Collagen V α1 Chain Decrease in Papillary Dermis from Early Systemic Sclerosis: A New Proposal in Cutaneous Fibrosis Molecular Structure.

Cutaneous fibrosis is one of the main features of systemic sclerosis (SSc). Recent findings correlated abnormal collagen V (Col V) deposition in dermis with skin thickening and disease activity in SSc. Considering that Col V is an important regulator of collagen fibrillogenesis, understanding the role of Col V in the first two years of the skin fibrosis in SSc (early SSc) can help to determine new targets for future treatments. In this study, we analyzed the morphological, ultrastructural and molecular features of α1(V) and α2(V) chains and the expression of their coding genes COL5A1 and COL5A2 in collagen fibrillogenesis in early-SSc. Skin biopsies were obtained from seven consecutive treatment-naïve patients with SSc-related fibrosis and four healthy controls. Our data showed increased α1(V) and α2(V) chain expression in the reticular dermis of early-SSc patients; however, immunofluorescence and ultrastructural immunogold staining determined a significant decreased expression of the α1(V) chain along the dermoepidermal junction in the papillary dermis from early-SSc-patients in relation to the control (12.77 ± 1.34 vs. 66.84 ± 3.36; p < 0.0001). The immunoblot confirmed the decreased expression of the α1(V) chain by the cutaneous fibroblasts of early-SSc, despite the increased COL5A1 and COL5A2 gene expression. In contrast, the α2(V) chain was overexpressed in the small vessels (63.18 ± 3.56 vs. 12.16 ± 0.81; p < 0.0001) and capillaries (60.88 ± 5.82 vs. 15.11 ± 3.80; p < 0.0001) in the reticular dermis of early-SSc patients. Furthermore, COLVA2 siRNA in SSc cutaneous fibroblasts resulted in a decreased α1(V) chain expression. These results highlight an intense decrease in the α1(V) chain along the dermoepidermal junction, suggesting an altered molecular histoarchitecture in the SSc papillary dermis, with a possible decrease in the expression of the α1(V)3 homotrimeric isoform, which could interfere with the thickening and cutaneous fibrosis related to SSc.

Resveratrol Downmodulates Neutrophil Extracellular Trap (NET) Generation by Neutrophils in Patients with Severe COVID-19.

The formation of microthrombi in lung autopsies indicates the involvement of NETs in the immunopathogenesis of severe COVID-19. Therefore, supplements inhibiting NET formation, in association with drugs with fewer adverse effects, should be a relevant strategy to attenuate the disease. Resveratrol (RESV) is a natural polyphenol with an important antiviral and antioxidant role. To modulate neutrophils from patients infected with SARS-CoV-2, we evaluated the in vitro effect of RESV on NET formation. Herein, we investigated 190 patients hospitalized with moderate, severe, and critical symptoms at Hospital das Clínicas, Brazil. We observed that neutrophilia in patients with severe COVID-19 infection is composed of neutrophils with activated profile able to release NET spontaneously. Notably, RESV decreased the neutrophil-activated status and the release of free DNA, inhibiting NET formation even under the specific PMA stimulus. At present, there is no evidence of the role of RESV in neutrophils from patients with COVID-19 infection. These findings suggest that adjunctive therapies with RESV may help decrease the inflammation of viral or bacterial infection, improving patient outcomes.

Immunization with SARS-CoV-2 Nucleocapsid protein triggers a pulmonary immune response in rats.

The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats (Rattus norvegicus); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection.

A randomized clinical trial on the effects of exercise on muscle remodelling following bariatric surgery.

BACKGROUND: Muscle atrophy and strength loss are common adverse outcomes following bariatric surgery. This randomized, controlled trial investigated the effects of exercise training on bariatric surgery-induced loss of muscle mass and function. Additionally, we investigated the effects of the intervention on molecular and histological mediators of muscle remodelling. METHODS: Eighty women with obesity were randomly assigned to a Roux-en-Y gastric bypass (RYGB: n = 40, age = 42 ± 8 years) or RYGB plus exercise training group (RYGB + ET: n = 40, age = 38 ± 7 years). Clinical and laboratory parameters were assessed at baseline, and 3 (POST3) and 9 months (POST9) after surgery. The 6 month, three-times-a-week, exercise intervention (resistance plus aerobic exercise) was initiated 3 months post-surgery (for RYGB + ET). A healthy, lean, age-matched control group was recruited to provide reference values for selected variables. RESULTS: Surgery resulted in a similar (P = 0.66) reduction in lower-limb muscle strength in RYGB and RYGB+ET (-26% vs. -31%), which was rescued to baseline values in RYGB + ET (P = 0.21 vs. baseline) but not in RYGB (P < 0.01 vs. baseline). Patients in RYGB+ET had greater absolute (214 vs. 120 kg, P < 0.01) and relative (2.4 vs. 1.4 kg/body mass, P < 0.01) muscle strength compared with RYGB alone at POST9. Exercise resulted in better performance in timed-up-and-go (6.3 vs. 7.1 s, P = 0.05) and timed-stand-test (18 vs. 14 repetitions, P < 0.01) compared with RYGB. Fat-free mass was lower (POST9-PRE) after RYBG than RYGB + ET (total: -7.9 vs. -4.9 kg, P < 0.01; lower-limb: -3.8 vs. -2.7 kg, P = 0.02). Surgery reduced Types I (~ - 21%; P = 0.99 between-group comparison) and II fibre cross-sectional areas (~ - 27%; P = 0.88 between-group comparison), which were rescued to baseline values in RYGB+ET (P > 0.05 vs. baseline) but not RYGB (P > 0.01 vs. baseline). RYGB + ET showed greater Type I (5187 vs. 3898 µm2 , P < 0.01) and Type II (5165 vs. 3565 µm2 , P < 0.01) fCSA than RYGB at POST9. RYGB + ET also resulted in increased capillarization (P < 0.01) and satellite cell content (P < 0.01) than RYGB at POST9. Gene-set normalized enrichment scores for the muscle transcriptome revealed that the ubiquitin-mediated proteolysis pathway was suppressed in RYGB + ET at POST9 vs. PRE (NES: -1.7; P < 0.01), but not in RYGB. Atrogin-1 gene expression was lower in RYGB + ET vs. RYGB at POST9 (0.18 vs. 0.71-fold change, P < 0.01). From both genotypic and phenotypic perspectives, the muscle of exercised patients resembled that of healthy lean individuals. CONCLUSIONS: This study provides compelling evidence-from gene to function-that strongly supports the incorporation of exercise into the recovery algorithm for bariatric patients so as to counteract the post-surgical loss of muscle mass and function.

Th17/Treg-Related Intracellular Signaling in Patients with Chronic Obstructive Pulmonary Disease: Comparison between Local and Systemic Responses.

Th17/Treg imbalance plays a pivotal role in COPD development and progression. We aimed to assess Th17/Treg-related intracellular signaling at different COPD stages in local and systemic responses. Lung tissue and/or peripheral blood samples were collected and divided into non-obstructed (NOS), COPD stages I and II, and COPD stages III and IV groups. Gene expression of STAT3 and -5, RORγt, Foxp3, interleukin (IL)-6, -17, -10, and TGF-ß was assessed by RT-qPCR. IL-6, -17, -10, and TGF-ß levels were determined by ELISA. We observed increased STAT3, RORγt, Foxp3, IL-6, and TGF-ß gene expression and IL-6 levels in the lungs of COPD I and II patients compared to those of NOS patients. Regarding the systemic response, we observed increased STAT3, RORγt, IL-6, and TGF-ß gene expression in the COPD III and IV group and increased IL-6 levels in the COPD I and II group. STAT5 was increased in COPD III and IV patients, although there was a decrease in Foxp3 expression and IL-10 levels in the COPD I and II and COPD III and IV groups, respectively. We demonstrated that an increase in Th17 intracellular signaling in the lungs precedes this increase in the systemic response, whereas Treg intracellular signaling varies between the compartments analyzed in different COPD stages.

Pathological pulmonary vascular remodeling is induced by type V collagen in a model of scleroderma.

OBJECTIVE: The pulmonary vascular remodeling in systemic sclerosis (SSc) is poorly understood and animal models are lacking. Type V collagen (COLV) is elevated in SSc and is implicated in the pathogenesis, and immunization with human COLV induces SSc-like skin and lung changes in rabbits and mice. Here we tested the hypothesis that COLV immunization will induce pathological and functional changes that phenocopy SSc-associated pulmonary vascular disease. METHODS: Pulmonary vascular changes in rabbits immunized with human COLV were extensively characterized by a combination of histology, electron microscopy and immunohistochemistry. Physiologic changes induced by COLV in explanted pulmonary artery rings were evaluated. The pattern of histopathologic alterations and gene expression induced in immunized rabbits were compared to those in SSc patients. RESULTS: COLV immunization was accompanied by striking pulmonary vascular abnormalities, characterized by reduced capillary density, perivascular inflammation, endothelial cell injury and collagen accumulation, that closely phenocopy changes seen in SSc patients. Moreover, pulmonary arteries from immunized rabbits showed impaired ex vivo vascular relaxation. Expression of COL5A2 was significantly increased in the lungs from immunized rabbits (p = 0.02), as well as in patients with SSc (P = 0.02). CONCLUSION: COLV immunity in rabbits is associated with marked vascular remodeling in the lung that phenocopies early-stage human SSc-associated pulmonary vascular disease. COLV immunization therefore represents a novel approach to model SSc pulmonary vascular pathology. Moreover, our findings suggest that COLV might represent a novel pathogenic autoantigen in SSc and future studies with the present model should be developed for possible association with PAH.

Exercise-Induced Increases in Insulin Sensitivity After Bariatric Surgery Are Mediated By Muscle Extracellular Matrix Remodeling.

Exercise seems to enhance the beneficial effect of bariatric (Roux-en-Y gastric bypass [RYGB]) surgery on insulin resistance. We hypothesized that skeletal muscle extracellular matrix (ECM) remodeling may underlie these benefits. Women were randomized to either a combined aerobic and resistance exercise training program following RYGB (RYGB + ET) or standard of care (RYGB). Insulin sensitivity was assessed by oral glucose tolerance test. Muscle biopsy specimens were obtained at baseline and 3 and 9 months after surgery and subjected to comprehensive phenotyping, transcriptome profiling, molecular pathway identification, and validation in vitro. Exercise training improved insulin sensitivity beyond surgery alone (e.g., Matsuda index: RYGB 123% vs. RYGB + ET 325%; P ≤ 0.0001). ECM remodeling was reduced by surgery alone, with an additive benefit of surgery and exercise training (e.g., collagen I: RYGB -41% vs. RYGB + ET -76%; P ≤ 0.0001). Exercise and RYGB had an additive effect on enhancing insulin sensitivity, but surgery alone did not resolve insulin resistance and ECM remodeling. We identified candidates modulated by exercise training that may become therapeutic targets for treating insulin resistance, in particular, the transforming growth factor-ß1/SMAD 2/3 pathway and its antagonist follistatin. Exercise-induced increases in insulin sensitivity after bariatric surgery are at least partially mediated by muscle ECM remodeling.

Evaluation of Radiosterilized Glyercerolated Amniotic Membranes as a Substrate for Cultured Human Epithelial Cells.

Human amniotic membrane (HAM) is a biomaterial with biological properties beneficial to tissue repair, serving as a substrate for cell cultivation. Irradiation is used for tissue sterilization, but can damage the HAM structure. The objective of this paper was to construct a skin substitute, composed of human keratinocytes cultured on glycerolated HAMs, and to evaluate the influence radiation on subsequent cell culture growth. Four batches of HAMs were glycerolated, and half of them were radio-sterilzed with 25 kGy. Non-irradiated glycerolated HAM (ni-HAM) and irradiated glycerolated HAM (i-HAM) samples were then de-epithelized and analyzed using optical microscopy (Picrossirius staining), immunofluorescence and electron microscopy. Subsequently, keratinocytes were cultured on ni- and i-HAMs, and either immersed or positioned at the air-liquid interface. The basement membranes of the ni-HAM group remained intact following de-epithelialization, whereas the i-HAM group displayed no evidence or remnant presence of these membranes. Concerning the keratinocyte cultures, the ni-HAM substrate promoted the growth of multi-layered and differentiated epithelia. Keratinocytes cultured on i-HAM formed epithelium composed of three layers of stratification and discrete cell differentiation. The glycerolated HAM was compatible with cultured epithelia, demonstrating its potential as a skin substitute. Irradiation at 25 kGy caused structural damage to the amnion.

Increased cytokine levels and histological changes in cartilage, synovial cells and synovial fluid after malleolar fractures.

BACKGROUND: Malleolar fractures are among the most common fractures in the human skeleton with a high risk of later development of post-traumatic osteoarthritis (OA). The acute ankle injury initiates a sequence of events potentially leading to progressive articular surface damage resulting from inflammatory changes in cartilage, synovial tissue and synovial fluid. We hypothesised that in the acute phase of ankle fracture, these changes occur at the same time in the different tissues. METHODS: Specimens of chondral tissue, synovial tissue and synovial fluid were collected from 16 patients with acute articular ankle fracture (study group). Additional samples were obtained from five male fresh cadavers within 12 hours of death (control group). Chondral tissue was assessed for cellularity, irregularities and chondrocyte disarray. Synovial tissue was assessed for synovitis, proteoglycans and collagen deposition. Synovial fluid was assessed for cytokines IL-2, IL-6, IL-10, IL-17, IFN-γ and TGF-ß1. RESULTS: Chondral tissue showed discontinuity in the tidemark between cartilage and subchondral bone, chondrocyte disarray, increased cellularity (both at the cartilage surface and subchondral bone), articular surface irregularities and increased deposition of proteoglycans and collagen fibres. Synovial tissue showed a statistically significant difference between the study and control groups in the concentration per tissue area of both thin collagen fibres (p=0.0274) and thick collagen fibres (p<0.0001). Cytokine concentrations in synovial fluid samples were significantly higher in ankle fracture tissue compared with controls for IL-2 (p=0.0002), IL-6 (p<0.0001), IL-10 (p=0.002) and IL-17 (p<0.0001). No statistically significant differences were observed for IFN-γ (p=0.06303) and TGF-ß1 (p=0.8832). CONCLUSION: We observed a pattern of simultaneous and interrelated pathological changes in cartilage, subchondral bone, synovial tissue and synovial fluid after acute malleolar fracture. As the observed inflammatory changes could lead to the development of OA, a more thorough knowledge of these early processes could be helpful to find strategies for prevention or delay of this common complication.

N-acetylcysteine Counteracts Adipose Tissue Macrophage Infiltration and Insulin Resistance Elicited by Advanced Glycated Albumin in Healthy Rats.

Background: Advanced glycation endproducts elicit inflammation. However, their role in adipocyte macrophage infiltration and in the development of insulin resistance, especially in the absence of the deleterious biochemical pathways that coexist in diabetes mellitus, remains unknown. We investigated the effect of chronic administration of advanced glycated albumin (AGE-albumin) in healthy rats, associated or not with N-acetylcysteine (NAC) treatment, on insulin sensitivity, adipose tissue transcriptome and macrophage infiltration and polarization. Methods: Male Wistar rats were intraperitoneally injected with control (C) or AGE-albumin alone, or, together with NAC in the drinking water. Biochemical parameters, lipid peroxidation, gene expression and protein contents were, respectively, determined by enzymatic techniques, reactive thiobarbituric acid substances, RT-qPCR and immunohistochemistry or immunoblot. Carboxymethyllysine (CML) and pyrraline (PYR) were determined by LC/mass spectrometry (LC-MS/MS) and ELISA. Results: CML and PYR were higher in AGE-albumin as compared to C. Food consumption, body weight, systolic blood pressure, plasma lipids, glucose, hepatic and renal function, adipose tissue relative weight and adipocyte number were similar among groups. In AGE-treated animals, insulin resistance, adipose macrophage infiltration and Col12a1 mRNA were increased with no changes in M1 and M2 phenotypes as compared to C-albumin-treated rats. Total GLUT4 content was reduced by AGE-albumin as compared to C-albumin. NAC improved insulin sensitivity, reduced urine TBARS, adipose macrophage number and Itgam and Mrc mRNA and increased Slc2a4 and Ppara. CD11b, CD206, Ager, Ddost, Cd36, Nfkb1, Il6, Tnf, Adipoq, Retn, Arg, and Il12 expressions were similar among groups. Conclusions: AGE-albumin sensitizes adipose tissue to inflammation due to macrophage infiltration and reduces GLUT4, contributing to insulin resistance in healthy rats. NAC antagonizes AGE-albumin and prevents insulin resistance. Therefore, it may be a useful tool in the prevention of AGE action on insulin resistance and long-term complications of DM.

Low-sodium diet induces atherogenesis regardless of lowering blood pressure in hypertensive hyperlipidemic mice.

This study investigated the influence of sodium restriction and antihypertensive drugs on atherogenesis utilizing hypertensive (H) low-density lipoprotein-receptor knockout mice treated or not with losartan (Los) or hydralazine (Hyd) and fed low-sodium (LS) or normal-sodium (NS) chow. Despite reducing the blood pressure (BP) of H-LS mice, the LS diet caused arterial lipid infiltration due to increased plasma total cholesterol (TC) and triglycerides (TG). Los and Hyd reduced the BP of H-LS mice, and Los effectively prevented arterial injury, likely by reducing plasma TG and nonesterified fatty acids. Aortic lipid infiltration was lower in Los-treated H-LS mice (H-LS+Los) than in normotensive (N)-LS and H-LS mice. Aortic angiotensin II type 1 (AT1) receptor content was greater in H-NS than H-LS mice and in H-LS+Hyd than H-LS+Los mice. Carboxymethyl-lysine (CML) and receptor for advanced glycation end products (RAGE) immunostaining was greater in H-LS than H-NS mice. CML and RAGE levels were lower in LS animals treated with antihypertensive drugs, and Hyd enhanced the AT1 receptor level. Hyd also increased the gene expression of F4/80 but not tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-10, intercellular adhesion molecule-1 or cluster of differentiation 66. The novelty of the current study is that in a murine model of simultaneous hypertension and hyperlipidemia, the pleiotropic effect of chronic, severe sodium restriction elicited aortic damage even with reduced BP. These negative effects on the arterial wall were reduced by AT1 receptor antagonism, demonstrating the influence of angiotensin II in atherogenesis induced by a severely LS diet.

Collagenase mRNA Overexpression and Decreased Extracellular Matrix Components Are Early Events in the Pathogenesis of Emphysema.

To describe the progression of parenchymal remodeling and metalloproteinases gene expression in earlier stages of emphysema, mice received porcine pancreatic elastase (PPE) instillation and Control groups received saline solution. After PPE instillation (1, 3, 6 hours, 3 and 21 days) we measured the mean linear intercept, the volume proportion of types I and III collagen, elastin, fibrillin and the MMP-1, -8, -12 and -13 gene expression. We observed an initial decrease in type I (at the 3rd day) and type III collagen (from the 6th hour until the 3rd day), in posterior time points in which we detected increased gene expression for MMP-8 and -13 in PPE groups. After 21 days, the type III collagen fibers increased and the type I collagen values returned to similar values compared to control groups. The MMP-12 gene expression was increased in earlier times (3 and 6 hours) to which we detected a reduced proportion of elastin (3 days) in PPE groups, reinforcing the already established importance of MMP-12 in the breakdown of ECM. Such findings will be useful to better elucidate the alterations in ECM components and the importance of not only metalloelastase but also collagenases in earlier emphysema stages, providing new clues to novel therapeutic targets.

Unusual remodeling of the hyalinization band in vulval lichen sclerosus by type V collagen and ECM 1 protein.

OBJECTIVES: The vulva is the primary site affected in lichen sclerosus, a chronic dermatosis in women that is histologically characterized by a zone of collagen remodeling in the superior dermis. The normal physiological properties of the vulva depend on the assembly of collagen types I (COLI), III (COLIII) and V (COLV), which form heterotypic fibers, and extracellular matrix protein interactions. COLV regulates the heterotypic fiber diameter, and the preservation of its properties is important for maintaining normal tissue architecture and function. In the current work, we analyzed the expression of COLV and its relationship with COLI, COLIII, elastic fibers and extracellular matrix protein 1 in vulvar biopsies from patients with lichen sclerosus. METHODS: Skin biopsies from 21 patients with lichen sclerosus, classified according to Hewitt histological criteria, were studied and compared to clinically normal vulvar tissue (N=21). Morphology, immunohistochemistry, immunofluorescence, 3D reconstruction and morphometric analysis of COLI, COLIII, COLV deposition, elastic fibers and extracellular matrix 1 expression in a zone of collagen remodeling in the superior dermis were performed. RESULTS: A significant decrease of elastic fibers and extracellular matrix 1 protein was present in the hyalinization zone of lichen sclerosus compared to healthy controls. The non-homogeneous distribution of collagen fibers visualized under immunofluorescence in the hyalinization zone of lichen sclerosus and control skin was confirmed by histomorphometry. Lichen sclerosus dermis shows a significant increase of COLI, COLIII and COLV expression compared to the healthy controls. Significant inverse associations were found between elastic fibers and COLV and between COLV and extracellular matrix 1 expression. A direct association was found between elastic fiber content and extracellular matrix 1 expression. Tridimensional reconstruction of the heterotypic fibers of the lichen sclerosus zone of collagen remodeling confirmed the presence of densely clustered COLV. CONCLUSIONS: Increased deposition of abnormal COLV and its correlation with extracellular matrix 1 and elastic fibers suggest that COLV may be a trigger in the pathogenesis of lichen sclerosus.

Unusual remodeling of the hyalinization band in vulval lichen sclerosus by type V collagen and ECM 1 protein

OBJECTIVES: The vulva is the primary site affected in lichen sclerosus, a chronic dermatosis in women that is histologically characterized by a zone of collagen remodeling in the superior dermis. The normal physiological properties of the vulva depend on the assembly of collagen types I (COLI), III (COLIII) and V (COLV), which form heterotypic fibers, and extracellular matrix protein interactions. COLV regulates the heterotypic fiber diameter, and the preservation of its properties is important for maintaining normal tissue architecture and function. In the current work, we analyzed the expression of COLV and its relationship with COLI, COLIII, elastic fibers and extracellular matrix protein 1 in vulvar biopsies from patients with lichen sclerosus. METHODS: Skin biopsies from 21 patients with lichen sclerosus, classified according to Hewitt histological criteria, were studied and compared to clinically normal vulvar tissue (N=21). Morphology, immunohistochemistry, immunofluorescence, 3D reconstruction and morphometric analysis of COLI, COLIII, COLV deposition, elastic fibers and extracellular matrix 1 expression in a zone of collagen remodeling in the superior dermis were performed. RESULTS: A significant decrease of elastic fibers and extracellular matrix 1 protein was present in the hyalinization zone of lichen sclerosus compared to healthy controls. The non-homogeneous distribution of collagen fibers visualized under immunofluorescence in the hyalinization zone of lichen sclerosus and control skin was confirmed by histomorphometry. Lichen sclerosus dermis shows a significant increase of COLI, COLIII and COLV expression compared to the healthy controls. Significant inverse associations were found between elastic fibers and COLV and between COLV and extracellular matrix 1 expression. A direct association was found between elastic fiber content and extracellular matrix 1 expression. Tridimensional reconstruction of the heterotypic fibers ...

The Th17 pathway in the peripheral lung microenvironment interacts with expression of collagen V in the late state of experimental pulmonary fibrosis.

BACKGROUND: Myofibroblasts derived from fibroblasts in the pathogenesis of pulmonary fibrosis causes excessive and disordered deposition of matrix proteins, including collagen V, which can cause a Th17-mediated immune response and lead to apoptosis. However, whether the intrinsic ability of lung FBs to produce the matrix depends on their site-specific variations is not known. AIM: To investigate the link between Th17 and collagen V that maintains pulmonary remodeling in the peripheral lung microenvironment during the late stage of experimental pulmonary fibrosis. METHODS: Young male mice including wild Balb/c mice (BALB, n=10), wild C57 Black/6J mice (C57, n=10) and IL-17 receptor A knockout mice (KO, n=8), were sacrificed 21 days after treatment with bleomycin. Picrosirius red staining, immunohistochemistry for IL-17-related markers and "in situ" detection of apoptosis, immunofluorescence for collagen types I and V, primary cell cultures from tissue lung explants for RT-PCR and electron microscopy were used. RESULTS: The peripheral deposition of extracellular matrix components by myofibroblasts during the late stage is maintained in C57 mice compared with that in Balb mice and is not changed in the absence of IL-17 receptor A; however, the absence of IL-17 receptor A induces overexpression of type V collagen, amplifies the peripheral expression of IL-17 and IL-17-related cytokines and reduces peripheral lung fibroblast apoptosis. CONCLUSION: A positive feedback loop between the expression patterns of collagen V and IL-17 may coordinate the maintenance of peripheral collagen I in the absence of IL-17 receptor A in fibrosis-susceptible strains in a site-specific manner.

Bone plasticity in response to exercise is sex-dependent in rats.

PURPOSE: To characterize the potential sexual dimorphism of bone in response to exercise. METHODS: Young male and female Wistar rats were either submitted to 12 weeks of exercise or remained sedentary. The training load was adjusted at the mid-trial (week 6) by the maximal speed test. A mechanical test was performed to measure the maximal force, resilience, stiffness, and fracture load. The bone structure, formation, and resorption were obtained by histomorphometric analyses. Type I collagen (COL I) mRNA expression and tartrate-resistant acid phosphatase (TRAP) mRNA expression were evaluated by quantitative real-time PCR (qPCR). RESULTS: The male and female trained rats significantly improved their maximum speed during the maximal exercise test (main effect of training; p<0.0001). The male rats were significantly heavier than the females, irrespective of training (main effect of sex; p<0.0001). Similarly, both the weight and length of the femur were greater for the male rats when compared with the females (main effect of sex; p<0.0001 and p<0.0001, respectively). The trabecular volume was positively affected by exercise in male and female rats (main effect of training; p = 0.001), whereas the trabecular thickness, resilience, mineral apposition rate, and bone formation rate increased only in the trained males (within-sex comparison; p<0.05 for all parameters), demonstrating the sexual dimorphism in response to exercise. Accordingly, the number of osteocytes increased significantly only in the trained males (within-sex comparison; p<0.05). Pearson's correlation analyses revealed that the COL I mRNA expression and TRAP mRNA expression were positively and negatively, respectively, related to the parameters of bone remodeling obtained from the histomorphometric analysis (r = 0.59 to 0.85; p<0.05). CONCLUSION: Exercise yielded differential adaptations with respect to bone structure, biomechanical proprieties, and molecular signaling in male and female rats.

Experimental diabetes modulates collagen remodelling of joints in rats.

UNLABELLED: The aim of this study was to evaluate extracellular matrix components in articular cartilage, ligaments and synovia in an experimental model of diabetes. Young Wistar rats were divided into a streptozotocin-induced (STZ; 35 mg/kg) diabetic group (DG; n=15) and a control group (CG; n=15). Weight, blood glucose and plasma anti-carboxymethyllysine were measured 70 days after STZ infusions. Knee joints, patellar ligaments, and lateral and medial collateral ligaments were isolated and stained with hematoxylin-eosin and Picrosirius. The total collagen content was determined by morphometry. Immunofluorescence was employed to evaluate types I, III, and V collagen in ligaments and synovial tissues and types II and XI collagen in cartilage. RESULTS: Higher blood glucose levels and plasma anti-carboxymethyllysine were observed in DG rats when compared to those in CG rats. The final weight was significantly lower in the DG rats than in the CG rats. Histomorphometric evaluation depicted a small quantity of collagen fibers in ligaments and articular cartilage in DG rats, as well as increased collagen in synovial tissue. There was a decrease in cartilage proteoglycans in DG rats when compared with CG rats. Immunofluorescence staining revealed an increase of collagen III and V in ligaments, collagen XI in cartilage, and collagen I in synovial tissue of DG rats compared with CG rats. CONCLUSION: The ligaments, cartilage and synovia are highly affected following STZ-induced diabetes in rats, due the remodeling of collagen types in these tissues. This process may promote the degradation of the extracellular matrix, thus compromising joint function. Our data may help to better understand the pathogenesis of joint involvement related to diabetes.

Abnormal collagen V deposition in dermis correlates with skin thickening and disease activity in systemic sclerosis.

OBJECTIVE: The physiological and mechanical properties of the skin, the primary tissue affected by systemic sclerosis, depend on the assembly of collagen types I, III and V, which form heterotypic fibers. Collagen V (COLV) regulates heterotypic fiber diameter, and the maintenance of its properties is important for maintaining normal tissue architecture and function. Based on a COLV-induced experimental SSc model, in which overexpression of abnormal COLV was a prominent feature, we assumed that this abnormality could be present in SSc patients and could be correlated to disease duration, skin thickening and disease activity. METHODS: Skin biopsies from 18 patients (6 early-stage and 12 late-stage) and 10 healthy controls were studied. Skin thickening assessment was performed with the Modified Rodnan Skin Score (MRSS), and activity was calculated using the Valentini Disease Activity Index. Morphology, morphometry of COLV deposition in dermis, as well as, quantitative RT-PCR and 3D-reconstruction of the dermal fibroblast culture were performed. RESULTS: Structurally abnormal COLV was overexpressed in SSc skin, mainly in the early stages of the disease, when compared to normal controls and late-stage. A positive correlation between COLV expression and MRSS and disease activity was observed. Collagen V alpha-1 and alpha-2 mRNA expression levels were higher in SSc. Tridimensional reconstruction of SSc dermal heterotypic fibers confirmed the presence of atypical COLV. CONCLUSION: Increased synthesis of abnormal COLV and its correlation with disease stage, activity and MRSS suggest that this collagen can be a possible trigger involved in the pathogenesis of SSc.

Role of focal adhesion kinase in lung remodeling of endotoxemic rats.

Despite significant advances in the care of critically ill patients, acute lung injury continues to be a complex problem with high mortality. The present study was designed to characterize early lipopolysaccharide (LPS)-induced pulmonary injury and small interfering RNA targeting focal adhesion kinase (FAK) as a possible therapeutic tool in the septic lung remodeling process. Male Wistar rats were assigned into endotoxemic group and control group. Total collagen deposition was performed 8, 16, and 24 h after LPS injection. Focal adhesion kinase expression, interstitial and vascular collagen deposition, and pulmonary mechanics were analyzed at 24 h. Intravenous injection of small interfering RNA targeting FAK was used to silence expression of the kinase in pulmonary tissue. Focal adhesion kinase, total collagen deposition, and pulmonary mechanics showed increased in LPS group. Types I, III, and V collagen showed increase in pulmonary parenchyma, but only type V increased in vessels 24 h after LPS injection. Focal adhesion kinase silencing prevented lung remodeling in pulmonary parenchyma at 24 h. In conclusion, LPS induced a precocious and important lung remodeling. There was fibrotic response in the lung characterized by increased amount in total and specific-type collagen. These data may explain the frequent clinical presentation during sepsis of reduced lung compliance, oxygen diffusion, and pulmonary hypertension. The fact that FAK silencing was protective against lung collagen deposition underscores the therapeutic potential of FAK targeting by small interfering RNA.