Deposition Of Collagen Fibrils Research Articles

Development of a Chimeric Protein BiPPB-mIFNγ-tTβRII for Improving the Anti-Fibrotic Activity in Vivo by Targeting Fibrotic Liver and Dual Inhibiting the TGF-β1/Smad Signaling Pathway.

Excessive production of transforming growth factor β1 (TGF-β1) in activated hepatic stellate cells (aHSCs) promotes liver fibrosis by activating the TGF-β1/Smad signaling pathway. Thus, specifically inhibiting the pro-fibrotic activity of TGF-β1 in aHSCs is an ideal strategy for treating liver fibrosis. Overexpression of platelet-derived growth factor β receptor (PDGFβR) has been demonstrated on the surface of aHSCs relative to normal cells in liver fibrosis. Interferon-gamma peptidomimetic (mIFNγ) and truncated TGF-β receptor type II (tTβRII) inhibit the TGF-β1/Smad signaling pathway by different mechanisms. In this study, we designed a chimeric protein by the conjugation of (1) mIFNγ and tTβRII coupled via plasma protease-cleavable linker sequences (FNPKTP) to (2) PDGFβR-recognizing peptide (BiPPB), namely BiPPB-mIFNγ-tTβRII. This novel protein BiPPB-mIFNγ-tTβRII was effectively prepared using Escherichia coli expression system. The active components BiPPB-mIFNγ and tTβRII were slowly released from BiPPB-mIFNγ-tTβRII by hydrolysis using the plasma protease thrombin in vitro. Moreover, BiPPB-mIFNγ-tTβRII highly targeted to fibrotic liver tissues, markedly ameliorated liver morphology and fibrotic responses in chronic liver fibrosis mice by both inhibiting the phosphorylation of Smad2/3 and inducing the expression of Smad7. Meanwhile, BiPPB-mIFNγ-tTβRII markedly reduced the deposition of collagen fibrils and expression of fibrosis-related proteins in acute liver fibrosis mice. Furthermore, BiPPB-mIFNγ-tTβRII showed a good safety performance in both liver fibrosis mice. Taken together, BiPPB-mIFNγ-tTβRII improved the in vivo anti-liver fibrotic activity due to its high fibrotic liver-targeting potential and the dual inhibition of the TGF-β1/Smad signaling pathway, which may be a potential candidate for targeting therapy on liver fibrosis.

#3553 TUBULE CELL-DERIVED ANGIOPOIETIN-2 EXACERBATES INFLAMMATION AND IS DETRIMENTAL TO RENAL FIBROSIS

Abstract Background and Aims Our previous studies have shown that angiogenic growth factors are dysregulated in fibrotic kidneys. In murine models of progressive renal disease, angiopoietin-2 (Angpt2) was upregulated in injured tubule epithelial cells. We hypothesize that overexpression of Angpt2 in tubule epithelial cells would exacerbate renal fibrosis and inflammation. Method The unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion injury (UIRI) are used as the murine model of chronic kidney disease. We use the tetracycline/doxycycline-controlled Tet-on system to induce tubule cell-specific overexpression of Angpt2 before the induction of renal fibrosis. Transgenic mice carrying both Pax8-rtTA and pTetos-hAngpt2 genes (double transgenic mice) are used for Angpt2 overexpression. Littermates that inherit single or no transgene serve as experimental controls. Doxycycline is administered to adult (8 weeks) mice to overexpress Angpt2 in tubule epithelial cells. Results Doxycycline administration induce overexpression of human Anpgt2 in the tubule epithelial cells of both sham and fibrotic kidneys. Compared to littermate controls after the UUO and UIRI surgery, the expression of fibrosis-related genes, Col1a1, Col3a1, and Acta2 are higher in the kidneys of double transgenic mice. Deposition of picrosirius red-stained collagen fibrils is significantly higher in double transgenic mice. Inflammatory cell infiltration is also significantly higher in double transgenic mice. Conclusion Overall, these findings suggest that increased Angpt2 in tubule epithelial cells deteriorate renal fibrosis through detrimental inflammation. Further studies are necessary to explore the participating cellular components and pathogenic mechanisms.

Effects of microRNA-21 on endothelial-to-mesenchymal transition and its role in the pathogenesis of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airflow restriction. This study aims to explore whether there is endothelial-to-mesenchymal transition (EndMT) in COPD mice and to explore the relationship between microRNA-21 (miR-21) and EndMT. We established the COPD and the miR-21 gene knockout COPD animal model (both cigarette smoke-induced). Mice were divided into 3 groups (n=4): a control group, a COPD group, and a miR-21 knockout COPD (miR-21-/--COPD) group. Masson trichrome staining was used to observe the deposition of collagen around the perivascular. The relative protein levels and positions of endothelial cell markers including vascular endothelial-cadherin (VE-cadherin), endothelial nitric oxide synthase (eNOS), and platelet endothelial cell adhesion molecule-1 (CD31) as well as mesenchymal cell markers including α-smooth muscle actin (α-SMA) and neural cadherin (N-cadherin) in lung tissues were observed by immunohistochemical staining. Compared with the control group, the area of collagen fibril deposition was increased in the COPD group (P<0.05), the expression levels of VE-cadherin, eNOS, and CD31 were all decreased (all P<0.05), and the expression levels of α-SMA and N-cadherin were increased (both P<0.05). Compared with the COPD group, the miR-21-/--COPD group had a reduced area of collagen fiber deposition (P<0.05), the expression levels of VE-cadherin, eNOS, and CD31 were all increased (all P<0.05), and the expression levels of α-SMA and N-cadherin were decreased (both P<0.05). There is a EndMT process in cigarette smoke-induced COPD animal models.MiR-21 gene knockdown could reduce collagen deposition area and inhibit the EndMT process in COPD mice.

Expression of procollagen C-proteinase enhancer 1 in human trabecular meshwork tissues and cells.

Glaucoma is a primary cause of progressive, irreversible blindness. One of the primary tissues involved in glaucoma pathology is the trabecular meshwork (TM). In glaucoma, the TM is a site of increased extracellular matrix (ECM) protein secretion, deposition, and accumulation, contributing to a disrupted TM architecture and increased resistance to the outflow of aqueous humor. The healthy TM structure is comprised of sheets and beams composed of multiple extracellular matrix proteins, including mature fibrillar collagens. In the glaucomatous eye, this structure is disrupted by the abnormal deposition of collagen fibrils and other ECM proteins in the TM. In this study, we determined whether procollagen C-proteinase enhancer 1 (PCOLCE1) - a protein typically involved in collagen fibril processing - is expressed in the human TM tissues and cells and whether its expression is altered in glaucomatous conditions. Using immunocytochemistry, qPCR, and western blot (WB) analyses, we found that PCOLCE1 is expressed and translated in human TM tissues and cells. Our data analysis suggests that PCOLCE1 expression by TM cells may be downregulated by TGFβ2 treatment, which warrants further investigation of a possible role for PCOLCE1 in glaucomatous pathology.

NRF2 Shortage in Human Skin Fibroblasts Dysregulates Matrisome Gene Expression and Affects Collagen Fibrillogenesis

NRF2 is a master regulator of the antioxidative response that was recently proposed as a potential regulator of extracellular matrix (ECM) gene expression. Fibroblasts are major ECM producers in all connective tissues, including the dermis. A better understanding of NRF2-mediated ECM regulation in skin fibroblasts is thus of great interest for skin homeostasis maintenance and aging protection. In this study, we investigate the impact of NRF2 downregulation on matrisome gene expression and ECM deposits in human primary dermal fibroblasts. RNA-sequencing‒based transcriptome analysis of NRF2 silenced dermal fibroblasts shows that ECM genes are the most regulated gene sets, highlighting the relevance of the NRF2-mediated matrisome program in these cells. Using complementary light and electron microscopy methods, we show that NRF2 deprivation in dermal fibroblasts results in reduced collagen I biosynthesis and impacts collagen fibril deposition. Moreover, we identify ZNF469, a putative transcriptional regulator of collagen biosynthesis, as a target of NRF2. Both ZNF469 silenced fibroblasts and fibroblasts derived from Brittle Corneal Syndrome patients carrying variants in ZNF469 gene show reduced collagen I gene expression. Our study shows that NRF2 orchestrates matrisome expression in human skin fibroblasts through direct or indirect transcriptional mechanisms that could be prioritized to target dermal ECM homeostasis in health and disease.

A cyclodextrin-based macrocyclic oligosaccharide cavitand with a dual functionality limits the collagen fibrillogenesis: A possible carbohydrate-based therapeutic molecule for fibrotic diseases

β-Cyclodextrin (β-CD), a macrocyclic oligosaccharide cavitand, is a well-known candidate for drug delivery and formulation. In this study, we extended the application of β-CD using a β-cyclodextrin sulfate (β-CDS) as a possible therapeutic for fibrotic diseases caused by excess deposition of collagen fibrils. We have strategically chosen β-CDS, which mimics the natural existence of dermatan sulfate in the extracellular matrix, for limiting collagen fibrillation. The hydrophobic nature of the inner core β-CDS is expected to form an inclusion complex with hydrophobic side chain amino acids with the simultaneous action of forming an ionic bond through a negative charge on sulfate group with positively charged amino acids side chain in collagen. Various results suggested that such dual action not only limited the collagen fibrillation but also reduced the fibril size formed in the presence of β-CDS. The contemporary results thus indicate that β-CDS can be explored as a therapeutic molecule in fibrotic diseases.

Additional Value of Second Harmonic Generation Microscopy in the Diagnosis of Feline Collagen Type III Glomerulopathy

A 1.5-year-old neutered female Domestic Shorthair cat was euthanized after the diagnosis of end-stage protein-losing nephropathy associated with the onset of nephrotic syndrome. At necropsy, both kidneys were diffusely pale and swollen with a granular cortex. Histologically, glomeruli had diffuse global mesangial and capillary wall expansion by homogeneous pale eosinophilic material. This material was Congo red negative, blue with Masson's trichrome stain, weakly positive with periodic acid-Schiff stain, bright red with Picrosirius red and birefringent under polarized light. Transmission electron microscopy and second harmonic generation (SHG) microscopy revealed mesangial and subendothelial collagen fibril deposition. Type III collagen deposition was confirmed by immunohistochemistry. This study provides an original and complete description of feline collagen type III glomerulopathy and emphasizes the possibility of directly diagnosing glomerular collagen deposition on unstained slides through SHG microscopy.

Abstract MP35: Single-cell Analysis Uncovers Multiple Adventitial Fibroblast Populations With Expression Of CAD GWAS Genes In Murine Atherosclerosis

The arterial adventitia is a layer of cells outside the external lamina of blood vessels that contribute to the progression of coronary artery disease (CAD) like atherosclerosis. Adventitial fibroblasts are known regulators of vascular remodeling through their deposition of collagen fibrils around vessels during atherosclerotic lesion development. However, the molecular identity and function of the participating fibroblasts have not been well studied. We used single cell RNA sequencing (scRNA-seq) to characterize the transcriptome of adventitial fibroblasts and other cell types at multiple timepoints during atherogenesis in hyperlipidemic Ldlr knockout mice. Specifically, scRNA-seq was applied to Ldlr KO aortic adventitia after 0, 9, and 16 weeks of western diet (WD) feeding. Unbiased clustering analysis uncovered 9 different cell clusters, including 5 different fibroblast populations (Fibro1-5), and a single cluster each of macrophages, T cells, SMCs, and ECs. We observed that, as atherosclerosis progressed from 9 to 16 weeks of feedings, the proportion of total cells that were Fibro1, 3, and 4 decreased (33, 40, and 13%), while populations of Fibro2 and 5 slightly increased (13 and 18%). All of non-fibroblast populations (Macrophage, T cells, SMCs and ECs) increased from 9-16wks of diet. Next, we asked whether genes associated with coronary artery disease by human genome-wide association studies (GWAS) are expressed in different adventitial fibroblast clusters. We determined the expression of 266 genes implicated in CAD by GWAS and found 201 genes (76%) were expressed in adventitial cells at either/both 9 or 16wks. 34 genes displayed large changes in expression from 9 to 16wks. We identified five genes that were highly expressed in the adventitial Fibro5 cluster specifically, and additional other CAD GWAS genes specifically expressed in adventitial macrophages, SMCs, and ECs. In summary, we present here a cell atlas defined by scRNA-seq that reveals the heterogeneity amongst the fibroblasts in adventitial cells during atherogenesis. Additionally, we find that many CAD GWAS genes are expressed in adventitial cell populations, raising the intriguing possibility that CAD GWAS are identifying adventitial mechanisms that contribute to CAD.

Verbascoside alleviates renal fibrosis in unilateral ureteral obstruction rats by inhibiting macrophage infiltration.

Objective(s): To explore the effect of verbascoside on renal fibrosis in unilateral ureteral obstruction (UUO) rats.Materials and Methods:Twenty Sprague-Dawley rats were randomly distributed into sham-operated, UUO, and UUO+Verbascoside groups. After two weeks of rat model construction, urine and blood samples were collected for biochemical analysis while kidney tissues were harvested for hematoxylin and eosin (H&E), Masson’s Trichrome, and immunohistochemistry staining. Pearson coefficient was used to analyze the correlation between the two proteins. Results:Verbascoside improved UUO-induced renal dysfunction as detected by decreased serum creatinine, urea nitrogen, and urinary protein excretion rate. In UUO rats, H&E staining result revealed increased total nucleated cell number, and Masson’s Trichrome staining results showed tubular interstitial fibrosis with the deposition of collagen fibrils. Besides, expressions of fibrosis-related proteins including collagen type I (COL-I), α-smooth muscle actin (a-SMA), and tissue inhibitor of metalloproteinase 2 (TIMP2) expressed higher in the UUO group. Moreover, macrophage infiltration-related factors such as CD68+, F4/80+ cells, and suppressor of cytokine signaling-3 (SOCS3) were significantly higher in the UUO group than in sham-operated rats. However, after administration with verbascoside, the accumulation of collagen fibrils and total nucleated cell numbers were mitigated. Likewise, macrophage infiltration was extenuated and fibrosis-related proteins were down-regulated in the UUO+Verbascoside rats. Correlation analysis indicated that macrophage infiltration-related markers were related to fibrosis-related factors.Conclusion:Verbascoside could alleviate renal fibrosis in UUO rats probably through ameliorating macrophage infiltration.

Pharmacokinetics-based comprehensive strategy to identify multiple effective components in Huangqi decoction against liver fibrosis

BackgroundHuangqi decoction (HQD) has been used to treat chronic liver diseases since the 11th century, but the effective components in HQD against liver fibrosis have not been definitively clarified. PurposeTo investigate and identify multiple effective components in HQD against liver fibrosis using a pharmacokinetics-based comprehensive strategy. MethodsThe absorbed representative components in HQD and their metabolites were detected in human plasma and urine using high-resolution mass spectrometry combined with a database-directed method, and then pharmacokinetics in multiple HQD components in human plasma was analyzed by ultra-performance liquid chromatography coupled with triple-quadruple mass spectrometry. Furthermore, the anti-fibrotic effect of potential effective HQD components was studied in LX-2 cells and that of a multi-component combination of HQD (MCHD) was verified in a mouse CCl4-induced hepatic fibrosis model. ResultsTwenty-four prototype components in HQD and 17 metabolites were identified in humans, and the pharmacokinetic characteristics of 14 components were elucidated. Among these components, astragaloside IV, cycloastragenol, glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin, and isoliquiritigenin downregulated the mRNA expression of α-SMA; cycloastragenol, calycosin-7-O-β-D-glucoside, formononetin, glycyrrhetinic acid, liquiritin, and isoliquiritin downregulated the mRNA expression of Col I; and calycosin, liquiritigenin, isoliquiritigenin, cycloastragenol, and glycyrrhetinic accelerated the apoptosis of LX-2 cells. MCHD reduced serum aminotransferase activity and hepatic collagen fibril deposition in mice with CCl4-induced hepatic fibrosis. ConclusionUsing the pharmacokinetics-based comprehensive strategy, we revealed that multiple effective HQD components act together against liver fibrosis.

Histone deacetylase 6 inhibition mitigates renal fibrosis by suppressing TGF-β and EGFR signaling pathways in obstructive nephropathy.

We have recently shown that histone deacetylase 6 (HDAC6) is critically involved in the pathogenesis of acute kidney injury. Its role in renal fibrosis, however, remains unclear. In this study, we examined the effect of ricolinostat (ACY-1215), a selective inhibitor of HDAC6, on the development of renal fibrosis in a murine model induced by unilateral ureteral obstruction (UUO). HDAC6 was highly expressed in the kidney following UUO injury, which was coincident with deposition of collagen fibrils and expression of α-smooth muscle actin, fibronectin, and collagen type III. Administration of ACY-1215 reduced these fibrotic changes and inhibited UUO-induced expression of transforming growth factor-β1 and phosphorylation of Smad3 while increasing expression of Smad7. ACY-1215 treatment also suppressed phosphorylation of epidermal growth factor receptor (EGFR) and several signaling molecules associated with renal fibrogenesis, including AKT, STAT3, and NF-κB in the injured kidney. Furthermore, ACY-1215 was effective in inhibiting dedifferentiation of renal fibroblasts to myofibroblasts and the fibrotic change of renal tubular epithelial cells in culture. Collectively, these results indicate that HDAC6 inhibition can attenuate development of renal fibrosis by suppression of transforming growth factor-β1 and EGFR signaling and suggest that HDAC6 would be a potential therapeutic target for the treatment of renal fibrosis.

Clinical, Serological, and Histopathological Similarities Between Severe COVID-19 and Acute Exacerbation of Connective Tissue Disease-Associated Interstitial Lung Disease (CTD-ILD).

Background and ObjectivesUnderstanding the pathophysiology of respiratory failure in coronavirus disease 2019 (COVID-19) is indispensable for development of therapeutic strategies. Since we observed similarities between COVID-19 and interstitial lung disease in connective tissue disease (CTD-ILD), we investigated features of autoimmunity in SARS-CoV-2-associated respiratory failure.MethodsWe prospectively enrolled 22 patients with RT-PCR-confirmed SARS-CoV-2 infection and 10 patients with non-COVID-19-associated pneumonia. Full laboratory testing was performed including autoantibody (AAB; ANA/ENA) screening using indirect immunofluorescence and immunoblot. Fifteen COVID-19 patients underwent high-resolution computed tomography. Transbronchial biopsies/autopsy tissue samples for histopathology and ultrastructural analyses were obtained from 4/3 cases, respectively.ResultsThirteen (59.1%) patients developed acute respiratory distress syndrome (ARDS), and five patients (22.7%) died from the disease. ANA titers ≥1:320 and/or positive ENA immunoblots were detected in 11/13 (84.6%) COVID-19 patients with ARDS, in 1/9 (11.1%) COVID-19 patients without ARDS (p = 0.002) and in 4/10 (40%) patients with non-COVID-19-associated pneumonias (p = 0.039). Detection of AABs was significantly associated with a need for intensive care treatment (83.3 vs. 10%; p = 0.002) and occurrence of severe complications (75 vs. 20%, p = 0.03). Radiological and histopathological findings were highly heterogeneous including patterns reminiscent of exacerbating CTD-ILD, while ultrastructural analyses revealed interstitial thickening, fibroblast activation, and deposition of collagen fibrils.ConclusionsWe are the first to report overlapping clinical, serological, and imaging features between severe COVID-19 and acute exacerbation of CTD-ILD. Our findings indicate that autoimmune mechanisms determine both clinical course and long-term sequelae after SARS-CoV-2 infection, and the presence of autoantibodies might predict adverse clinical course in COVID-19 patients.

Bone Morphogenetic Protein-9 Is a Potent Chondrogenic and Morphogenic Factor for Articular Cartilage Chondroprogenitors.

Articular cartilage contains a subpopulation of tissue-specific progenitors that are an ideal cell type for cell therapies and generating neocartilage for tissue engineering applications. However, it is unclear whether the standard chondrogenic medium using transforming growth factor beta (TGFβ) isoforms is optimal to differentiate these cells. We therefore used pellet culture to screen progenitors from immature bovine articular cartilage with a number of chondrogenic factors and discovered that bone morphogenetic protein-9 (BMP9) precociously induces their differentiation. This difference was apparent with toluidine blue staining and confirmed by biochemical and transcriptional analyses with BMP9-treated progenitors exhibiting 11-fold and 5-fold greater aggrecan and collagen type II (COL2A1) gene expression than TGFβ1-treated progenitors. Quantitative gene expression analysis over 14 days highlighted the rapid and phased nature of BMP9-induced chondrogenesis with sequential activation of aggrecan then collagen type II, and negligible collagen type X gene expression. The extracellular matrix of TGFβ1-treated progenitors analyzed using atomic force microscopy was fibrillar and stiff whist BMP9-induced matrix of cells more compliant and correspondingly less fibrillar. Polarized light microscopy revealed an annular pattern of collagen fibril deposition typified by TGFβ1-treated pellets, whereas BMP9-treated pellets displayed a birefringence pattern that was more anisotropic. Remarkably, differentiated immature chondrocytes incubated as high-density cultures in vitro with BMP9 generated a pronounced anisotropic organization of collagen fibrils indistinguishable from mature adult articular cartilage, with cells in deeper zones arranged in columnar manner. This contrasted with cells grown with TGFβ1, where a concentric pattern of collagen fibrils was visualized within tissue pellets. In summary, BMP9 is a potent chondrogenic factor for articular cartilage progenitors and is also capable of inducing morphogenesis of adult-like cartilage, a highly desirable attribute for in vitro tissue-engineered cartilage.

Hedgehog signaling pathway regulates hexavalent chromium-induced liver fibrosis by activation of hepatic stellate cells

With the spread of hexavalent chromium [Cr(VI)] contamination, risk of exposure in non-occupational populations is increasing. The liver is the main target organ for Cr(VI) accumulation; however, the effect of long-term Cr(VI) exposure on liver toxicity is largely unknown. In this study, we investigated the effect of chronic Cr(VI) exposure on liver fibrosis and its possible mechanism. Mice were injected with Cr(VI) for two months, and our results showed Cr(VI) treatment caused liver toxicity characterized by liver structure disorganization, liver dysfunction, and antioxidant enzyme system inhibition. The development of liver fibrosis was also found via the emergence of collagen fibril deposition, increased expression of extracellular matrix-related genes, activation of hepatic stellate cells (HSCs) and increase the expression levels of Hedgehog (Hh) signaling pathway-related molecules. To demonstrate the role of Hh signaling in the regulation of Cr(VI)-induced liver fibrosis, genetically modified mice with heterozygous deficiency of Shh (Shh+/−) were used. In the Shh+/− mice, Hh signaling, HSCs activation and liver fibrosis development were all ameliorated. In conclusion, we demonstrated that Cr(VI)-induced liver fibrosis development resulted from Hh pathway-mediated HSCs activation. Our findings strongly suggest that inhibition of Hh pathway may help in the development of new strategies for Cr(VI)-associated liver fibrosis.

Surfactant dysfunction and alveolar collapse are linked with fibrotic septal wall remodeling in the TGF-β1-induced mouse model of pulmonary fibrosis

In human idiopathic pulmonary fibrosis (IPF), collapse of distal airspaces occurs in areas of the lung not (yet) remodeled. Mice lungs overexpressing transforming growth factor-β1 (TGF-β1) recapitulate this abnormality: surfactant dysfunction results in alveolar collapse preceding fibrosis and loss of alveolar epithelial type II (AE2) cells' apical membrane surface area. Here we examined whether surfactant dysfunction-related alveolar collapse due to TGF-β1 overexpression is linked to septal wall remodeling and AE2 cell abnormalities. Three and 6 days after gene transfer of TGF-β1, mice received either intratracheal surfactant (Surf-groups: Curosurf®, 100 mg/kg bodyweight) or 0.9% NaCl (Saline-groups). On days 7 (D7) and 14 (D14), lung mechanics were assessed followed by design-based stereology at light and electron microscopic level to quantify structures. Compared with Saline, Surf showed significantly improved tissue elastance, increased numbers of open alveoli, as well as reduced alveolar size heterogeneity on D7. Deterioration in lung mechanics was highly correlated to the loss of open alveoli. On D14, lung mechanics, number of open alveoli, and alveolar size heterogeneity remained significantly improved in the Surf-group. Volumes of extracellular matrix and collagen fibrils in septal walls were significantly reduced, whereas the apical membrane surface area of AE2 cells was increased in Surf compared with Saline. In remodeled tissue with collapsed alveoli, three-dimensional reconstruction of AE2 cells based on scanning electron microscopy array tomography revealed that AE2 cells were trapped without contact to airspaces in the TGF-β1 mouse model. Similar observations were made in human IPF. Based on correlation analyses, the number of open alveoli and of alveolar size heterogeneity were highly linked with the loss of apical membrane surface area of AE2 cells and deposition of collagen fibrils in septal walls on D14. In conclusion, surfactant replacement therapy stabilizes alveoli and prevents extracellular matrix deposition in septal walls in the TGF-β1 model.

Curcumin ameliorates glyoxylate-induced calcium oxalate deposition and renal injuries in mice

BackgroundNephrolithiasis is one of the most common and frequent urologic diseases worldwide. Several pathophysiological mechanisms are involved in stone formation, including oxidative stress, inflammation, apoptosis, fibrosis and autophagy. Curcumin, the predominant active component of turmeric, has been shown to have pleiotropic biological and pharmacological properties, such as antioxidant, anti-inflammatory and antifibrotic effects. PurposeThe current study proposed to systematically investigate the protective effects and the underlying mechanisms of curcumin in a calcium oxalate (CaOx) nephrolithiasis mouse model. MethodsThe animal model was established in male C57BL/6 mice by successive intraperitoneal injection of glyoxylate (100 mg/kg) for 1 week. Curcumin was orally given to mice 7 days before the injection of glyoxylate and for a total of 14 days at 50 mg/kg or 100 mg/kg. Bilateral renal tissue was harvested and processed for oxidative stress index detection, histopathological examinations and other analyses. ResultsCoadministration of curcumin could significantly reduce glyoxylate-induced CaOx deposition and simultaneous tissue injury in mouse kidneys. Meanwhile, curcumin alleviated the oxidative stress response via reducing MDA content and increasing SOD, CAT, GPx, GR and GSH levels in this animal model. Moreover, treatment with curcumin significantly inhibited apoptosis and autophagy induced by hyperoxaluria. Curcumin also attenuated the high expression of IL-6, MCP-1, OPN, CD44, α-SMA, Collagen I and collagen fibril deposition, which were elevated by hyperoxaluria. Furthermore, the results revealed that both the total expression and nuclear accumulation of Nrf2, as well as its main downstream products such as HO-1, NQO1 and UGT, were decreased in the kidneys of mice in the crystal group, while treatment with curcumin could rescue this deterioration. ConclusionCurcumin could significantly alleviate CaOx crystal deposition in the mouse kidney and the concurrent renal tissue injury. The underlying mechanism involved the combination of antioxidant, anti-apoptotic, inhibiting autophagy, anti-inflammatory, and antifibrotic activity and the ability to decrease expression of OPN and CD44 through the Nrf2 signaling pathway. The pleiotropic antilithic properties, combined with the minimal side effects, make curcumin a good potential choice to prevent and treat new or recurrent nephrolithiasis.

Cord Blood-Derived Stem Cells Suppress Fibrosis and May Prevent Malignant Progression in Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by mutations in the Col7a1 gene. Patients with RDEB suffer from recurrent erosions in skin and mucous membranes and have a high risk for developing cutaneous squamous cell carcinoma (cSCCs). TGFβ signaling has been associated with fibrosis and malignancy in RDEB. In this study, the activation of TGFβ signaling was demonstrated in col7a1-/- mice as early as a week after birth starting in the interdigital folds of the paws, accompanied by increased deposition of collagen fibrils and elevated dermal expression of matrix metalloproteinase (MMP)-9 and MMP-13. Furthermore, human cord blood-derived unrestricted somatic stem cells (USSCs) that we previously demonstrated to significantly improve wound healing and prolong the survival of col7a1-/- mice showed the ability to suppress TGFβ signaling and MMP-9 and MMP-13 expression meanwhile upregulating anti-fibrotic TGFβ3 and decorin. In parallel, we cocultured USSCs in a transwell with RDEB patient-derived fibroblasts, keratinocytes, and cSCC, respectively. The patient-derived cells were constitutively active for STAT, but not TGFβ signaling. Moreover, the levels of MMP-9 and MMP-13 were significantly elevated in the patient derived-keratinocytes and cSCCs. Although USSC coculture did not inhibit STAT signaling, it significantly suppressed the secretion of MMP-9 and MMP-13, and interferon (IFN)-γ from RDEB patient-derived cells. Since epithelial expression of these MMPs is a biomarker of malignant transformation and correlates with the degree of tumor invasion, these results suggest a potential role for USSCs in mitigating epithelial malignancy, in addition to their anti-inflammatory and anti-fibrotic functions. Stem Cells 2018;36:1839-12.

Gastrointestinal Involvement in Systemic Sclerosis: An Update.

Gastrointestinal Involvement in Systemic Sclerosis: An Update.

Lysolecithin as feed additive enhances collagen expression and villus length in the jejunum of broiler chickens

Adding lysolecithin to feed has reportedly improved the performance of broiler chickens. Lysolecithin is generated by phospholipase catalyzed hydrolysis of lecithin. The enzymatic reaction converts various phospholipids into the corresponding lysophospholipids, with lysophosphatidylcholine (LPC) one of the primary products. Here we compared supplementation with a commercial lysolecithin (Lysoforte®) with comparable levels of highly purified LPC for effects on broilers. Despite no differences in weight gain during the starter period, we discovered a significant increase in average villus length with lysolecithin and an increase in villus width with purified LPC. High-throughput gene expression microarray analyses revealed many more genes were regulated in the epithelium of the jejunum by lysolecithin compared to purified LPC. The most up-regulated genes and pathways were for collagen, extracellular matrix, and integrins. Staining sections of the jejunum with Picrosirius Red confirmed the increased deposition of collagen fibrils in the villi of broilers fed lysolecithin, but not purified LPC. Thus, lysolecithin elicits gene expression in the intestinal epithelium, leading to enhanced collagen deposition and villus length. Purified LPC alone as a supplement does not mimic these responses. Feed supplementation with lysolecithin triggers changes in the intestinal epithelium with the potential to improve overall gut health and performance.

Abstract 13854: Micro-RNA Regulation of Collagen Production by Vascular Smooth Muscle Cells Mediated by La Ribonucleoprotein Domain Family Member 6: Potential Mechanisms Underlying Stable Phenotype of Atherosclerotic Plaque by Insulin-like Growth Factor 1

Collagen is a major constituent of the extracellular matrix in atherosclerotic plaques and supports plaque stability. La ribonucleoprotein domain family member 6 (LARP6) is an RNA binding protein, which assists translation of fibrillar collagen, thereby promoting mature collagen deposition. In Apoe-null mice, insulin-like growth factor 1 (IGF1) promoted stable features of plaques by increasing smooth muscle cell (SMC) numbers and mature collagen fibril deposition without increasing plaque burden. To study mechanisms underlying the IGF1’s plaque stabilizing effects, we exposed human aortic SMCs to proatherogenic oxidized LDL (oxLDL) or IGF1 and determined collagen expression levels. OxLDL downregulated collagen (I) by 36.0±3.2% (P&lt;0.05), along with LARP6 (40.9±10.4 % decrease, P&lt;0.05) and procollagen (I) (49.0±8.9 % decrease, P&lt;0.05), which are consistent with decreased translation of procollagen (I) mRNA. IGF1 abolished oxLDL effects, reversing LARP6 and procollagen (I) protein levels, and thus increased collagen (I) to the levels comparable with native LDL-treated control. As we found previously, the IGF1 upregulation of collagen (I) was mediated by enhanced expression of LARP6, however IGF1 did not alter LARP6 mRNA levels, suggesting translational/post-translational mechanisms. Micro RNA regulates translation of mRNAs; in fact, miR-126, miR-744, and miR-1976 target LARP6 mRNA. In human aortic SMCs, oxLDL upregulated miR-1976 by 40.4±0.5 %, implying miR-mediated suppression of LARP6 translation. Intriguingly, IGF1 abrogated the oxLDL upregulation of miR-1976; moreover, IGF1 decreased miR-126 (by 25.0±1.0 %, P&lt;0.05) and miR-744 (by 24.0±0.4 %, P&lt;0.01), further suggesting that downregulation of miRs mediated IGF1 upregulation of LARP6. To evaluate LARP6 expression in human atherosclerotic plaques, atherosclerotic carotid artery sections were stained for LARP6, and it was detected mainly in SMCs in the medial layer and in the plaque fibrous cap, where collagen fibrils are integral components supporting plaque stability. Our findings suggest a novel mechanism of collagen accumulation by miR-dependent LARP6 expression, and provide a basis for a novel therapeutic approach to enhance plaque stability by targeting miR.