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

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

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

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

Increased camptothecin toxicity induced in mammalian cells expressing Saccharomyces cerevisiae DNA topoisomerase I

The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I. To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells. Camptothecin preferentially induced apoptosis in cells expressing wild-type eScTop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant. Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not. Thus, the enzyme retained catalytic activity while tethered to a support. Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabilization of the covalent enzyme-DNA intermediate, induced apoptosis in COS cells in the absence of camptothecin. This correlated with increased DNA cleavage in immunoprecipitates of eScTop1T722Ap, in the absence of the drug. The observation that the phenotypic consequences of expressing wild-type and mutant yeast enzymes were reiterated in mammalian cells suggests that the mechanisms underlying cellular responses to DNA topoisomerase I-mediated DNA damage are conserved between yeast and mammalian cells

Prospects and limitations of improving skeletal growth in a mouse model of spondyloepiphyseal dysplasia caused by R992C (p.R1192C) substitution in collagen II.

Skeletal dysplasias form a group of skeletal disorders caused by mutations in macromolecules of cartilage and bone. The severity of skeletal dysplasias ranges from precocious arthropathy to perinatal lethality. Although the pathomechanisms of these disorders are generally well defined, the feasibility of repairing established aberrant skeletal tissues that developed in the presence of mutant molecules is currently unknown. Here, we employed a validated mouse model of spondyloepiphyseal dysplasia (SED) that enables temporal control of the production of the R992C (p.R1192C) collagen II mutant that causes this disease. Although in our earlier studies we determined that blocking the expression of this mutant at the early prenatal stages prevents a SED phenotype, the utility of blocking the R992C collagen II at the postnatal stages is not known. Here, by switching off the expression of R992C collagen II at various postnatal stages of skeletal development, we determined that significant improvements of cartilage and bone morphology were achieved only when blocking the production of the mutant molecules was initiated in newborn mice. Our study indicates that future therapies of skeletal dysplasias may require defining a specific time window when interventions should be applied to be successful

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

Abstract 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 β1 (TGFβ1). We compared TGFβ1‐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 TGFβ1‐dependent increases in the production of CTGF, HSP47, αSMA, procollagen I, and fibronectin in fibroblasts from both groups of patients. In contrast, TGFβ1‐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

BiP-specific immunostaining of growth plates of 10-week-old mice.

<p>A, ^(-)DoxR992C-ProGFP(+) mice; B, ^NB-DoxR992C-ProGFP(+) mice; C, ^1w-DoxR992C-ProGFP(+) mice; D, ^2w-DoxR992C-ProGFP(+) mice. Bars = 20 μm.</p

A graphic representation of measurements of the height of the hypertrophic zones and (A) the acellular areas (B).

<p>Data from the NB-Dox, the 1w-Dox, and the 2w-Dox mice from the 7-week-old and the 10-week-old age groups are presented. Data from the ^(-)DoxR992C-ProGFP(+) and ^(-)DoxR992C-ProGFP(-) mice are also shown (-Dox). The means with 95% CI are indicated. Symbols: ■ R992C-ProGFP(+) 7-week-old; ● R992C-ProGFP(-) 7-week-old; □ R992C-ProGFP(+) 10-week-old; ○ R992C-ProGFP(-) 10-week-old.</p