Collagen Subtypes Research Articles

Doxycycline alters collagen composition following ventral hernia repair.

Doxycycline, a nonspecific metalloproteinase (MMP) inhibitor, has been demonstrated to impact the strength of the polypropylene (PP) mesh-repaired hernia with an increase in the deposition of collagen type 1. The impact of doxycycline with porcine acellular dermal matrices (PADM) is unknown; therefore, we evaluated the impact of doxycycline administration upon hernia repair with PP and PADM mesh. Sprague-Dawley rats weighing ~400g underwent laparotomy with creation of a midline ventral hernia. After a 27-day recovery, animals were randomly assigned to four groups of eight and underwent intraperitoneal underlay hernia repair with either PP or PADM. Groups were assigned to daily normal saline (S) or daily doxycycline in normal saline 10mg/kg (D) via oral gavage for 8weeks beginning 24h preoperatively. Animals were euthanized at 8weeks and underwent tensiometric testing of the abdominal wall and western blot analyses for collagen subtypes and MMPs. Thirty-two animals underwent successful hernia creation and repair with either PADM or PP. At 8weeks, 15 of 16 PP-implanted animals survived with only 12 of 16 PADM-implanted animals surviving. There were no differences in the mesh to fascial interface tensiometric strength between groups. Densitometric counts in the PADM-D group demonstrated increased collagen type 1 compared to PP-S (PADM-D [1286.5], PADM-S [906.9], PP-S [700.4], p=0.037) and decreased collagen type 3 compared to PP-S (PADM-D [7446.9], PADM-S [8507.6], PP-S [11,297.1], p=0.01). MMP-9 levels were increased in PADM-D (PP-S vs. PADM-D, p=0.04), while MMP-2 levels were similar between PADM-D and PADM-S, respectively. Collagen type 1 deposition at the mesh to fascial interface is enhanced following administration of doxycycline in ventral hernia repairs with porcine acellular dermal matrices. Doxycycline administration may have implications for enhancing hernia repair outcomes using biologic mesh.

Efficacy and Safety of the Collagenase of the Bacterium Clostridium Histolyticum for the Treatment of Capsular Contracture after Silicone Implants: Ex-Vivo Study on Human Tissue

BackgroundThe fibrotic capsule that surrounds silicone implants consists mainly of collagen. The FDA-approved collagenase of the bacterium clostridium histolyticum provides a reasonable treatment option. Safety and efficacy at the female breast site must be evaluated before clinical utilization.Materials and MethodsWe incubated 20 samples of fibrotic capsule as well as 12 full thickness skin grafts harvested from the female breast site for 24 hours with different doses of collagenase. Outcome measures involved histological assessment of thickness and density of the capsule tissue as well as the skin grafts. Furthermore, we performed a collagen assay and immunohistochemistry staining for collagen subtypes.ResultsCollagenase treatment was able to degrade human capsule contracture tissue ex-vivo. The remaining collagen subtype after degradation was type 4 only. 0.3 mg/ml of collagenase was most effective in reducing capsule thickness when compared with higher concentrations. Of note, effectiveness was inversely related to capsule density, such that there was less reduction in thickness with higher capsule densities and vice versa. Furthermore, the application of 0.3mg/ml collagenase did not lead to thinning or perforation of full thickness skin grafts.ConclusionAdjustment of collagenase dose will depend on thickness and density of the contracted capsule. A concentration of 0.3mg/ml seems to be safe and effective in an ex-vivo setting. The remaining collagen subtype 4 is suitable to serve as a neo-capsule/acellular tissue matrix. Collagenase treatment for capsular contracture may soon become a clinical reality.

English

With increased recent interest in the utilization of industrial by-products, finding different sources, optimizing extracting conditions and characterization of collagen extracts have recently become important research topics. This study addresses the isolation of acid-soluble and pepsin-soluble forms of collagen from dried jellyfish and squid, and their partial characterization. The properties of these proteins have been studied and a comparison made of the protein patterns of collagen extracted from marine organisms with those from other organisms, to determine which collagen subtypes are present, and in what proportions. Pepsin-soluble collagen (PSC) from dried jellyfish and dried squid contained a collagen form classified as type I, of molecular composition comparable with that of collagen type I from rat tail. Peptide maps of collagens digested by achromopeptidase were slightly different, indicating some differences in amino acid sequence or conformation. The collagen showed high solubility at acidic pH (4-5) but its solubility markedly decreased in the presence of sodium chloride (NaCl) up to 2%. Collagen type I from dried jellyfish and dried squid could be a useful alternative to mammalian collagen, with potential use in the biomedical, pharmaceutical and nutraceuticals industries. Key words: Collagen, pepsin-soluble form, acid-soluble form, partial characterization.

Novel Method for Measuring Hepatic Collagen Turnover Rate in Nonalcoholic Fatty Liver Disease (NAFLD): Comparison of Blood and Liver Biopsy Biomarkers of Fibrogenesis

Background and ObjectivesExcess collagen synthesis in the liver plays a causal role in progression from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). Markers that identify a patient's risk for progression, characterize disease activity and provide early assessment of treatment efficacy are needed. In particular, the availability of a non‐invasive metric of the rates of liver collagen synthesis (fibrogenesis) and turnover (fibrolysis) would be extremely valuable. We evaluated novel clinical biomarkers that directly quantify hepatic fibrogenesis rate in well‐characterized patients with biopsy‐proven NAFLD.MethodsPatients with suspected NAFLD and a clinical indication for liver biopsy received the stable, non‐radioactive tracer heavy water (2H2O, 50 ml, 2–3 times daily) by mouth for 20–35 days prior to biopsy. A plasma sample was collected weekly. Collagen fractional synthesis rate (FSR) from liver biopsy tissue and FSR of lumican (a proteoglycan involved in collagen fibril assembly that exhibits increased synthesis when collagen synthesis is increased) from plasma were measured by 2H incorporation, using tandem mass spectrometry (LC‐MS/MS). Magnetic resonance elastography (MRE) measurements of liver stiffness were obtained within 2 weeks of biopsy.ResultsFSR of hepatic type I collagen (Col1a1) was significantly elevated in NASH relative to NAFL (0.97% vs 0.43 %/day, p=0.03). Liver Col1a1 FSR also significantly correlated with fibrosis severity by histological score (r= 0.63), with liver stiffness by MRE (r=0.65), and with HbA1c levels (r=0.47). FSRs of fibrillar hepatic collagen subtypes (I and III) were highly correlated (r= 0.94). FSR of circulating plasma lumican correlated strongly with liver collagen FSR (r=0.69) and with liver stiffness (r=0.71).ConclusionWe conclude that hepatic collagen synthesis rates increase with fibrosis severity in NAFLD patients, indicating not only markedly increased absolute rates of collagen synthesis with disease progression but also suggesting that hepatic scar is dynamic even in advanced disease and may therefore be potentially reversible. Importantly, plasma lumican FSR correlates closely with liver collagen FSR and with liver stiffness. Moreover, established clinical risk factors for disease progression (NASH diagnosis, histologic fibrosis score, diabetes) were associated with higher collagen FSR. Non‐invasive kinetic measurements of fibrogenesis, such as plasma lumican FSR, may be helpful as markers of risk for disease progression and as early read outs of treatment response in anti‐fibrotic trials in NASH. Prospective studies are required to establish the predictive power of fibrogenesis rates for disease progression or reversal.Support or Funding InformationKineMed, Inc.

Nur77-deficiency in bone marrow-derived macrophages modulates inflammatory responses, extracellular matrix homeostasis, phagocytosis and tolerance.

BackgroundThe nuclear orphan receptor Nur77 (NR4A1, TR3, or NGFI-B) has been shown to modulate the inflammatory response of macrophages. To further elucidate the role of Nur77 in macrophage physiology, we compared the transcriptome of bone marrow-derived macrophages (BMM) from wild-type (WT) and Nur77-knockout (KO) mice.ResultsIn line with previous observations, SDF-1α (CXCL12) was among the most upregulated genes in Nur77-deficient BMM and we demonstrated that Nur77 binds directly to the SDF-1α promoter, resulting in inhibition of SDF-1α expression. The cytokine receptor CX3CR1 was strongly downregulated in Nur77-KO BMM, implying involvement of Nur77 in macrophage tolerance. Ingenuity pathway analyses (IPA) to identify canonical pathways regulation and gene set enrichment analyses (GSEA) revealed a potential role for Nur77 in extracellular matrix homeostasis. Nur77-deficiency increased the collagen content of macrophage extracellular matrix through enhanced expression of several collagen subtypes and diminished matrix metalloproteinase (MMP)-9 activity. IPA upstream regulator analyses discerned the small GTPase Rac1 as a novel regulator of Nur77-mediated gene expression. We identified an inhibitory feedback loop with increased Rac1 activity in Nur77-KO BMM, which may explain the augmented phagocytic activity of these cells. Finally, we predict multiple chronic inflammatory diseases to be influenced by macrophage Nur77 expression. GSEA and IPA associated Nur77 to osteoarthritis, chronic obstructive pulmonary disease, rheumatoid arthritis, psoriasis, and allergic airway inflammatory diseases.ConclusionsAltogether these data identify Nur77 as a modulator of macrophage function and an interesting target to treat chronic inflammatory disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2469-9) contains supplementary material, which is available to authorized users.

Extracellular matrix microarrays to study inductive signaling for endoderm specification

During tissue development, stem and progenitor cells are faced with fate decisions coordinated by microenvironmental cues. Although insights have been gained from in vitro and in vivo studies, the role of the microenvironment remains poorly understood due to the inability to systematically explore combinations of stimuli at a large scale. To overcome such restrictions, we implemented an extracellular matrix (ECM) array platform that facilitates the study of 741 distinct combinations of 38 different ECM components in a systematic, unbiased and high-throughput manner. Using embryonic stem cells as a model system, we derived definitive endoderm progenitors and applied them to the array platform to study the influence of ECM, including the interactions of ECM with growth factor signaling, on the specification of definitive endoderm cells towards the liver and pancreas fates. We identified ECM combinations that influence endoderm fate decisions towards these lineages, and demonstrated the utility of this platform for studying ECM-mediated modifications to signal activation during liver specification. In particular, defined combinations of fibronectin and laminin isoforms, as well as combinations of distinct collagen subtypes, were shown to influence SMAD pathway activation and the degree of hepatic differentiation. Overall, our systematic high-throughput approach suggests that ECM components of the microenvironment have modulatory effects on endoderm differentiation, including effects on lineage fate choice and cell adhesion and survival during the differentiation process. This platform represents a robust tool for analyzing effects of ECM composition towards the continued improvement of stem cell differentiation protocols and further elucidation of tissue development processes. Statement of SignificanceCellular microarrays can provide the capability to perform high-throughput investigations into the role of microenvironmental signals in a variety of cell functions. This study demonstrates the utility of a high-throughput cellular microarray approach for analyzing the effects of extracellular matrix (ECM) in liver and pancreas differentiation of endoderm progenitor cells. Despite an appreciation that ECM is likely involved in these processes, the influence of ECM, particularly combinations of matrix proteins, had not been systematically explored. In addition to the identification of relevant ECM compositions, this study illustrates the capability of the cellular microarray platform to be integrated with a diverse range of cell fate measurements, which could be broadly applied towards the investigation of cell fate regulation in other tissue development and disease contexts.

Colorectal anastomotic healing: why the biological processes that lead to anastomotic leakage should be revealed prior to conducting intervention studies.

BackgroundAnastomotic leakage (AL) remains the most dreaded complication after colorectal surgery and causes high morbidity and mortality. The pathophysiology of AL remains unclear, despite numerous studies that have been conducted on animals and humans, probably due to the undetermined healing process of colorectal anastomoses. Increasing basic knowledge on this healing process may shed more light on causal factors of AL, and additionally reduce the quantity and accelerate the quality of experimental studies. In this debate article, our aim was to provide different perspectives on what is known about the colorectal healing process in relation to wound healing and AL.DiscussionSince knowledge on anastomotic healing is lacking, it remains difficult to conclude which factors are essential in preventing AL. This is essential information in the framework of humane animal research, where the focus should lie on Replacement, Reduction and Refinement (3Rs). While many researchers compare anastomotic healing with wound healing in the skin, there are substantial recognized differences, e.g. other collagen subtypes and different components involved.SummaryBased on our findings in literature as well as discussions with experts, we advocate stop considering anastomotic healing in the gastrointestinal tract and cutaneous healing as a similar process. Furthermore, intervention studies should at least address the anastomotic healing process in terms of histology and certain surrogate markers. Finally, the anastomotic healing process ought to be further elucidated – with modern techniques to achieve 3Rs in animal research - to provide starting points for potential interventions that can prevent AL.

Impact of Prolapse Meshes on the Metabolism of Vaginal Extracellular Matrix in Rhesus Macaque

Synthetic meshes are commonly used to repair pelvic organ prolapse. Increasing evidence of mesh-related complications, including mesh erosion and mesh exposure, led the Food and Drug Administration to issue warnings about use of synthetic mesh and to call for mechanistic studies. A recent study (by some of the same authors) that used a nonhuman primate sacrocolpopexy model showed that heavier-weight, lower-porosity, and higher-stiffness meshes result in thinner and biomechanically inferior vaginas. The aim of this study was to define and compare alterations in collagen and elastin metabolism following the implantation of 3 synthetic meshes into the vagina of nonhuman primates with prolapse. The impact of the heavier, less porous, and stiffer prolapse mesh (Gynemesh PS) was compared with 2 lighter, more porous, and less-stiff meshes (UltraPro and Restorelle). Following hysterectomy, 39 middle-aged parous rhesus macaques were implanted via sacrocolpopexy with Gynemesh PS (n = 12), UltraPro with its blue line perpendicular to the longitudinal axis of vagina (low stiffness direction, n = 11), UltraPro with its blue line parallel to the longitudinal axis of vagina (high stiffness direction, n = 8), or Restorelle (n = 8). Sham-operated animals (n = 10) served as controls. The mesh-tissue complex was removed 12 weeks after surgery and analyzed. Relative to sham, Gynemesh PS increased degradation of both collagen and elastin, whereas UltraPro Perpendicular and UltraPro Parallel induced an increase only in elastin degradation. The mesh with the least impact was Restorelle. Compared with sham, increased degradation of collagen and elastin in vaginas implanted with Gynemesh PS was associated with a simultaneous increase in active matrix metalloproteinase 1 (MMP-1), MMP-8, MMP-13, and total MMP-2 and MMP-9 (all comparisons, P < 0.05). Increased degradation of elastin (tropoelastin and mature elastin) in the UltraPro-implanted vagina was associated with a concomitant increase of MMP-2 and MMP-9 (all comparisons, P < 0.05). Relative to sham, the ratio of collagen subtype III/I was higher in the Gynemesh PS and UltraPro perpendicular groups (both, P < 0.05). These data show that implantation of the heavier, less porous, and stiffer mesh, Gynemesh PS, degrades vaginal collagen and elastin, most likely as a result of increased MMP activity, and weakens the vaginal wall, predisposing to mesh exposure development.

Pirfenidone attenuates bladder fibrosis and mitigates deterioration of bladder function in a rat model of partial bladder outlet obstruction.

To investigate the effects of pirfenidone (PFD) on the attenuation of bladder remodeling, and the associated functional changes caused by partial bladder outlet obstruction (pBOO), the present study performed surgery on adult male Sprague‑Dawley rats produce a model of pBOO. The rats in the pBOO group were administered a placebo and, in the CMC group, PFD mixed with the placebo was administered orally at 500 mg/kg body weight each day for 5 weeks, from 1 week after surgery. The rat bladders were harvested for biochemical analysis following cystometry at the end of the 6 week period. The histopathology was determined using Masson's trichrome staining. The mRNA and protein levels of pro‑fibrotic growth factors and extracellular matrix subtypes were assessed. pBOO debilitated bladder function and caused the parameters from cystometry to increase significantly compared with those in the control group (P<0.05), which were mitigated significantly following PFD treatment (P<0.05). In terms of the histology, the rats in the pBOO group exhibited significant increases in bladder weight, muscle hypertrophy and deposition of collagens, which were suppressed by PFD treatment (P<0.05). Based on the biochemical analysis, significant increases in the mRNA levels of collagen subtypes and growth factors, and protein levels of profibrotic growth factors and α‑smooth muscle actin in the bladders of rats in the pBOO group were reduced following PFD treatment. PFD prevented bladder remodeling and attenuated bladder fibrosis and, therefore, mitigated the deterioration of bladder function during the initial stage of pBOO.

Biomechanical evaluation of human and porcine auricular cartilage.

The mechanical properties of normal auricular cartilage provide a benchmark against which to characterize changes in auricular structure/function due to genetic defects creating phenotypic abnormalities in collagen subtypes. Such properties also provide inputs/targets for auricular reconstruction scaffold design. Several studies report the biomechanical properties for septal, costal, and articular cartilage. However, analogous data for auricular cartilage are lacking. Therefore, our aim in this study was to characterize both whole-ear and auricular cartilage mechanics by mechanically testing specimens and fitting the results to nonlinear constitutive models. Mechanical testing of whole ears and auricular cartilage punch biopsies. Whole human cadaveric ear and auricular cartilage punch biopsies from both porcine and human cartilage were subjected to whole-ear helix-down compression and quasistatic unconfined compression tests. Common hyperelastic constitutive laws (widely used to characterize soft tissue mechanics) were evaluated for their ability to represent the stress-strain behavior of auricular cartilage. Load displacement curves for whole ear testing exhibited compliant linear behavior until after significant displacement where nonlinear stiffening occurred. All five commonly used two-term hyperelastic soft tissue constitutive models successfully fit both human and porcine nonlinear elastic behavior (mean R(2) fit >0.95). Auricular cartilage exhibits nonlinear strain-stiffening elastic behavior that is similar to other soft tissues in the body. The whole ear exhibits compliant behavior with strain stiffening at high displacement. The constants from the hyperelastic model fits provide quantitative baselines for both human and porcine (a commonly used animal model for auricular tissue engineering) auricular mechanics. NA

The Small Leucine-Rich Proteoglycan BGN Accumulates in CADASIL and Binds to NOTCH3

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited form of cerebral small vessel disease caused by mutations in conserved residues of NOTCH3. Affected arteries of CADASIL feature fibrosis and accumulation of NOTCH3. A variety of collagen subtypes (types I, III, IV, and VI) have been identified in fibrotic CADASIL vessels. Biglycan (BGN) and decorin (DCN) are class I members of the small leucine-rich proteoglycan (SLRP) family that regulate collagen fibril size. Because DCN has been shown to deposit in arteries in cerebral small vessel disease, we tested whether BGN accumulates in arteries of CADASIL brains. BGN was strongly expressed in both small penetrating and leptomeningeal arteries of CADASIL brain. BGN protein was localized to all three layers of arteries (intima, media, and adventitia). Substantially, more immunoreactivity was observed in CADASIL brains compared to controls. Immunoblotting of brain lysates showed a fourfold increase in CADASIL brains (compared to controls). Messenger RNA encoding BGN was also increased in CADASIL and was localized by in situ hybridization to all three vascular layers in CADASIL. Human cerebrovascular smooth muscle cells exposed to purified NOTCH3 ectodomain upregulated BGN, DCN, and COL4A1 through mechanisms that are sensitive to rapamycin, a potent mTOR inhibitor. In addition, BGN protein interacted directly with NOTCH3 protein in cell culture and in direct protein interaction assays. In conclusion, BGN is a CADASIL-enriched protein that potentially accumulates in vessels by mTOR-mediated transcriptional activation and/or post-translational accumulation via protein interactions with NOTCH3 and collagen.

Expression of type I and type III collagens in oral submucous fibrosis: An immunohistochemical study

Background: Oral submucous fibrosis (OSMF) is a potentially malignant collagen - metabolic disorder linked to consumption of betel quid and areca nut. The deposition of collagen and its major subtypes have been the subject of intense scrutiny in the etiopathogenesis of the disorder. Aims and Objectives: The present study was planned to immunohistochemically identify the expression of collagens I and III (COL I and III) in different grades of OSF and compare it with normal oral mucosa and scar tissue. Materials and Methods: Archival paraffin sections of 72 cases of various grades of OSMF, ten cases of normal mucosa as controls and four cases of scar tissue were stained with antibodies to COL I and III (BioGenex Laboratories, CA, USA) to evaluate the collagen subtypes on paraffin sections. The expression was quantified by image analysis software (Jenoptik Optical System, ProgRes ® Capture Pro, version 2.8.8) and statistically analyzed. Results: COL I and III stained all the tissues ubiquitously. COL I was more in ratio and quantity in all the grades of OSMF, normal mucosa, and scar tissue. The proportion of COL I to COL III seemed to increase with progressive grades of OSF. Interestingly, during the process of fibrosis COL III seems to be deposited earlier and gradually replaced by COL I resulting in a skewed ratio vis a vis normal oral mucosa and scar tissue. Conclusions: COL I expression was found to be proportionate with advancing grades of OSF while COL III expression increased in Grade I but subsequently decreased as severity of OSF increased. The increase in COL I at the expense of COL III showed a similar pattern in the submucosa while in the deeper muscle only Grade III cases reflected the trend. While all cases of OSF revealed excessive expression in comparison with normal oral mucosa, the comparison with scar tissue was variable.

Effect of vaginal or systemic estrogen on dynamics of collagen assembly in the rat vaginal wall.

The objective of this study was to compare the effects of systemic and local estrogen treatment on collagen assembly and biomechanical properties of the vaginal wall. Ovariectomized nulliparous rats were treated with estradiol or conjugated equine estrogens (CEEs) either systemically, vaginal CEE, or vaginal placebo cream for 4 wk. Low-dose local CEE treatment resulted in increased vaginal epithelial thickness and significant vaginal growth without uterine hyperplasia. Furthermore, vaginal wall distensibility increased without compromise of maximal force at failure. Systemic estradiol resulted in modest increases in collagen type I with no change in collagen type III mRNA. Low-dose vaginal treatment, however, resulted in dramatic increases in both collagen subtypes whereas moderate and high dose local therapies were less effective. Consistent with the mRNA results, low-dose vaginal estrogen resulted in increased total and cross-linked collagen content. The inverse relationship between vaginal dose and collagen expression may be explained in part by progressive downregulation of estrogen receptor-alpha mRNA with increasing estrogen dose. We conclude that, in this menopausal rat model, local estrogen treatment increased total and cross-linked collagen content and markedly stimulated collagen mRNA expression in an inverse dose-effect relationship. High-dose vaginal estrogen resulted in downregulation of estrogen receptor-alpha and loss of estrogen-induced increases in vaginal collagen. These results may have important clinical implications regarding the use of local vaginal estrogen therapy and its role as an adjunctive treatment in women with loss of vaginal support.

Impact of prolapse meshes on the metabolism of vaginal extracellular matrix in rhesus macaque

The impact of polypropylene mesh implantation on vaginal collagen and elastin metabolism was analyzed using a nonhuman primate model to further delineate the mechanism of mesh induced complications. Forty-nine middle-aged parous rhesus macaques underwent surgical implantation of 3 synthetic meshes via sacrocolpopexy. Gynemesh PS (n = 12) (Ethicon, Somerville, NJ) and 2 lower-weight, higher-porosity, lower-stiffness meshes (UltraPro [n = 19] [Ethicon] and Restorelle [n = 8] [Coloplast, Minneapolis, MN]) were implanted, in which UltraPro was implanted with its blue orientation lines perpendicular (low stiffness direction, n = 11) and parallel (high stiffness direction, n = 8) to the longitudinal axis of the vagina. Sham-operated animals were used as controls (n = 10). Twelve weeks after surgery, the mesh-tissue complex was excised and analyzed. Relative to sham, Gynemesh PS had a negative impact on the metabolism of both collagen and elastin-favoring catabolic reactions, whereas UltraPro induced an increase only in elastin degradation. Restorelle had the least impact. As compared with sham, the degradation of collagen and elastin in the vagina implanted with Gynemesh PS was increased with a simultaneous increase in active matrix metalloproteinase (MMP)-1, -8, -13, and total MMP-2 and -9 (all P < .05). The degradation of elastin (tropoelastin and mature elastin) was increased in the UltraPro-implanted vagina with a concomitant increase of MMP-2, and -9 (all P < .05). Collagen subtype ratio III/I was increased in Gynemesh PS and UltraPro perpendicular groups (P < .05). Following implantation with the heavier, less porous, and stiffer mesh, Gynemesh PS, the degradation of vaginal collagen and elastin exceeded synthesis, most likely as a result of increased activity of MMPs, resulting in a structurally compromised tissue.

Doxycycline administration improves fascial interface in hernia repair

BackgroundDespite improvements in ventral hernia repair techniques, their recurrence rates are unacceptably high. Increased levels of matrix metalloproteinases (MMPs) and reduced collagen-1 to -3 ratios are implicated in incisional hernia formation. We have recently shown doxycycline treatment for 4 wk after hernia repair reduced MMP levels, significantly increased collagen-1 to -3 ratios, and increased tensile strength of repaired interface fascia. However, this increase was not statistically significant. In this study, we extended treatment duration to determine whether this would impact the tensile strength of the repaired interface fascia. Materials and methodsThirty-two male Sprague–Dawley rats underwent incision hernia creation and subsequent repair with polypropylene mesh. The animals received either saline (n = 16) or doxycycline (n = 16) beginning from 1 day before hernia repair until the end of survival time of 6 wk (n = 16) or 12 wk (n = 16). Tissue samples were investigated for MMPs and collagen subtypes using Western blot procedures, and tensiometric analysis was performed. ResultsAt both 6 and 12 wk after hernia repair, the tensiometric strength of doxycycline-treated mesh to fascia interface (MFI) tissue showed a statistically significant increase when compared with untreated control MFI. In both groups, collagen-1, -2, and -3 ratios were remarkably increased in doxycycline-treated MFI. At 6 wk, the doxycycline-treated MFI group showed a significant decrease in MMP-2, an increase in MMP-3, and no change in MMP-9. At 12 wk, MMP-9 showed a remarkable reduction, whereas MMP-2 and -3 protein levels increased in the doxycycline-treated MFI group. ConclusionsDoxycycline administration results in significantly improved strength of repaired fascial interface tissue along with a remarkable increase in collagen-1, -2, and -3 ratios.

Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta

Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.

Development of peptidomic assays for discovery of endogenously processed peptides from the healthy and juvenile idiopathic arthritis human synovial fluid (981.9)

Juvenile idiopathic arthritis (JIA) is the most common chronic condition seen by pediatric rheumatologists. We and others hypothesized that joint destruction could be partly due to an adaptive immune response mediated by antibodies, and that the characterization of the peptidome (together with the degradome) found in arthritic synovial fluid (SF) (from children with JIA) will help to advance the present studies and shed new molecular mechanisms on the pathogenesis of JIA. Herein, we present the development of peptidomics assays coupled with high resolution nanoLC‐MS/MS on LTQ/Orbitrap Velos mass spectrometer aimed to characterize the endogenously processed peptides from the SF of children with JIA and control patients. The SF peptidome was fractionated by ultrafiltration and peptides with MW&lt;5 kDa were sequenced by HCD/ETD nanoLC Orbitrap‐ESI‐MS/MS or CID nanoLC LTQ‐ESI‐MS/MS. The MS/MS spectra were analyzed with two searching engines, Mascot and PEAKS, against SwissProt database. The peptidome analysis highlighted the breakdown products of the fibrinogen alpha and beta chains and cartilage, mainly proteoglycan 4, fibronectins and multiple collagen subtypes (I, II, III, IV, V, among others). Ingenuity Pathway Analysis (IPA) revealed that the SF from JIA patients is enriched with acute phase reactants, as well as structural components having more than 80% of the proteome connected into the pathways characterizing the inflammatory phenotype: activation of the complement, coagulation systems, intrinsic snd extrinsic prothrombin and the amplification of the acute phase signaling. We proposed that the peptidome biomarkers found in the JIA‐SF could help to elucidate the mechanism for the amplification of the autoimmune process during inflammation. Moreover, the peptidomic assays described herein could be applied to other biological samples of biomedical interest and further used to investigate the proteases (the degradome) responsible for regulating the bioactive peptides.

Apoptosis, mast cell degranulation and collagen breakdown in the pathogenesis of loxoscelism in subcutaneously implanted sponges

Envenomation by the Loxosceles spider causes loxoscelism, a pattern of signs and symptoms that primarily manifests in the dermonecrotic form. Our studies have shown that a mouse subcutaneous sponge implantation model may be useful in evaluating the effects of Loxosceles similis venom. This model provides an ideal microenvironment in which to study loxoscelism; however, it is still important to evaluate its pathogenesis and to observe the effects of L. similis venom for longer time periods than those in previous studies of this model. The aims of this study are: (1) to histologically characterize the effects of L. similis crude venom in a subcutaneous sponge implant; (2) to quantify the mast cells present in the implant and to measure their degranulation activity; (3) to quantify collagen subtypes I and III; and (4) to verify, quantify, and evaluate the effects of apoptosis in the implant on the pathogenesis of loxoscelism at 1 h, 4 h, and 24 h after injecting the venom. Thirty Swiss mice (6–8 weeks old, male) were subcutaneously implanted with polyester-polyurethane sponge discs. Fourteen days post-implantation, the animals were divided into six groups (5 animals per group): three control groups (C1h, C4h, and C24h), in which the mice received 30 μl injections of intra-implant saline, and three treated groups (T1h, T4h, and T24h), in which the mice received 30 μl (0.5 μg) injections of L. similis crude venom at 1 h, 4 h, and 24 h intervals. After each time interval, the animals were euthanized, and the implants were harvested and processed for light and electron microscopic analyses. The following results were observed in the implants harvested from the treated groups: acute inflammation with marked edema, thrombus, and vasculitis, as well as increased levels of mast cells and mast cell degranulation, and apoptosis in giant cells. Furthermore, degradation of collagen types I and III was observed. An analysis of the ultrastructure revealed apoptosis in various cell types. The present results suggest that apoptosis in some cell types associated with an increase in mast cell degranulation and the degradation of collagen fibers are important in the pathogenesis of loxoscelism therefore may explain the difficulty in repairing the ulcer is commonly observed in severe cases of loxoscelism cutaneous in humans.

The Collagenopathies: Review of Clinical Phenotypes and Molecular Correlations

Genetic defects of collagen formation (the collagenopathies) affect almost every organ system and tissue in the body. They can be grouped by clinical phenotype, which usually correlates with the tissue distribution of the affected collagen subtype. Many of these conditions present in childhood; however, milder phenotypes presenting in adulthood are increasingly recognized. Many are difficult to differentiate clinically. Precise diagnosis by means of genetic testing assists in providing prognosis information, family counseling, and individualized treatment. This review provides an overview of the current range of clinical presentations associated with collagen defects, and the molecular mechanisms important to understanding how the results of genetic testing affect medical care.

Stromal matrix metalloprotease-13 knockout alters Collagen I structure at the tumor-host interface and increases lung metastasis of C57BL/6 syngeneic E0771 mammary tumor cells

BackgroundMatrix metalloproteases and collagen are key participants in breast cancer, but their precise roles in cancer etiology and progression remain unclear. MMP13 helps regulate collagen structure and has been ascribed largely harmful roles in cancer, but some studies demonstrate that MMP13 may also protect against tumor pathology. Other studies indicate that collagen’s organizational patterns at the breast tumor-host interface influence metastatic potential. Therefore we investigated how MMP13 modulates collagen I, a principal collagen subtype in breast tissue, and affects tumor pathology and metastasis in a mouse model of breast cancer.MethodsTumors were implanted into murine mammary tissues, and their growth analyzed in Wildtype and MMP13 KO mice. Following extraction, tumors were analyzed for collagen I levels and collagen I macro- and micro-structural properties at the tumor-host boundary using immunocytochemistry and two-photon and second harmonic generation microscopy. Lungs were analyzed for metastases counts, to correlate collagen I changes with a clinically significant functional parameter. Statistical analyses were performed by t-test, analysis of variance, or Wilcoxon-Mann–Whitney tests as appropriate.ResultsWe found that genetic ablation of host stromal MMP13 led to: 1. Increased mammary tumor collagen I content, 2. Marked changes in collagen I spatial organization, and 3. Altered collagen I microstructure at the tumor-host boundary, as well as 4. Increased metastasis from the primary mammary tumor to lungs.ConclusionsThese results implicate host MMP13 as a key regulator of collagen I structure and metastasis in mammary tumors, thus making it an attractive potential therapeutic target by which we might alter metastatic potential, one of the chief determinants of clinical outcome in breast cancer. In addition to identifying stromal MMP13 is an important regulator of the tumor microenvironment and metastasis, these results also suggest that stromal MMP13 may protect against breast cancer pathology under some conditions, a finding with important implications for development of chemotherapies directed against matrix metalloproteases.