Smoking induces increased apoptosis in osteoblasts: changes in bone matrix organic components.

Clinical studies demonstrate the impact of smoking on bone tissue fragility and higher incidence of fractures. However, it is not totally understood which physiological mechanisms could be involved in these events. Previously, we showed important changes in bone tissue components in experimental model of cigarette smoke (CS) exposure. CS exposure induces worsening in bone mineralization and a decrease in collagen type I deposition, leading to bone fragility. Considering that the majority of clinical studies described bone structural changes by radiographic images, in this study we performed analyses "in situ" using tissue samples from smokers, former smokers and non-smokers to better understand how the increase in inflammatory mediators induced by smoking exposure could interfere in bone cells activity leading bone structural changes. We observed increased levels of IL-1ß, IL-6 and TNF-α in bone tissue homogenates with a concomitant increase in osteoblast apoptosis in smokers and former smokers compared with non-smokers. Histological changes in both smokers and former smokers were characterized by reduction in collagen type I. Only in smokers, it was observed decrease in trabecular area, suggesting increased bone resorption and increase in collagen type V. These results showed that osteoblasts apoptosis in association with increased bone resorption leads bone structural changes in smokers.

Modeling extracellular matrix through histo-molecular gradient in NSCLC for clinical decisions.

Lung cancer still represents a global health problem, being the main type of tumor responsible for cancer deaths. In this context, the tumor microenvironment, and the extracellular matrix (ECM) pose as extremely relevant. Thus, this study aimed to explore the prognostic value of epithelial-to-mesenchymal transition (EMT), Wnt signaling, and ECM proteins expression in patients with non-small-cell lung carcinoma (NSCLC) with clinical stages I-IIIA. For that, we used 120 tissue sections from patients and evaluated the immunohistochemical, immunofluorescence, and transmission electron microscopy (TEM) to each of these markers. We also used in silico analysis to validate our data. We found a strong expression of E-cadherin and ß-catenin, which reflects the differential ECM invasion process. Therefore, we also noticed a strong expression of chondroitin sulfate (CS) and collagens III and V. This suggests that, after EMT, the basal membrane (BM) enhanced the motility of invasive cells. EMT proteins were directly associated with WNT5A, and collagens III and V, which suggests that the WNT pathway drives them. On the other hand, heparan sulfate (HS) was associated with WNT3A and SPARC, while WNT1 was associated with CS. Interestingly, the association between WNT1 and Col IV suggested negative feedback of WNT1 along the BM. In our cohort, WNT3A, WNT5A, heparan sulfate and SPARC played an important role in the Cox regression model, influencing the overall survival (OS) of patients, be it directly or indirectly, with the SPARC expression stratifying the OS into two groups: 97 months for high expression; and 65 for low expression. In conclusion, the present study identified a set of proteins that may play a significant role in predicting the prognosis of NSCLC patients with clinical stages I-IIIA.

Histoarchitecture of the fibrillary matrix of human fetal posterior tibial tendons.

Adult tendons are highly differentiated. In mature individuals, tendon healing after an injury occurs through fibrotic tissue formation. Understanding the intrinsic reparative properties of fetal tendons would help to understand the maturation tissue process and tendon tissue repair. The present study evaluated the evolution of histoarchitecture, cellularity and the distribution of collagens I, III and V in the posterior tibial tendon in human fetuses at different gestational ages. Morphological profiles were assessed in nine fresh spontaneously aborted fetuses (Group I: five fetuses aged between 22 and 28 weeks of gestation; Group II: four fetuses aged between 32 and 38 weeks of gestation), characterized by a combination of histology, fluorescence and immunohistochemistry. In Group I, the posterior tibial tendon showed statistically significant greater cellularity and presence of collagen III and V than in Group II tendon, which showed a predominance of collagenous I and a better organization of the extracellular matrix compared with Group I tendons. In addition, a statistically significant higher rate of CD90, a marker of mesenchymal cells, was found in Group I tendons. In fetuses with gestational age between 22 and 28 weeks, the posterior tibialis tendons showed a thin and disorganized fibrillar structure, with an increase in collagen III and V fibers and mesenchymal cells. In the posterior tibialis tendons of fetuses with gestational age between 32 and 38 weeks, the fibrillar structure was thicker with a statistically significant increase in type I collagen and decreased cellularity.

Effects of different fluid management on lung and kidney during pressure-controlled and pressure-support ventilation in experimental acute lung injury.

Optimal fluid management is critical during mechanical ventilation to mitigate lung damage. Under normovolemia and protective ventilation, pulmonary tensile stress during pressure-support ventilation (PSV) results in comparable lung protection to compressive stress during pressure-controlled ventilation (PCV) in experimental acute lung injury (ALI). It is not yet known whether tensile stress can lead to comparable protection to compressive stress in ALI under a liberal fluid strategy (LF). A conservative fluid strategy (CF) was compared with LF during PSV and PCV on lungs and kidneys in an established model of ALI. Twenty-eight male Wistar rats received endotoxin intratracheally. After 24 h, they were treated with CF (minimum volume of Ringer's lactate to maintain normovolemia and mean arterial pressure ≥70 mmHg) or LF (~4 times higher than CF) combined with PSV or PCV (VT  = 6 ml/kg, PEEP = 3 cmH2 O) for 1 h. Nonventilated animals (n = 4) were used for molecular biology analyses. CF-PSV compared with LF-PSV: (1) decreased the diffuse alveolar damage score (10 [7.8-12] vs. 25 [23-31.5], p = 0.006), mainly due to edema in axial and alveolar parenchyma; (2) increased birefringence for occludin and claudin-4 in lung tissue and expression of zonula-occludens-1 and metalloproteinase-9 in lung. LF compared with CF reduced neutrophil gelatinase-associated lipocalin and interleukin-6 expression in the kidneys in PSV and PCV. In conclusion, CF compared with LF combined with PSV yielded less lung epithelial cell damage in the current model of ALI. However, LF compared with CF resulted in less kidney injury markers, regardless of the ventilatory strategy.

Dissecting and Reconstructing Matrix in Malignant Mesothelioma Through Histocell-Histochemistry Gradients for Clinical Applications.

Background: Malignant pleural mesotheliomas (MM) are known for their heterogenous histology and clinical behavior. MM histology reveals three major tumor cell populations: epithelioid, sarcomatoid, and biphasic. Using a dissecting approach, we showed that histochemical gradients help us better understand tumor heterogeneity and reconsider its histologic classifications. We also showed that this method to characterize MM tumor cell populations provides a better understanding of the underlying mechanisms for invasion and disease progression. Methods: In a cohort of 87 patients with surgically excised MM, we used hematoxylin and eosin to characterize tumor cell populations and Movat's pentachrome staining to dissect the ECM matrisome. Next, we developed a computerized semi-assisted protocol to quantify and reconstruct the ECM in 3D and examined the clinical association between the matricellular factors and patient outcome. Results: Epithelioid cells had a higher matrix composition of elastin and fibrin, whereas, in the sarcomatoid type, hyaluronic acid and total collagen were most prevalent. The 3D reconstruction exposed the collagen I and III that form channels surrounding the neoplastic cell blocks. The estimated volume of the two collagen fractions was 14% of the total volume, consistent with the median estimated area of total collagen (12.05 mm2) for epithelioid MM. Conclusion: Differential patterns in matricellular phenotypes in MM could be used in translational studies to improve patient outcome. More importantly, our data raise the possibility that cancer cells can use the matrisome for disease expansion and could be effectively targeted by anti-collagen, anti-elastin, and/or anti-hyaluronic acid therapies.

Sterilized human skin graft with a dose of 25 kGy provides a privileged immune and collagen microenvironment in the adhesion of Nude mice wounds.

This study aimed to report the effects of different doses of ionizing radiation on inflammatory and repair stage of human skin graft adherence in Nude mice wounds. Animals were divided into transplanted with irradiated human skin grafts (IHSG) at 25 and 50 kGy (IHSG 25 kGy; IHSG 50 kGy) and non-IHSG and euthanized on the 3rd, 7th and 21st days after the surgery, by gross and microscopic changes, immunostaining for human type I collagen (Col I) and mouse Col I and Col III and inflammatory cells. We found an effectiveness of human split-thickness graft adherence in mice transplanted with IHSG 25 kGy, as well decrease in dermo-epidermal necrosis and neutrophils, lower loss of skin thickness, epithelization and neo-vascularization. Day 21 post-transplantation with IHSG 25 kGy was observed a well-preserved human skin in the border of the graft, a prominent granulation tissue in an organization by proliferated fibroblasts, Col III deposition and increased B-cells and macrophages. A complete adherence of human skin graft occurred with IHSG 25 kGy. We suggest that the ionizing radiation at 25 kGy mediates inflammation and the repair stage of human skin graft adherence in murine model, thus emerging as a potential tool in healing cutaneous wounds.

In Situ Overexpression of Matricellular Mechanical Proteins Demands Functional Immune Signature and Mitigates Non-Small Cell Lung Cancer Progression.

Non-small cell lung carcinoma (NSCLC) is a complex cancer biome composed of malignant cells embedded in a sophisticated tumor microenvironment (TME) combined with different initiating cell types, including immune cells and cancer-associated fibroblasts (CAFs), and extracellular matrix (ECM) proteins. However, little is known about these tumors' immune-matricellular relationship as functional and mechanical barriers. This study investigated 120 patients with NSCLC to describe the immune-matricellular phenotypes of their TME and their relationship with malignant cells. Immunohistochemistry (IHC) was performed to characterize immune checkpoints (PD-L1, LAG-3, CTLA-4+, VISTA 1), T cells (CD3+), cytotoxic T cells (CD8+, Granzyme B), macrophages (CD68+), regulatory T cells (FOXP3+, CD4+), natural killer cells (CD57+), and B lymphocytes (CD20+), whereas CAFs and collagen types I, III, and V were characterized by immunofluorescence (IF). We observed two distinct functional immune-cellular barriers-the first of which showed proximity between malignant cells and cytotoxic T cells, and the second of which showed distant proximity between non-cohesive nests of malignant cells and regulatory T cells. We also identified three tumor-associated matricellular barriers: the first, with a localized increase in CAFs and a low deposition of Col V, the second with increased CAFs, Col III and Col I fibers, and the third with a high amount of Col fibers and CAFs bundled and aligned perpendicularly to the tumor border. The Cox regression analysis was designed in two steps. First, we investigated the relationship between the immune-matricellular components and tumor pathological stage (I, II, and IIIA), and better survival rates were seen in patients whose tumors expressed collagen type III > 24.89 fibers/mm². Then, we included patients who had progressed to pathological stage IV and found an association between poor survival and tumor VISTA 1 expression > 52.86 cells/mm² and CD3+ ≤ 278.5 cells/mm². We thus concluded that differential patterns in the distribution of immune-matricellular phenotypes in the TME of NSCLC patients could be used in translational studies to predict new treatment strategies and improve patient outcome. These data raise the possibility that proteins with mechanical barrier function in NSCLC may be used by cancer cells to protect them from immune cell infiltration and immune-mediated destruction, which can otherwise be targeted effectively with immunotherapy or collagen therapy.

The Fibrosis-Targeted Collagen/Integrins Gene Profile Predicts Risk of Metastasis in Pulmonary Neuroendocrine Neoplasms.

Recently, collagen/integrin genes have shown promise as predictors of metastasis mainly in non-small cell lung cancer and breast cancer. However, it is unknown if these gene expression profiling differ in metastatic potential of pulmonary neuroendocrine neoplasms (PNENs). In this study, we sought to identify differentially expressed collagen/integrin genes in PNENs in order to understand the molecular mechanisms underlying the development of stroma-associated fibrosis for invasion and metastasis. We compared collagen/integrin gene expression profiling between PNE tumors (PNETs) and PNE carcinomas (PNECs) using a two-stage design. First, we used PCR Array System for 84 ECM-related genes, and among them, we found COL1A2, COL3A1, COL5A2, ITGA5, ITGAV, and ITGB1 functionally involved in the formation of the stroma-associated fibrosis among PNENs histological subtypes. Second, we examined the clinical association between the six collagen/integrin genes in tumor tissues from 24 patients with surgically excised PNENs. However, the pathological exam of their resected tissues demonstrated that 10 developed lymph node metastasis and 7 distant metastasis. We demonstrated and validated up regulation of the six fibrogenic genes in PNECs and down regulation in PNETs that were significantly associated with metastasis-free and overall survival (P<0.05). Our study implicates up regulation of fibrogenic genes as a critical molecular event leading to lymph node and distant metastasis in PNENs.

Post-Adipose-Derived Stem Cells (ADSC) Stimulated by Collagen Type V (Col V) Mitigate the Progression of Osteoarthritic Rabbit Articular Cartilage.

Collagen is essential for cartilage adhesion and formation. In the present study, histology, immunofluorescence, morphometry, and qRT-PCR suggested that adipose-derived stem cells (ADSCs) stimulated by type V collagen (Col V) induce a significant increase of type II collagen (Col II) in the degenerative area of surgical-induced osteoarthritic rabbit articular cartilage (OA). In vitro, the effects of Col V on the proliferation and differentiation of ADSC were investigated. The expression of the cartilage-related genes Col2a1 and Acan was significantly upregulated and Pou5fl was downregulated post-ADSC/Col V treatment. Post-ADSC/Col V treatment, in vivo analyses revealed that rabbits showed typical signs of osteoarthritic articular cartilage regeneration by hematoxylin and eosin (H&E) and Safranin O/Fast Green staining. Immunohistochemical staining demonstrated that the volume of Col II fibers and the expression of Col II protein were significantly increased, and apoptosis Fas ligand positive significantly decreased post-ADSC/Col V treatment. In conclusion, the expression of Col II was higher in rabbits with surgical-induced osteoarthritic articular cartilage; hence, ADSC/Col V may be a promising therapeutic target for OA treatment.

Photobiomodulation therapy increases collagen II after tendon experimental injury.

A tendon is a mechanosensitive tissue that transmits muscle-derived forces to bones. Photobiomodulation (PBM), also known as low-level laser therapy (LLLT), has been used in therapeutic approaches in tendon lesions, but uncertainties regarding its mechanisms of action have prevented its widespread use. We investigated the response of PBM therapy in experimental lesions of the Achilles tendon in rats. Thirty adult male Wistar rats weighing 250 to 300 g were surgically submitted to bilateral partial transverse section of the Achilles tendon. The right tendon was treated with PBM, whereas the left tendon served as a control. On the third postoperative day, the rats were divided into three experimental groups consisting of ten rats each, which were treated with PBM (Konf, Aculas - HB 750), 780 nm and 80 mW for 20 seconds, three times/week for 7, 14 and 28 days. The rats were sacrificed at the end of the therapeutic time period. The Sca-1 was examined by immunohistochemistry and histomorphometry, and COLA1, COLA2 and COLA3 gene expression was examined by qRT-PCR. COLA2 gene expression was higher in PBM treated tendons than in the control group. The histomorphometric analysis coincided with increased number of mesenchymal cells, characterized by Sca-1 expression in the lesion region (p<0.001). PBM effectively interferes in tendon tissue repair after injury by stimulating mesenchymal cell proliferation and the synthesis of collagen type II, which is suggested to provide structural support to the interstitial tissues during the healing process of the Achilles tendon. Further studies are needed to confirm the role of PBM in tendon healing.

Different histological patterns of type-V collagen levels confer a matrices-privileged tissue microenvironment for invasion in malignant tumors with prognostic value.

Previous studies have reported a close relationship between type V collagen (Col V) and tumor invasion and motility in both breast cancer (BC) and lung cancer (LC). The present work aims to determine whether the extracellular-matrix (ECM)-defined microenvironment influences patient clinical outcome and investigate to which extent histological patterns of Col V expression in malignant cells have a prognostic effect in patients. To that end, we examined the expression of Col V in the tissues of 174 primary tumors (MM, N = 82; LC, N = 41; and BC, N = 46) by immunohistochemistry. We found: (1) diffuse strong green birefringence in membrane and cytoplasm individualizing malignant cells in MM; (2) a focal and weak birefringence mainly in cytoplasmic membrane involving groups of malignant cells in LC and BC; (3) higher average H-score of Col V in MM than in LC and BC samples; (4) a direct correlation between Col V histologic pattern and TNM stage IV, status and median overall survival; (5) patients with LC in TNM stage I, and Col V ≤ 41.7 IOD/mm2 had a low risk of death and a median survival time more than 20 months; (6) patients with MM in TNM stage IV and Col V > 41.7 IOD/mm2 presented a high risk of death and a median survival time of just 20 months. These findings suggest that high levels of Col V individualizing malignant cells, as observed in MM, and low levels grouping malignant cells, as observed in LC and BC, confers different immune-privileged tissue microenvironment for tumor invasion with impact on prognosis of the patients.

In situ Evidence of Collagen V and Interleukin-6/Interleukin-17 Activation in Vascular Remodeling of Experimental Pulmonary Hypertension.

Several studies have reported the pathophysiologic and molecular mechanisms responsible for pulmonary arterial hypertension (PAH). However, the in situ evidence of collagen V (Col V) and interleukin-17 (IL-17)/interleukin-6 (IL-6) activation in PAH has not been fully elucidated. We analyzed the effects of collagen I (Col I), Col V, IL-6, and IL-17 on vascular remodeling and hemodynamics and its possible mechanisms of action in monocrotaline (MCT)-induced PAH. Twenty male Wistar rats were randomly divided into two groups. In the PAH group, animals received MCT 60 mg/kg intraperitoneally, whereas the control group (CTRL) received saline. On day 21, the pulmonary blood pressure (PAP) and right ventricular systolic pressure (RVSP) were determined. Lung histology (smooth muscle cell proliferation [α-smooth muscle actin; α-SMA] and periadventitial fibrosis), immunofluorescence (Col I, Col V, and α-SMA), immunohistochemistry (IL-6, IL-17, and transforming growth factor-beta [TGF-ß]), and transmission electron microscopy to detect fibronexus were evaluated. The RVSP (40 ± 2 vs. 24 ± 1 mm Hg, respectively; p < 0.0001), right ventricle hypertrophy index (65 ± 9 and 25 ± 5%, respectively; p < 0.0001), vascular periadventitial Col I and Col V, smooth muscle cell α-SMA+, fibronexus, IL-6, IL-17, and TGF-ß were higher in the MCT group than in the CTRL group. In conclusion, our findings indicate in situ evidence of Col V and IL-6/IL-17 activation in vascular remodeling and suggest that increase of Col V may yield potential therapeutic targets for treating patients with PAH.

In situ evidence of collagen V and signaling pathway of found inflammatory zone 1 (FIZZ1) is associated with silicotic granuloma in lung mice.

Inhalation of silica particles causes silicosis: an occupational lung disease characterized by persistent inflammation with granuloma formation that leads to tissue remodeling and impairment of lung function. Although silicosis has been studied intensely, little is known about the crucial cellular mechanisms that initiate and drive the process of inflammation and fibrosis. Recently, found in inflammatory zone 1 (FIZZ1) protein, produced by alveolar macrophages and fibroblasts have been shown to induce the proliferation of myofibroblasts and their transdifferentiation, causing tissue fibrosis. Moreover, autoimmunogenic collagen V, produced by alveolar epithelial cells and fibroblasts, is involved in the pathophysiology of interstitial pulmonary fibrosis and bleomycin-induced lung fibrosis. Based on the aforementioned we hypothesized that FIZZ1 and collagen V may be involved in the silicotic granuloma process in mice lungs. Male C57BL/6 mice (N = 20) received intratracheal administration of silica particles (Silica; 20 mg in 50 µL saline) or saline (Control; 50 µL). After 15 days, the lung histology was performed through immunohistochemistry and morphometric analysis. Within silicotic granulomas, collagen V and FIZZ1 increased, while peroxisome proliferator-activated receptor gamma (PPARγ) positive cells decreased. In addition, the expression of proteins Notch-1, alpha smooth muscle actin (α-SMA) and macrophages163 (CD163) were higher in silicotic granulomas than control lungs. A significant positive correlation was found between collagen V and FIZZ1 (r = 0.70; p < 0.05), collagen V and Notch-1 (r = 0.72; p < 0.05), whereas Collagen V was inversely associated with peroxisome proliferator-activated receptor gamma (r=-0.69; p < 0.05). These findings suggested that collagen V association with FIZZ1, Notch-1 and PPARγ might be a key pathogenic mechanism for silicotic granulomas in mice lungs.

Decreased Bone Type I Collagen in the Early Stages of Chronic Obstructive Pulmonary Disease (COPD).

Smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and is known to have deleterious effects on bone metabolism. However, the effects on bone collagen matrix during the development of COPD are unclear. The aim of this study was to evaluate the temporal effect of cigarette smoke exposure on bone type I collagen during COPD development in a cigarette smoke-induced model. C57BL/6 mice were allocated to three groups: control (C), animals exposed to filtered air for 1, 3 and 6 months; cigarette smoke (S), animals exposed to cigarette smoke for 1, 3 and 6 months; provisional smoking (PS), animals exposed to cigarette smoke for 3 months, followed by another 3 months of filtered air exposure. Evaluation of the respiratory mechanics and alveolar enlargement were performed. Femoral and tibial extraction was also performed to evaluate the type I collagen by immunofluorescence and COL1A1 gene expression. Exposure to cigarette smoke led to an alveolar enlargement and progressive reduction in lung tissue resistance and elastance, progressive reduction of type I collagen and reduction in COL1A1 gene expression. Although we did not observe any improvement in the functional and histological parameters in the provisional smoking group, we detected an increase in COL1A1 gene expression. A worsening in bone collagen matrix is part of the initial physiopathological events during COPD development and the smoking cessation induced an evident recovery of COL1A1 expression, possibly to attempt at tissue repair.

An integrative histopathologic clustering model based on immuno-matrix elements to predict the risk of death in malignant mesothelioma.

OBJECTIVE: Previous studies have reported a close relationship between malignant mesothelioma (MM) and the immune matricial microenvironment (IMM). One of the major problems in these studies is the lack of adequate adjustment for potential confounders. Therefore, the aim of this study was to identify and quantify risk factors such as IMM and various tumor characteristics and their association with the subtype of MM and survival. METHODS: We examined IMM and other tumor markers in tumor tissues from 82 patients with MM. These markers were evaluated by histochemistry, immunohistochemistry, immunofluorescence, and morphometry. Logistic regression analysis, cluster analysis, and Cox regression analysis were performed. RESULTS: Hierarchical cluster analysis revealed two clusters of MM that were independent of clinicopathologic features. The high-risk cluster included MM with high tumor cellularity, high type V collagen (Col V) fiber density, and low CD8+ T lymphocyte density in the IMM. Our results showed that the risk of death was increased for patients with MM with high tumor cellularity (OR = 1.63, 95% CI = 1.29-2.89, P = .02), overexpression of Col V (OR = 2.60, 95% CI = 0.98-6.84, P = .04), and decreased CD8 T lymphocytes (OR = 1.001, 95% CI = 0.995-1.007, P = .008). The hazard ratio for the high-risk cluster was 2.19 (95% CI = 0.54-3.03, P < .01) for mortality from MM at 40 months. CONCLUSION: Morphometric analysis of Col V, CD8+ T lymphocytes, and tumor cellularity can be used to identify patients with high risk of death from MM.

Nrf2 positively regulates autophagy antioxidant response in human bronchial epithelial cells exposed to diesel exhaust particles.

Diesel exhaust particles (DEP) are known to generate reactive oxygen species in the respiratory system, triggering cells to activate antioxidant defence mechanisms, such as Keap1-Nrf2 signalling and autophagy. The aim of this study was to investigate the relationship between the Keap1-Nrf2 signalling and autophagy pathways after DEP exposure. BEAS-2B cells were transfected with silencing RNA (siRNA) specific to Nrf2 and exposed to DEP. The relative levels of mRNA for Nrf2, NQO1, HO-1, LC3B, p62 and Atg5 were determined using RT-PCR, while the levels of LCB3, Nrf2, and p62 protein were determined using Western blotting. The autophagy inhibitor bafilomycin caused a significant decrease in the production of Nrf2, HO-1 and NQO1 compared to DEPs treatment, whereas the Nrf2 activator sulforaphane increased the LC3B (p = 0.020) levels. BEAS-2B cells exposed to DEP at a concentration of 50 µg/mL for 2 h showed a significant increase in the expression of LC3B (p = 0.001), p62 (p = 0.008), Nrf2 (p = 0.003), HO-1 (p = 0.001) and NQO1 (p = 0.015) genes compared to control. In siRNA-transfected cells, the LC3B (p < 0.001), p62 (p = 0.001) and Atg5 (p = 0.024) mRNA levels and the p62 and LC3II protein levels were decreased, indicating that Nrf2 modulated the expression of autophagy markers (R < 1). These results imply that, in bronchial cells exposed to DEP, the Nrf2 system positively regulates autophagy to maintain cellular homeostasis.

Identification of Autoimmunity to Peptides of Collagen V α1 Chain as Newly Biomarkers of Early Stage of Systemic Sclerosis.

Patients with Systemic sclerosis (SSc) presents immune dysregulation, vasculopathy, and fibrosis of the skin and various internal organs. Pulmonary fibrosis leads to SSc-associated interstitial lung disease (ILD), which is the main cause of morbidity and mortality in SSc. Recently autoimmunity to type V collagen (Col V) has been characterized in idiopathic pulmonary fibrosis and show promise to be related to the development in SSc. Our aim was to evaluate autoimmunity to Col V α1(V) and α2(V) chains and to the antigenic peptides of these Col V chains in early-SSc sera employing lung tissue of SSc-ILD, as antigen source. We found that sera samples from patients with early-SSc were reactive to Col V (41.18%) and presented immunoreactivity for Col5A1(1.049) and Col5A1(1.439) peptides. The IgG isolated from early-SSc patients-anti-Col V positive sera (anti-ColV IgG) was adsorbed with α1(V) chain (anti-ColV IgG/ads-α1(V)) and α2(V) chain (anti-ColV IgG/ads-α2(V)) and biotinylated to evaluate the spectrum of reactivity in SSc-ILD patients lung biopsies by immunofluorescence. The SSc-ILD lung tissue samples immunostained with anti-ColV IgG showed increased green fluorescence in the vascular basement membrane, bronchiolar smooth muscle, and adventitial layer, contrasting with the tenue immunostaining in control lungs. Col V protein expression in these pulmonary compartments immunostained with early-SSc anti-ColV IgG was confirmed by immune colocalization assays with commercial anti-human Col V antibodies. In addition, SSc-ILD lung tissues immunostained with anti-ColV IgG/ads-α1(V) (sample in which Col V α1 chain-specific antibodies were removed) showed decreased green fluorescence compared to anti-ColV IgG and anti-ColV IgG/ads-α2(V). Our data show that autoimmunity to Col V in early-SSc was related to peptides of the α1(V) chain, suggesting that these antibodies could be biomarkers of SSc stages and potential target of immunotherapy with Col V immunogenic peptides.

The deleterious effects of smoking in bone mineralization and fibrillar matrix composition.

INTRODUCTION: This study aimed to verify the effects of cigarette smoke exposure in bone mineralization and fibrillar matrix composition as well as in bone healing after tibial fracture induction. METHODS: C57Bl/6 Mice were assigned according to exposure and surgery: C room air; F room air and tibia open osteotomy; CS cigarette smoke; FCS cigarette smoke and tibia open osteotomy. In order to study fracture healing we performed, under anesthesia, a bone injury through a tibial shaft osteotomy. Bone samples were obtained to evaluate bone histomorphometry, trabecular morphology and volume, trabecular collagen types composition and presence of inflammatory cytokines and growth factors. RESULTS: CS exposure significantly reduced the thickness of bone trabeculae associated with decrease in mineralizing surface and mineral deposition rate, leading a lower bone formation rate and longer mineralization time. Resorption surface and osteoclastic surface were greater in the CS group, attesting increased resorptive action. There was a decrease in type I collagen deposition and genes expression in the CS and FCS groups compared to C group and in contrast there was an increase in type V collagen deposition and genes expression in the CS, FC and FSC groups compared to C group. Also, CS exposure induced a decrease in bone forming cytokines and an increase in inflammatory associated cytokines, and these changes were intensified under fracture conditions. CONCLUSION: Cigarette smoke exposure alters bone matrix composition and worsens bone mineralization, leading to bone fragility by increasing collagen V synthesis and deposition and impairing collagen I fibril forming and assembling. And these deleterious effects contributed to the worsening in fracture healing after tibia osteotomy.

Proposition of a novel animal model of systemic sclerosis induced by type V collagen in C57BL/6 mice that reproduces fibrosis, vasculopathy and autoimmunity.

BACKGROUND: Type V collagen (Col V) has the potential to become an autoantigen and has been associated with the pathogenesis of systemic sclerosis (SSc). We characterized serological, functional, and histopathological features of the skin and lung in a novel SSc murine model induced by Col V immunization. METHODS: Female C57BL/6 mice (n = 19, IMU-COLV) were subcutaneously immunized with two doses of Col V (125 µg) emulsified in complete Freund adjuvant, followed by two intramuscular boosters. The control group (n = 19) did not receive Col V. After 120 days, we examined the respiratory mechanics, serum autoantibodies, and vascular manifestations of the mice. The skin and lung inflammatory processes and the collagen gene/protein expressions were analyzed. RESULTS: Vascular manifestations were characterized by endothelial cell activity and apoptosis, as shown by the increased expression of VEGF, endothelin-1, and caspase-3 in endothelial cells. The IMU-COLV mice presented with increased tissue elastance and a nonspecific interstitial pneumonia (NSIP) histologic pattern in the lung, combined with the thickening of the small and medium intrapulmonary arteries, increased Col V fibers, and increased COL1A1, COL1A2, COL3A1, COL5A1, and COL5A2 gene expression. The skin of the IMU-COLV mice showed thickness, epidermal rectification, decreased papillary dermis, atrophied appendages, and increased collagen, COL5A1, and COL5A2 gene expression. Anti-collagen III and IV and ANA antibodies were detected in the sera of the IMU-COLV mice. CONCLUSION: We demonstrated that cutaneous, vascular, and pulmonary remodeling are mimicked in the Col V-induced SSc mouse model, which thus represents a suitable preclinical model to study the mechanisms and therapeutic approaches for SSc.

Heart remodeling produced by aortic stenosis promotes cardiomyocyte apoptosis mediated by collagen V imbalance.

Cardiac remodeling (CR) is a structural change of the heart due to chronic hemodynamic overload related to changes in both myocyte and extracellular matrix (ECM). We investigated that the imbalance of collagen V promotes cardiomyocyte apoptosis that contributes to heart failure and cell death. Aortic stenosis was induced surgically and male Wistar rats were randomized to 18 weeks (Sham 18 w, n = 12; AoS 18 w, n = 12) and severe of heart failure (Sham HF, n = 12; AoS HF, n = 12) groups. Functional and structural echocardiogram, immunohistochemistry for Ki-67, TUNEL assay and Immunofluorescence for collagen were performed. Our main results were: (1) Progressive reduction of cardiac functional capacity due to cardiac remodeling with decreased eject fraction in heart failure; (2) Imbalance of collagen deposition with increased, crowded and irregular collagen I in situ expression; (3) Dysregulation of dynamic control of collagen fibers with exposed epitopes of collagen V; (4) Additional apoptosis that are dependent to cardiac injury. The collagen V expression in cardiac remodeling is for the first time described and may be related to additional apoptosis and autoimmune response. Our findings suggest a critical role of collagen V in cardiac remodeling to modulate and promote heart failure and death.