COL1A1 mRNA Research Articles

Upregulated Collagen COL10A1 Remodels the Extracellular Matrix and Promotes Malignant Progression in Lung Adenocarcinoma.

Collagens are major components of the ECM in various organs, including the lungs. Ectopic expression of collagens can regulate the tumor progression and disease outcome through remodeling of the extracellular matrix (ECM). However, it remains largely unexplored whether collagens are involved in the tumor progression of lung adenocarcinoma (LUAD). Analysis of three LUAD transcriptional expression profiles showed that COL10A1 mRNA expression was up-regulated and associated with poor prognosis. Gain- and loss-of-function studies were performed to observe that up-regulated COL10A1 promotes LUAD cell proliferation and invasion in vitro and in vivo. In molecular mechanism study, we found that COL10A1 interacts with DDR2 and affects the downstream FAK signaling pathway to regulate LUAD cell progression. The expression of COL10A1 on tissue microarray (TMA) was also measured to explore the association between COL10A1 expression and patient outcome. The results addressed that COL10A1 is up-regulated and positively correlated with lymph node metastasis in lung adenocarcinoma, and the COL10A1 expression is also an independent prognostic factor. In summary, the up-regulated COL10A1 remodels the ECM and the COL10A1/DDR2/FAK axis regulates the proliferation and metastasis of LUAD cells, implying that COL10A1 is a promising therapeutic target and prognostic marker for LUAD patients.

Collagen release by human hepatic stellate cells requires vitamin C and is efficiently blocked by hydroxylase inhibition.

Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFβ) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFβ. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.

COL6A3 expression in adipose tissue cells is associated with levels of the homeobox transcription factor PRRX1

Fibrillar collagen COL6α3 in adipose tissue has been associated with obesity, inflammation, insulin resistance and cancer. We here aimed to identify novel transcriptional regulators of COL6A3 expression. Based on a transcriptome dataset of adipose tissue, we identified strong correlations for 56 genes with COL6A3 mRNA, including targets of TGF-β/SMAD signaling. Among the identified candidates, the homeobox transcription factor PRRX1 showed a particularly striking co-expression with COL6A3, validated across several different cohorts, including patients with extreme obesity, insulin sensitive and resistant obesity (subcutaneous and omental), after profound fat loss (subcutaneous), and lean controls (subcutaneous). In human and mouse adipose cells, PRRX1 knockdown reduced COL6A3 mRNA and PRRX1 overexpression transactivated a reporter construct with the endogenous human COL6A3 promoter. Stable PRRX1 overexpression in 3T3-L1 cells induced Col6a3 mRNA threefold specifically after adipogenic induction, whereas TGF-β1 treatment upregulated Col6a3 mRNA also in the preadipocyte state. Interestingly, pro-inflammatory stimulus (i.e., TNF-α treatment) decreased PRRX1-mediated Col6a3 transactivation and mRNA expression, supporting a role for this mechanism in the regulation of adipose tissue inflammation. In conclusion, we identified the homeobox factor PRRX1 as a novel transcriptional regulator associated with COL6A3 expression, providing new insight into the regulatory mechanisms of altered adipose tissue function in obesity and insulin resistance.

Abstract 12570: Association Between Left Atrial Global Peak Longitudinal Strain and Tgfβ1 in Cardiovascular Surgery Patients

Introduction: Atrial fibrosis plays an important role in atrial remodeling and the development of atrial fibrillation (AF). Transforming growth factor β1 (TGFβ1) is one of the major factors that accelerate atrial fibrosis. Left atrium global peak longitudinal strain (GPLS) measured by two-dimensional speckle tracking (2DS) method has been reported to be a useful method to predict the degree of atrial fibrosis. Hypothesis: To investigate the association between GPLS, Af and atrial fibrotic remodeling by analyzing fibrosis-relating gene expression including collagen type 1 (COL1A1), and TGFβ1 in cardiovascular surgery patients. Methods: Conventional echocardiography and 2DS method were performed in 117 patients (36 atrial fibrillation (Af) patients, 81 patients with sinus rhythm (SR), average age 69.7 years, 69 males) before cardiovascular surgery including cardiac valve replacement, and CABG. The fibrosis-relating gene expression such as collagen type 1 (COL1A1), COL4A1, TGFβ1, and connective tissue growth factor (CTGF) mRNA were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) in 39 cases in which LA tissues were removed from the appendage during surgery. Results: GPLS was significantly negatively correlated with left atrial diameter (LAD), E/e‘, and left atrial volume index (LAVI). Expression of TGFβ1 in the atrium was positively correlated with expression level of Col1A1, Col4A1, and CTGF mRNA. GPLS was significantly negatively correlated with the expression of Col1A1 (r = -0.418, p = 0.015), TGFβ1 (r = -0.557, p = 0.000), and Col4A1 (r = -0.411, p = 0.013). However, no clear correlation was observed with LAD and LAVI. In multivariate analysis, unlike LAD and LAVI, GSLS was a clear predictor of Col1A1 expression, even when corrected with gender, age, brain natriuretic peptide (BNP), and type of heart rhythm (Af, sinus rhythm). On the other hand, TGFβ1 concentration in blood did not correlate with TGFβ1, Col1A1 and Col41A mRNA expression level in the atrium. Conclusions: Low LA GPLS exhibits increased atrial fibrosis reflected by collagen and TGFβ1 expression , irrespectively of heart rhythm (Af or sinus rhythm), and becomes an extremely useful marker for predicting LA fibrosis in cardiovascular surgery patients.

Abstract 15013: Ischemic Mitral Regurgitation is Associated With Impaired Transforming Growth Factor/Bone Morphogenic Protein Signaling and Increased CD45+ Cell Presence in the Mitral Valve Interstitial Cell Population

Introduction: Ischemic mitral regurgitation (IMR) develops in a fraction of patients in response to left ventricular (LV) remodeling after ischemia. IMR independently predicts adverse events and doubles mortality. While largely driven by LV distortion, the susceptibility of the mitral valve (MV) to undergo remodeling may play a role in determining whether MR develops. We hypothesized that impaired cellular response in MV cells may contribute to MR development by inducing maladaptive tissue remodeling. Methods: Dorsett hybrid sheep (n=32) were subjected to ligation of the circumflex coronary artery to induce myocardial infarction (MI). Development of MR was reported by echocardiography. MV leaflets and isolated MV interstitial cells (MVIC) were collected at baseline (Control), 4-, and 8-weeks post MI. RNAseq (150 paired end reads, 20 million reads/sample) was performed on MV leaflets from all groups. MVICs were treated with for 24h with 10ng/ml TGFβ1 of 50ng/ml BMP4. Results: Gene set enrichment analysis using pre-defined gene sets from the Gene Ontology project, considering genes with significant changes (p<0.05) between MV with/without MR showed enrichment of cardiac contraction biological processes. TGFβ/BMP-dependent SMAD7 was the main hub in the protein-protein-interaction network of the top contributing genes to the enriched pathways in MR. In vitro, collagen matrix-contracting ability of MR MVICs was higher than non-MR MI MVICs. MVICs from MI sheep were less responsive to BMP4 pro-osteogenic stimulation (as reflected by blunted increased in ID2, osteopontin and osteocalcin mRNA) than control. MVICs from sheep with MR had a diminished COL1A1 mRNA response to TGFβ1 and enhanced pro-chondrogenic RUNX2 and SOX9 mRNA response to BMP4 vs. non-MR MVICs. Baseline CD45 expression was detectable only in MR MVICs. Upon TGFβ1 stimulation, CD45 expression was detected in all groups. Immunostaining confirmed increased presence of CD45+ cells in IMR MV interstitium. Conclusions: MVs from sheep developing MR had altered TGFβ/BMP response, which may be partially mediated by increased CD45+ fibrocyte presence. Pharmacological strategies aimed to modulate TGFβ/BMP signaling after MI may protect from pathological MV remodeling leading to IMR.

Interleukin-1β Modulation of the Mechanobiology of Primary Human Pulmonary Fibroblasts: Potential Implications in Lung Repair.

Pro-inflammatory cytokines like interleukin-1β (IL-1β) are upregulated during early responses to tissue damage and are expected to transiently compromise the mechanical microenvironment. Fibroblasts are key regulators of tissue mechanics in the lungs and other organs. However, the effects of IL-1β on fibroblast mechanics and functions remain unclear. Here we treated human pulmonary fibroblasts from control donors with IL-1β and used Atomic Force Microscopy to unveil that IL-1β significantly reduces the stiffness of fibroblasts concomitantly with a downregulation of filamentous actin (F-actin) and alpha-smooth muscle (α-SMA). Likewise, COL1A1 mRNA was reduced, whereas that of collagenases MMP1 and MMP2 were upregulated, favoring a reduction of type-I collagen. These mechanobiology changes were functionally associated with reduced proliferation and enhanced migration upon IL-1β stimulation, which could facilitate lung repair by drawing fibroblasts to sites of tissue damage. Our observations reveal that IL-1β may reduce local tissue rigidity by acting both intracellularly and extracellularly through the downregulation of fibroblast contractility and type I collagen deposition, respectively. These IL-1β-dependent mechanical effects may enhance lung repair further by locally increasing pulmonary tissue compliance to preserve normal lung distension and function. Moreover, our results support that IL-1β provides innate anti-fibrotic protection that may be relevant during the early stages of lung repair.

GDF11 induces mild hepatic fibrosis independent of metabolic health.

Background & aims: Growth Differentiation Factor 11 (GDF11) is an anti-aging factor, yet its role in liver diseases is not established. We evaluated the role of GDF11 in healthy conditions and in the transition from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH).Results: GDF11 mRNA levels positively correlated with NAFLD activity score and with CPT1, SREBP, PPARγ and Col1A1 mRNA levels, and associated to portal fibrosis, in morbidly obese patients with NAFLD/NASH. GDF11-treated mice showed mildly exacerbated hepatic collagen deposition, accompanied by weight loss and without changes in liver steatosis or inflammation. GDF11 triggered ALK5-dependent SMAD2/3 nuclear translocation and the pro-fibrogenic activation of HSC.Conclusions: GDF11 supplementation promotes mild liver fibrosis. Even considering its beneficial metabolic effects, caution should be taken when considering therapeutics that regulate GDF11.Methods: We analyzed liver biopsies from a cohort of 33 morbidly obese adults with NAFLD/NASH. We determined the correlations in mRNA expression levels between GDF11 and genes involved in NAFLD-to-NASH progression and with pathological features. We also exposed wild type or obese mice with NAFLD to recombinant GDF11 by daily intra-peritoneal injection and monitor the hepatic pathological changes. Finally, we analyzed GDF11-activated signaling pathways in hepatic stellate cells (HSC).

Interdependence between Chromogranin-A, Alternatively Activated Macrophages, Tight Junction Proteins and the Epithelial Functions. A Human and In-Vivo/In-Vitro Descriptive Study.

Background: Ulcerative colitis (UC) is characterized by altered chromogranin-A (CHGA), alternatively activated macrophages (M2) and intestinal epithelial cells (IECs). We previously demonstrated that CHGA is implicated in colitis progression by regulating the macrophages. Here, we investigated the interplay between CHGA, M2, tight junctions (TJ) and IECs in an inflammatory environment. Methods: Correlations between CHGA mRNA expression of and TJ proteins mRNA expressions of (Occludin [OCLN], zonula occludens-1 [ZO1], Claudin-1 [CLDN1]), epithelial associated cytokines (interleukin [IL]-8, IL-18), and collagen (COL1A2) were determined in human colonic mucosal biopsies isolated from active UC and healthy patients. Acute UC-like colitis (5% dextran sulphate sodium [DSS], five days) was induced in Chga-C57BL/6-deficient (Chga−/−) and wild type (Chga+/+) mice. Col1a2 TJ proteins, Il-18 mRNA expression and collagen deposition were determined in whole colonic sections. Naïve Chga−/− and Chga+/+ peritoneal macrophages were isolated and exposed six hours to IL-4/IL-13 (20 ng/mL) to promote M2 and generate M2-conditioned supernatant. Caco-2 epithelial cells were cultured in the presence of Chga−/− and Chga+/+ non- or M2-conditioned supernatant for 24 h then exposed to 5% DSS for 24 h, and their functional properties were assessed. Results: In humans, CHGA mRNA correlated positively with COL1A2, IL-8 and IL-18, and negatively with TJ proteins mRNA markers. In the experimental model, the deletion of Chga reduced IL-18 mRNA and its release, COL1A2 mRNA and colonic collagen deposition, and maintained colonic TJ proteins. Chga−/− M2-conditioned supernatant protected caco-2 cells from DSS and oxidative stress injuries by improving caco-2 cells functions (proliferation, viability, wound healing) and by decreasing the release of IL-8 and IL-18 and by maintaining the levels of TJ proteins, and when compared with Chga+/+ M2-conditioned supernatant. Conclusions: CHGA contributes to the development of intestinal inflammation through the regulation of M2 and epithelial cells. Targeting CHGA may lead to novel biomarkers and therapeutic strategies in UC.

A retrotransposon gag-like-3 gene RTL3 and SOX-9 co-regulate the expression of COL2A1 in chondrocytes

ABSTRACT Purpose Transposable elements are known to remodel gene structure and provide a known source of genetic variation. Retrotransposon gag-like-3 (RTL3) is a mammalian retrotransposon-derived transcript (MART) whose function in the skeletal tissue is unknown. This study aimed to elucidate the biological significance of RTL3 in chondrogenesis and type-II collagen (COL2A1) gene expression in chondrocytes. Materials and Methods Expression of RTL3, SOX-9 and COL2A1 mRNAs was determined by TaqMan assays and the protein expression by immunoblotting. RTL3 and Sox-9 depletion in human chondrocytes was achieved using validated siRNAs. An RTL3 mutant (∆RTL3) lacking the zinc finger domain was created using in vitro mutagenesis. Forced expression of RTL3, ∆RTL3, and SOX-9 was achieved using CMV promoter containing expression plasmids. CRISPR-Cas9 was utilized to delete Rtl3 and create a stable ATDC5Rlt3-/- cell line. Matrix deposition and Col2a1 quantification during chondrogenesis were determined by Alcian blue staining and Sircol™ Soluble Collagen Assay, respectively. Results RTL3 is not ubiquitously expressed but showed strong expression in cartilage, chondrocytes and synoviocytes but not in muscle, brain, or other tissues analyzed. Loss-of-function and gain-of-function studies demonstrated a critical role of RTL3 in the regulation of SOX-9 and COL2A1 expression and matrix synthesis during chondrogenesis. Both RTL3 and SOX-9 displayed co-regulated expression in chondrocytes. Gene regulatory activity of RTL3 requires the c-terminal CCHC zinc-finger binding domain. Conclusions Our results identify a novel regulatory mechanism of COL2A1 expression in chondrocytes that may help to further understand the skeletal development and the pathogenesis of diseases with altered COL2A1 expression.

Efficacy of resveratrol in the wound healing process by reducing oxidative stress and promoting fibroblast cell proliferation and migration.

Oxidative stress (OS) is implicated in the pathogenesis of diabetic, chronic, and inflamed wounds. In these cases, wound healing is difficult and remains a big clinical challenge. As fibroblasts are crucial in wound healing by producing extracellular matrix and wound contraction, the researchers investigated the resveratrol (RES) effects as a potential therapeutic alternative, on an in vitro OS and wound healing model established by 3T3 Swiss Albino fibroblasts. OS was induced by H2 O2 , and 1-100 μM RES doses were applied on cells for up to 48 hours. The cell proliferation was assessed by the population doubling time and bromodeoxyuridine immunocytochemistry. The OS levels were determined by fluorescence staining and all values were calculated by the ImageJ program. The collagen-I expression (COL1A2 mRNA) was evaluated by a real-time PCR. Cell migration was evaluated by wound closure assay and the ultrastructure was evaluated by electron microscopy. The cell proliferation index significantly decreased in H2 O2 incubation and increased with RES application. RES application reduced OS levels and stabilized cell proliferation, but no differences were found in COL1A2 mRNA expression. RES-treated cells showed better proliferation, migration rates, and ultrastructural preservation. RES administration decreases OS levels and improves the healing process by increasing cell proliferation and migration quality, suggesting it is a powerful candidate for the treatment of skin wounds.

Glucagon-Like Peptide-2 Analogue ZP1849 Augments Colonic Anastomotic Wound Healing

Background The enteroendocrine hormone glucagon-like peptide- (GLP-) 2 is a potent trophic factor in the gastrointestinal tract. The GLP-2 receptor (GLP-2R) is expressed in the stroma of the large bowel wall, which is the major therapeutic target area to prevent anastomotic leakage. We investigated the efficacy of the long-acting GLP-2 analogue ZP1849 on colonic anastomotic wound healing. Methods Eighty-seven male Wistar rats were stratified into four groups and received daily treatment with vehicle or ZP1849 starting one day before (day -1) end-to-end anastomosis was constructed in the left colon on day 0, and on days 0 (resected colon segment), 3, and 5, gene expressions of GLP-2R, Ki67, insulin-like growth factor- (IGF-) 1, type I (COL1A1) and type III (COL3A1) procollagens, cyclooxygenase- (COX-) 1, COX-2, and matrix metalloproteinase- (MMP-) 7 were quantified by RT-qPCR. Breaking strength, myeloperoxidase (MPO), transforming growth factor- (TGF-) β1, and soluble collagen proteins were measured on days 3 and 5. Results ZP1849 treatment increased Ki67 (P < 0.0001) and IGF-1 (P < 0.05) mRNA levels in noninjured colon day 0, and postoperatively in the anastomotic wounds compared to vehicle-treated rats. ZP1849-treated rats had increased (P = 0.042) anastomotic breaking strength at day 5 compared with vehicle. COL1A1 and COL3A1 mRNA levels (P < 0.0001) and soluble collagen proteins (P < 0.05) increased from day 3 to day 5 in ZP1849-treated rats, but not in vehicle-treated rats. COX-2 mRNA and MPO protein levels decreased from day 3 to day 5 (P < 0.001) in both groups. ZP1849 treatment reduced TGF-β1 protein levels on day 5 (P < 0.001) but did not impact MMP-7 transcription. Conclusions The GLP-2 analogue ZP1849 increased breaking strength, IGF-1 expression, and cell proliferation, which may be beneficial for colonic anastomotic wound healing.

Collagen XI regulates the acquisition of collagen fibril structure, organization and functional properties in tendon

Collagen XI is a fibril-forming collagen that regulates collagen fibrillogenesis. Collagen XI is normally associated with collagen II-containing tissues such as cartilage, but it also is expressed broadly during development in collagen I-containing tissues, including tendons. The goals of this study are to define the roles of collagen XI in regulation of tendon fibrillar structure and the relationship to function. A conditional Col11a1-null mouse model was created to permit the spatial and temporal manipulation of Col11a1 expression. We hypothesize that collagen XI functions to regulate fibril assembly, organization and, therefore, tendon function. Previous work using cho mice with ablated Col11a1 alleles supported roles for collagen XI in tendon fibril assembly. Homozygous cho/cho mice have a perinatal lethal phenotype that limited the studies. To circumvent this, a conditional Col11a1flox/flox mouse model was created where exon 3 was flanked with loxP sites. Breeding with Scleraxis-Cre (Scx-Cre) mice yielded a tendon-specific Col11a1-null mouse line, Col11a1Δten/Δten. Col11a1flox/flox mice had no phenotype compared to wild type C57BL/6 mice and other control mice, e.g., Col11a1flox/flox and Scx-Cre. Col11a1flox/flox mice expressed Col11a1 mRNA at levels comparable to wild type and Scx-Cre mice. In contrast, in Col11a1Δten/Δten mice, Col11a1 mRNA expression decreased to baseline in flexor digitorum longus tendons (FDL). Collagen XI protein expression was absent in Col11a1Δten/Δten FDLs, and at ~50% in Col11a1+/Δten compared to controls. Phenotypically, Col11a1Δten/Δten mice had significantly decreased body weights (p < 0.001), grip strengths (p < 0.001), and with age developed gait impairment becoming hypomobile. In the absence of Col11a1, the tendon collagen fibrillar matrix was abnormal when analyzed using transmission electron microscopy. Reducing Col11a1 and, therefore collagen XI content, resulted in abnormal fibril structure, loss of normal fibril diameter control with a significant shift to small diameters and disrupted parallel alignment of fibrils. These alterations in matrix structure were observed in developing (day 4), maturing (day 30) and mature (day 60) mice. Altering the time of knockdown using inducible I-Col11a1−/− mice indicated that the primary regulatory foci for collagen XI was in development. In mature Col11a1Δten/Δten FDLs a significant decrease in the biomechanical properties was observed. The decrease in maximum stress and modulus suggest that fundamental differences in the material properties in the absence of Col11a1 expression underlie the mechanical deficiencies. These data demonstrate an essential role for collagen XI in regulation of tendon fibril assembly and organization occurring primarily during development.

Suppressing PLCγ1 enhances osteogenic and chondrogenic potential of BMSCs

Phosphatidylcholine-specific phospholipase Cγ1 (PLCγ1) is involved in regulating cell metabolism. However, little is known how PLCγ1 directs BMSC differentiation. Here, we investigated the role of PLCγ1 in rat BMSC differentiation into osteoblasts and chondrocytes. The results of Alizarin red and Alcian blue staining showed that PLCγ1 inhibitor U73122 significantly enhanced the mineralization capacity and proteoglycan deposition of BMSCs. The results of qPCR technique and Western blot analysis showed that long-term treatment of U73122 enhanced COL1A1 and OPG mRNA levels and Collagen 1A1, BMP2, and p-Smad1/5/9 protein levels and that short-term treatment of U73122 enhanced COL2A1 and SOX9 mRNA levels and Collagen 2, SOX9, Aggrecan, TGF-β3, and p-Smad2/3 protein levels. Decreased p-mTOR and p-P38 contributed to enhanced osteogenic potentials of BMSCs and increased p-P38 contributed to enhanced chondrogenic potentials of BMSCs. The scaffold transplantation with U73122+BMSC was more efficacious than BMSC alone for osteochondral defect repair in a rat model. Therefore, suppressing PLCγ1 could improve the capacity to effectively use BMSCs for cell therapy of osteochondral defect.

Targeting Collagen Type III in Proteinuric Kidney Disease: Informing Drug Potential Using the Jaccard–Tanimoto Index

Collagenofibrotic glomerulopathy, a collagen type III kidney disease, is associated with proteinuria and accumulation ofcollagen type III in the glomerulus specifically the mesangium and/or capillary walls. The puromcyin aminonucleoside (PAN) nephropathy model was evaluated to examine the relation between COL3A1 mRNA and proteinuria. In Wistar rats administered PAN, a robust increase in proteinuria was accompanied by glomerular hypertrophy and expansion of both the Bowman’s capsule and Bowman’s space. An ~4-fold increase in renal COL3A1 mRNA was observed in the PAN cohort with urine protein exhibiting a direct (r = 0.8) and significant correlation with kidney COL3A1 mRNA level. Both Picrosirius red polarized microscopy and immunohistochemical analysis showed localization of collagen type III to the glomerular mesangium. Gene ontology-driven transcriptomic analysis reveals a robust COL3A1 network in the glomerular compartment.

NOTCH2 participates in Jagged1-induced osteogenic differentiation in human periodontal ligament cells

Jagged1 activates Notch signaling and subsequently promotes osteogenic differentiation in human periodontal ligament cells (hPDLs). The present study investigated the participation of the Notch receptor, NOTCH2, in the Jagged1-induced osteogenic differentiation in hPDLs. NOTCH2 and NOTCH4 mRNA expression levels increased during hPDL osteogenic differentiation. However, the endogenous NOTCH2 expression levels were markedly higher compared with NOTCH4. NOTCH2 expression knockdown using shRNA in hPDLs did not dramatically alter their proliferation or osteogenic differentiation compared with the shRNA control. After seeding on Jagged1-immobilized surfaces and maintaining the hPDLs in osteogenic medium, HES1 and HEY1 mRNA levels were markedly reduced in the shNOTCH2-transduced cells compared with the shControl group. Further, shNOTCH2-transduced cells exhibited less alkaline phosphatase enzymatic activity and in vitro mineralization than the shControl cells when exposed to Jagged1. MSX2 and COL1A1 mRNA expression after Jagged1 activation were reduced in shNOTCH2-transduced cells. Endogenous Notch signaling inhibition using a γ-secretase inhibitor (DAPT) attenuated mineralization in hPDLs. DAPT treatment significantly promoted TWIST1, but decreased ALP, mRNA expression, compared with the control. In conclusion, Notch signaling is involved in hPDL osteogenic differentiation. Moreover, NOTCH2 participates in the mechanism by which Jagged1 induced osteogenic differentiation in hPDLs.

Macrophage Subpopulations and the Acute Inflammatory Response of Elderly Human Skeletal Muscle to Physiological Resistance Exercise.

The current model for repair of damaged tissue includes immune cells, mediating the progression from a pro-inflammatory to an anti-inflammatory environment. How this process changes with aging in human skeletal muscle under conditions of physiological exercise loading remains unclear. To investigate this, 25 elderly males (mean age 70 ± SD 7 years), as well as 12 young (23 ± 3 years) and 12 elderly (74 ± 3 years) females, performed a unilateral bout of heavy resistance leg extension exercise. Biopsies were collected from the vastus lateralis muscle of the rested (control) leg, and post exercise from the exercised leg at 4.5 h, and on days 1, 4, and 7 for the male participants, or on day 5 for the female participants. Total macrophages (CD68+) as well as pro- (CD11b+) and anti-inflammatory (CD163+, CD206+) subpopulations were identified on sections by immunohistochemistry. Gene expression levels of COL1A1, TNF-a, CD68, myostatin, TCF7L2, IL-1B, IL-1R, IL-10, and Ki67 were determined by real-time RT-PCR. At rest, the muscle tissue from the elderly vs. young females was characterized by higher gene expression levels of CD68, IL-10, lower myostatin mRNA, and trends for a greater number of macrophages, while COL1A1 mRNA post exercise values were greater in the elderly vs young. For the male participants, mRNA levels of the inflammatory cytokines IL-1B, IL-1R were elevated in the early phase following exercise, followed by increases in COL1A1 and Ki67 on days 4 and 7. In general, exercise induced increases in all types of macrophages counted in the elderly, but not in young, individuals. Cells expressing CD68, CD11b, and CD206 simultaneously were the most frequently observed cell type, which raises the possibility that pure pro- and anti-inflammatory macrophages populations do not exist in healthy human skeletal muscle within the spectrum of tissue remodeling induced by physiological exercise designed to induce hypertrophy. Together these data provide insight into the time course of macrophage activity and associated molecular targets in human skeletal muscle in the context of aging and exercise.

Remodeling process in bone of aged rats in response to resistance training

AimsWe investigate the effects of RT on the mechanical function, gene, and protein expression of key factors involved in bone remodeling during aging. Main methodsMale rats of 3 and 21 months of age were randomly allocated into four groups (8 per group): young sedentary (YS), young trained (YT), old sedentary (OS), and old trained (OT). RT was performed three times per week (12 weeks). Bone tenacity and stiffness were measured by biomechanical tests and mRNA levels of COL1A1, MEPE, SOST, OPG, BMP-2, PPAR-y, MMP-2-9-13, and TIMP-1 were evaluated by quantitative PCR. COL1A1 protein and MMP-2 activity were detected by western blotting and zymography assays. Key findingsAging increased stiffness, while BMP-2, OPG, COL1A1 and MMP-2 mRNA levels reduced (OS vs YS; p ≤ 0.05). RT increased the tenacity of the femur and reduced PPAR-γ regardless of age (YT vs. YS; OT vs. OS; p ≤ 0.05). RT downregulated SOST mRNA levels only in the OT group (vs. OS group, p ≤ 0.05). RT mitigated the age-associated increase in MMP-9 mRNA levels (p ≤ 0.05). In young animals, upregulation in MEPE, MMP-13, TIMP-1 were observed after RT, as well an increase in COL1A1 protein and MMP-2 activity (p ≤ 0.05). SignificanceRT improved bone tenacity independent of aging, which is relevant for mechanical function, while, at protein levels, RT upregulated MMP-2 activity and collagen 1 only in young rats. This study highlights the importance of exercise on bone health and identifies specific molecular changes in response to RT. Our findings provide insights into the mechanisms involved in age-related changes.

Development of a novel in vitro insulin resistance model in primary human tenocytes for diabetic tendinopathy research.

BackgroundType 2 diabetes mellitus (T2DM) had been reported to be associated with tendinopathy. However, the underlying mechanisms of diabetic tendinopathy still remain largely to be discovered. The purpose of this study was to develop insulin resistance (IR) model on primary human tenocytes (hTeno) culture with tumour necrosis factor-alpha (TNF-α) treatment to study tenocytes homeostasis as an implication for diabetic tendinopathy.MethodshTenowere isolated from human hamstring tendon. Presence of insulin receptor beta (INSR-β) on normal tendon tissues and the hTeno monolayer culture were analyzed by immunofluorescence staining. The presence of Glucose Transporter Type 1 (GLUT1) and Glucose Transporter Type 4 (GLUT4) on the hTeno monolayer culture were also analyzed by immunofluorescence staining. Primary hTeno were treated with 0.008, 0.08, 0.8 and 8.0 µM of TNF-α, with and without insulin supplement. Outcome measures include 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) assay to determine the glucose uptake activity; colourimetric total collagen assay to quantify the total collagen expression levels; COL-I ELISA assay to measure the COL-I expression levels and real-time qPCR to analyze the mRNA gene expressions levels of Scleraxis (SCX), Mohawk (MKX), type I collagen (COL1A1), type III collagen (COL3A1), matrix metalloproteinases (MMP)-9 and MMP-13 in hTeno when treated with TNF-α. Apoptosis assay for hTeno induced with TNF-α was conducted using Annexin-V FITC flow cytometry analysis.ResultsImmunofluorescence imaging showed the presence of INSR-β on the hTeno in the human Achilles tendon tissues and in the hTeno in monolayer culture. GLUT1 and GLUT4 were both positively expressed in the hTeno. TNF-α significantly reduced the insulin-mediated 2-NBDG uptake in all the tested concentrations, especially at 0.008 µM. Total collagen expression levels and COL-I expression levels in hTeno were also significantly reduced in hTeno treated with 0.008 µM of TNF-α. The SCX, MKX and COL1A1 mRNA expression levels were significantly downregulated in all TNF-α treated hTeno, whereas the COL3A1, MMP-9 and MMP-13 were significantly upregulated in the TNF–α treated cells. TNF-α progressively increased the apoptotic cells at 48 and 72 h.ConclusionAt 0.008 µM of TNF-α, an IR condition was induced in hTeno, supported with the significant reduction in glucose uptake, as well as significantly reduced total collagen, specifically COL-I expression levels, downregulation of candidate tenogenic markers genes (SCX and MKX), and upregulation of ECM catabolic genes (MMP-9 and MMP-13). Development of novel IR model in hTeno provides an insight on how tendon homeostasis could be affected and can be used as a tool for further discovering the effects on downstream molecular pathways, as the implication for diabetic tendinopathy.

P0852PERICYTE-SPECIFIC MANIPULATION OF HYPOXIA-INDUCIBLE FACTORS REGULATES ERYTHROPOIESIS WITHOUT AGGRAVATING RENAL FIBROSIS

Abstract Background and Aims Stabilizers of hypoxia-inducible factor (HIF) have been shown to be effective on treatment of anemia in patients with chronic kidney disease (CKD). Increased erythropoietin (EPO) production and enhanced erythropoiesis are known to be the major mechanisms responsible for the treatment effects. However, the effect of HIF stabilization on renal fibrosis is controversial. We created animal models characterized by CKD and pericyte-specific stabilization of HIF to examine the effects of HIF on renal fibrosis and erythropoiesis. Method Gli1CreERT2/+;Egln1F/F, Gli1CreERT2/+;VhlF/F, and Gli1CreERT2/+;Hif1aF/F;Hif2aF/F mice were generated to study the effects of pericyte-specific overexpression or knockout of Hif. Unilateral ureteral obstruction (UUO) was used to induce CKD. The severity of fibrosis was determined by Picrosirius red stain and Col1a1 mRNA level in the kidney. Results Pericyte-specific stabilization of HIF resulted in increased serum EPO level, augmented splenic erythropoiesis, and polycythemia, while the severity of renal fibrosis was not affected. In line with these findings, pericyte-specific knockout of Hif1a or Hif2a did not result in significant change of renal fibrosis. Conclusion Our study endorses the neutral effects of pericyte-specific HIF stabilization on renal fibrosis.

Prostaglandins effect on matrix metallopeptidases and collagen in mare endometrial fibroblasts

An increasing number of studies have shown that prostaglandins (PGs) exert multiple regulatory actions in the processes associated to tissue remodeling and fibrosis. Extracellular matrix (ECM) turnover is mediated by matrix metallopeptidases (MMPs). The knowledge about the regulation of their expression in mare endometrium is still limited. Thus, the aim of this study was to investigate whether: (i) profibrotic transforming growth factor (TGF)-β1 modulates PG production in equine endometrium; and (ii) PGE2 and PGF2α modulate MMPs, their tissue inhibitors (TIMPs), and collagen 1 (COL1) expression. In experiment 1, the effect of TGF-β1 (5 ng/mL) on PG secretion and PG synthases mRNA transcription, after 24 and 48 h treatment of mare endometrial fibroblast and epithelial cells was investigated using ELISA and qPCR. In experiment 2, the effects of PGE2 and PGF2α in doses 10-7M and 10-8M on secretion and MMP1, 2, 9, 13, TIMP1, 2, and COL1A1 mRNA transcription in mare endometrial fibroblasts were assessed. Transforming growth factor-β1 treatment decreased secretion of PGF2α by endometrial fibroblasts (P < 0.05) and PGF2α and PGE2 by endometrial epithelial cells (P < 0.05). Prostaglandin E2 increased MMP-2 and MMP-9, and decreased MMP-13 secretion by endometrial fibroblasts (P < 0.05). Additionally, PGF2α treatment increased MMP-2, MMP-13 and COL1, but decreased MMP-1 secretion by endometrial fibroblasts (P < 0.05). Prostaglandins may be involved in the processes associated to pathological endometrial remodeling by their effect on MMP expression. The effect of PGF2α on COL1 secretion from fibroblasts suggests its profibrotic role in pathological endometrial remodeling.