COL1A1 Research Articles

P029 Histopathological characterization of a chronic DSS-induced mouse model of IBD with intestinal fibrosis

Abstract Background Inflammatory bowel disease (IBD) comprises a group of intestinal disorders, including ulcerative colitis and Crohn's disease. Intestinal fibrosis, as result of chronic inflammation, is a common complication in IBD. High treatment failure rates associated with existing interventions indicate a high unmet need for more effective drugs to improve the management and outcomes of IBD. Consequently, translational animal models of IBD demonstrating chronic, progressive colonic fibrosis are important tools in preclinical drug discovery for IBD. The aim of the present study was to characterize intestinal histopathology in a chronic DSS-induced mouse model of IBD. Methods Ten-weeks-old male C57BL/6JRj mice were randomized into study groups based on body weight and received 3 cycles of DSS (2.5 % w/v) in the drinking water (DSS-IBD) or normal water (CTRL). Terminal endpoints included colon morphometry and quantitative histological markers of inflammation and fibrosis assessed in proximal, middle and distal colonic segments. Using RNA sequencing, transcriptome signatures were profiled in the middle colon segment. Results Compared to healthy controls, the chronic DSS-IBD mouse model demonstrated hallmarks of IBD, including mild-to-moderate weight loss and colonic hypertrophy. Histopathological analysis indicated increased deposition of inflammatory (CD45 and CD11b), fibrogenesis (alpha-smooth muscle actin) and fibrosis markers (collagen 1a1, Picrosirius red) in the middle and distal colon. Severe fibrosis was observed throughout all layers of the middle colon. Histological findings were supported by significantly upregulated gene expression markers of inflammation and fibrosis. Conclusion The DSS-IBD mouse is a translational preclinical model suitable for testing novel drug therapies for IBD with intestinal fibrosis.

Potential role of endoplasmic reticulum stress in broiler woody breast myopathy.

Although broiler (meat-type) chickens are one of the most efficient protein sources that supports the livelihoods and food security of billions of people worldwide, they are facing several challenges. Due to its unknown etiology and heavy economic impact, woody breast (WB) myopathy is one of the most challenging problems facing the poultry industry, and for which there is no effective solution. Here, using a primary chicken myotube culture model, we show that hypoxia and endoplasmic reticulum (ER) stress are an integral component of the etiology of the myopathy. Multiple components of the ER stress response are significantly upregulated in WB as compared with normal muscle, and this response was mimicked by hypoxic conditions in chicken primary myotube culture. In addition, apoptotic pathways were activated as indicated by increases in active caspase 3 protein levels in both WB-affected tissues and hypoxic myotube culture, and caspase 3 activity and apoptosis in hypoxic myotube culture. Finally, as a phenotypic hallmark of WB is enhanced fibrosis and increased collagen aggregation, here, we show that hypoxic conditions increase collagen 1A1 and 1A2 gene expression, as well as collagen 1 protein levels in primary myotubes. These effects were partially reversed by tauroursodeoxycholic acid (TUDCA), an ER-stress inhibitor, in myotube culture. Taken together, these findings indicate that hypoxia and ER stress are present in WB, hypoxia can upregulate the cell death arm of the unfolded protein response (UPR) and lead to collagen production in a culture model of WB. This opens new vistas for potential mechanistic targets for future effective interventions to mitigate this myopathy.

HISTOPATHOLOGICAL CHARACTERIZATION OF A CHRONIC DSS-INDUCED MOUSE MODEL OF IBD WITH INTESTINAL FIBROSIS

Abstract RATIONALE Inflammatory bowel disease (IBD) comprises a group of intestinal disorders, including ulcerative colitis and Crohn's disease. Intestinal fibrosis, as result of chronic inflammation, is a common complication in IBD. High treatment failure rates associated with existing interventions indicate a high unmet need for more effective drugs to improve the management and outcomes of IBD. Consequently, translational animal models of IBD demonstrating chronic, progressive colonic fibrosis are important tools in preclinical drug discovery for IBD. The aim of the present study was to characterize intestinal histopathology in a chronic DSS-induced mouse model of IBD. METHODS Ten-weeks-old male C57BL/6JRj mice were randomized into study groups based on body weight and received 3 cycles of DSS (2.5 % w/v) in the drinking water (DSS-IBD) or normal water (CTRL). Terminal endpoints included colon morphometry and quantitative histological markers of inflammation and fibrosis assessed in proximal, middle and distal colonic segments. Using RNA sequencing, transcriptome signatures were profiled in the middle colon segment. RESULTS Compared to healthy controls, the chronic DSS-IBD mouse model demonstrated hallmarks of IBD, including mild-to-moderate weight loss and colonic hypertrophy. Histopathological analysis indicated increased deposition of inflammatory (CD45 and CD11b), fibrogenesis (alpha-smooth muscle actin) and fibrosis markers (collagen 1a1, Picrosirius red) in the middle and distal colon. Severe fibrosis was observed throughout all layers of the middle colon. Histological findings were supported by significantly upregulated gene expression markers of inflammation and fibrosis. CONCLUSIONS The DSS-IBD mouse is a translational preclinical model suitable for testing novel drug therapies for IBD with intestinal fibrosis.

Temporal changes of genes associated with intestinal homeostasis in broiler chickens following a single infection with Eimeria acervulina

Infection with the protozoan parasite Eimeria can cause the economically devastating disease coccidiosis, which is characterized by gross tissue damage and inflammation resulting in blunted villi and altered intestinal homeostasis. Male broiler chickens at 21 d of age were given a single challenge with Eimeria acervulina. Temporal changes in intestinal morphology and gene expression were investigated at 0, 3, 5, 7, 10, and 14 d postinfection (dpi). There were increased crypt depths for chickens infected with E. acervulina starting at 3 dpi and continuing to 14 dpi. At 5 and 7 dpi, infected chickens had decreased Mucin2 (Muc2), and Avian beta defensin (AvBD) 6 mRNA at 5 and 7 dpi and decreased AvBD10 mRNA at 7 dpi compared to uninfected chickens. Liver-enriched antimicrobial peptide 2 (LEAP2) mRNA was decreased at 3, 5, 7, and 14 dpi compared to uninfected chickens. After 7 dpi, there was increased Collagen 3a1 and Notch 1 mRNA compared to uninfected chickens. Marker of proliferation Ki67 mRNA was increased in infected chickens from 3 to 10 dpi. In addition, the presence of E. acervulina was visualized by in situ hybridization (ISH) with an E. acervulina sporozoite surface antigen (Ea-SAG) probe. In E. acervulina infected chickens, Ea-SAG mRNA was only detectable on 5 and 7 dpi by both ISH and qPCR. To further investigate the site of E. acervulina infection, Ea-SAG and Muc2 probes were examined on serial sections. The Muc2 ISH signal was decreased in regions where the Ea-SAG ISH signal was present, suggesting that the decrease in Muc2 by qPCR may be caused by the loss of Muc2 in the localized regions where the E. acervulina had invaded the tissue. Eimeria acervulina appears to manipulate host cells by decreasing their defensive capabilities and thereby allows the infection to propagate freely. Following infection, the intestinal cells upregulate genes that may support regeneration of damaged intestinal tissue.

TRPA1 as a potential factor and drug target in scleroderma: dermal fibrosis and alternative macrophage activation are attenuated in TRPA1-deficient mice in bleomycin-induced experimental model of scleroderma

BackgroundSystemic sclerosis is a rheumatoid disease best known for its fibrotic skin manifestations called scleroderma. Alternatively activated (M2-type) macrophages are normally involved in the resolution of inflammation and wound healing but also in fibrosing diseases such as scleroderma. TRPA1 is a non-selective cation channel, activation of which causes pain and neurogenic inflammation. In the present study, we investigated the role of TRPA1 in bleomycin-induced skin fibrosis mimicking scleroderma.MethodsWild type and TRPA1-deficient mice were challenged with intradermal bleomycin injections to induce a scleroderma-mimicking disease. Macrophages were investigated in vitro to evaluate the underlying mechanisms.ResultsBleomycin induced dermal thickening and collagen accumulation in wild type mice and that was significantly attenuated in TRPA1-deficient animals. Accordingly, the expression of collagens 1A1, 1A2, and 3A1 as well as pro-fibrotic factors TGF-beta, CTGF, fibronectin-1 and YKL-40, and M2 macrophage markers Arg1 and MRC1 were lower in TRPA1-deficient than wild type mice. Furthermore, bleomycin was discovered to significantly enhance M2-marker expression particularly in the presence of IL-4 in wild type macrophages in vitro, but not in macrophages harvested from TRPA1-deficient mice. IL-4-induced PPARγ-expression in macrophages was increased by bleomycin, providing a possible mechanism behind the phenomenon.ConclusionsIn conclusion, the results indicate that interfering TRPA1 attenuates fibrotic and inflammatory responses in bleomycin-induced scleroderma. Therefore, TRPA1-blocking treatment could potentially alleviate M2 macrophage driven diseases like systemic sclerosis and scleroderma.

Oxy210, a Semi-Synthetic Oxysterol, Inhibits Profibrotic Signaling in Cellular Models of Lung and Kidney Fibrosis.

Oxy210, a semi-synthetic oxysterol derivative, displays cell-selective inhibition of Hedgehog (Hh) and transforming growth factor beta (TGF-β) signaling in epithelial cells, fibroblasts, and macrophages as well as antifibrotic and anti-inflammatory efficacy in models of liver fibrosis. In the present report, we examine the effects of Oxy210 in cellular models of lung and kidney fibrosis, such as human lung fibroblast cell lines IMR-90, derived from healthy lung tissue, and LL97A, derived from an idiopathic pulmonary fibrosis (IPF) patient. In addition, we examine the effects of Oxy210 in primary human renal fibroblasts, pericytes, mesangial cells, and renal tubular epithelial cells, known for their involvement in chronic kidney disease (CKD) and kidney fibrosis. We demonstrate in fibroblasts that the expression of several profibrotic TGF-β target genes, including fibronectin (FN), collagen 1A1 (COL1A1), and connective tissue growth factor (CTGF) are inhibited by Oxy210, both at the basal level and following TGF-β stimulation in a statistically significant manner. The inhibition of COL1A1 gene expression translated directly to significantly reduced COL1A1 protein expression. In human primary small airway epithelial cells (HSAECs) and renal tubular epithelial cells, Oxy210 significantly inhibited TGF-β target gene expression associated with epithelial-mesenchymal transition (EMT). Oxy210 also inhibited the proliferation of fibroblasts, pericytes, and mesangial cells in a dose-dependent and statistically significant manner.

Dietary oxidized frying oil activates hepatic stellate cells and accelerates the severity of carbon tetrachloride- and thioacetamide-induced liver fibrosis in mice

Deep-frying is a common cooking practice worldwide, and after repeated heating's, the oil undergoes various chemical reactions, including hydrolysis, polymerization, lipid oxidation, and the Maillard reaction. Studies have pointed out that oxidized dietary frying oil may cause teratogenesis in mice and increase cancer and cardiovascular risks. The liver is the main organ involved in dietary nutrient catabolism, detoxification, bile production, and lipid metabolism. Nevertheless, the effects of oxidized frying oil exposure on the activation of hepatic stellate cells (HSCs) and liver fibrosis are still unclear. In this study, we showed that exposure to oxidized frying oil enhanced the sensitivity of HSCs to transforming growth factor (TGF)-β1-induced α-smooth muscle actin (α-SMA), collagen 1a2, collagen 1a1, metalloproteinase-2, and phosphorylated smad2/3 activation. In both carbon tetrachloride (CCl4)- and thioacetamide (TAA)-induced liver fibrosis mouse models, we showed that long-term administration of a 10% fried oil-containing diet significantly upregulated fibrogenesis genes expression and deposition of hepatic collagen. Furthermore, long-term fried oil exposure not only promoted macrophage infiltration and increased inflammatory-related gene expression, but also accumulated excess cholesterol and lipid peroxidation in the liver tissues. In conclusion, our study demonstrated that feeding a fried oil-containing diet may trigger TGF-β1-induced HSCs activation and thereby promote liver damage and fibrosis progression through enhancing the inflammatory response and lipid peroxidation.

Potential implications of the glycosylation patterns in collagen α1(I) and α2(I) chains for fibril assembly and growth

O-Glycosylation of hydroxylysine (Hyl) in collagen occurs at an early stage of biosynthesis before the triple-helix has formed. This simple post-translational modification (PTM) of lysine by either a galactosyl or glucosylgalactosyl moiety is highly conserved in collagens and depends on the species, type of tissue and the collagen amino acid sequence. The structural/functional reason why only specific lysines are modified is poorly understood, and has led to increased efforts to map the sites of PTMs on collagen sequences from different species and to ascertain their potential role in vivo. To investigate this, we purified collagen type I (Col1) from the skins of four animals, then used mass spectrometry and proteomic techniques to identify lysines that were oxidised, galactosylated, glucosylgalactosylated, or glycated in its mature sequence. We found 18 out of the 38 lysines in collagen type Iα1, (Col1A1) and 7 of the 30 lysines in collagen type Iα2 (Col1A2) were glycosylated. Six of these modifications had not been reported before, and included a lysine involved in crosslinking collagen molecules. A Fourier transform analysis of the positions of the glycosylated hydroxylysines showed they display a regular axial distribution with the same d-period observed in collagen fibrils. The significance of this finding in terms of the assembly of collagen molecules into fibrils and of potential restrictions on the growth of the collagen fibrils is discussed.

Full physicochemical and biocompatibility characterization of a supercritical CO2 sterilized nano-hydroxyapatite/chitosan biodegradable scaffold for periodontal bone regeneration

Despite bone's innate self-renewal capability, some periodontal pathologic and traumatic defects' size inhibits full spontaneous regeneration. This current research characterized a 3D porous biodegradable nano-hydroxyapatite/chitosan (nHAp/CS, 70/30) scaffold for periodontal bone regeneration, which preparation method includes the final solvent extraction and sterilization through supercritical CO2 (scCO2).Micro-CT analysis revealed the fully interconnected porous microstructure of the nHAp/CS scaffold (total porosity 78 %, medium pore size 200 μm) which is critical for bone regeneration. Scanning electron microscopy (SEM) showed HAp crystals forming on the surface of the nHAp/CS scaffold after 21 days in simulated body fluid, demonstrating its bioactivity in vitro. The presence of nHAp in the scaffolds promoted a significantly lower biodegradation rate compared to a plain CS scaffold in PBS. Dynamic mechanical analysis confirmed their viscoelasticity, but the presence of nHAp significantly enhanced the storage modulus (42.34 ± 6.09 kPa at 10 Hz after 28 days in PBS), showing that it may support bone ingrowth at low-load bearing bone defects. Both scaffold types significantly inhibited the growth, attachment and colony formation abilities of S. aureus and E. coli, enhancing the relevance of chitosan in the grafts' composition for the naturally contaminated oral environment. At SEM and laser scanning confocal microscopy, MG63 cells showed normal morphology and could adhere and proliferate inside the biomaterials' porous structure, especially for the nHAp/CS scaffold, reaching higher proliferative rate at day 14. MG63 cells seeded within nHAp/CS scaffolds presented a higher expression of RUNX2, collagen A1 and Sp7 osteogenic genes compared to the CS samples.The in vivo subcutaneous implantation in mice of both scaffold types showed lower biodegradability with the preservation of the scaffolds porous structure that allowed the ingrowth of connective tissue until 5 weeks. Histology shows an intensive and progressive ingrowth of new vessels and collagen between the 3rd and the 5th week, especially for the nHAp/CS scaffold.So far, the scCO2 method enabled the production of a cost-effective and environment-friendly ready-to-use nHAp/CS scaffold with microstructural, chemical, mechanical and biocompatibility features that make it a suitable bone graft alternative for defect sites in an adverse environment as in periodontitis and peri-implantitis.

Aspirin alleviates pulmonary fibrosis through PI3K/AKT/mTOR-mediated autophagy pathway

Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible lung disease with limited therapeutic options. Aspirin can alleviate liver, kidney, and cardiac fibrosis. However, its role in lung fibrosis is unclear. This study aims to investigate the effects of aspirin on lung fibroblast differentiation and pulmonary fibrosis. TGF-β1-induced human embryonic lung fibroblasts, IPF lung fibroblasts, and bleomycin-induced lung fibrosis mouse model were used in this study. The results showed that aspirin significantly decreased the expression of Collagen 1A1, Fibronectin, Alpha-smooth muscle actin, and equestosome1, and increased the ratio of light chain 3 beta II/I and the number of autophagosome in vivo and in vitro; reduced bleomycin-induced lung fibrosis. Aspirin also decreased the ratios of phosphorylated phosphatidylinositol 3 kinase (p-PI3K)/PI3K, protein kinase B (p-AKT)/AKT, and mechanistic target of rapamycin (p-mTOR)/mTOR in vitro. Autophagy inhibitor 3-methyladenine, bafilomycin-A1, and AKT activator SC-79 abrogated the effects of aspirin. These findings indicate that aspirin ameliorates pulmonary fibrosis through a PI3K/AKT/mTOR-dependent autophagy pathway.

Kalkitoxin attenuates calcification of vascular smooth muscle cells via RUNX-2 signaling pathways.

Kalkitoxin (KT) is an active lipopeptide isolated from the cyanobacterium Lyngbya majuscula found in the bed of the coral reef. Although KT suppresses cell division and inflammation, KT's mechanism of action in vascular smooth muscle cells (VSMCs) is unidentified. Therefore, our main aim was to investigate the impact of KT on vascular calcification for the treatment of cardiovascular disease. Using diverse calcification media, we studied the effect of KT on VSMC calcification and the underlying mechanism of this effect. VSMC was isolated from the 6 weeks ICR mice. Then VSMCs were treated with different concentrations of KT to check the cell viability. Alizarin red and von Kossa staining were carried out to examine the calcium deposition on VSMC. Thoracic aorta of 6 weeks mice were taken and treated with different concentrations of KT, and H and E staining was performed. Real-time polymerase chain reaction and western blot were performed to examine KT's effect on VSMC mineralization. Calcium deposition on VSMC was examined with a calcium deposition quantification kit. Calcium deposition, Alizarin red, and von Kossa staining revealed that KT reduced inorganic phosphate-induced calcification phenotypes. KT also reduced Ca++-induced calcification by inhibiting genes that regulate osteoblast differentiation, such as runt-related transcription factor 2 (RUNX-2), SMAD family member 4, osterix, collagen 1α, and osteopontin. Also, KT repressed Ca2+-induced bone morphogenetic protein 2, RUNX-2, collagen 1α, osteoprotegerin, and smooth muscle actin protein expression. Likewise, Alizarin red and von Kossa staining showed that KT markedly decreased the calcification of ex vivo ring formation in the mouse thoracic aorta. This experiment demonstrated that KT decreases vascular calcification and may be developed as a new therapeutic treatment for vascular calcification and arteriosclerosis.

Lectroacupuncture stimulation at CV4 sites prevents breast cancer-induced osteoporosis by activating the Wnt/β-catenin signaling pathway

Osteoporosis is a common condition among breast cancer patients, characterized by decreased bone density due to chemotherapy and hormone therapy. However, the application of electroacupuncture (EA) stimulation at Guanyuan (CV4) sites has been found to be beneficial in enhancing bone mineral density. In this study, we aimed to investigate the efficacy of EA stimulation at CV4 sites in breast cancer-induced osteoporosis and elucidate the potential underlying mechanisms. A breast cancer model in mice was established, EA stimulation at CV4 sites was performed, and hematoxylin-eosin (HE) stain was conducted to assess the bone mineral density and trabecular bone volume of femur tissues. Additionally, Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify and visualize osteoclasts. Furthermore, Western blots were used to evaluate the expression levels of various proteins, including c-src, Cathepsin K (CtSK), matrix metalloproteinase 9 (MMP9), Collagen 1A1 (COL1A1), Osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), Wnt3a and β-catenin, and Enzyme Linked Immunosorbent Assay (ELISA) to measure the concentration of Alkaline phosphatase (ALP), NO synthase 2 (NOS2), glutathione reductase (GSR), superoxide dismutase (SOD), reactive oxygen species (ROS) and lactate dehydrogenase (LDH) in the serum. The results revealed that EA stimulation in the breast cancer model resulted in higher bone mineral density and trabecular bone volume, along with a reduction in the number of osteoclasts. EA stimulation at CV4 site significantly decreased the protein levels of c-src, CtSK and MMP9, while also reducing ROS and LDH production. Conversely, it promoted the expression of COL1A1, OPN, RUNX2, Wnt3a and β-catenin. Additionally, it elevated the concentration of ALP, NOS2, GSR and SOD in the serum. Overall, EA stimulation at the CV4 site may inhibit bone loss and promote bone differentiation by activating the Wnt/β-catenin pathway, indicating its potential use for breast cancer-induced osteoporosis treatment.

Sensitivity of the amide I band to matrix manipulation in bone: a Raman micro-spectroscopy and spatially offset Raman spectroscopy study.

The fracture resistance of bone arises from the hierarchical arrangement of minerals, collagen fibrils (i.e., cross-linked triple helices of α1 and α2 collagen I chains), non-collagenous proteins, and water. Raman spectroscopy (RS) is not only sensitive to the relative fractions of these constituents, but also to the secondary structure of bone proteins. To assess the ability of RS to detect differences in the protein structure, we quantified the effect of sequentially autoclaving (AC) human cortical bone at 100 °C (∼34.47 kPa) and then at 120 °C (∼117.21 kPa) on the amide I band using a commercial Raman micro-spectroscopy (μRS) instrument and custom spatially offset RS (SORS) instrument in which rings of collection fiber optics are offset from the central excitation fiber optics within a hand-held, cylindrical probe. Being clinically viable, measurements by SORS involved collecting Raman spectra of cadaveric femur mid-shafts (5 male & 5 female donors) through layers of a tissue mimic. Otherwise, μRS and SORS measurements were acquired directly from each bone. AC-related changes in the helical status of collagen I were assessed using amide I sub-peak ratios (intensity, I, at ∼1670 cm-1 relative to intensities at ∼1610 cm-1 and ∼1640 cm-1). The autoclaving manipulation significantly decreased the selected amide I sub-peak ratios as well as shifted peaks at ∼1605 cm-1 (μRS), ∼1636 cm-1 (SORS) and ∼1667 cm-1 in both μRS and SORS. Compared to μRS, SORS detected more significant differences in the amide I sub-peak ratios when the fiber optic probe was directly applied to bone. SORS also detected AC-related decreases in I1670/I1610 and I1670/I1640 when spectra were acquired through layers of the tissue mimic with a thickness ≤2 mm by the 7 mm offset ring, but not with the 5 mm or 6 mm offset ring. Overall, the SORS instrument was more sensitive than the conventional μRS instrument to pressure- and temperature-related changes in the organic matrix that affect the fracture resistance of bone, but SORS analysis of the amide I band is limited to an overlying thickness layer of 2 mm.

PS-B04-12: DEVELOPMENT OF VACCINE AGAINST CONNECTIVE TISSUE GROWTH FACTOR TO PREVENT THE PROGRESSION OF RENAL FIBROSIS

Objective: Hypertension is closely related with chronic kidney disease (CKD); Hypertension and CKD can cause and exacerbate each other, forming a vicious cycle. There is a critical need for efficient treatment of CKD. Renal fibrosis is a final common pathway to end-stage renal disease, and connective tissue growth factor (CTGF) is a well-recognized central profibrotic factor in fibrosis of various organ systems. Here, we developed a novel peptide vaccine against CTGF to prevent the progression of renal fibrosis. Design and method: Among sequences of CTGF, the sequence common to mice and humans in the interaction region with transforming growth factor-β; (TGF-β;) and bone morphogenic proteins was selected as the epitope for the vaccine. The efficacy of this vaccine was verified in two renal fibrosis models using male C57BL/6J mice: adenine-induced CKD and unilateral ureteral obstruction (UUO) models. Moreover, the functions of CTGF vaccine-elicited IgG antibodies were evaluated in an in vitro study. Results: Three immunizations with this CTGF vaccine at 2-week intervals elicited serum IgG antibodies specifically binding to human full-length CTGF, and the antigen-specific antibody titers were maintained at high level for > 30 weeks. In adenine-induced CKD model, histological analyses showed that immunization with the CTGF vaccine significantly attenuated renal interstitial fibrosis compared to the vehicle treatment (8.5 ± 0.8% vs 12.3 ± 0.8%; P < 0.01). The CTGF vaccination also suppressed the increased mRNA expressions of collagen 1α and α-smooth muscle actin (α-SMA) in kidney tissue samples. Moreover, vaccinated mice showed lower levels of serum urea nitrogen (UN) and creatinine and lower urine albumin-creatinine ratio compared with vehicle-treated mice (UN: 77.7 ± 5.0 vs 104.0 ± 6.8 mg/dL; P < 0.01. creatinine: 0.42 ± 0.02 vs 0.51 ± 0.04 mg/dL; P = 0.06. albumin-creatinine ratio: 74.7 ± 13.8 vs 132.0 ± 15.3 μg/mg; P < 0.05). In UUO model, the CTGF vaccination also ameliorated the expansion of the interstitial fibrotic areas (7.7 ± 0.4 vs 9.8 ± 0.7%; P < 0.05). In an in vitro study, CTGF vaccine-elicited IgG antibodies efficiently suppressed CTGF-induced- and TGF-β;-induced α-SMA expression in cultured kidney fibroblasts. Conclusions: The CTGF vaccine has the potential to prevent the progression of renal fibrosis and delay the deterioration of renal function with a low frequency of administration. Thus, this CTGF vaccine could be a promising preventive strategy for CKD patients.

Photo-crosslinked GelMA loaded with dental pulp stem cells and VEGF to repair critical-sized soft tissue defects in rats

BackgroundTissue engineering of skin and mucosa is essential for the esthetic and functional reconstruction of individuals disfigured by trauma, resection surgery, or severe burns while overcoming the limited amount of autograft and donor site morbidity. PurposeWe aimed to determine whether a combination of Gelatin-methacryloyl (GelMA) hydrogel scaffold alone or loaded with either dental pulp stem cells (DPSCs) and/or vascular endothelial growth factor (VEGF) could improve skin wound healing in rats. Materials and MethodsFour 10 mm full-thickness skin defects were created on the dorsum of 15 Sprague-Dawley rats. The wounds were treated with GelMA alone, GelMA+DPSCs, or GelMA+DPSCs+VEGF. Unprotected wounds were used as controls. Animals were euthanized at 1-, 2-, and 4 weeks post-surgery, and the healing wounds were harvested for clinical, histological, and RT-PCR analysis. ResultsNo signs of clinical inflammation were observed among all groups. Few and sparse mononuclear inflammatory cells were observed in GelMA+DPSCs and GelMA+DPSCs+VEGF groups at 2 weeks, with complete epithelialization of the wounds. At 4 weeks, the epidermis in GelMA+DPSCs and GelMA+DPSCs+VEGF groups was indistinguishable from the empty defect and GelMA groups. The decrease in cellularity and increase in density of collagen fibers were observed over time in both GelMA+DPSCs and GelMA+DPSCs+VEGF groups but were more evident in the GelMA+DPSCs+VEGF group. The GelMA+DPSCs+VEGF group showed a higher expression of the KER 10 gene at all time points compared with the other groups. Expression of Col1 A1 and TGFβ-1 were not statistically different over time neither among the groups. ConclusionGelMA scaffolds loaded with DPSCs, and VEGF accelerated the re-epithelialization of skin wounds.

Caffeic Acid Phenethyl Ester Loaded PEG-PLGA Nanoparticles Enhance Wound Healing in Diabetic Rats.

Delayed wound healing is a serious complication of diabetes and a main reason for foot amputation. Caffeic acid phenethyl ester (CAPE) is a main active constituent of honeybee propolis with reported appealing pharmacological activities. In the current study, CAPE was loaded onto PEG-PLGA nanoparticles and showed a particle size of 198 ± 7.3 nm and polydispersity index of 0.43 ± 0.04. An in vivo study was performed to appraise the wound-healing activity of CAPE-loaded PEG-PLGA nanoparticles (CAPE-NPs) in diabetic rats. Wound closure was significantly accelerated in rats treated with CAPE-NPs. This was confirmed via histological examinations of skin tissues that indicated expedited healing and enhanced collagen deposition. This was accompanied by observed antioxidant activity as evidenced by the prevention of lipid peroxidation and the exhaustion of superoxide dismutase (SOD) and catalase (CAT) activities. In addition, CAPE-NPs showed superior anti-inflammatory activity as compared with the regular formula of CAPE, as they prevented the expression of interleukin-6 (IL-6) as well as tumor necrosis-alpha (TNF-α). The pro-collagen actions of CAPE-NPs were highlighted by the enhanced hyroxyproline content and up-regulation of Col 1A1 mRNA expression. Furthermore, the immunohistochemial assessment of skin tissues indicated that CAPE-NPs enhance proliferation and angiogenesis, as shown by the increased expression of transforming growth factor β1 (TGF-β1) and platelet-derived growth factor subunit B (PDGF-B). In conclusion, CAPE-loaded PEG-PLGA nanoparticles possess potent healing effects in diabetic wounds. This is mediated, at least partially, by its antioxidant, anti-inflammatory, and pro-collagen as well as angiogenic activities.

The quest for a generic bird target to detect the presence of bird in food products and considerations for paleoprotein analysis.

It can be important for consumers to know whether food products contain animal material and, if so, of which species. Food products with animal material as an ingredient often contain collagen type 1. LC-MS/MS (Liquid Chromatography-tandem Mass Spectrometry) was applied as technique to generically detect bird. Unlike for example fish, that have experienced longer divergence times, it is still possible to find generic LC-MS targets for avian type 1 collagen. After theoretical target selection using 83 collagen 1α2 bird sequences of 33 orders and construction of a common ancestor sequence of birds, experimental evidence was provided by analyzing extracts from 10 extant bird species. Two suitable options have been identified. The combination of VGPIGPAGNR and VGPIGAAGNR (pheasant only) covers all investigated birds and was not found in other species. The peptide EGPVGFpGADGR covers all investigated birds, but also occurs in several species of crocodiles and turtles. The presence of the generic peptide (combination) was confirmed in food products, proving the principle, and can therefore be used to detect the presence of bird. Furthermore, it is shown how the use of constructed ancestor sequences could benefit the field of paleoproteomics, in the interpretation of collagen MS/MS spectra of ancient species. Our theoretical analysis and assessment of reported Brachylophosaurus canadensis collagen 1α2 MS/MS data provided support for several previous peptide sequence assignments, but we also propose that our constructed ancestral bird sequence GPpGESGAVGPAGPIGSR may fit the MS/MS data better than the original assignment GLPGESGAVGPAGPpGSR.

Longitudinal Evaluation of Biomarkers in Wound Fluids from Venous Leg Ulcers and Split-thickness Skin Graft Donor Site Wounds Treated with a Protease-modulating Wound Dressing.

Venous leg ulcers represent a clinical challenge and impair the quality of life of patients. This study examines impaired wound healing in venous leg ulcers at the molecular level. Protein expression patterns for biomarkers were analysed in venous leg ulcer wound fluids from 57 patients treated with a protease-modulating polyacrylate wound dressing for 12 weeks, and compared with exudates from 10 acute split-thickness wounds. Wound healing improved in the venous leg ulcer wounds: 61.4% of the 57 patients with venous leg ulcer achieved a relative wound area reduction of ≥ 40%, and 50.9% of the total 57 patients achieved a relative wound area reduction of ≥ 60%. Within the first 14 days, abundances of S100A8, S100A9, neutrophil elastase, matrix metalloproteinase-2, and fibronectin in venous leg ulcer exudates decreased significantly and remained stable, yet higher than in acute wounds. Interleukin-1β, tumour necrosis factor alpha, and matrix metalloproteinase-9 abundance ranges were similar in venous leg ulcers and acute wound fluids. Collagen (I) α1 abundance was higher in venous leg ulcer wound fluids and was not significantly regulated. Overall, significant biomarker changes occurred in the first 14 days before a clinically robust healing response in the venous leg ulcer cohort.

Type IV collagen α5 chain promotes luminal breast cancer progression through c-Myc-driven glycolysis.

Cancer cell metabolism reprogramming is one of the hallmarks of cancer. Cancer cells preferentially utilize aerobic glycolysis, which is regulated by activated oncogenes and the tumor microenvironment. Extracellular matrix (ECM) in the tumor microenvironment, including the basement membranes (BMs), is dynamically remodeled. However, whether and how ECM regulates tumor glycolysis is largely unknown. We show that type IV collagens, components of BMs essential for the tissue integrity and proper function, are differentially expressed in breast cancer subtypes that α5 chain (α5(IV)) is preferentially expressed in the luminal-type breast cancer and is regulated by estrogen receptor-α. α5(IV) is indispensable for luminal breast cancer development. Ablation of α5(IV) significantly reduces the growth of luminal-type breast cancer cells and impedes the development of luminal-type breast cancer. Impaired cell growth and tumor development capability of α5(IV)-ablated luminal breast cancer cells is attributed to the reduced expression of glucose transporter and glycolytic enzymes and impaired glycolysis in luminal breast cancer cells. Non-integrin collagen receptor discoidin domain receptor-1 (DDR1) expression and p38 mitogen-activated protein kinase activation are attenuated in α5(IV)-ablated luminal breast cancer cells, resulting in reduced c-Myc oncogene expression and phosphorylation. Ectopic expression of constitutively active DDR1 or c-Myc restores the expression of glucose transporter and glycolytic enzymes, and thereafter restores aerobic glycolysis, cell proliferation, and tumor growth of luminal breast cancer. Thus, type IV collagen α5 chain is a luminal-type breast cancer-specific microenvironmental regulator modulating cancer cell metabolism.

Glycyrrhizic acid alleviates liver fibrosis in vitro and in vivo via activating CUGBP1-mediated IFN-γ/STAT1/Smad7 pathway

BackgroundHepatic fibrosis, a common pathological feature of chronic liver injuries, is a serious public health problem and lacks effective therapy. Glycyrrhizic acid (GA) is a bioactive ingredient in the root of traditional Chinese medicine licorice, and exhibits remarkable anti-viral, anti-inflammatory and hepatoprotective actions. PurposeHere we aimed to investigated whether GA provided a therapeutic efficacy in hepatic fibrosis and uncover its molecular mechanisms. Study design and methodsWe investigated the anti-fibrosis effects of GA using CCl4-induced mouse mode of liver fibrosis as well as TGF-β1-activated human LX-2 cells and primary hepatic stellate cells (HSCs). CUGBP1-mediated IFN-γ/STAT1/Smad7 signaling was examined with immunofluorescence staining and western blot analysis. We designed and studied the binding of GA to CUGBP1 using in silico docking, and validated by microscale thermophoresis (MST) assay. ResultsGA obviously attenuated CCl4-induced liver histological damage, and reduced serum ALT and AST levels. Meanwhile, GA decreased liver fibrogenesis markers such as α-SMA, collagen α1, HA, COL-III, and LN in the hepatic tissues. Mechanistically, GA remarkably elevated the levels of IFN-γ, p-STAT1, Smad7, and decreased CUGBP1 in vivo and in vitro. Over-expression of CUGBP1 completely abolished the anti-fibrotic effect of GA and regulation on IFN-γ/STAT1/Smad7 pathway in LX-2 cells and primary HSCs, confirming CUGBP1 played a pivotal role in the protection by GA from liver fibrosis. Further molecular docking and MST assay indicated that GA had a good binding affinity with the CUGBP1 protein. The dissociation constant (Kd) of GA and CUGBP1 was 0.293 μM. ConclusionOur study demonstrated for the first time that GA attenuated liver fibrosis and hepatic stellate cell activation by promoting CUGBP1-mediated IFN-γ/STAT1/Smad7 signalling pathways. GA may be a potential candidate compound for preventing or reliving liver fibrosis.