Fibronectin Protein Levels Research Articles

O100 IL15RA MEDIATES BLOOD PRESSURE ELEVATION, INFLAMMATION AND VASCULAR DAMAGE IN ANGIOTENSIN II-INDUCED HYPERTENSION

Cytokines are key mediators regulating blood pressure (BP) and target organ damage in hypertension. IL15 levels increase in cardiovascular diseases, but the role of IL15 and its receptor alpha (IL15R) in hypertension and hypertension-mediated inflammation and vascular damage is still unknown. Wild-type and IL15Rα-/-mice (n=5-9) were infused with vehicle or angiotensin II (AngII, 490ng/min/kg). Recombinant IL15 was administered to WT mice (1ug, daily, 14 days) during AngII infusion. Telemetry, wire myography, luminometry, PSR staining, qPCR, immunoblotting, flow cytometry, and primary cell lines were used. Hypertensive patients have increased levels of IL15Rα in arteries, which significantly correlated with systolic (r=0.33, p=0.002), diastolic (r=0.27, p=0.02) pressure and maximal constriction to phenylephrine ex vivo (r=0.27, p=0.03). Similarly, AngII infusion significantly increased serum IL15/IL15Rα (8.8±0.7pg/ml vs 12.1±0.7pg/ml, p<0.01), IL15Rα mRNA (1±0.1 vs 1.8±0.1, p<0.01) and protein (1±0.03 vs 1.8±0.28, p<0.01) levels in the aortas. BP lowering using hydralazine and hydrochlorothiazide treatment prevented these changes in vasculature. IL15Rα was induced by pathophysiological stretch (15%) or interferon-gamma in adventitial fibroblasts, vascular smooth muscles, and endothelial cells in vitro. In contrast, AngII, other inflammatory cytokines or oxidative stress mediators have no such direct effect. Il15Rα-/- blunted AngII-induced hypertension (199±5.6mmHg vs 148±7.3mmHg, p<0.001), protected against endothelial dysfunction (p<0.01) and aortic superoxide production (59.4±7.8 vs 36.4±7.6 RLU/sec/mg) compared to wild-type mice. This was accompanied by reduced perivascular inflammation, lack of immune cell recruitment and effector and memory T cell formation. Similarly, T cell interferon-gamma and interleukin-17 production was attenuated in Il15Rα-/- hypertensive mice. In contrast, simultaneous administration of AngII and rIL15 into WT mice enhanced these outcomes. Perivascular fibrosis was observed in WT, but not Il15Rα-/- hypertensive animals (111036±19117 μm2 vs 48689±5515 μm2, p<0.01) and was in line with significantly lower mRNA of collagen and fibronectin protein levels in Il15Rα-/- aortas. IL15Rα plays a crucial role in immune activation, vascular damage, and AngII-induced hypertension. Targeting this pathway may offer potential therapeutic benefits for controlling hypertension and preventing associated vascular complications.

In Vivo and In Vitro Pro-Fibrotic Response of Lung-Resident Mesenchymal Stem Cells from Patients with Idiopathic Pulmonary Fibrosis.

Lung-resident mesenchymal stem cells (LR-MSC) are thought to participate in idiopathic pulmonary fibrosis (IPF) by differentiating into myofibroblasts. On the other hand, LR-MSC in IPF patients present senescence-related features. It is unclear how they respond to a profibrotic environment. Here, we investigated the profibrotic response of LR-MSC isolated from IPF and control (CON) patients. LR-MSC were inoculated in mice 48 h after bleomycin (BLM) instillation to analyze their contribution to lung damage. In vitro, LR-MSC were exposed to TGFβ. Mice inoculated with IPF LR-MSC exhibited worse maintenance of their body weight. The instillation of either IPF or CON LR-MSC sustained BLM-induced histological lung damage, bronchoalveolar lavage fluid cell count, and the expression of the myofibroblast marker, extracellular matrix (ECM) proteins, and proinflammatory cytokines in the lungs. In vitro, IPF LR-MSC displayed higher basal protein levels of aSMA and fibronectin than CON LR-MSC. However, the TGFβ response in the expression of TGFβ, aSMA, and ECM genes was attenuated in IPF LR-MSC. In conclusion, IPF LR-MSC have acquired myofibroblastic features, but their capacity to further respond to profibrotic stimuli seems to be attenuated. In an advanced stage of the disease, LR-MSC may participate in disease progression owing to their limited ability to repair epithelial damage.

Fluvoxamine supresses transforming growth factor beta‐2‐induced fibrotic changes in trabecular meshwork cells

Aims/Purpose: Primary open‐angle glaucoma is the leading cause of blindness worldwide, but no definitive treatment is available. Fibrotic‐like changes of trabecular meshwork (TM) results in increased stiffness and impaired outflow leading to increased IOP. Transforming growth factor‐β2 (TGF‐β2), a main driver of eye fibrosis, is increased in the aqueous humour of glaucoma patients. Recently, we proved that the specific sigma‐1 receptor agonist fluvoxamine (FLU) is antifibrotic in the kidney but its role in the TM is unknown. Therefore, we investigated the effects of FLU on the TGF‐β2‐induced fibrotic response in TM cells.Methods: Fibrosis of the AS was induced in C57BL/6J mice by intracameral TGF‐β2 injection. AS were collected and F‐actin was visualized with Phalloidin and detected with confocal microscopy. The effect of FLU on TGF‐β2‐induced cell proliferation, cytoskeletal rearrangement, and fibrosis‐related proteins was investigated by MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide) assay, Phalloidin staining and Western blotting, respectively.Results: TGF‐β2 injection resulted in F‐actin enhancement in vivo, indicating fibrotic‐like changes. In vitro, TGF‐β2 induced cytoskeletal rearrangement with increased F‐actin‐ bundles and clumps formation (p < 0.001) which were reduced by FLU (p < 0.001). Furthermore, FLU also inhibited cell proliferation (p < 0.05) in TGF‐β2‐treated cells. The profibrotic protein levels of connective tissue growth factor, fibronectin and collagen type IV were elevated by TGF‐β2 treatment (p < 0.001), while FLU prevented the elevation of these elements (p < 0.01; p < 0.001). FLU also hindered the TGF‐β2 caused decrease of Matrix Metalloproteinase‐2 (p < 0.05) thus facilitating ECM degradation.Conclusions: FLU may reduce the fibrotic response of trabecular meshwork and could be a potential candidate for the treatment of fibrosis‐induced ocular hypertension.Grants: OTKA‐ K135398, LP2021‐3/2021, TKP2021‐EGA‐24, Stipendium Hungaricum.

The renoprotective effects of taurine against diabetic nephropathy via the p38 MAPK and TGF-β/Smad2/3 signaling pathways.

Diabetic nephropathy (DN), a severe diabetes complication, causes kidney morphological and structural changes due to extracellular matrix accumulation. This accumulation is caused mainly by oxidative stress. Semi-essential amino acid derivative taurine has powerful antioxidant and antifibrotic effects. The aim of this study was to investigate the renoprotective effects of taurine through its possible roles in oxidative stress, extracellular matrix proteins, and the signaling pathways associated with the accumulation of extracellular matrix proteins in DN rats. 29 Wistar albino rats were randomly separated into control, taurine, diabetes, and diabetes + taurine groups. Diabetes animals were injected 45mg/kg streptozosine. Taurine is given by adding to drinking water as 1% (w/v). Urine, serum, and kidney tissue were collected from rats for biochemical and histological analysis after 12weeks. According to the studies, taurine significantly reduces the levels of malondialdehyde (MDA), total oxidant status (TOS), and protein expression of NADPH oxidase 4 (NOX4) that increase in diabetic kidney tissue. Also, decreased superoxide dismutase (SOD) activity levels significantly increased with taurine in diabetic rats. Moreover, increased mRNA and protein levels of fibronectin decreased with taurine. The matrix metalloproteinase (MMP)-2 and MMP-9 activities and their mRNA levels increased significantly, and this increase was significantly summed with taurine. There was a decrease in mRNA expression of Extracellular matrix metalloproteinase inducer (EMMPRIN). Taurine significantly increased this decrease. Diabetes increased mRNA expressions of transforming growth factor (TGF)-β and Smad2/3. Taurine significantly reduced this induction. TGF-β protein expression, p38, and Smad2/3 activations were also inhibited, but taurine was suppressed significantly. All these findings indicate that taurine may be an effective practical strategy to prevent renal diabetic injury.

MicroRNA-145-5p suppresses cell proliferation, migration, and invasion in upper tract urothelial carcinoma by targeting 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase.

Upper tract urothelial carcinoma (UTUC), including renal, pelvic, and ureteral carcinoma, has a high incidence rate in Taiwan, which is different from that in Western countries. Therefore, it is imperative to elucidate the mechanisms underlying UTUC growth and metastasis. To explore the function of miR-145-5p in UTUC, we transfected the BFTC909 cell line with miR-145-5p mimics and analyzed the differences in protein levels by performing two-dimensional polyacrylamide gel electrophoresis. Real-time polymerase chain reaction and Western blot analysis were used to analyze 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inositol monophosphate cyclohydrolase (ATIC) messenger RNA and protein levels. A dual-luciferase assay was performed to identify the target of miR-145-5p in ATIC. The effects of miR-145-5p and ATIC expression by cell transfection on cell proliferation, migration, and invasion were also assessed. miR-145-5p downregulated ATIC protein expression. High ATIC expression is associated with tumor stage, metastasis, recurrence, and a poor prognosis in patients with UTUC. Cell function assays revealed that ATIC knockdown inhibited the proliferation, migration, and invasive abilities of UTUC cells. In contrast, miR-145-5p affected the proliferation, migration, and invasive abilities of UTUC cells by directly targeting the 3'-untranslated regions of ATIC. Furthermore, we used RNA sequencing and Ingenuity Pathway Analysis to identify possible downstream genes regulated by ATIC and found that miR-145-5p regulated the protein levels of fibronectin 1, Slug, cyclin A2, cyclin B1, P57, and interferon-induced transmembrane 1 via ATIC. ATIC may be a valuable predictor of prognosis and a potential therapeutic target for UTUC.

Sigma-1 Receptor Activation Is Protective against TGFβ2-Induced Extracellular Matrix Changes in Human Trabecular Meshwork Cells.

The trabecular meshwork (TM) route is the principal outflow egress of the aqueous humor. Actin cytoskeletal remodeling in the TM and extracellular matrix (ECM) deposition increase TM stiffness, outflow resistance, and elevate intraocular pressure (IOP). These alterations are strongly linked to transforming growth factor-β2 (TGFβ2), a known profibrotic cytokine that is markedly elevated in the aqueous humor of glaucomatous eyes. Sigma-1 receptor (S1R) has been shown to have neuroprotective effects in the retina, but data are lacking about its role in the TM. In this study, we identified the presence of S1R in mouse TM tissue and investigated the effect of an S1R agonist fluvoxamine (FLU) on TGFβ2-induced human TM cells regarding cell proliferation; ECM-related functions, including F-actin reorganization; and the accumulation of ECM elements. TGFβ2 increased the proliferation, cytoskeletal remodeling, and protein levels of fibronectin, collagen type IV, and connective tissue growth factor, and decreased the level of matrix metalloproteinase-2. Most importantly, FLU reversed all these effects of TGFβ2, suggesting that S1R agonists could be potential candidates for preserving TM function and thus maintaining normal IOP.

Long non-coding ribonucleic acid zinc finger E-box binding homeobox 1 antisense RNA 1 regulates myocardial fibrosis in diabetes through the Hippo-Yes-associated protein signaling pathway.

Fibrosis is the principle reason for heart failure in diabetes. Regarding the involvement of long non-coding ribonucleic acid zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) in diabetic myocardial fibrosis, we explored its specific mechanism. Human cardiac fibroblasts (HCF) were treated with high glucose (HG) and manipulated with plasmid cloning deoxyribonucleic acid 3.1-ZEB1-AS1/microribonucleic acid (miR)-181c-5p mimic/short hairpin RNA specific to sirtuin 1 (sh-SIRT1). ZEB1-AS1, miR-181c-5p expression patterns, cell viability, collagen I and III, α-smooth muscle actin (α-SMA), fibronectin levels and cell migration were assessed by reverse transcription quantitative polymerase chain reaction, cell counting kit-8, western blot and scratch tests. Nuclear/cytosol fractionation assay verified ZEB1-AS1 subcellular localization. The binding sites between ZEB1-AS1 and miR-181c-5p, and between miR-181c-5p and SIRT1 were predicted and verified by Starbase and dual-luciferase assays. The binding of SIRT1 to Yes-associated protein (YAP) and YAP acetylation levels were detected by co-immunoprecipitation. Diabetic mouse models were established. SIRT1, collagen I, collagen III, α-SMA and fibronectin levels, mouse myocardium morphology and collagen deposition were determined by western blot, and hematoxylin-eosin and Masson trichrome staining. Zinc finger E-box binding homeobox 1 antisense 1 was repressed in HG-induced HCFs. ZEB1-AS1 overexpression inhibited HG-induced HCF excessive proliferation, migration and fibrosis, and diminished collagen I, collagen III, α-SMA and fibronectin protein levels in cells. miR-181c-5p had targeted binding sites with ZEB1-AS1 and SIRT1. SIRT1 silencing/miR-181c-5p overexpression abrogated ZEB1-AS1-inhibited HG-induced HCF proliferation, migration and fibrosis. ZEB1-AS1 suppressed HG-induced HCF fibrosis through SIRT1-mediated YAP deacetylation. ZEB1-AS1 and SIRT1 were repressed in diabetic mice, and miR-181c-5p was promoted. ZEB1-AS1 overexpression improved myocardial fibrosis in diabetic mice, and reduced collagen I, collagen III, α-SMA and fibronectin protein levels in myocardial tissues. Long non-coding ribonucleic acid ZEB1-AS1 alleviated myocardial fibrosis through the miR-181c-5p-SIRT1-YAP axis in diabetic mice.

Enhanced reparatory effect of EI1 on dental pulp via extracellular matrix remodeling by miR-181b-2-3p inhibitor

Background/purposeExtracellular matrix (ECM) is crucial for dental pulp repair. The aim of this paper is to investigate the ECM remodeling effect of miR-181b-2-3p (a microRNA) and to verify the reparatory effect of EI1 (an epigenetic drug) and miR-181b-2-3p inhibitor on dental pulp. Materials and methodsLevels of ECM-related factors in EI1-treated human dental pulp cells (hDPCs) were measured by qRT-PCR and Western blot. The anti-inflammation effect of EI1 was examined in Lipopolysaccharide-stimulated hDPCs. miR-181b-2-3p mimics or inhibitors were transfected into hDPCs and then the cells’ functions were detected. A dual luciferase reporter assay was used to identify the targets of miR-181b-2-3p. Pulpotomy using miR-181b-2-3p antagomirs and EI1 as pulp capping materials was performed in male six-week-old Sprague-Dawley rats. ResultsEI1 upregulated ECM-related genes expression in hDPCs, but failed to upregulate the collagen1A1 (COL1A1) protein level. Pro-inflammatory factors were downregulated by EI1 in Lipopolysaccharide-stimulated hDPCs. Overexpression of miR-181b-2-3p downregulated the expression of transforming growth factor-β2 (TGF-β2) and fibronectin type III domain-containing protein 5 precursor (FNDC5), while the inhibition had the opposite effect. Dual luciferase reporter assays demonstrated that miR-181b-2-3p targets TGF-β2, FNDC5 and integrin alpha 4 protein (ITGA4). Compared to EI1 was used alone, EI1 combined with the inhibitor upregulated the protein levels of COL1A1, fibronectin (FN1) and TGF-β2 in hDPCs, promoted hDPCs migration, and exhibited reparatory effects on inflamed rat pulp tissue. ConclusionmiR-181b-2-3p inhibitor could enhance the reparatory effect of EI1 via ECM remodeling in dental pulp both in vitro and in vivo.

Simvastatin Attenuates Glucocorticoid-Induced Human Trabecular Meshwork Cell Dysfunction via YAP/TAZ Inactivation

Purpose Impairment of the trabecular meshwork (TM) is the principal cause of increased outflow resistance in the glaucomatous eye. Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) are emerging as potential mediators of TM cell/tissue dysfunction. Furthermore, YAP/TAZ activity was recently found to be controlled by the mevalonate pathway in non-ocular cells. Clinically used statins block the mevalonate cascade and were shown to improve TM cell pathobiology; yet, the link to YAP/TAZ signaling was not investigated. In this study, we hypothesized that simvastatin attenuates glucocorticoid-induced human TM (HTM) cell dysfunction via YAP/TAZ inactivation. Methods Primary HTM cells were seeded atop or encapsulated within bioengineered extracellular matrix (ECM) hydrogels. Dexamethasone was used to induce a pathologic phenotype in HTM cells in the absence or presence of simvastatin. Changes in YAP/TAZ activity, actin cytoskeletal organization, phospho-myosin light chain levels, hydrogel contraction/stiffness, and fibronectin deposition were assessed. Results Simvastatin potently blocked pathologic YAP/TAZ nuclear localization/activity, actin stress fiber formation, and myosin light chain phosphorylation in HTM cells. Importantly, simvastatin co-treatment significantly attenuated dexamethasone-induced ECM contraction/stiffening and fibronectin mRNA and protein levels. Sequential treatment was similarly effective but did not match clinically-used Rho kinase inhibition. Conclusions YAP/TAZ inactivation with simvastatin attenuates HTM cell pathobiology in a tissue-mimetic ECM microenvironment. Our data may help explain the association of statin use with a reduced risk of developing glaucoma via indirect YAP/TAZ inhibition as a proposed regulatory mechanism.

Keloid Core Factor CTRP3 Overexpression Significantly Controlled TGF-β1-Induced Propagation and Migration in Keloid Fibroblasts.

Keloid is a type of benign fibrous proliferative tumor characterized by excessive scarring. C1q/TNF-related protein 3 (CTRP3) has been proven to possess antifibrotic effect. Here, we explored the role of CTRP3 in keloid. In the current research, we examined the influence of CTRP3 on keloid fibroblasts (KFs) and investigated the potential molecular mechanism. KF tissue specimens and adjacent normal fibroblast (NF) tissues were collected cultured from 10 keloid participants. For the TGF-β1 stimulation group, KFs were processed with human recombinant TGF-β1. Cell transfection of pcDNA3.1-CTRP3 or pcDNA3.1 was performed. The siRNA of CTRP3 (si-CTRP3) or negative control siRNA (si-scramble) was transfected into KFs. CTRP3 was downregulated in keloid tissues and KFs. CTRP3 overexpression significantly controlled TGF-β1-induced propagation and migration in KFs. Col I, α-SMA, and fibronectin mRNA and protein levels were enhanced by TGF-β1 stimulation, whereas they were inhibited by CTRP3 overexpression. In contrast, CTRP3 knockdown exhibited the opposite effect. In addition, CTRP3 attenuated TGF-β receptors TRI and TRII in TGF-β1-induced KFs. Furthermore, CTRP3 prevented TGF-β1-stimulated nuclear translocation of smad2 and smad3 and suppressed the expression levels of p-smad2 and p-smad3 in KFs. CTRP3 exerted an antifibrotic role through inhibiting proliferation, migration, and ECM accumulation of KFs via regulating TGF-β1/Smad signal path.

Long-term exercise preserves pancreatic islet structure and β-cell mass through attenuation of islet inflammation and fibrosis.

Islet fibrosis is associated with the disruption of islet structure and contributes to β-cell dysfunction, playing an essential role in the pathogenesis of type 2 diabetes. Physical exercise has been shown to attenuate fibrosis in various organs; however, the effect of exercise on islet fibrosis has not been defined. Male Sprague-Dawley rats were divided into four groups: normal diet sedentary [N-Sed], normal diet + exercise [N-Ex], high-fat diet sedentary [H-Sed], and high-fat diet + exercise [H-Ex]. After 60 weeks of exercise, 4452 islets from Masson-stained slides were analyzed. Exercise led to a 68% and 45% reduction in islet fibrosis in the normal and high-fat diet groups and was correlated with a lower serum blood glucose. Fibrotic islets were characterized by irregular shapes and substantial loss of β-cell mass, which were significantly reduced in the exercise groups. Remarkably, the islets from exercised rats at week 60 were morphologically comparable to those of sedentary rats at 26 weeks. In addition, the protein and RNA levels of collagen and fibronectin, and the protein levels of hydroxyproline in the islets were also attenuated by exercise. This was accompanied by a significant reduction in inflammatory markers in the circulation Interleukin-1 beta (IL-1β)] and pancreas [IL-1β, Tumor Necrosis Factor-alpha, Transforming Growth Factor-β, and Phosphorylated Nuclear Factor Kappa-B p65subunit], lower macrophage infiltration, and stellate cell activation in the islets of exercised rats. In conclusion, we have demonstrated that long-term exercise preserves pancreatic islet structure and β-cell mass through anti-inflammatory and anti-fibrotic actions, suggesting additional rationales for the success of exercise training in the prevention and treatment of type 2 diabetes that should be further explored.

Effects of long-term tubular HIF-2α overexpression on progressive renal fibrosis in a chronic kidney disease model

Renal fibrosis is the final manifestation of chronic kidney disease (CKD) regardless of etiology. Hypoxia-inducible factor-2 alpha (HIF-2α) is an important regulator of chronic hypoxia, and the late-stage renal tubular HIF-2α activation exerts protective effects against renal fibrosis. However, its specific role in progressive renal fibrosis remains unclear. Here, we investigated the effects of the long-term tubular activation of HIF-2α on renal function and fibrosis, using in vivo and in vitro models of renal fibrosis. Progressive renal fibrosis was induced in renal tubular epithelial cells (TECs) of tetracycline-controlled HIF-2α transgenic (Tg) mice and wild-type (WT) controls through a 6-week adenine diet. Tg mice were maintained on doxycycline (DOX) for the diet period to induce Tg HIF-2α expression. Primary TECs isolated from Tg mice were treated with DOX (5 μg/ml), transforming growth factor-β1 (TGF-β1) (10 ng/ml), and a combination of both for 24, 48, and 72 hr. Blood was collected to analyze creatinine (Cr) and blood urea nitrogen (BUN) levels. Pathological changes in the kidney tissues were observed using hematoxylin and eosin, Masson's trichrome, and Sirius Red staining. Meanwhile, the expression of fibronectin, E-cadherin and α-smooth muscle actin (α-SMA) and the phosphorylation of p38 mitogenactivated protein kinase (MAPK) was observed using western blotting. Our data showed that serum Cr and BUN levels were significantly lower in Tg mice than in WT mice following the adenine diet. Moreover, the protein levels of fibronectin and E-cadherin and the phosphorylation of p38 MAPK were markedly reduced in the kidneys of adenine-fed Tg mice. These results were accompanied by attenuated fibrosis in Tg mice following adenine administration. Consistent with these findings, HIF-2α overexpression significantly decreased the expression of fibronectin in TECs, whereas an increase in α-SMA protein levels was observed after TGF-β1 stimulation for 72 hr. Taken together, these results indicate that long-term HIF-2α activation in CKD may inhibit the progression of renal fibrosis and improve renal function, suggesting that long-term renal HIF-2α activation may be used as a novel therapeutic strategy for the treatment of CKD. [BMB Reports 2023; 56(3): 196-201].

Effects of knockdown ACC1 on glioma U251 cell migration and its mechanisms

Objective: To investigate the effects of ACC1 knockdown on human glioma U251 cell migration and its molecular mechanisms. Methods: Human glioma U251 cell line was used. The experiment was carried out in three steps. Experiment 1: knockdown of ACC1 in U251 cells (shACC1) and its control (NC) U251 cells were established by transfection of shACC1 lentivirus and negative control virus. The cell migration was detected by Transwell migration assay and scratch test. Western blot (WB) was performed to detect the levels of ACC1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Experiment 2: RT-qPCR and WB were performed to verify the RNA-seq result, upregulation effect of ACC1 knockdown on PAI-1 in U251 cells. The cells then were treated with PAI-1 inhibitor PAI-039, and the cell migration was detected by Transwell migration assay and scratch assay. The protein levels of ACC1, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug were examined by WB. Experiment 3: the molecular mechanisms of knocking down ACC1 to increase PAI-1 were explored. The cells were treated with acetyltransferase inhibitor C646, and cell migration was examined by Transwell migration assay and scratch assay. WB was conducted to test the levels of ACC1, H3K9ac, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Each experiment was repeated three times. Results: Experiment 1: lentivirus transfection was performed on glioma U251 cells. Compared with NC group, the expression level of ACC1 in shACC1 group was decreased significantly, indicating that lentivirus transfection was successful (P＜0.01), and the number of migrated cells in shACC1 group was increased significantly (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Experiment 2: Compared with NC group, PAI-1 mRNA level in shACC1 group was up-regulated. Compared with control group, cell migration in shACC1+PAI-039 group was decreased (P＜0.01), and migration-related proteins Vimentin, Fibronectin, N-cadherin, and Slug were up-regulated. E-cadherin expression was down-regulated (P＜0.01). Experiment 3: Compared with NC group, the concentration of acetyl-coA and the expression level of H3K9ac in shACC1 group were increased significantly (P＜0.01); After further treatment with histone acetyl transferase inhibitor C646, PAI-1 mRNA level was decreased, cell migration number and H3K9ac expression level were decreased in shACC1+C646 group compared with control group (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Conclusion: Knockdown of ACC1 promotes the migration of human glioma U251 cells by increasing histone acetylation which elevates the level of PAI-1.

Experimental Study on Danggui Shaoyao San Improving Renal Fibrosis by Promoting Autophagy.

Renal fibrosis could lead to chronic kidney disease (CKD) developing into the end-stage with its pathological manifestation is the deposition of extracellular matrix (ECM). Danggui Shaoyao San (DSS) is one of the widely used herbal formulas in ancient China, which has been proven to have efficacy in the treatment of CKD. The experiment employed TGF-β1 to stimulate the NRK-52E cells to establish a renal fibrosis model. With rapamycin (RAPA) used as the positive control, we detected the expression of fibronectin (FN), caspase-3, and autophagy-related proteins in the NRK-52E cells treated with DSS by Western blot and immunofluorescence assay. In order to further verify autophagy-promoting effects of DSS, we adopted 3-MA to inhibit autophagy. The experiment has found that DSS can lower the protein levels of FN and caspase-3 in the NRK-52E cells induced by TGF-β1. After TGF-β1 stimulation, the expression of LC3 II/I and Beclin 1 has decreased, and the protein levels of mTOR and p62 have increased. Consistent with rapamycin, DSS has significantly reduced these effects of TGF-β1. It has also been found that DSS can increase the expression of LC3 II/I and Beclin 1 proteins and can reduce the level of mTOR in cells treated with 3-MA, suggesting that DSS can promote autophagy. In conclusion, DSS has been proved to reduce the apoptosis and fibrosis of NRK-52E cells induced by TGF-β1, which may be achieved by promoting autophagy.

TRPM7 restrains plasmin activity and promotes transforming growth factor-β1 signaling in primary human lung fibroblasts

Sustained exposure of the lung to various environmental or occupational toxins may eventually lead to pulmonary fibrosis, a devastating disease with no cure. Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins such as fibronectin and collagens. The peptidase plasmin degrades the ECM, but protein levels of the plasmin activator inhibitor-1 (PAI-1) are increased in fibrotic lung tissue, thereby dampening plasmin activity. Transforming growth factor-β1 (TGF-β1)-induced activation of SMAD transcription factors promotes ECM deposition by enhancing collagen, fibronectin and PAI-1 levels in pulmonary fibroblasts. Hence, counteracting TGF-β1-induced signaling is a promising approach for the therapy of pulmonary fibrosis. Transient receptor potential cation channel subfamily M Member 7 (TRPM7) supports TGF-β1-promoted SMAD signaling in T-lymphocytes and the progression of fibrosis in kidney and heart. Thus, we investigated possible effects of TRPM7 on plasmin activity, ECM levels and TGF-β1 signaling in primary human pulmonary fibroblasts (pHPF). We found that two structurally unrelated TRPM7 blockers enhanced plasmin activity and reduced fibronectin or PAI-1 protein levels in pHPF under basal conditions. Further, TRPM7 blockade strongly inhibited fibronectin and collagen deposition induced by sustained TGF-β1 stimulation. In line with these data, inhibition of TRPM7 activity diminished TGF-β1-triggered phosphorylation of SMAD-2, SMAD-3/4-dependent reporter activation and PAI-1 mRNA levels. Overall, we uncover TRPM7 as a novel supporter of TGF-β1 signaling in pHPF and propose TRPM7 blockers as new candidates to control excessive ECM levels under pathophysiological conditions conducive to pulmonary fibrosis.

Multidirectional activity of bakuchiol against cellular mechanisms of facial ageing - Experimental evidence for a holistic treatment approach.

ObjectiveSkin ageing is a multifactorial process involving formation of reactive oxygen species, consecutive inflammation with reduced epidermal and dermal cell viability and resulting damage to the extracellular matrix. Effective dermocosmetic treatment modalities should ideally address these hallmarks in a holistic approach. Here, we determined the corresponding activity profile of bakuchiol, a plant‐derived meroterpene, in an array of in vitro, ex vivo and in vivo studies and compared it to retinol, currently considered as gold standard in topical antiageing cosmetics.MethodsThe antioxidative capacity and power of bakuchiol and retinol were analysed by measuring 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH) reduction via its absorption decay and electron spin resonance spectroscopy, respectively. Effects on prostaglandin E2 (PGE2), macrophage migration inhibitory factor (MIF), fibroblast growth factor 7 (FGF7), collagen type I and VII (COL1A1, COL7A1), fibronectin (FN) levels as well as the metabolization of water‐soluble tetrazolium 1 (WST‐1) were determined in human dermal fibroblasts. Epidermal regeneration was assessed utilizing an in vitro wound healing model. FN protein levels were analysed ex vivo after treatment with a formulation containing bakuchiol, retinol or vehicle using suction blister fluid. Skin condition improvement was determined in vivo in a split‐face comparison study after application of bakuchiol or vehicle.ResultsIn contrast to retinol, bakuchiol demonstrated high antioxidative efficacy. Levels of PGE2 and MIF were significantly decreased by both bakuchiol and retinol. Bakuchiol but not retinol significantly increased FGF7 protein levels. WST‐1 metabolization levels were significantly augmented by bakuchiol and retinol. Bakuchiol and retinol application led to a significant augmentation of COL1A1, COL7A1 and FN protein levels. Wounds supplemented with bakuchiol but not retinol displayed a significant increase in epidermis regeneration. Clinically, areas treated with a bakuchiol‐containing formulation showed a statistically significant increase in FN protein values after a 4‐week application compared to untreated areas and areas treated with vehicle.ConclusionThese data provide evidence for the multidirectional efficacy of bakuchiol against cellular hallmarks of skin ageing. Its activity profile shares some common features with retinol but demonstrates several hitherto unknown biopositive effects in our studies, namely stimulation of the critical extracellular matrix component FN, and accelerated epidermal regeneration and wound healing.

Meleagrin Isolated from the Red Sea Fungus Penicillium chrysogenum Protects against Bleomycin-Induced Pulmonary Fibrosis in Mice.

The Red Sea marine fungus Penicillium chrysogenum (Family: Ascomycota) comprises a panel of chemically diverse natural metabolites. A meleagrin alkaloid was isolated from deep-sediment-derived P. chrysogenum Strain S003 and has been reported to exert antibacterial and cytotoxic activities. The present study aimed to explore the therapeutic potential of meleagrin on pulmonary fibrosis. Lung fibrosis was induced in mice by a single intratracheal instillation of 2.5 mg/kg bleomycin. Mice were given 5 mg/kg meleagrin daily either for 3 weeks after bleomycin administration in the treatment group or 2 weeks before and 3 weeks after bleomycin administration in the protection group. Bleomycin triggered excessive ROS production, inflammatory infiltration, collagen overproduction and fibrosis. Bleomycin-induced pulmonary fibrosis was attenuated by meleagrin. Meleagrin was noted to restore the oxidant–antioxidant balance, as evidenced by lower MDA contents and higher levels of SOD and catalase activities and GSH content compared to the bleomycin group. Meleagrin also activated the Nrf2/HO-1 antioxidant signaling pathway and inhibited TLR4 and NF-κB gene expression, with a subsequent decreased release of pro-inflammatory cytokines (TNF-α, IL-6 and IFN-γ). Additionally, meleagrin inhibited bleomycin-induced apoptosis by abating the activities of pro-apoptotic proteins Bax and caspase-3 while elevating Bcl2. Furthermore, it suppressed the gene expression of α-SMA, TGF-β1, Smad-2, type I collagen and MMP-9, with a concomitant decrease in the protein levels of TGF-β1, α-SMA, phosphorylated Smad-2, MMP-9, elastin and fibronectin. This study revealed that meleagrin’s protective effects against bleomycin-induced pulmonary fibrosis are attributed to its antioxidant, anti-inflammatory, anti-apoptotic and antifibrotic properties. Notably, the use of meleagrin as a protective agent against bleomycin-induced lung fibrosis was more efficient than its use as a treatment agent.

FC089: The Absence of Sting Prevents Peritoneal Damage in a Murine Model of Peritoneal Fibrosis

Abstract BACKGROUND AND AIMS Peritoneal dialysis (PD) is the current replacement therapy for end-stage renal disease (ESRD) patients until renal transplantation can be achieved. However, chronic exposure to non-physiological PD fluids (PDF) and occasional infection episodes may lead to peritoneal damage and membrane transport failure. Chronic inflammation, together with mesothelial-to-mesenchymal transition (MMT) and submesothelial fibrosis, is a hallmark of PDF-associated peritoneal damage whose intrinsic mechanisms are still expected to be disclosed [1–2]. Activation of the innate immunity is the cornerstone for infectious agents’ elimination, also enrolled in response to damage-associated molecular patterns (DAMPs) released from stressed or injured host cells. Leakage of DNA from stressed mitochondria and damaged genomic DNA may act as DAMPs that are recognized by cytosolic double-strand DNA sensors, like the stimulator of interferon gene (STING), to mount an inflammatory response [3]. Excessive activation of STING is involved in autoinflammatory, autoimmune, and increasingly related to several chronic inflammatory conditions [4]. Thus, this study aimed to investigate the role of STING in experimental peritoneal damage. METHOD We induced peritoneal fibrosis in wild-type and STING-KO mice by intraperitoneal administration of 0.1% chlorhexidine gluconate (CHX) daily for 4 weeks. No treated WT and STING-KO mice were used as control. Then, mice were euthanized, and peritoneal tissue and peritoneal lavage fluids were recollected for the following experiments. STING mRNA (Tmem173) and protein levels in control and CHX-treated wild-type were measured by RT-qPCR, western blot and immunohistochemistry. Peritoneal levels of the STING downstream proteins TBK1 and IRF3 and their phosphorylated isoforms were assayed by western blot. Peritoneal membrane thickness was measured in formalin-fixed paraffin-embedded tissues stained with Masson's trichrome stain. Fibronectin protein levels were evaluated by western blot and MMT, fibrosis, and inflammation markers were determined by RT-qPCR. Inflammatory cells present in peritoneal effluents were assayed by flow cytometry. RESULTS We found that in the peritoneum of CHX-treated wild-type mice both Tmem173 expression and STING protein levels were increased. Interestingly, STING-positive cells were located in areas of peritoneal thickness and immune infiltration. Moreover, the protein levels of IRF3, p-IRF3, TBK1 and p-TBK1 were increased, indicating that the STING pathway is activated in experimental peritoneal damage. In STING-KO mice, CHX-induced peritoneal damage was prevented. STING-KO mice under CHX treatment showed decreased peritoneal membrane thickness and attenuated CHX-induced expression of fibrosis and MMT markers, such as Tgb1, Snai1, Cdh2, Col1a1, Fn1 and fibronectin protein when compared with CHX-treated WT mice. The absence of STING also prevented the gene expression of cytokines (Il1b, Il6 and Ifng) and chemokines (Ccl5, Ccl2 and Ccl19) and macrophages infiltration into the peritoneum. Inflammatory cell recruitment into the peritoneal cavity was also decreased in STING-KO mice, including CD3+CD4+ lymphocytes and CD11b+F4/80+ macrophages. CONCLUSION In summary, STING absence prevents peritoneal membrane thickness, fibrosis, and inflammation induced by CHX in a mice model. These results suggest that STING plays a key role in peritoneal membrane injury. Therefore, STING may become a new potential therapeutic target in PD-associated peritoneal damage.

Sigmar1 Deficiency Results in Pulmonary Fibrosis Associated with Increased Pulmonary Inflammation and Surfactant Protein Levels in Mice Lung

BackgroundSigmar1 is a ubiquitously expressed multifunctional molecular chaperone protein. Extensive studies demonstrated Sigmar1’s molecular functions in the central nervous system, and mutations in the Sigmar1 gene showed association with different neurodegenerative diseases, including amyotrophic lateral sclerosis and distal hereditary motor neuropathy. Studies also showed the protective roles of Sigmar1 in cardiovascular diseases in animal models of cardiac hypertrophy and heart failure. Recent studies focused on Sigmar1 in COVID‐19 drug repurposing as Sigmar1 was identified to interact with SARS‐Cov‐2 replication proteins using proteomic screening and Sigmar1 ligands showed promising protective effects in clinical trials. Despite all these studies showing a function of Sigmar1 in the respiratory system, the Sigmar1’s expression, localization, molecular and physiological functions in the lung remained unknown.ObjectivesThe main objective of this study was to characterize the expression and localization of Sigmar1 in human and mouse lungs. We also investigated the pathophysiological role of Sigmar1 in the lung using Sigmar1 null mice.ResultsUsing a combination of immunohistochemistry, immunofluorescence, and biochemical experiments, we confirmed the expression of Sigmar1 in rodent and human lungs. Hematoxylin and Eosin staining showed that Sigmar1 global knockout (Sigmar1‐/‐) mice lungs develop altered alveolar structure and organization and higher immune‐cells infiltration compared to Wildtype (Wt) littermate controls. This was associated with an upregulated protein level of phospho‐NFκB in Sigmar1‐/‐ mice lungs. Sirius red and Masson’s trichrome staining of lung tissue sections showed increased collagen deposition, pulmonary fibrotic remodeling associated with elevated fibronectin and phospho‐SMAD2 protein levels in Sigmar1‐/‐ mice lungs. Ultrastructural analysis by transmission electron microscopy (TEM) showed structural alteration in the multilamellar bodies with partial loss of lipid lamellae in alveolar‐type II epithelial cells (AV‐II) in Sigmar1‐/‐ mice lungs. Multilamellar bodies in AV‐II cells serve as secretory organelles releasing pulmonary surfactants into the extracellular space. Elevated levels of surfactant proteins (SP), SP‐A and SP–D, were reported to serve as biomarkers for acute respiratory failure and lung inflammation. Consistent with the TEM data, we also found increased SP (A, B, C, and D) protein levels in the lungs indicating the AV‐II pneumocytes dysfunction in Sigmar1‐/‐ mice.ConclusionOur findings suggested that Sigmar1’s lack of function leads to increased pulmonary inflammation, pulmonary fibrotic remodeling, altered structure and functions of multilamellar bodies, and AV‐II pneumocytes dysfunction in Sigmar1‐/‐mice.

Regulation of Extracellular Matrix Gene Expression by Desmosomal Cadherins

Desmosomes are protein complexes crucial for maintaining cell‐cell adhesion and integrity of tissues. These complexes are made up of proteins from three families: transmembrane cadherins (Desmoglein and Desmocollin) link adjacent cells in the extracellular space, armadillo proteins (Plakophilin and Plakoglobin) stabilize the intracellular plaque, and the cytolinker Desmoplakin (DP) connects the plaque to the intermediate filament network. Desmosomal proteins have also been shown to coordinate gene expression pathways required for processes such as proliferation, differentiation and cell migration. In particular, several lines of evidence have linked desmosomal proteins to gene expression of extracellular matrix (ECM) proteins. Loss of Plakophilin‐2 or Desmoplakin causes increases in expression of fibronectin and collagen, while in contrast, loss of plakoglobin results in a significant decrease in expression of fibronectin. These data indicate that individual components of the desmosome control ECM gene expression via distinct cellular signaling networks. In our study, we sought to investigate the role of desmosomal cadherins in ECM gene expression, via use of A431 cells lacking Desmoglein‐2, generated via CRISPR‐mediated knock‐out. Compared to control cells, Dsg2 KO cells demonstrated a dramatic increase in expression of Fibronectin (Fn1) mRNA, and relatively minor changes in expression of Collagen 1 (Col1a1) and Collagen 2 (Col2a1) mRNA. An increase in expression of Fibronectin protein levels in Dsg2‐deficient cells was also observed via western blot. We show that loss of Dsg2 KO also caused a significant increase in mRNA expression of the pro‐fibrotic signaling molecule transforming growth factor beta 2 (Tgfb2), but not Tgfb1 or Tgfb3. Increased expression of Fn1, Col1a1, Col2a1 and Tgfb2 was also observed upon siRNA‐mediated knockdown of Dsg2 in both A431 cells and HaCaT keratinocytes, verifying that these changes are not clone‐specific or due to off‐target CRISPR effects. siRNA‐mediated knockdown of other desmosomal proteins could not rescue Fn1 or Tgfb2 increases in Dsg2 KO cells, indicating that changes are not due to mislocalization of other desmosomal components. An extensive analysis of signaling pathways known to be involved in regulation of ECM gene expression uncovered an important role for PI3‐Kinase and Akt signaling, as inhibition of either signaling protein resulted in a rescue of Dg2KO‐mediated increases in Fn1 gene expression. Moreover, we have documented a significant increase in phosphorylation of Akt in Dsg2‐deficient cells. Taken together, our study highlights a novel role for Dsg2 in mediating ECM gene expression via PI3‐K/Akt signaling, adding significant insight into the mechanisms by which desmosomal cadherins control the adhesive behavior of cancer cells.