Search Results (514)

Galectin-3 is a multifunctional protein that is associated with diseases of the chorioretinal interface, in which the retinal pigment epithelium (RPE) plays a central role in disease development and progression. Since galectin-3 can function extracellularly as well as intracellularly via different mechanisms, we developed an immortalized human RPE cell line (ARPE-19) with a knockdown for galectin-3 expression (ARPE-19/LGALS3^+/−) using a sgRNA/Cas9 all-in-one expression vector. By Western blot analysis, a reduced galectin-3 expression of approximately 48 to 60% in heterozygous ARPE-19/LGALS3^+/− cells was observed when compared to native controls. Furthermore, ARPE-19/LGALS3^+/− cells displayed a flattened, elongated phenotype with decreased E-cadherin as well as enhanced N-cadherin and α-smooth muscle actin mRNA expression, indicating an epithelial–mesenchymal transition of the cells. Compared to wildtype controls, ARPE-19/LGALS3^+/− cells had significantly reduced metabolic activity to 86% and a substantially decreased proliferation to 73%. Furthermore, an enhanced cell adhesion and a diminished migration of immortalized galectin-3 knockdown RPE cells was observed compared to native ARPE-19 cells. Finally, by Western blot analysis, reduced pAKT, pERK1/2, and β-catenin signaling were detected in ARPE-19/LGALS3^+/− cells when compared to wildtype controls. In summary, in RPE cells, endogenous galectin-3 appears to be essential for maintaining the epithelial phenotype as well as cell biological functions such as metabolism, proliferation, or migration, effects that might be mediated via a decreased activity of the AKT, ERK1/2, and β-catenin signaling pathways. Full article

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive extracellular matrix (ECM) deposition driven by aberrant fibroblast-to-myofibroblast transition (FMT). However, the upstream regulators and downstream effectors of this process remain incompletely understood. Here, we identify acyl-CoA synthetase long-chain family member 4 (ACSL4), a lipid metabolic enzyme, as a critical mediator linking complement component 5a (C5a)/C5a receptor 1 (C5aR1) signaling to FMT via calcium signaling. In bleomycin (BLM)-induced pulmonary fibrosis of C57BL/6JGpt mice, and in C5a-stimulated primary lung fibroblasts, the expression of ACSL4 was markedly upregulated. Pharmacological inhibition of ACSL4 (PRGL493) or C5aR1 (PMX53) attenuated the deposition of ECM and suppressed the expression of fibrotic markers in vivo and in vitro. Mechanistically, the activation of C5a/C5aR1 signaling increased intracellular calcium levels and promoted the expression of ACSL4, while inhibition of calcium signaling (FK506) reversed the upregulation of ACSL4 and FMT-related changes, including the expression of α-smooth muscle actin (αSMA) and the migration of fibroblasts. Notably, inhibition of ACSL4 did not affect the proliferation of fibroblasts, suggesting its specific role in phenotypic transition. These findings demonstrate that ACSL4 functions downstream of C5a/C5aR1-induced calcium signaling to promote FMT and the progression of pulmonary fibrosis. Targeting ACSL4 may therefore offer a novel therapeutic strategy for IPF. Full article

(1) Background: The sophisticated function of the aortic root relies on the coordinated movement of its constituent components. This study examines the extracellular components of the interleaflet triangles (ILTs) and characterises the cells that are present within this region of the aortic root. (2) Methods: A total of 10 human aortic valves and 6 porcine aortic valves were processed for immunohistochemical staining, scanning, and transmission electron microscopy. (3) Results: The three ILTs differed in size and macroscopic appearance. Each triangle comprised up to five distinct layers of tissue: an innermost endothelial layer, an inner elastin-rich layer, a thicker outer layer comprising densely packed layers of collagen and glycosaminoglycans, and an outer layer of intermingled myocardial and adipose tissue. A band of cells near the luminal surfaces of all ILTs expressed smooth muscle cell α-actin with variable expression of smooth muscle myosin heavy chain. In all the ILTs, there was evidence of neurofilament staining, indicating the presence of nerve fibres. (4) Conclusions: Each ILT is unique in its structure and organisation, with differing amounts of elastin and collagen, as well as myocardial, adipose, and fibrous content. The ILTs contain multiple cell types in varying abundance. Functional studies are required to determine the role of the different cells and their organisation in contributing to the sophisticated, dynamic behaviour of the aortic root. Full article

Wound healing involves complex interplay between cellular and molecular events. In this study, we investigated the therapeutic potential of the bovine amniotic membrane (BAM) in wound healing using a mouse model. Twelve male C57BL/6 mice were divided into four groups: negative control (Vehicle), positive control (DuoDERM Extra Thin^®), amniotic membrane attachment (Amniotic Membrane), and compressed amniotic membrane attachment (Amniotic Membrane with Compression). The dorsal skin of each mouse was excised and wound-healing parameters were assessed over a two-week period. Our results revealed that the Amniotic Membrane and Amniotic Membrane with Compression groups demonstrated significant sustained reductions in the wound area compared to the Vehicle group. These reductions were more pronounced than those observed in the DuoDERM group. Histopathological analysis revealed advanced wound healing characteristics in the BAM-treated groups. Immunohistochemical analysis demonstrated elevated expression levels of wound healing markers (including α-smooth muscle actin, collagen type III, SMAD 1/5/8, and SMAD 2/3) in the BAM-treated groups compared to the control and DuoDERM groups. Conversely, cluster of differentiation 4 levels were significantly lower in BAM-treated groups. Overall, our findings highlight the therapeutic efficacy of BAM and compression in promoting wound healing. Thus, BAM offers a promising therapeutic approach for enhancing wound healing outcomes in clinical settings, potentially by modulating key wound healing pathways and processes. Full article

Background: Gallbladder hypomotility is a key pathogenic factor in cholelithiasis. Non-invasive interventions to enhance gallbladder contractility remain limited. Ultrasound therapy has shown promise in various muscular disorders, but its effects on gallbladder function are unexplored. Methods: This study employed low-intensity pulsed ultrasound (LIPUS) at a 3 MHz frequency and 0.8 W/cm² intensity with a 20% duty cycle to irradiate the gallbladder region of fasting guinea pigs. Gallbladder contractile function was evaluated through multiple complementary approaches: in vivo assessment via two-dimensional/three-dimensional ultrasound imaging to monitor volumetric changes; quantitative functional evaluation using nuclear medicine scintigraphy (^99mTc-HIDA); and ex vivo experiments including isolated gallbladder muscle strip tension measurements, histopathological analysis, α-smooth muscle actin (α-SMA) immunohistochemistry, and intracellular calcium fluorescence imaging. Results: Ultrasound significantly enhanced gallbladder emptying, evidenced by the volume reduction and increased ejection fraction. Scintigraphy confirmed accelerated bile transport in treated animals. Ex vivo analyses demonstrated augmented contractile force, amplitude, and frequency in ultrasound-treated smooth muscle. Histological examination revealed smooth muscle hypertrophy, α-SMA upregulation, and elevated intracellular calcium levels. Extended ultrasound exposure produced sustained functional improvements without tissue damage. Conclusions: Ultrasound effectively enhances gallbladder contractile function through mechanisms involving smooth muscle structural modification and calcium signaling modulation. These findings establish the experimental foundation for ultrasound as a promising non-invasive therapeutic approach to improve gallbladder motility and potentially prevent gallstone formation. Full article

Wound healing remains a significant clinical challenge worldwide, and effective management strategies are essential for improving outcomes. This study evaluated SCderm Matrix, a novel acellular dermal matrix (ADM) patch developed using supercritical carbon dioxide (sCO₂) processing of human skin tissue. This innovative processing method preserves structural integrity while enhancing biocompatibility, resulting in a patch characterized by porous architecture, uniform thickness, excellent tensile strength, and optical transparency. In vivo wound healing experiments using full-thickness skin wounds in Sprague–Dawley rats demonstrated the patch’s superior performance. Treatment with the sCO₂ ADM patch accelerated wound closure, reduced inflammation, and enhanced granulation tissue formation compared to both untreated controls and two commercially available ADM products. Histological analysis revealed improved re-epithelialization and collagen deposition, while molecular and immunohistochemical assessments showed decreased reactive oxygen species (ROS) and pro-inflammatory cytokines. Simultaneously, the treatment upregulated key proliferation and remodeling markers including alpha smooth muscle actin (α-SMA), vimentin, and transforming growth factor beta 1 (TGF-β1). These findings demonstrate that the SCderm Matrix promotes wound healing through multiple mechanisms: modulating inflammatory responses, enhancing antioxidant defenses, and supporting tissue regeneration. The results suggest this biomaterial has significant potential as an effective and versatile solution for clinical wound care applications. Full article

This study investigates the pathophysiological process of healed perforated corneal ulcers (HPCUs) in humans. All subjects underwent keratoplasty due to opacities or leakage from HPCUs. Half of each specimen was fixed with 4% glutaraldehyde for transmission electron microscope (TEM) examination. The other half was fixed in 10% formaldehyde for immunofluorescence (IF) examination. TEM identified layered structures with two cell types (polygonal and elongated) connected by gap or adherent junctions during early stage of healing. Both apoptotic and mitotic changes were found in both types of cells. There were no endothelial cells or Descemet’s membrane (DM) present in early stage of healing. During the intermediate stage, the healed area comprised three layers: epithelium, Bowman’s layer, and stroma, with an increase in stromal collagen. Later, adjacent endothelial cells crept in, forming DM and completing the cornea’s 5-layer structure. IF examinations revealed that vimentin⁺ and α-smooth muscle actin (αSMA)⁺ myofibroblasts gathered around the damaged site. Proliferating cell nuclear antigen⁺ cells, which indicated cell proliferation, were found in both cells. Anti-phospho-histone H2AX antibodies were found in some epithelial cells. CK14-positive cells were only found in superficial polygonal cells. Corneal wound healing is a complex process that includes apoptosis, cell migration, mitosis, differentiation, and extracellular matrix remodeling. Full article

During mouse pregnancy, the pubic symphysis (PS) undergoes a gradual transitioning into an interpubic ligament (IpL) for a successful delivery. After birth, this IpL is rapidly remodeled, returning to the non-pregnant morphology. The PS fibrocartilaginous cells acquire a myofibroblast-like phenotype, characterized by extracellular matrix (ECM) secretion, expression of α-smooth muscle actin (α-SMA), and vimentin. While the presence of myofibroblast-like cells during the IpL remodeling is well described, cell–cell interactions and how this might contribute to the delivery remains poorly understood. This study uses ultrastructure and molecular approaches to investigate cell–cell and cell–ECM junctions during mouse pregnancy and postpartum. Our findings reveal that the intercellular contacts between adjacent IpL myofibroblast-like cells, particularly at late pregnancy stages, are characterized as adherens and GAP junctions. The acquisition of contractile elements by IpL cells, coupled with neighboring cells and the surrounding ECM via junctional complexes, suggests an important role in supporting changes in the mechanical forces generated by pubic bone movements during mouse pregnancy and also in tying the pelvic bones together, which may help the birth canal closure after delivery. Further studies in PS biology may investigate fibroblast to myofibroblast differentiation signaling cascades, which regulate the expression of pro-fibrotic proteins and may provide new insights for preterm labor. Full article

In fibrotic skeletal muscles, excessive extracellular matrix (ECM) deposition is a result of increased activation and decreased apoptosis of myofibroblasts. The aim of this study is to investigate whether treatment with quercetin, kaempferol or capsaicin can reduce the transforming growth factor-beta 1 (TGF-β1)-induced myofibroblast differentiation and fibrotic ECM expression in differentiated C2C12 cells. Two-day-differentiated C2C12 cells were treated with TGF-β1 for 48 h to induce myofibroblast differentiation. Twenty-four hours before (pre-treatment) and for forty-eight hours with (co-treatment) TGF-β1 treatment, cells were exposed to quercetin (25, 50 µM), kaempferol (10, 25, 50 µM) or capsaicin (25, 50 µM). The immunofluorescence intensity of alpha smooth muscle actin (αSMA) and collagen type I/III gene expression were assessed as myofibroblast markers. MyoD immunofluorescence intensity was measured as a myogenic marker. Co-treatment of TGF-β1 with the phytochemicals was most effective, resulting in a decreased number of αSMA-positive cells (all three compounds), decreased collagen type I (kaempferol, capsaicin) and type III (kaempferol) gene expression, and increased MyoD (kaempferol, capsaicin) protein expression compared to TGF-β1 treatment. This study demonstrates that treatment with quercetin, kaempferol or capsaicin can reduce myofibroblast markers. This suggests a possible anti-fibrotic effect of the phytochemicals in skeletal muscle. Full article

Prolonged exposure to fine particulate matter (PM_2.5) has been implicated in accelerated aging, including organ fibrosis. This study aimed to investigate whether prenatal and postnatal PM_2.5 exposure promotes renal fibrogenesis in adulthood and whether long-term vitamin D supplementation alleviates associated renal injury. Pregnant Sprague-Dawley rats were randomly assigned to three groups: control (normal saline, NS), PM_2.5 exposure, and PM_2.5 exposure with vitamin D supplementation during gestation and lactation (n = 3/group). Male offspring were subsequently exposed to the same conditions from postnatal weeks 3 to 8 (n = 7/group). On postnatal day 56, PM_2.5-exposed rats showed lower body weight and more severe glomerular and tubulointerstitial damage compared to controls. Serum calcium levels were elevated in the PM_2.5 group. The expression of intrarenal renin, transforming growth factor-β1, α-smooth muscle actin, and vimentin was upregulated, accompanied by increased collagen deposition. Long-term vitamin D supplementation reversed most of these changes, except for intrarenal vimentin expression and serum calcium levels. These findings indicate that prenatal and postnatal PM_2.5 exposure can activate intrarenal renin signaling and fibrogenic pathways, contributing to renal fibrosis later in life. Long-term vitamin D supplementation may provide partial protective effects against PM_2.5-induced renal fibrogenesis. Full article

Background: Although endothelial mesenchymal transition (EndMT) has been characterized as a basic process in embryogenesis, EndMT is the mechanism that accelerates the development of cardiovascular diseases, including heart failure, aging, and complications of diabetes or hypertension as well. Endothelial cells lose their distinct markers and take on a mesenchymal phenotype during EndMT, expressing distinct products. Methods: In this study, type 1 Diabetes mellitus (T1DM) was induced in rats with streptozotocin (STZ) by intraperitoneal injection at a 60 mg/kg dose. Diabetic rats were randomly divided into two groups, namely, control and diabetic rats, for 4 weeks. Heart, aorta, and plasma samples were collected at the end of 4 weeks. Sequentially, biochemical parameters, cytokines, reactive oxygen species (ROS), protein expression of EndMT markers (Chemokine C-X-C motif ligand-1 (CXCL-1), vimentin, citrullinated histone H3 (H3Cit), α-smooth muscle actin (α-SMA), and transforming growth factor beta (TGF-β) and versican), components of the extracellular matrix (matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinase-1(TIMP-1), and discoidin domain tyrosine kinase receptor 2 (DDR-2)) were detected by ELISA or Western blot, respectively. Results: Cytokines and ROS were increased in diabetic hearts, which induced partial EndMT. Among EndMT markers, histone citrullination, α-SMA, and CXCL-1 were increased; vimentin was decreased in DM. The endothelial marker endothelin-1 was significantly higher in the aortas of DM rats. Interestingly, TGF-β showed a significant decrease in the diabetic heart, plasma, and aorta. Additionally, MMP-2/TIMP-1 levels also decreased in DM. Conclusions: To sum up, the identification of molecules and regulatory pathways involved in EndMT provided novel therapeutic approaches for cardiac pathophysiological conditions. Full article

Background/Objectives: Under physiological conditions, vascular smooth muscle cells (VSMCs) are in a quiescent contractile state, but under pathological conditions, such as atherosclerosis, they change their phenotype to synthetic, characterized by increased proliferation, migration, and production of an extracellular matrix. Furthermore, VSMCs can undergo calcification, switching to an osteoblast-like phenotype, contributing to plaque instability. Methods: In this study, we analyzed the phenotypic changes in VSMCs during the transition from a physiological to a pathological state, a key process in the progression of atherosclerosis, using confocal and transmission electron microscopy, real-time PCR, and intracellular calcium quantification. Results: Confocal and transmission electron microscopy revealed a prominent remodeling of the actin cytoskeleton, increasing autophagic vacuoles in synthetic VSMCs and the deposition of calcium microcrystals in calcified cells. Immunofluorescence analysis revealed differential expression of α-SMA (contractile marker) and galectin-3 (synthetic marker), confirming the phenotypic changes. Real-time PCR further validated these changes, showing upregulation of RUNX-2, a marker of osteogenic transition, in calcified VSMCs. Conclusions: This study highlights the dynamic plasticity of VSMCs and their role in atherosclerosis progression. Understanding the characteristics of these phenotypic transitions can help develop targeted therapies to mitigate vascular calcification and plaque instability, potentially countering cardiovascular disease. Full article

Vitamin D has been shown to improve immunity as well as vascular function. We investigated the effect of cholecalciferol on T-cell phenotype in cultured peripheral blood mononuclear cells (PBMCs) from twenty vitamin D-deficient, non-diabetic chronic kidney disease (CKD) subjects. We also studied vitamin D effects on endothelial and vascular function markers in human aortic endothelial cells (HAECs) and in human aortic smooth muscle cells (HASMCs), respectively. We studied endothelial nitric oxide synthase (eNOS), mitogen-activated protein kinase 38 (p38 Map kinase), protein kinase B (Akt), and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) in HAECs and α-smooth muscle actin (α-SMA), smooth muscle calponin (SM-Calponin), smooth muscle myosin heavy chain (SM-MHC), and calcium-sensing receptor (CaSR) in HASMCs. Vitamin D receptors (VDRs) and CYP27B1 were studied in both cell types. In cultured PBMCs isolated from CKD subjects, the percentage of T helper 1(TH1) cells significantly decreased while that of T helper 2 (TH2) cells increased after cholecalciferol treatment. No significant change in intracellular and surface markers of T helper 17 (TH17) and T regulatory (Treg) cells was observed. In vitro treatment of HASMCs and HAECs with cholecalciferol led to significant and favorable alterations in mRNA expression of markers of vascular smooth muscle cells, i.e., α-SMA, SM-Calponin, and SM-MHC. Regarding endothelial cell markers, mRNA encoding eNOS, p38 Map kinase, protein kinase B (Akt), NADPH oxidase, VDR, and CYP27B1 were also significantly changed. Finally, the expression levels of the following proteins were notably altered: NADPH oxidase and protein kinase B (Akt) (in HAECs); SM-MHC and SM-Calponin (in HASMCs). In vitro treatment of PBMCs with cholecalciferol led to a favorable change in T-cell population, decreasing TH1 and increasing TH2 cell percentage, along with beneficial alterations in mRNA expression of HASMCs and HAECs’ cell markers. This study provides evidence that cholecalciferol can influence immune and vascular function in CKD. Full article

Multipotent vascular stem cells (MVSCs) are found in the vascular wall and surrounding tissues and possess the ability to differentiate into mesenchymal lineages. Previous studies have shown that MVSCs can be activated in response to vascular injury and differentiate into vascular smooth muscle cells (SMCs), contributing to vascular remodeling and microvessel formation. However, it remains unclear as to whether and how microenvironmental changes in the extracellular matrix, such as substrate stiffness, modulates MVSC differentiation under pathological conditions. This study demonstrated that MVSCs cultured on stiff substrates exhibited increased cell spreading, stronger cell adhesion, and a higher expression of SMC markers, including myosin heavy chain (MHC), myocardin (MYCD), calponin 1 (CNN1), and smooth muscle α-actin (SMA). In contrast, MVSCs on soft substrates showed an elevated expression of the chondrogenic markers aggrecan 1 (AGC1) and collagen-II (COL2A1). The presence of TGF-β1 further increased the expression of SMC markers on stiff substrates and chondrogenic markers on the soft substrates. Collectively, these results establish substrate stiffness as a key regulator of MVSC lineage commitment through cytoskeletal reorganization, with TGF-β1 acting as a biochemical amplifier. Our findings highlight the substrate-stiffness-dependent differentiation of MVSCs and provide mechanistic insights into the role of MVSCs in vascular remodeling during atherosclerosis development and blood vessel regeneration. Full article

Background/Objectives: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by excessive extracellular matrix (ECM) production and tissue stiffening, resulting in impaired lung function. Sodium hydrogen exchanger isoform 1 (NHE1) is a key mediator of intracellular and extracellular pH regulation, influencing fibroblast activation, motility, and proliferative pathways. This study investigates the role of NHE1 in actin stress fiber formation, fibroblast-to-myofibroblast differentiation, and cytokine secretion in IPF progression. Methods: Fibroblasts were treated with profibrotic agonists, including transforming growth factor-beta (TGFβ), lysophosphatidic acid (LPA), and serotonin (THT), in the presence or absence of the NHE1-specific inhibitor, EIPA. Actin stress fibers were visualized using phalloidin staining, while α-smooth muscle actin (α-SMA) expression and cytokine secretion (TGFβ, IL-6, and IL-8) were quantified using immunostaining and ELISA. Intracellular pH changes were measured using BCECF-AM fluorescence. Results: Profibrotic agonists induced significant actin stress fiber formation and α-SMA expression in fibroblasts, both of which were abolished by EIPA. NHE1 activity was shown to mediate intracellular alkalization, a critical factor for fibroblast activation. Cytokine secretion, including TGFβ, IL-6, and IL-8, was enhanced by agonist treatments but reduced with NHE1 inhibition. Chronic TGFβ exposure increased intracellular pH and sustained myofibroblast differentiation, which was partially reversed by EIPA. Conclusions: NHE1 is indicated to play a novel and potential role in processes supporting profibrotic agonists driving fibroblast activation and IPF progression. Targeting NHE1 could present a potential therapeutic approach to disrupt profibrotic pathways and mitigate IPF severity. Full article