Increased Expression Of α-SMA Research Articles

SR9009 attenuates TGF-β1-induced renal fibrotic responses by inhibiting the NOX4/p38 signaling pathway in NRK-49F cells

The circadian clock protein reverse erythroblastosis virus (REV)-ERBα is implicated in the pathogenesis of various diseases, including cancer and myocardial infarction. Emerging evidence suggests that SR9009, an agonist of REV-ERBα, regulates multiple signaling molecules independent or dependent of REV-ERBα. However, the impact of SR9009 on renal fibrosis remains largely unevaluated. In this study, we investigated the effects of SR9009 on transforming growth factor (TGF)-β1-induced fibrotic responses and elucidated the mechanisms involved. Masson's trichome staining revealed that in the unilateral ureteral obstruction groups, there was a decrease in REV-ERBα expression, accompanied by increased levels of the profibrotic factor TGF-β1 and the fibrosis marker α-smooth muscle actin (α-SMA). REV-ERBα knockdown significantly increased α-SMA expression in NRK-49F cells. SR9009 significantly attenuated unilateral ureteral obstruction-induced fibrosis and TGF-β1-induced fibrotic responses in normal rat kidney fibroblasts (NRK-49F cells). Conversely, the REV-ERBα antagonist SR8278 did not affect TGF-β1-induced fibrotic responses. Mechanistic studies revealed that SR9009 significantly inhibited the phosphorylation of ERK and p38, concomitant with reduced α-SMA levels, suppressing TGF-β1-induced NADPH oxidase 4 (NOX4) mRNA expression in NRK-49F cells. Notably, SR9009 did not influence the expression of dual specificity phosphatase 4, which dephosphorylates MAPKs, including p38. Furthermore, REV-ERBα knockdown did not affect the ability of SR9009 to inhibit TGF-β1-induced fibrotic responses and NOX4 expression in NRK-49F cells. In conclusion, SR9009 exerts a protective role against renal fibrosis independent of REV-ERBα. Therefore, SR9009 is a promising therapeutic agent for the prevention and treatment of renal fibrosis associated with renal failure.

3D in vitro modelling of post-partum cardiovascular health reveals unique characteristics and signatures following hypertensive disorders in pregnancy

BackgroundHypertensive disorders of pregnancy (HDP) affect 2–8% of pregnancies and are associated postpartum with increased cardiovascular disease (CVD) risk, although mechanisms are poorly understood.MethodsHuman induced pluripotent stem cells (iPSC)-derived cardiomyocytes, cardiac fibroblasts and coronary artery endothelial cells were cocultured to form cardiac spheroids (CSs) in collagen type-1 hydrogels containing 10% patient plasma collected five years postpartum [n = 5 per group: normotensive control, gestational hypertension (GH) and preeclampsia (PE)]. Plasma-treated CSs were assessed for cell viability and contractile function and subjected to immunofluorescence staining and imaging. A quantitative proteomic analysis of plasma samples was conducted (controls n = 21; GH n = 5; PE n = 12).ResultsContraction frequency (CF) was increased in PE-treated CSs (CF: 45.5 ± 3.4 contractions/minute, p < 0.001) and GH-treated CSs (CF: 45.7 ± 4.0 contractions/minute, p < 0.001), compared to controls (CF = 21.8 ± 2.6 contractions/min). Only PE-treated CSs presented significantly increased fractional shortening (FS) % (9.95 ± 1.8%, p < 0.05) compared to controls (3.7 ± 1.1%). GH-treated CSs showed a reduction in cell viability (p < 0.05) and an increase in α-SMA expression (p < 0.05). Proteomics analyses identified twenty differentially abundant proteins, with hemoglobin A2 being the only protein perturbed in both GH and PE versus control plasma (p < 0.05).ConclusionsThe innovative patient-relevant CS platforms led to the discovery of biomarkers/targets linked to cell death signaling and cardiac remodeling in GH-induced CVD and vascular/endothelial cell dysfunction in PE-induced CVD.

Short-Term Culture of Human Hyalocytes Retains Their Initial Phenotype and Displays Their Contraction Abilities.

Hyalocytes are the main vitreal cell types with critical functions in health and vitreoretinal diseases. Our aim was to develop cultures of human hyalocytes and verify the retention of their initial cellular features after 3 and 6 days of culturing (3 d and 6 d) by analyzing and comparing a few morphological and functional parameters. Vitreous samples (n = 22) were collected and vitreous cells and bead-enriched hyalocytes were developed and compared (3 d vs. 6 d cultures). Vitreous and conditioned media were tested for collagen, vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGFβ1), nerve growth factor (NGF), matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and alpha-smooth muscle actin (αSMA) expression (ELISA, array/IP/WB, RT-PCR). Cells were observed at light and fluorescent microscopy (phenotypical properties) and tested for their 3D collagen gel contraction abilities. An increased expression of collagens, vimentin, fibronectin, and the MMP9/TIMP1 ratio were observed in vitreous tissues. In 3 d cultures, collagens and MMP9 were upregulated while the related tissue-enzymes were deregulated. Vitreous samples also showed high levels of TGFβ1, VEGF, and NGF, and this protein signature was retained at 3 d while decreased at 6 d. The original phenotype (low αSMA) was retained at 3 d from seeding while an increased αSMA expression was observed at 6 d; NGF/trkANGFR was expressed in cultured hyalocytes and partially drives the collagen retraction. The vitreous print comparison between untouched and cultured hyalocytes allowed us, on one side, to select 3 d cultures and, on the other, to highlight the neuroprotective/contractile NGF in vitro hyalocytes effects. The possibility of scoring reactive hyalocytes would represent an interesting aspect of screening the vitreoretinal interface severity.

Harnessing the regenerative effects of human amniotic stem cells (hAFSCs) on restoring erectile function in a bilateral cavernous nerve crush (BCNC) injury rat model.

Intracavernous (IC) injections of stem cells has been shown to ameliorate cavernous nerve (CN)-induced erectile dysfunction (ED). However, the regenerative effects underlying the recovery of erectile function (EF) in human amniotic fluid-derived stem cells (hAFSCs) remain unclear. In the bilateral cavernous nerve crushing (BCNC) injury rat paradigm, we sought to ascertain the effects of hAFSC treatment on EF recovery during the incipient phase. Three groups of 45 male rats were used in this study: sham (Group 1), saline IC injection after BCNC (Group 2), and hAFSC intracavernous injection (ICI) after BCNC (Group 3). hAFSCs from the fourth passage showed potential to differentiate into trilineage cells. All animals were subjected to EF analysis on the 28th day post-injection and tissues were retrieved for histopathological and immunohistochemical analyses. IC injections of hAFSC significantly improved EF parameters in BCNC-ED rats at 28 days post-injury. AFSC treatment enhanced the smooth muscle condition and increased the smooth muscle/collagen ratio, as evidenced by histological analysis. Immunohistology revealed increased expression of 𝛼-SMA andvWf in the corpus cavernosum and enhanced expression of nNOS in the dorsal penile nerve in BCNC-ED rats (p < 0.05). Western blotting showed that hAFSC treatment significantly increased α-SMA expression in the hAFSC group compared with that in the BCNC group. Electron microscopy revealed significantly elevated myelination in the CN (p < 0.05), maintenance of smooth muscle structures, and restoration of EF in BCNC-ED rats treated with hAFSC. hAFSC treatment increased EF in BCNC-ED rats at a single dose. As BCNC-ED resembles ED caused by radical prostatectomy (RP), this therapy has high potential for ED patients after RP surgery.

Preventive Effect of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene Triazole on Pulmonary Fibrosis through Inhibition of Inflammation and Down-regulation of TGF-b Signaling Pathway.

In the present study effect of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene triazole (STT) on plmonary fibrosis development was investigated in vitro in primary lung fibroblasts as well as in vivo in mice model. The results demonstrated that STT treatment effectively inhibited the TGF-β1 induced increase in expression of α-SMA and collagen I proteins in PLFs. STT treatment effectively reversed the TGF-β1 induced increase in expression of LOXL2 protein and phosphorylation of Smad2/3 proteins. Treatment of PLFs with STT reversed the TGF-β1-induced increase in expression of NOX4 and suppression of p-AMPK protein. In mice model of pulmonary fibrosis STT treatment significantly inhibited the BLM-mediated decrease in body weight and survival rate. The BLM induced increase in pulmonary index in mice was also effectively inhibited on treatment with STT. Treatment of the mice with STT inhibited the BLM-induced increase in α-SMA and Col I protein expression in pulmonary tissues. The BLM-induced increase in TGF-β1 protein expression in pulmonary tissues of the mice was inhibited on treatment with STT. Treatment with STT effectively promoted the AMPK activation in lung tissues of the BLM administered mice. In summary, the present study demonstrates that STT treatment prevents TGF-β1 induced up-regulation of α-SMA, collagen I, LOXL2 protein expression and targets phosphorylation of Smad2/3 proteins in PLFs. Moreover, it inhibits TGF-β1-induced increase in expression of NOX4 and reverses TGF-β1-mediated suppression in expression of p-AMPK protein. Therefore, STT inhibits fibrosis development in vitro as well as in vivo and therefore can be investigated further as a therapeutic agent for the treatment of lung fibrosis.

Specific peptides targeting the myocardiocyte are prognostic markers for heart attack: Function of α-SMA protein

Myocardial infarction (MI) is a serious cardiovascular disease that often results in a significant decline in heart function and associated complications. α-SMA (α-smooth muscle cell actin) is an important biomarker in the process of cardiac remodeling and repair, and its expression level is closely related to myocardial remodeling and prognosis. Therefore, the purpose of this study was to investigate the potential of nanoparticles containing cardiomyocyte targeting peptides in predicting prognosis and α-SMA protein expression after myocardial infarction, with a view to providing new therapeutic strategies and clinical guidelines. In this study, a novel targeting nanoparticle was constructed, using cardiomyocyte specific peptides as targeting ligands, and characterized by loading different drugs. Subsequently, a mouse model of myocardial infarction was used to systematically evaluate the effect of nanoparticles on α-SMA protein expression and prognosis prediction ability after MI. The expression level of α-SMA was analyzed by Western blot and immunohistochemistry, and the prognosis was evaluated by cardiac function assessment. The study found that nanoparticles containing cardiomyocyte targeting peptides significantly increased α-SMA expression levels and improved heart function in animal models of myocardial infarction. Compared with the control group, the application of targeted nanoparticles was closely related to the level of myocardial cell repair and fibrosis, and could effectively predict the prognosis after myocardial infarction. Therefore, nanoparticles containing cardiomyocyte targeting peptides can not only effectively improve the expression of α-SMA, but also serve as an important prognostic tool after myocardial infarction.

Nesfatin-1 regulates the phenotype transition of cavernous smooth muscle cells by activating PI3K/AKT/mTOR signaling pathway to improve diabetic erectile dysfunction.

This study aims to explore the impact of Nesfatin-1 on type 2 diabetic erectile dysfunction (T2DMED) and its underlying mechanism in regulating the phenotypic switching of corpus cavernosum smooth muscle cells (CCSMCs). Twenty-four 4-week-old male C57 wild-type mice were randomly assigned to the control group, model group, and Nesfatin-1 treatment group. Monitoring included body weight, blood glucose levels, and penile cavernous pressure (ICP). Histochemistry and Western blot analyses were conducted to assess the expressions of α-SMA, OPN, and factors related to the PI3K/AKT/mTOR signaling pathway. CCSMCs were categorized into the control group, high glucose and high oleic acid group (GO group), Nesfatin-1 treatment group (GO+N group), sildenafil positive control group (GO+S group), and PI3K inhibitor group (GO+N+E group). Changes in phenotypic markers, cell morphology, and the PI3K/AKT/mTOR signaling pathway were observed in each group. (1) Nesfatin-1 significantly ameliorated the body size, body weight, blood glucose, glucose tolerance, and insulin resistance in T2DMED mice. (2) Following Nesfatin-1 treatment, the ICP/MSBP ratio and the peak of the ICP curve demonstrated a significant increase. (3) Nesfatin-1 significantly enhanced smooth muscle and reduced collagen fibers in the corpus cavernosum. (4) Nesfatin-1 notably increased α-SMA expression and decreased OPN expression in CCSMCs. (5) Nesfatin-1 elevated PI3K, p-AKT/AKT, and p-mTOR/mTOR levels in penile cavernous tissue. Nesfatin-1 not only effectively improves body weight and blood glucose levels in diabetic mice but also enhances erectile function and regulates the phenotypic switching of corpus cavernosum smooth muscle. The potential mechanism involves Nesfatin-1 activating the PI3K/AKT/mTOR signaling pathway to induce the conversion of CCSMCs to a contractile phenotype.

Immunohistochemical and Ultrastructural Characterization of Telocytes in Normal and Diabetic Human Kidneys.

Background: Telocytes are interstitial stromal cells identified in various human organs, including the kidney. Their presence and role in human diabetic kidney disease remain unknown. Methods: To identify and localize telocytes in glomerular and tubule-interstitial compartments, both normal and diabetic human renal tissues were examined using immunohistochemistry, immunofluorescence, and transmission electron microscopy. Results: Renal telocytes are elongated interstitial cells with long, thin telopodes, showing alternating thin and thick segments. They expressed CD34, Nestin, α-SMA, and Vimentin markers. Occasionally, c-Kit expression was observed in some rounded and spindle cells, while no positivity was detected for PDGFR-β and NG2. Telocytes were identified around Bowman's capsule, tubules, and peritubular capillaries in both normal and diabetic conditions. In diabetic renal samples, there was a significant increase in α-SMA expressing telocytes, leading to periglomerular fibrosis. These telocytes also exhibited a synthetic phenotype with proteoglycan deposition in the extracellular matrix and, in some cases, showed pre-adipocytic differentiation. Conclusions: Telocytes were identified in normal and diabetic human kidneys. These cells form an elastic mechanical scaffold in the interstitium and are present in all renal cortical compartments. In diabetic samples, their increased α-SMA expression and synthetic phenotype suggest their potential role in the pathogenesis of diabetic nephropathy.

Signalling of the neuropeptide calcitonin gene-related peptide (CGRP) through RAMP1 promotes liver fibrosis via TGFβ1/Smad2 and YAP pathways

The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGFβ1 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGFβ1 production and downstream signalling as well as HSC activation documented by increased α-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGFβ1/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGFβ1/Smad2 and YAP activity.

Role and mechanism of epithelial-mesenchymal transition in a rat model of bronchopulmonary dysplasia induced by hyperoxia exposure

To investigate the role and mechanism of epithelial-mesenchymal transition (EMT) in a rat model of bronchopulmonary dysplasia (BPD). The experiment consisted of two parts. (1) Forty-eight preterm rats were randomly divided into a normoxia group and a hyperoxia group, with 24 rats in each group. The hyperoxia group was exposed to 85% oxygen to establish a BPD model, while the normoxia group was kept in room air at normal pressure. Lung tissue samples were collected on days 1, 4, 7, and 14 of the experiment. (2) Rat type II alveolar epithelial cells (RLE-6TN) were randomly divided into a normoxia group (cultured in air) and a hyperoxia group (cultured in 95% oxygen), and cell samples were collected 12, 24, and 48 hours after hyperoxia exposure. Hematoxylin-eosin staining was used to observe alveolarization in preterm rat lungs, and immunofluorescence was used to detect the co-localization of surfactant protein C (SPC) and α-smooth muscle actin (α-SMA) in preterm rat lung tissue and RLE-6TN cells. Quantitative real-time polymerase chain reaction and protein immunoblotting were used to detect the expression levels of EMT-related mRNA and proteins in preterm rat lung tissue and RLE-6TN cells. (1) Compared with the normoxia group, the hyperoxia group showed blocked alveolarization and simplified alveolar structure after 7 days of hyperoxia exposure. Co-localization of SPC and α-SMA was observed in lung tissue, with decreased SPC expression and increased α-SMA expression in the hyperoxia group at 7 and 14 days of hyperoxia exposure compared to the normoxia group. In the hyperoxia group, the mRNA and protein levels of TGF-β1, α-SMA, and N-cadherin were increased, while the mRNA and protein levels of SPC and E-cadherin were decreased at 7 and 14 days of hyperoxia exposure compared to the normoxia group (P<0.05). (2) SPC and α-SMA was observed in RLE-6TN cells, with decreased SPC expression and increased α-SMA expression in the hyperoxia group at 24 and 48 hours of hyperoxia exposure compared to the normoxia group. Compared to the normoxia group, the mRNA and protein levels of SPC and E-cadherin in the hyperoxia group were decreased, while the mRNA and protein levels of TGF-β1, α-SMA, and E-cadherin in the hyperoxia group increased at 48 hours of hyperoxia exposure (P<0.05). EMT disrupts the tight connections between alveolar epithelial cells in a preterm rat model of BPD, leading to simplified alveolar structure and abnormal development, and is involved in the development of BPD. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 765-773.

Nesfatin-1 regulates the phenotype transition of cavernous smooth muscle cells by activating PI3K/AKT/mTOR signaling pathway to improve diabetic erectile dysfunction

ObjectiveThis study aims to explore the impact of Nesfatin-1 on type 2 diabetic erectile dysfunction (T2DMED) and its underlying mechanism in regulating the phenotypic switching of corpus cavernosum smooth muscle cells (CCSMCs). MethodsTwenty-four 4-week-old male C57 wild-type mice were randomly assigned to the control group, model group, and Nesfatin-1 treatment group. Monitoring included body weight, blood glucose levels, and penile cavernous pressure (ICP). Histochemistry and Western blot analyses were conducted to assess the expressions of α-SMA, OPN, and factors related to the PI3K/AKT/mTOR signaling pathway. CCSMCs were categorized into the control group, high glucose and high oleic acid group (GO group), Nesfatin-1 treatment group (GO + N group), sildenafil positive control group (GO + S group), and PI3K inhibitor group (GO + N + E group). Changes in phenotypic markers, cell morphology, and the PI3K/AKT/mTOR signaling pathway were observed in each group. Results(1) Nesfatin-1 significantly ameliorated the body size, body weight, blood glucose, glucose tolerance, and insulin resistance in T2DMED mice. (2) Following Nesfatin-1 treatment, the ICP/MSBP ratio and the peak of the ICP curve demonstrated a significant increase. (3) Nesfatin-1 significantly enhanced smooth muscle and reduced collagen fibers in the corpus cavernosum. (4) Nesfatin-1 notably increased α-SMA expression and decreased OPN expression in CCSMCs. (5) Nesfatin-1 elevated PI3K, p-AKT/AKT, and p-mTOR/mTOR levels in penile cavernous tissue. ConclusionsNesfatin-1 not only effectively improves body weight and blood glucose levels in diabetic mice but also enhances erectile function and regulates the phenotypic switching of corpus cavernosum smooth muscle. The potential mechanism involves Nesfatin-1 activating the PI3K/AKT/mTOR signaling pathway to induce the conversion of CCSMCs to a contractile phenotype.

Endothelial-to-Mesenchymal Transition in Human and Murine Models of Congenital Diaphragmatic Hernia

Introduction: Congenital diaphragmatic hernia (CDH) is a complex congenital disorder, characterized by pulmonary hypertension (PH) and hypoplasia. PH secondary to CDH (CDH-PH) features devastating morbidity and mortality (25–30%) among neonates. An unmet need is determining mechanisms triggering CDH-PH to save infants. Prior data suggest abnormal remodeling of the pulmonary vascular extracellular matrix (ECM), presumed to be driven by endothelial-to-mesenchymal transition (EndoMT), hinders postnatal vasodilation and limits anti-PH therapy in CDH. There are limited data on the role of EndoMT in CDH-PH. Methods: The purpose of the study was to investigate how EndoMT contributes to CDH-PH by identifying cells undergoing EndoMT noted by alpha smooth muscle actin (α-SMA) expression in human umbilical vein endothelial cells (HUVECs) and lung tissue obtained from murine pups using the nitrofen model. N = 8 CDH, N = 8 control HUVECs were stained for α-SMA and CD31 after being exposed for 24 h to TGFB, a known EndoMT promoter. N = 8 nitrofen, N = 8 control murine pup lungs were also stained for α-SMA and CD31. α-SMA and CD31 expression was quantified in HUVECs and murine tissue using Fiji imaging software and normalized to the total number of cells per slide noted by DAPI staining. Results: CDH HUVECs demonstrated a 1.1-fold increase in α-SMA expression (p = 0.02). The murine model did not show statistical significance between nitrofen and control pup lungs; however, there was a 0.4-fold increase in α-SMA expression with a 0.8-fold decrease in CD31 expression in the nitrofen pup lungs when compared to controls. Conclusion: These results suggest that EndoMT could potentially play a role in the ECM remodeling seen in CDH-PH.

Effects of Bone Marrow Sparing and TGF-β3 Treatment in Total Body Irradiation of C57BL/6J Mice

Introduction: Mortality from acute radiation syndrome is frequently caused by hematopoietic or gastrointestinal radiotoxicity, the latter of which currently has no effective treatment. Transforming growth factor-beta 3 (TGF-β3) may decrease the severity of radiation-induced gastrointestinal damage in mice. In addition, treatment with TGF-β3 may alleviate radiation-induced fibrosis. Objectives: The current study aimed to investigate the effect of TGF-β3 treatment on acute and late radiotoxicity in whole body irradiated mice. Methods: C57BL/6J mice were total body irradiated with 8.5 Gy X-rays with or without shielding of one hind leg to alleviate hematopoietic radiotoxicity. The effects of intravenous TGF-β3 treatment were investigated. Body weight and pain expression were monitored. Intestine, lung, and liver tissues were preserved and analyzed. Alpha smooth muscle actin (α-SMA) expression in MRC-5 cells after 3.5 Gy X-irradiation combined with TGF-β3 treatment was analyzed using flow cytometry. Results: All total body irradiated animals died within ten days after irradiation. Ninety-three percent of femur-shielded mice survived until sampling or termination. No effect of TGF-β3 treatment was observed in either group. No increase in collagen content was detected in the lungs or liver from irradiated mice regardless of TGF-β3 treatment. In vitro, α-SMA expression increased synergistically after irradiation and TGF-β3 treatment. Conclusions: Shielding of the femur during total body irradiation decreased acute gastrointestinal radiation toxicity and increased survival. TGF-β3 treatment did not impact symptoms or survival. TGF-β3 treatment and irradiation increased α-SMA expression in MRC-5 cells synergistically.

Effect of Benidipine Alone and in Combination With Bosentan and Sildenafil in Amelioration of Pulmonary Arterial Hypertension in Experimental Model in Rats.

Pulmonary arterial hypertension (PAH) is a persistent condition affecting the pulmonary arteries' endothelium. Benidipine, a calcium channel blocker, possesses vasodilatory, anti-inflammatory activity, reduces oxidative stress, and inhibits the activity of Transforming growth factor-β (TGF-β) and α-smooth muscle actin (α-SMA). The present study was designed to investigate the effect of benidipine alone and in combination with bosentan and sildenafil on monocrotaline (MCT)-induced pulmonary hypertension in a rat model. PAH was induced by a single-dose administration of MCT in rats. Animals were randomized into different groups and treated with benidipine alone and in combination with bosentan or sildenafil. Various parameters such as hemodynamic parameters, Fulton's index and oxidative stress parameters were performed. Additionally, histopathology of lung and right ventricular of heart tissue, immunohistochemistry, expression of α-SMA, endothelial nitric oxide synthase (eNOS), TGF-β, and RT-PCR, and an in vitro study using human umbilical vein endothelial cells (HUVECs) was also carried out. Treatment of benidipine and its combination exhibited better prevention in the elevated right ventricular systolic pressure, right ventricular hypertrophy, rise in oxidative stress, and increase in expression of α-SMA and TGF-β receptor 1 compared with MCT control group rats. In HUVECs, the expression of α-SMA was increased, whereas that of eNOS decreased after TGF-β exposure and was substantially reversed after pretreatment with benidipine. We concluded that benidipine and its combination with bosentan and sildenafil exhibit beneficial effects in MCT-induced PAH through the eNOS/TGF-β/α-SMA signaling pathway.

P094 Lack of DNA methyltransferase DNMT3A in fibroblasts impairs cell growth and wound healing

Abstract Background Fibrotic scarring is an untreatable pathological wound-healing complication that invalidates correct organ function. However, pathomechanisms leading to intestinal fibrosis during inflammatory bowel disease (IBD) are not fully understood. Since the de novo DNA methyltransferase DNMT3A has been associated with increased risk for IBD and alters epithelial homeostasis, we sought to characterize how DNMT3A loss might affect fibroblasts, the key mesenchymal cells involved in wound healing. Methods We stably depleted Dnmt3a in 3T3 fibroblast cell line using CRISPR-Cas9 technology and subsequently selected GFP-positive clones using FACS sorting. Cell cultures were performed in vessels with or without 0.2% gelatin coat. Reverse transcription quantitative PCR, western blot, and immunofluorescence were used to confirm Dnmt3a gene deletion and characterize wild-type (WT) and knockout cells (KO). Wound healing assays in the presence or absence of tissue repair cytokine TGF-β served to quantify and assess cell capacity of wound closure. Results 3T3 fibroblasts lacking Dnmt3a acquire an activated myofibroblast phenotype (characterized by increased α-SMA expression) that also overexpress tissue remodelling matrix metalloproteinase Mmp3, and the inflammation-induced necroptosis marker RIPK3 when compared to their WT counterparts. In addition, Dnmt3a-depleted 3T3 cells grow overlapped in tightly aggregated foci (fig. 1), and when seeded in vessels without the gelatin coat, they form spheroids. To assess healing properties, the closure of a wound (gap) in a cell monolayer was monitored. While WT 3T3 fibroblasts closed the gap within 48h, Dnmt3a KO 3T3 cells were unable to refill the empty space and formed aggregated foci in the vicinity of the wound (fig. 1). When the wound healing assay was repeated in the presence of TGF-β, WT cells nearly closed the gap within 24h whereas Dnmt3a KO 3T3 still failed to grow. Conclusion We conclude that Dnmt3a is crucial for fibroblast homeostasis and correct wound healing. Furthermore, TGF-β signalling is impaired when Dnmt3a is lacking. Thus, additional characterization of Dnmt3a depletion in fibroblasts will deepen the understanding of fibrosis and point towards new therapeutic approaches for this untreatable complication.

Effector T Cells Promote Fibrosis in Corneal Transplantation Failure.

To evaluate whether fibrosis contributes to corneal transplant failure and to determine whether effector CD4+ T cells, the key immune cells in corneal transplant rejection, play a direct role in fibrosis formation. Allogeneic corneal transplantation was performed in mice. Graft opacity was evaluated by slit-lamp biomicroscopy, and fibrosis was assessed by in vivo confocal microscopy. Expression of alpha-smooth muscle actin (α-SMA) in both accepted and failed grafts was assessed by real-time PCR and immunohistochemistry. Frequencies of graft-infiltrating CD4+ T cells, neutrophils, and macrophages were assessed using flow cytometry. In vitro, MK/T-1 corneal fibroblasts were co-cultured with activated CD4+CD25- effector T cells isolated from corneal transplant recipient mice, and α-SMA expression was quantified by real-time PCR and ELISA. Neutralizing antibody was used to evaluate the role of interferon gamma (IFN-γ) in promoting α-SMA expression. The majority of failed grafts demonstrated clinical signs of fibrosis which became most evident at week 6 after corneal transplantation. Failed grafts showed higher expression of α-SMA as compared to accepted grafts. Flow cytometry analysis showed a significant increase in CD4+ T cells in failed grafts compared to accepted grafts. Co-culture of activated CD4+CD25- effector T cells with corneal fibroblasts led to an increase in α-SMA expression by fibroblasts. Inhibition of IFN-γ in culture significantly suppressed this increase in α-SMA expression as compared to immunoglobulin G control. Fibrosis contributes to graft opacity in corneal transplant failure and is mediated at least in part by effector CD4+ T cells via IFN-γ.

&lt;b&gt;Direct and indirect effects of PGF2α administration in male wistar rats based on increased expression of α-SMA and androgen receptor&lt;/b&gt;

Background: PGF2α hormone administration can improve semen quality through increased contractility of smooth muscle cells in testicular tissue and increased testosterone hormone. Immunohistochemically, the increase in contractility of smooth muscle cells and testosterone hormone can be measured by an increase in the expression of alpha-smooth muscle actin (α-SMA) and androgen receptor (AR). Aim: This study aims to determine the distribution and increased α-SMA expression and AR in the testis of Wistar rats after administration of PGF2α which detected using the immunohistochemistry (IHC) method. Methods: A total of 15 male Wistar rats (Rattus norvegicus) with body weight 200-250 g and 8-10 weeks old were used in this study. All rats were acclimated for two weeks. The rats were divided into 5 treatment groups (n=3). The rat in the control group (P0), testicular collection was carried out 30 minutes after injection of 0.5 ml NaCl. In group P1, P2, P3, and P4, the rats were intraperitoneal injected with 2.5 mg/kg BW of PGF2α (Lutalyze, Zoetis, USA), and the testis collection was performed 30 minutes, 60 minutes , 90 minutes , and 120 minutes after PGF2α injection, respectively. were processed into histology preparations and stained with immunohistochemistry staining using avidin-biotin complex peroxidase method (ABC method). The distribution of α-SMA and AR was analyzed descriptively, while the score difference of α-SMA and AR expression was analyzed using the Kruskal-Wallis test and the Mann-Whitney U Test. Results: The results showed that α-SMA was positively detected in peritubular myoid cells on the basal membrane of seminiferous tubules, blood vessel walls, and connective tissue. Statistical analysis showed that α-SMA expression in peritubular myoid cells and testicular connective tissue in P2 (60-minute interval) was significantly different than other treatment groups (P0, P1, P3, and P4) (P&lt;0.05), while no significant difference was found in the blood vessel walls in all treatment groups (P&gt;0.05). AR was positively detected in connective tissue, peritubular myoid cells, Leydig cells, Sertoli cells, and blood vessels. Statistical analysis showed significant differences in AR expression in peritubular myoid cells and Sertoli cells (P&lt;0.05) between P0 and P2, P1 and P2, and P2 and P3 or P4. Conclusion: It can be concluded that the administration of PGF2α increase the expression of -SMA and AR, with the optimal administration interval is 60 minutes for peritubular myoid cells and Sertoli cells in Wistar rats testis.

Abstract 461: Alcohol Modulates Chemokine- And Hypoxia-induced Endothelial-to-mesenchymal Transition In Vitro And Following Ligation Injury In Vivo

Endothelial-to-mesenchymal transition (EndMT) plays a role in arteriosclerotic cardiovascular disease (CVD). Low-to-moderate alcohol consumption (i.e., 1-3 drinks/d range) protects against endothelial dysfunction and arteriosclerosis, in part by stimulating Notch, but whether it does so by influencing EndMT is unknown. Our aim was to address this question. Human coronary artery endothelial cells (HCAEC) (p5-p14) were treated (7 d) with transforming growth factor β (TGFβ, 10 ng/ml) or exposed to hypoxia (5% O 2 ), in the absence or presence of ‘moderate’ level EtOH (25 mM), or the Notch ligand DLL4 +/- the Notch inhibitor DAPT. EndMT was assessed by determining endothelial marker CD31 and smooth muscle cell marker αSMA expression by immunohistochemistry and qRT-PCR, as well as by cell morphology and extracellular matrix protein Fibronectin production. Both TGFβ and hypoxia induced EndMT of HCAEC as determined by simultaneously decreased CD31 and increased αSMA expression, together with a change in morphology from cobblestone (typical of endothelial cells) to spindle-shaped (typical of mesenchymal cells), and increased fibronectin expression. EtOH inhibited EndMT caused by both TGFβ and hypoxia as indicated by maintenance of CD31 expression and cobblestone morphology, and attenuated αSMA and fibronectin expression in the EtOH treated group vs controls. Similar to EtOH, treatment with DLL4 inhibited TGFβ-induced myogenic αSMA and fibronectin mRNA expression in HCAEC, effects blocked by DAPT. Following carotid ligation in C57Bl/6 mice, ~25% of neointimal and medial cells in the remodeled vessel co-expressed αSMA and CD31 (indicative of myo-endothelial cells), suggesting the involvement of EndMT. In the ‘daily moderate’ alcohol group (2 drinks per day equivalent; peak BAC=15 mM) carotid neointimal hyperplasia was reduced compared to controls concomitant with fewer (&lt;10%) αSMA + /CD31 + myo-endothelial cells suggesting repression of EndMT in that cohort. In conclusion, these data illuminate an inhibitory effect of alcohol on EndMT, potentially mediated via Notch. This effect on endothelial transdifferentiation may underlie, in part, the protective effect of moderate alcohol consumption on CVD.

MP03-19 ROLE OF MICROFIBRILLAR-ASSOCIATED PROTEIN 4 IN PARTIAL BLADDER OUTFLOW OBSTRUCTION-RELATED BLADDER FIBROSIS

You have accessJournal of UrologyCME1 Apr 2023MP03-19 ROLE OF MICROFIBRILLAR-ASSOCIATED PROTEIN 4 IN PARTIAL BLADDER OUTFLOW OBSTRUCTION-RELATED BLADDER FIBROSIS Reine Kanaan, Anders Schlosser, Grith Lykke Sorensen, and Peter Zvara Reine KanaanReine Kanaan More articles by this author , Anders SchlosserAnders Schlosser More articles by this author , Grith Lykke SorensenGrith Lykke Sorensen More articles by this author , and Peter ZvaraPeter Zvara More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Partial bladder outflow obstruction (pBOO) causes remodeling of the bladder wall, with smooth muscle hypertrophy and an excessive deposition of extracellular matrix (ECM) proteins, leading to bladder fibrosis. Our understanding of the pathophysiology of this process is limited and no effective treatment to prevent or reverse bladder fibrosis exists. Microfibrillar-associated protein 4 (MFAP4) is an ECM glycoprotein, localized to elastic fiber-rich regions. It binds ECM components including elastin and collagen and contributes to the process of elastogenesis in healthy tissues. Emerging data suggest the involvement of MFAP4 in the pathogenesis of disease-induced tissue remodeling, including fibrosis. To determine its value as a potential therapeutic target, we compared MFAP4 expression in sham-operated bladders to bladders 6 weeks following pBOO. MFAP4 levels were then correlated with bladder function. METHODS: Partial BOO, was induced in 9 male Sprague Dawley rats, 5 underwent sham surgery. Six weeks following surgery, rats were placed in a metabolic cage overnight to record 12-hour micturition frequency and volume per void. Gene expression levels of MFAP4, alpha-smooth muscle actin (α-SMA) and collagen I were quantified in the bladder tissue using real-time PCR. RESULTS: The bladder/body ratio was significantly higher in the pBOO group (0.07±0.04) when compared to the SHAM group (0.024±0.003). Obstructed rats with increased MFAP4 expression demonstrated an increase in α-SMA expression. No significant increase in collagen I levels was recorded in the pBOO group. Animals with bladder overactivity as determined by frequent low volume micturitions (≥4 micturitions with <0.1 mL per void/12 hours) had higher MFAP4 expression than animals in the SHAM group, which exhibited normal micturition pattern. CONCLUSIONS: These initial observations show that pBOO lasting 6 weeks results in detrusor hypertrophy. Although bladder wall fibrosis did not develop within the 6-week timeframe, our data suggest that bladder remodeling following pBOO is associated with increased MFAP4 levels and that MFAP4 might be involved in the pathogenesis of pBOO-induced bladder dysfunction. Experiments addressing processes extending beyond 6 weeks post-pBOO and possible involvement of MFAP4 in their pathophysiology are ongoing. Source of Funding: Odense University hospital © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e31 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Reine Kanaan More articles by this author Anders Schlosser More articles by this author Grith Lykke Sorensen More articles by this author Peter Zvara More articles by this author Expand All Advertisement PDF downloadLoading ...

Poly(2-hydroxyethyl methacrylate) surface chemistry and modulus differentially modulate neutrophils and lens epithelial cells-possible implications in cellular responses to intraocular lenses.

During cataract surgery, diseased lenses in the eye are surgically removed and replaced with polymeric artificial intraocular lenses (IOLs). Patients can experience a complication called posterior capsular opacification (PCO) that is corrected through the removal of part of the posterior capsule using a neodymium: yttrium-aluminum-garnet (Nd-YAG) laser to restore the optical path. These interventions have increased costs and can damage the retina and the IOL. PCO develops when lens epithelial cells (LECs) proliferate, migrate, and undergo epithelial-to-mesenchymal transition. Neutrophils involved in the immune response triggered during implantation impact LEC behavior and produce damaging neutrophil extracellular traps (NETs). In this research, poly(2-hydroxyethyl methacrylate) (PHEMA) -based disks were synthesized with varying amounts of comonomer (HEMA with 0, 2, and 12 mol% MMA) and functionalized with carboxyl and amine groups, yielding nine different hydrogels. Material and chemical properties of the disks were characterized, and neutrophil-like HL60 cells and B3 LECs were incubated with the disks. HL60 cell behavior was more strongly influenced by chemical functionalization than by mechanical properties with increases in adherence and NET accumulation. Conversely, the behavior and viability of B3 LECs were more strongly influenced by mechanical properties with increases in cell adhesion and α-SMA expression with increasing compressive moduli. Interestingly, B3 LECs had decreased viability and increased α-SMA expression when cultured on PHEMA2 disks pretreated with isolated NETs. Critical to the understanding of PCO and its prevention are both surface chemistry and mechanics as well as the inflammatory response.