSP1-LAP association and subsequent TGF-ß activation. Our study suggests a previously unreported mechanism for increased TGF-ß signaling in the absence of collagen VII in RDEB patient skin. Moreover, these data identify TSP1 as a possible target for reducing fibrosis in the tumor-promoting dermal microenvironment of RDEB patients.

Novel mutation in Teneurin 3 found to co-segregate in all affecteds in a multi-generation family with developmental dysplasia of the hip.

DDH is a debilitating condition characterized by incomplete formation of the acetabulum leading to dislocation of the hip, suboptimal joint function and accelerated wear of the articular cartilage resulting in early onset crippling arthritis of the hip in 20-40 year olds. Current diagnostic tests in newborns using physical manipulation of the femur or ultrasound either under or over-diagnose this condition. Developing an accurate, cost effective diagnostic test is a goal of this study. To better understand the biologic pathways involved in acetabular development, DNA from severely affected individuals in a four generation family that showed inter-generational transmission of the disorder was isolated and whole exome sequenced. A novel A to C transversion at position 183721398 on human chromosome four was found to co-segregate with the affected phenotype in this family. This mutation encodes a glutamine to proline change at position 2665 in the Teneurin 3 (TENM3) gene and was judged damaging by four prediction programs. Eight week old knock-in mutant mice show delayed development of the left acetabulum and the left glenoid fossa as shown by the presence of more Alcian blue staining on the socket rims of both the hip and the shoulder. We hypothesize that mutated TENM3 will slow chondrogenesis. MMP13 has been shown to impair extracellular matrix remodeling and suppress differentiation. Bone marrow cells from the knock-in mouse were found to overexpress MMP13 with or without BMP2 stimulation. This variant may elucidate pathways responsible for normal hip development and become part of an accurate test for DDH. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells.

Nucleus pulposus (NP) cells reside in the hypoxic niche of the intervertebral disc. Studies have demonstrated that RNA-binding protein HuR modulates hypoxic signaling in several cancers, however, its function in the disc is unknown. HuR did not show cytoplasmic translocation in hypoxia and its silencing did not alter levels of Hif-1α or HIF-targets in NP cells. RNA-Sequencing data revealed that important extracellular matrix-related genes including several collagens, MMPs, aggrecan, Tgf-ß3 and Sdc4 were regulated by HuR. Further analysis of HuR-silenced NP cells confirmed that HuR maintained expression of these matrix genes. We confirmed decreased levels of secreted collagen I and Sdc4 and increased pro-MMP13 in HuR-knockdown cells. In addition, messenger ribonucleoprotein immunoprecipitation demonstrated HuR binding to Tgf-ß3 and Sdc4 mRNAs. Interestingly, while HuR bound to Hif-1α and Vegf mRNAs, it was clear that compensatory mechanisms sustained their expression when HuR was silenced. Noteworthy, despite the presence of multiple HuR-binding sites and reported interaction in other cell types, HuR showed no binding to Pgk1, Eno1, Pdk1 and Pfkfb3 in NP cells. Metabolic studies showed a significant decrease in the extracellular acidification rate (ECAR) and mitochondrial oxygen consumption rate (OCR) and acidic pH in HuR-silenced NP cells, without appreciable change in total OCR. These changes were likely due to decreased Ca12 expression in HuR silenced cells. Taken together, our study demonstrates for the first time that HuR regulates extracellular matrix (ECM) and pH homeostasis of NP cells and has important implications in the maintenance of intervertebral disc health.

Fibroblasts from recurrent fibrotic overgrowths reveal high rate of proliferation in vitro - findings from the study of hereditary and idiopathic gingival fibromatosis.

PURPOSE: Investigate the content of fibrotic fibrils in gingival tissue and the proliferation of fibroblasts collected from recurrent and non-recurrent hereditary gingival fibromatosis (HGF) and idiopathic gingival fibromatosis (IGF). METHODS: Gingival biopsies were collected from HGF (n = 3) and IGF (n = 3) donors with recurrent and non-recurrent gingival overgrowths and from a control group (Ctrl, n = 3). Hematoxylin staining was performed to evaluate the histomorphology of gingival tissue. Heidenhain's AZAN trichrome staining served for visualization of fibrotic fibrils in gingiva. Quantitative analysis of the content of fibrotic fibrils in gingival tissue was performed using a polarized light microscope. Proliferation was evaluated at 24 h, 48 h, and 72 h in fibroblast cultures using a cell proliferation ELISA assay based on 5-bromo-2'-deoxyuridine (BrdU). RESULTS: Numerous blood vessels and fibroblasts were observed in recurrent overgrowths, whereas moderate blood vessels and moderate to scanty fibroblasts were detected in non-recurrent overgrowths. Heidenhain's staining revealed numerous collagen fibers in both recurrent and non-recurrent overgrowths. Quantitative analysis in a polarizing microscope showed significant accumulation of fibrotic fibrils exclusively in the overgrowths with the recurrence. In all time-points, increased proliferation of cells from all recurrent overgrowths was observed, but not from overgrowths which do not reoccur. CONCLUSIONS: The study revealed that recurrent gingival overgrowths consist of highly fibrotic and dense connective tissue with numerous blood vessels and abundant fibroblasts. We also demonstrated that unlike fibroblasts derived from overgrowths, which did not present recurrence, fibroblasts derived from highly fibrotic and recurrent overgrowths maintain high rate of proliferation in vitro.

Functional and structural studies of tolloid-like 1 mutants associated with atrial-septal defect 6.

Inactive mammalian tolloid-like 1 (tll1) and mutations detected in tolloid-like 1 (TLL1) have been linked to the lack of the heart septa formation in mice and to a similar human inborn condition called atrial-septal defect 6 (ASD6; OMIM 613087, formerly ASD II). Previously, we reported four point mutations in TLL1 found in approximately 20% of ASD6 patients. Three mutations in the coding sequence were M182L, V238A, and I629V. In this work, we present the effects of these mutations on TLL1 function. Three recombinant cDNA constructs carrying the mutations and one wild-type construct were prepared and then expressed in HT-1080 cells. Corresponding recombinant proteins were analyzed for their metalloendopeptidase activity using a native substrate, chordin. The results of these assays demonstrated that in comparison with the native TLL1, mutants cleaved chordin and procollagen I at significantly lower rates. CD analyses revealed significant structural differences between the higher order structure of wild-type and mutant variants. Moreover, biosensor-based assays of binding interactions between TLL1 variants and chordin demonstrated a significant decrease in the binding affinities of the mutated variants. The results from this work indicate that mutations detected in TLL1 of ASD6 patients altered its metalloendopeptidase activity, structure, and substrate-binding properties, thereby suggesting a possible pathomechanism of ASD6.

TIMP-1 association with collagen type I overproduction in hereditary gingival fibromatosis.

OBJECTIVES: To investigate the processes associated with the excessive production of collagen I in hereditary gingival fibromatosis (HGF). MATERIALS AND METHODS: Three HGF subjects and five controls were enrolled in the study. Histomorphological and immunohistological analyses were performed on gingival tissues. The expression of heat-shock protein 47 (HSP47), collagen I, transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) by gingival fibroblasts isolated from HGF and controls was analysed using qRT-PCR, Western blotting and ELISA. RESULTS: Considerable accumulation of fibrotic fibrils and increased synthesis of HSP47 were noted in HGF gingival tissues. The synthesis of collagen I, HSP47, TGF-ß1, CTGF and TIMP-1 was significantly elevated in HGF gingival fibroblasts compared with controls, while the production of MMP-1 was decreased. CONCLUSIONS: We report that fibrosis in HGF gingival tissues is associated with increased synthesis of HSP47. This finding was confirmed by an in vitro study, where excessive production of collagen I was associated with increased synthesis of HSP47, TGF-ß1 and CTGF by HGF gingival fibroblasts. Moreover, the shift in the TIMP-1/MMP-1 ratio identifies increased synthesis of TIMP-1 as one of the processes associated with collagen I overproduction in HGF fibroblasts.