Epithelial-mesenchymal Transdifferentiation Research Articles

Acidic fibroblast growth factor inhibits reactive oxygen species-induced epithelial-mesenchymal transdifferentiation in vascular endothelial cells via the miR-155-5p/SIRT1/Nrf2/HO-1 pathway to promote wound healing in diabetic mice.

Diabetic chronic wounds are among the most common and serious complications of diabetes and are associated with significant morbidity and mortality. Endothelial-to-mesenchymal transition (EndMT) is a specific pathological state in which endothelial cells are transformed into mesenchymal cells in response to various stimuli, such as high glucose levels and high oxidative stress. Acidic fibroblast growth factor (aFGF), which is a member of the fibroblast growth factor family, possesses strong antioxidant properties and can promote the differentiation of mesenchymal stem cells into angiogenic cells. Therefore, we investigated the role of aFGF in EndMT in diabetic wounds and analysed the underlying mechanisms. A diabetic mouse model was used to verify the effect of aFGF on wound healing, and the effect of aFGF on vascular endothelial cells in a high-glucose environment was examined in vitro. We examined the expression of miR-155-5p in a high-glucose environment and the miR-155 downstream target gene SIRT1 by luciferase reporter assays. aFGF promoted wound closure and neovascularization in a mouse model of type 2 diabetes. In vitro, aFGF inhibited the production of total and mitochondrial reactive oxygen species (ROS) in vascular endothelial cells and alleviated epithelial-mesenchymal transdifferentiation in a high-glucose environment. Mechanistically, aFGF promoted the expression of SIRT1 and the downstream targets Nrf2 and HO-1 by negatively regulating miR-155-5p, thereby reducing ROS generation. In conclusion, our results suggest that aFGF inhibits ROS-induced epithelial-mesenchymal transdifferentiation in diabetic vascular endothelial cells via the miR-155-5p/SIRT1/Nrf2/HO-1 axis, thereby promoting wound healing.

Inhibition of EZH2 mitigates peritoneal fibrosis and lipid precipitation in peritoneal mesothelial cells mediated by klotho

Background Peritoneal fibrosis caused by long-term peritoneal dialysis (PD) is the main reason why patients withdraw from PD treatment. Lipid accumulation in the peritoneum was shown to participate in fibrosis, and klotho is a molecule involved in lipid metabolism. GSK343 (enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) inhibitor) has been verified to inhibit epithelial mesenchymal transdifferentiation (EMT) and peritoneal fibrosis, but its related mechanism remains unclear. This study aimed to investigate whether lipid accumulation was involved in the effect of GSK343 and its related mechanism. Materials and Methods First, the expression of EZH2, klotho and EMT indices in human peritoneal mesothelial cells (HMrSV5) incubated with high glucose (HG) levels was detected. After EZH2 was inhibited by GSK343, Western blot (WB), wound healing and Transwell assays were used to explore the effect of GSK343. EZH2 and klotho expression was also detected. Oil red O and Nile red staining and triglyceride (TG) detection kits were used to detect lipid accumulation. A rescue experiment with small interfering RNA specific for klotho (si-klotho) on the basis of GSK343 was also conducted to verify that GSK343 exerted its effect via klotho. In in vivo experiments, rats were administered GSK343, and the related index was assessed. Results In our study, we revealed that the expression of EZH2 was significantly upregulated and klotho was significantly downregulated in HMrSV5 cells induced by high glucose. With the aid of GSK343, we found that lipid deposition caused by HG was significantly decreased. In addition, EMT and fibrosis were also significantly alleviated. Moreover, GSK343 could also restore the downregulation of klotho. To further verify whether klotho mediated the effect of EZH2, a rescue experiment with si-klotho was also conducted. The results showed that si-klotho could counteract the protective effect of GSK343 on high glucose-induced lipid accumulation and fibrosis. In vivo experiments also revealed that GSK343 could relieve peritoneal fibrosis, lipid deposition and EMT by mitigating EZH2 and restoring klotho expression. Conclusions Combining these findings, we found that EZH2 regulated lipid deposition, peritoneal fibrosis, and EMT mediated by klotho. To our knowledge, this is the first study to demonstrate the effect of the EZH2-klotho interaction on peritoneal fibrosis. Hence, EZH2 and klotho could act as potential targets for the treatment of peritoneal fibrosis.

MALAT1-miR-30c-5p-CTGF/ATG5 axis regulates silica-induced experimental silicosis by mediating EMT in alveolar epithelial cells

Epithelial-mesenchymal transdifferentiation of alveolar type Ⅱ epithelial cells is a vital source of pulmonary myofibroblasts, and myofibroblasts formation is recognized as an important phase in the pathological process of silicosis. miR-30c-5p has been determined to be relevant in the activation of the epithelial-mesenchymal transition (EMT) in numerous disease processes. However, elucidating the role played by miR-30c-5p in the silicosis-associated EMT process remains a great challenge. In this work, based on the establishment of mouse silicosis and A549 cells EMT models, miR-30c-5p was interfered with in vivo and in vitro models to reveal its effects on EMT and autophagy. Moreover, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), connective tissue growth factor (CTGF), autophagy-related gene 5 (ATG5), and autophagy were further interfered with in the A549 cells models to uncover the possible molecular mechanism through which miR-30c-5p inhibits silicosis associated EMT. The results demonstrated the targeted binding of miR-30c-5p to CTGF, ATG5, and MALAT1, and showed that miR-30c-5p could prevent EMT in lung epithelial cells by acting on CTGF and ATG5-associated autophagy, thereby inhibiting the silicosis fibrosis process. Furthermore, we also found that lncRNA MALAT1 might competitively absorb miR-30c-5p and affect the EMT of lung epithelial cells. In a word, interfering with miR-30c-5p and its related molecules (MALAT1, CTGF, and ATG5-associated autophagy) may provide a reference point for the application of silicosis intervention-related targets.

Celastrol attenuates renal injury in 5/6 nephrectomized rats via inhibiting epithelial-mesenchymal transition and transforming growth factor-β1/Smad3 pathway.

Renal injury is an important factor in the development of chronic kidney diseases that pathologically manifested as renal fibrosis and podocyte damage. In the disease state, renal fibroblasts lead to high expression levels of α-smooth muscle actin (α-SMA), while podocytes undergo epithelial-mesenchymal transition, leading to proteinuria. Celastrol, a bioactive compound in the medicinal plant Tripterygium wilfordii, was found to delay the progression of early diabetic nephropathy and attenuate renal fibrosis in mice with unilateral ureteral obstruction. However, its effect on the renal system in 5/6 nephrectomized (Nx) rats remains unknown. The aim of this study was to explore the protective effects of celastrol and its underlying mechanisms in 5/6 Nx rats. We found that 24 h proteinuria and levels of blood urea nitrogen, serum creatinine, triglycerides, serum P, renal index and cholesterol significantly increased (P < 0.05), while that of serum albumin decreased significantly in 5/6 Nx rats. After intervention with celastrol, 24 h proteinuria and levels of blood urea nitrogen, serum creatinine, triglycerides, serum P, renal index, and cholesterol significantly decreased, while that of serum albumin significantly increased. Renal tissue pathological staining and transmission electron microscopy showed that celastrol ameliorated kidney injury and glomerular podocyte foot injury and induced significant anti-inflammatory effects. Quantitative polymerase chain reaction (PCR) and western blotting results revealed that nephrin and NEPH1 expression levels were upregulated, whereas α-SMA and Col4a1 expression levels were downregulated in the celastrol group. Celastrol inhibited the expression of transforming growth factor (TGF)-β1/Smad3 signaling pathway-related molecules such as TGF-β1 and P-Smad3. In summary, celastrol contributes to renal protection by inhibiting the epithelial-mesenchymal transdifferentiation and TGF-β1/Smad3 pathways.

Losartan ameliorates renal interstitial fibrosis through metabolic pathway and Smurfs-TGF-β/Smad

The genesis and development of renal fibrosis involve a variety of pathways closely related to inflammation, cytokines, oxidative stress and metabolic abnormalities. Renal fibrosis is the result of a complex combination of a variety of lesions. Epithelial-mesenchymal transdifferentiation (EMT) of renal tubular epithelial cells is considered the key to renal fibrosis. Losartan is a typical Angiotensin II (ANG II) receptor antagonist and relaxes blood vessels. In this study, we investigated the effects of losartan on Unilateral Ureteral Obstruction (UUO) model mice by studying the changes in the TGF-β/Smad and metabolomics. Male C57BL/6 J mice were intervened with the UUO model and given losartan (10, 20, 30 mg/kg/d) for 28 consecutive days. The results showed that losartan could reduce UUO-induced abnormal serum metabolic spectrum and renal function. It could also improve renal tubular-interstitial injury and fibrosis by reducing tubulointerstitial dilation and collagen deposition. In addition, losartan promoted the expression of Smurf2 and Smurf1, i.e., Smad7 and E3 ubiquitin-linked enzymes, in the nucleus to degrade the type I receptor of TGF-β1 (TβR-I) and P-Smad2/3 to inhibit renal tubular epithelial cells EMT. In summary, these findings indicated that losartan could regulate the TGF-β/Smad and metabolic pathway in UUO model mice through ubiquitination to reduce renal fibrosis.

Role of IGF-1R in epithelial-mesenchymal transdifferentiation of human peritoneal mesothelial cells.

Peritoneal fibrosis (PF) is caused by epithelial-mesenchymal transdifferentiation (EMT) in the peritoneum under high glucose (HG) conditions. The study aimed to explored the role of Insulin-like growth factor 1 receptor (IGF-1R) in the regulation of EMT in human peritoneal mesothelial cells (HPMCs). We used HG peritoneal dialysis fluid (PDF) to induce in vivo PF in mice, and treated HPMCs with HG in vitro to stimulate EMT. In the mice, the higher the glucose concentration in the dialysate, the more obvious the peritoneal tissue thickening and the more that collagen was deposited. The in vitro study indicated that the expression of IGF-1R, α-SMA, vimentin was upregulated, while the expression of occludin, ZO-1, and E-cadherin was downregulated in HPMCs under HG and IGF-1R overexpression conditions. Conversely, the expression of IGF-1R, α-SMA, and vimentin was downregulated, while the expression of occludin, ZO-1, and E-cadherin was upregulated in IGF-1R-underexpressed HPMCs under HG conditions. The cell migration abilities were increased, while the cell adhesion abilities were reduced in HPMCs under HG and IGF-1R overexpression conditions. In contrast, cell migration abilities were reduced, while cell adhesion abilities were increased in IGF-1Runderexpressed HPMCs under HG conditions. Targeting at IGF-1R may provide novel insights into the prevention and treatment of PF.

Inhibition of PI3K/AKT/mTOR Signalling Pathway Activates Autophagy and Suppresses Peritoneal Fibrosis in the Process of Peritoneal Dialysis.

Peritoneal dialysis (PD) is an important part of replacement therapy for kidney failure. However, long-term PD treatment can cause peritoneal fibrosis. Autophagy may be involved in the pathological mechanism of peritoneal fibrosis (PF). Although autophagy is currently known to be involved in course of PF, its specific effects still lack in-depth research. In this experiment, a high-glucose (HG)-induced peritoneal fibrosis rat model was successfully established via intraperitoneal injection of HG peritoneal dialysate, and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the mechanistic target of rapamycin (mTOR) inhibitor rapamycin were used to treat peritoneal fibrosis rats. In addition, in vitro studies of high glucose-induced peritoneal fibrosis were performed using rat peritoneal mesothelial cells (PMCs). In vivo and in vitro experiments showed that LY294002 and rapamycin effectively inhibited the process of PF induced by high glucose. In addition, LY294002 and rapamycin were found to alleviate fibrosis by eliminating intracellular reactive oxygen species (ROS) levels, promoting the expression of the epithelial mesenchymal transdifferentiation proteins zonula occludens-1 (ZO-1) and E-cadherin, and inhibiting the expression of p-PI3K, PI3K, p-mTOR, mTOR, the fibroblast-specific proteins ferroptosis suppressor protein 1 (FSP1), and alpha-smooth muscle actin (α-SMA). Moreover, LY294002 and rapamycin promoted expression of autophagy-related proteins LC3-II/I, p62, and beclin-1. The current data indicated that inhibition of PI3K/AKT/mTOR signalling pathway activated autophagy and suppressed PF in the process of PD. Therefore, intervention in this signalling pathway may become a research goal for the prevention and treatment of PF, which has important clinical significance.

Effects of Long-Chain Non-Coding Ribonucleic Acid Hox Tran Antisense RNA on Proliferation and Migration of Epithelial Cells of Cataract Lens

In order to explore effects of long-chain non-coding ribonucleic acid (RNA) HOTAIR on proliferation and migration of human lens epithelial cells, SRA01/04 cells were selected as the research strain in this study and divided into S1 group (no HOTAIR transfection), S2 group (siHOTAIR transfection), S3 group (siHOTAIR+10 ng/mL TGF-β2), and S4 group (no HOTAIR transfection+10 ng/mL TGF-β2) according to the presence or absence of transforming growth factor (TGF)-β2 and silent HOTAIR treatment. 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) colorimetric method was applied to detect cell proliferation.Western blot was used for detection of E-cadherin, zonula occluden-1 (ZO-1), Vimentin, α-smooth muscle actin (SMA), Snail, Slug, zinc finger E-box binding homeobox 1 (ZEB1), and Smad-2 expressions. Results showed that the number of transmembrane cells in S4 group was higher markedly than that of the other groups, but that of S2 group dropped steeply compared with the other groups (P &lt;0.05); E-cadherin (2.59±0.58) and ZO-1 (1.95±0.56) of S2 group increased hugely compared with the other groups, while Vimentin (0.57±0.14) and α-SMA (0.64±0.28) decreased sharply compared with the other groups (P &lt; 0.05); Snail (2.51±0.59), Slug (2.11±0.47), and ZEB1 (2.83±0.53) of S4 group rose obviously compared with the other groups, but the above of S2 group reduced hugely compared with the other groups (P &lt; 0.05); pSmad-2 and pSmad-3 of S4 group elevated greatly compared with the other groups, and those of S2 group reduced hugely compared with the other groups (P &lt; 0.05). In conclusion, HOTAIR high expression could promote TGF-β2-induced SRA01/04 cell proliferation, migration, invasion, and epithelial-mesenchymal trans-differentiation, which was related to TGF-β/Smad signaling pathway.

Multiple Antitumor Molecular Mechanisms Are Activated by a Fully Synthetic and Stabilized Pharmaceutical Product Delivering the Active Compound Sulforaphane (SFX-01) in Preclinical Model of Human Glioblastoma

Frequent relapses and therapeutic resistance make the management of glioblastoma (GBM, grade IV glioma), extremely difficult. Therefore, it is necessary to develop new pharmacological compounds to be used as a single treatment or in combination with current therapies in order to improve their effectiveness and reduce cytotoxicity for non-tumor cells. SFX-01 is a fully synthetic and stabilized pharmaceutical product containing the α-cyclodextrin that delivers the active compound 1-isothiocyanato-4-methyl-sulfinylbutane (SFN) and maintains biological activities of SFN. In this study, we verified whether SFX-01 was active in GBM preclinical models. Our data demonstrate that SFX-01 reduced cell proliferation and increased cell death in GBM cell lines and patient-derived glioma initiating cells (GICs) with a stem cell phenotype. The antiproliferative effects of SFX-01 were associated with a reduction in the stemness of GICs and reversion of neural-to-mesenchymal trans-differentiation (PMT) closely related to epithelial-to-mesenchymal trans-differentiation (EMT) of epithelial tumors. Commonly, PMT reversion decreases the invasive capacity of tumor cells and increases the sensitivity to pharmacological and instrumental therapies. SFX-01 induced caspase-dependent apoptosis, through both mitochondrion-mediated intrinsic and death-receptor-associated extrinsic pathways. Here, we demonstrate the involvement of reactive oxygen species (ROS) through mediating the reduction in the activity of essential molecular pathways, such as PI3K/Akt/mTOR, ERK, and STAT-3. SFX-01 also reduced the in vivo tumor growth of subcutaneous xenografts and increased the disease-free survival (DFS) and overall survival (OS), when tested in orthotopic intracranial GBM models. These effects were associated with reduced expression of HIF1α which, in turn, down-regulates neo-angiogenesis. So, SFX-01 may have potent anti-glioma effects, regulating important aspects of the biology of this neoplasia, such as hypoxia, stemness, and EMT reversion, which are commonly activated in this neoplasia and are responsible for therapeutic resistance and glioma recurrence. SFX-01 deserves to be considered as an emerging anticancer agent for the treatment of GBM. The possible radio- and chemo sensitization potential of SFX-01 should also be evaluated in further preclinical and clinical studies.

Comparative study on the biological characteristics of menstrual blood- and endometrium-derived endometrial cells.

During a woman's reproductive period, the endometrial tissue is shed and regenerated every month to prepare for pregnancy or for the next cycle. The aim of the present study was to isolate, culture and characterize human endometrial cells (ECs) derived from menstrual blood (MB) and the endometrium (E). MB-derived ECs (MB-ECs) were isolated from women's MB. E-derived ECs (E-ECs) were isolated from women's endometrial tissues. The present study investigated the epithelial cell marker cytokeratin 18 (CK18) in MB-ECs and E-ECs. Cell proliferation analyses indicated that E-ECs (population doubling time, 20.85 h) grew faster than MB-ECs (population doubling time, 22.05 h; P<0.05). Cell migration ability was found to be significantly greater for MB-ECs than for E-ECs at 48 h (P<0.01). MB-ECs incubated with TGF-β1 (3 ng/ml) exhibited significantly decreased CK18 mRNA expression (P<0.01), and significantly increased vimentin (Vim) mRNA (P<0.05) and protein (P<0.01) expression at 6 and 12 h, respectively. E-EC incubation with TGF-β1 (3 ng/ml) significantly decreased CK18 mRNA expression (P<0.01) at 12 h and significantly increased Vim mRNA (P<0.01) and protein expression (P<0.05) at 6 h. The present results indicated that MB-ECs and E-ECs were biologically different, and that epithelial-mesenchymal transdifferentiation could be induced by TGF-β1 treatment.

Silibinin alleviates silica-induced pulmonary fibrosis: Potential role in modulating inflammation and epithelial-mesenchymal transition.

Pulmonary fibrosis (PF) is a devastating interstitial lung disease resulting from indefinite causes with very few limited, those too ineffective therapeutic options. Earlier evidence reported inflammation and epithelial-mesenchymal transition (EMT) are the major threats in PF. The present study was aimed to examine the anti-fibrotic activity of silibinin (SB) in PF. PF was induced by administering oropharyngeal 1.5 mg/mice silica on day 1, followed by treatment with and without oral SB for 14 days. Lung injury was assessed by x-ray analysis on day 14 and all the animals were sacrificed on day 15. The results showed that silica remarkably altered the histoarchitecture and induced the expression of inflammatory components in BALF and pulmonary tissue. Immunoblotting investigation quantified the expression of TGF-β, p-smad2/3, collagen-I, fibronectin, and α-SMA in the pulmonary tissue. To this end, treatment with SB alleviated inflammatory components, including IL-1β, IL-6, and TNF-α in the fibrotic tissue. Moreover, SB harnessed the tissue architecture, improved diffusive scattering of x-ray signals, and modulated epithelial-mesenchymal phenotypic alterations, including TGF-β, p-smad2/3, and collagen-I. Altogether, the significant reduction of inflammatory signaling, collagen deposition, and epithelial-mesenchymal transdifferentiation by SB suggested that it could be used as a potential therapeutic candidate to treat pulmonary inflammation and fibrosis.

Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity in vitro and in vivo.

It has been a long-standing challenge to obtain from cell cultures adequate amounts of mouse corneal epithelial cells (mCEC) to perform transplantation surgery. This limitation is attributable to the passage dependent declines in their proliferative activity. We describe here development of a novel 6C medium that contains six different modulators of different signaling pathways, which control proliferative mCEC activity. Its usage shortens the time and effort required to obtain epithelial sheets for hastening healing of an epithelial wound in an experimental animal model. This serum-free 6C medium contains:Y27632, forskolin, SB431542, DAPT, IWP-2, LDN-193189 and also DermaLife K keratinocyte calcium. Their inclusion inhibits rises in four specific markers of epithelial mesenchymal transdifferentiation:ZEB1/2, Snail, β-catenin and α-SMA. This medium is applied in a feeder-free air-lifted system to obtain sufficient populations of epithelial progenitor cells whose procurement is facilitated due to suppression of progenitor epithelial cell transdifferentiation into epithelial-mesenchymal cells. Diminution of this decline in transdifferentiation was confirmed based on the invariance of P63, K14, Pax6, and K12 gene expression levels. This cell culture technique is expected to facilitate ex vivo characterization of mechanisms underlying cell fate determination. Furthermore, its implementation will improve yields of progenitor mouse corneal epithelial cells, which increases the likelihood of using these cells as a source to generate epithelial sheets for performing transplantation surgery to treat limbal stem cell deficiency in a clinical setting. In addition, the novel insight obtainable from such studies is expected to improve the outcomes of corneal regenerative medicine.

Pathology and pathogenic pathways in fabry nephropathy.

The pathophysiology of renal damage in Fabry nephropathy involves a complex biological mechanism. The intracellular deposition globotriaosylceramide (Gb3) is just the first step of the mechanism. The glycolipid deposition occurs in all renal cells (endothelial, epithelial and mesangial cells). It stimulates many biological processes, including cytokine release, epithelial-mesenchymal transdifferentiation, oxidative stress and the remodelling of vascular walls, resulting in subtle initial inflammation and eventually tissue fibrosis. It has been hypothesized that the processes activated by Gb3 deposition can subsequently progress independently of cellular deposition and that even Gb3 clearance by specific therapy cannot retard or stop these pathways. This review aims to gather the reported evidence of these cellular alterations and the resulting histological changes. Our approach is similar to a routine study of kidney biopsy. In the first part of the review, "histology" section, we describe the structures involved (glomeruli, vessels, tubules and interstitium) from a histological point of view. While in the second part, "pathogenesis" section, we present some interpretations about the implicated pathways based on the up-to-date available evidence.

Impact of the Tumor Microenvironment on Tumor Heterogeneity and Consequences for Cancer Cell Plasticity and Stemness.

Simple SummaryThe cancer cells in solid tumors are embedded in a complex connective tissue matrix composed of various other cell types, i.e., mesenchymal stroma/stem-like cells (MSCs) and tumor-associated macrophages (TAMs). This tumor microenvironment (TME) is considered the major cause of tumor heterogeneity, which in turn accounts for treatment failure in current cancer therapies. Physical and chemical signals from the TME as well as factors secreted by MSCs and TAMs can induce epigenetic alterations in the cancer cells that alter their phenotypic plasticity, eventually resulting in the generation of cancer stem cells (CSCs). Phenotype switching of CSCs involves processes such as epithelial-mesenchymal transition, transdifferentiation, retrodifferentiation, or spontaneous cell fusion of cancer cells with stromal cells, particularly MSCs. Principally, phenotype plasticity of cancer (stem) cells may be targeted pharmacologically to reduce tumor heterogeneity and hence resistance to therapy.Tumor heterogeneity is considered the major cause of treatment failure in current cancer therapies. This feature of solid tumors is not only the result of clonal outgrowth of cells with genetic mutations, but also of epigenetic alterations induced by physical and chemical signals from the tumor microenvironment (TME). Besides fibroblasts, endothelial and immune cells, mesenchymal stroma/stem-like cells (MSCs) and tumor-associated macrophages (TAMs) intimately crosstalk with cancer cells and can exhibit both anti- and pro-tumorigenic effects. MSCs can alter cancer cellular phenotypes to increase cancer cell plasticity, eventually resulting in the generation of cancer stem cells (CSCs). The shift between different phenotypic states (phenotype switching) of CSCs is controlled via both genetic programs, such as epithelial-mesenchymal transdifferentiation or retrodifferentiation, and epigenetic alterations triggered by signals from the TME, like hypoxia, spatial heterogeneity or stromal cell-derived chemokines. Finally, we highlight the role of spontaneous cancer cell fusion with various types of stromal cells. i.e., MSCs in shaping CSC plasticity. A better understanding of cell plasticity and phenotype shifting in CSCs is a prerequisite for exploiting this phenomenon to reduce tumor heterogeneity, thereby improving the chance for therapy success.

Research Progress on the Pathological Mechanisms of Podocytes in Diabetic Nephropathy.

Diabetic nephropathy (DN) is not only an important microvascular complication of diabetes but also the main cause of end-stage renal disease. Studies have shown that the occurrence and development of DN are closely related to morphological and functional changes in podocytes. A series of morphological changes after podocyte injury in DN mainly include podocyte hypertrophy, podocyte epithelial-mesenchymal transdifferentiation, podocyte detachment, and podocyte apoptosis; functional changes mainly involve podocyte autophagy. More and more studies have shown that multiple signaling pathways play important roles in the progression of podocyte injury in DN. Here, we review research progress on the pathological mechanism of morphological and functional changes in podocytes associated with DN, to provide a new target for delaying the occurrence and development of this disorder.

Increased Regeneration Following Stress-Induced Lung Injury in Bleomycin-Treated Chimeric Mice with CD44 Knockout Mesenchymal Cells.

CD44, an adhesion-molecule promoting cell-migration, is shown here to increase in stress conditions following bleomycin-induced apoptosis in alveolar epithelial cells (AECs), a main target of lung injury. In vivo, it inhibits tissue regeneration and leads to fibrosis. We show that some AECs survive by the ataxia-telangiectasia mutated kinase/ATM pathway, and undergo a CD44-mediated epithelial-mesenchymal transdifferentiation (EMT) with migratory capacities in vitro, and in vivo. We assessed apoptosis vs. proliferation of AECs following bleomycin, ATM/P53 signaling pathway in AECs, and CD44 involvement in EMT, cell motility and tissue regeneration in vitro and in vivo. Expression of survival genes, CD44, and ATM/p53 pathway was elevated in AECs surviving bleomycin injury, as were the markers of EMT (downregulation of E-cadherin, upregulation of N-cadherin and vimentin, nuclear translocation of β-catenin). Inhibition of CD44 decreased AECs transdifferentiation. Bleomycin-treated chimeric CD44KO-mice had decreased EMT markers, ATM, and mesenchymal cells (α-SMA+) accumulation in lung, increased surfactant-b, diminished lung mesenchymal cell motility, and increased lung tissue regenerative capacity following bleomycin injury, as indicated by lung collagen content and semiquantitave morphological index scoring. Thus, AECs surviving lung injury are plastic and undergo ATM-mediated, CD44-dependent transdifferentiation, preventing tissue regeneration and promoting fibrosis. Synthetic or natural compounds that downregulate CD44 may improve tissue regeneration following injury.

C/EBP\u03b1 deficiency in podocytes aggravates podocyte senescence and kidney injury in aging mice

Kidney aging leads to an increased incidence of end-stage renal disease (ESRD) in the elderly, and aging is a complex biological process controlled by signaling pathways and transcription factors. Podocyte senescence plays a central role in injury resulting from kidney aging. Here, we demonstrated the critical role of C/EBPα in podocyte senescence and kidney aging by generating a genetically modified mouse model of chronological aging in which C/EBPα was selectively deleted in podocytes and by overexpressing C/EBPα in cultured podocytes, in which premature senescence was induced by treatment with adriamycin. Moreover, we illuminated the mechanisms by which podocyte senescence causes tubular impairment by stimulating HK-2 cells with bovine serum albumin (BSA) and chloroquine. Our findings suggest that C/EBPα knockout in podocytes aggravates podocyte senescence through the AMPK/mTOR pathway, leading to glomerulosclerosis, and that subsequent albuminuria exacerbates the epithelial–mesenchymal transdifferentiation of senescent tubular cells by suppressing autophagy. These observations highlight the importance of C/EBPα as a new potential target in kidney aging.

Upregulation of Tissue Transglutaminase in Transforming Growth Factor-b Induced Epithelial Mesenchymal TG2Transdifferentiation In Rat Tubular Epithelial Cells

AbstractBackground: Epithelial-Mesenchymal Transdifferentiation (EMT) is believed to be a key step in the course of glomerular and tubulointerstitial fibrosis. Many of the regulators of this process have been identified and Transforming Growth Factor-b (TGF-b) plays a central role. Tissue transglutaminase (TG2) has a crucial role in extracellular matrix formation and stabi-lization.Aim of Study: This study was designed to explore whether TG2 is essential in EMT induced by TGF-b in renal tubular epithelial cells.Material and Methods: EMT was achieved by treating NRK52-E renal tubular epithelial cells with high concentration of rhTGF-b (10ng/ml) for 24, 48 and 72h. Development of mesenchymal cell markers Fibroblast Specific Protein (FSP-1) and a Smooth Muscle Actin (a-SMA) and decrease in tubular epithelial cell surface marker E-Cadherin were used to confirm EMT. The NTU283; TG2 inhibitor was used for the inhibition assays. TG2 activity was quantified using a 14C putrescine incorporation assay. Protein expression and localisation of different markers were assessed by western blot and immunofluorescent dual immunofluorescent staining. Phase analysis was used to quantify the amount of staining for each of the markers used.Results: A significant increase in TG2 activity and protein expression were both observed with TGF-b induced transdif-ferentiation at 48h. This observation was associated with loss of epithelial cell marker E-Cadherin and appearance of intra-cellular a-SMA and FSP-1, characteristic of myofibroblasts. Dual staining showed high TG2 levels in cells (0.254±0.07), compared to the control (0.02±0.008). This also showed loss of epithelial phenotype, decrease of E-cadherin and acquisition of myofibroblast characteristics a-SMA (0.671±0.13) com-pared to the control (0.076±0.016) and FSP-1 (0.492±0.017) compared to control (0.03±0.017). Cells also demonstrated high degree of co-localization of Collagen I (ColI) and a-SMA with TG2. The NTU283; TG2 inhibitor ameliorated TGF-b induced EMT. This was also associated with changed pattern of collagen I and a-SMA co-localization.Conclusion: This data demonstrated upregulation of TG2 during the process of TGF-b induced EMT. TG2 inhibitor showed non-significant reduction of both TG2 and mesenchy-mal markers but demonstrated changes in the molecular configuration of mesenchymal markers a-SMA and Col I, raising the possibility of a role of TG2 in EMT.

Epithelial-Mesenchymal Transdifferentiation in Pediatric Lens Epithelial Cells.

Posterior capsule opacification (PCO) is a complication after cataract surgery, particularly in children. Epithelial-mesenchymal transition (EMT) of lens epithelial cells, mediated by transforming growth factor beta (TGFβ), contributes to PCO. However, its pathogenesis in children is poorly understood. We correlated cell growth in culture with patient characteristics, studied gene expression of pediatric lens epithelial cells (pLEC), and examined the effects of TGFβ-2 on these cells in vitro. Clinical characteristics of children with cataracts correlated with growth behavior of pLEC in vitro. mRNA expression of epithelial (αB-crystallin, connexin-43) and mesenchymal (αV-integrin, α-smooth muscle actin, collagen-Iα2, fibronectin-1) markers was quantified in pLEC and in cell line HLE-B3 in the presence and absence of TGFβ-2. Fifty-four anterior lens capsules from 40 children aged 1 to 180 months were obtained. Cell outgrowth occurred in 44% of the capsules from patients ≤ 12 months and in 33% of capsules from children aged 13 to 60 months, but in only 6% of capsules from children over 60 months. TGFβ-2 significantly upregulated expression of αB-crystallin (HLE-B3), αV-integrin (HLE-B3), collagen-Iα2, and fibronectin-1 (in pLEC and HLE-B3 cells). Patient characteristics correlated with growth behavior of pLEC in vitro, paralleling a higher clinical incidence of PCO in younger children. Gene expression profiles of pLEC and HLE-B3 suggest that upregulation of αV-integrin, collagen-Iα2, and fibronectin-1 are involved in EMT.

Seminal plasma (SP) induces a rapid transforming growth factor beta 1 (TGFβ1)-independent up-regulation of epithelial-mesenchymal transdifferentiation (EMT) and myofibroblastic metaplasia-markers in endometriotic (EM) and endometrial cells.

To study if short-term exposure (2h and 6h) of endometrial/endometriotic tissues and cells to 10% seminal plasma (SP) can induce EMT/metaplasia. Basic research experimental study was carried out in a University hospital-based fertility center. Semen samples, peritoneal fluid (PF) from endometriosis patients, endometrial biopsy from premenopausal women, immortalized endometriotic epithelial cell line (12Z), and immortalized endometrial stromal cell line (St-T1b) were studied. Rapid stain identification test (RSID), TGFβ1 immunofluorescence of washed sperms, TGFβ1-ELISA of SP and PF, in vitro study (2h and 6h incubation) and real-time PCR of endometrial tissue and cell lines to analyze gene expression of EMT/metaplasia markers and mediators were done. SP is still detectable in washed semen. TGFβ1 was expressed on the plasma membrane of the sperms and was significantly more concentrated in SP (88.17ng/ml) than PF. 10% SP induced an up-regulation of alpha smooth muscle actin expression in endometrial tissue (p = 0.008) and in 12Z cells (p = 0.05), mostly TGFβ1-independent. TWIST expression was persistently significantly down-regulated while Snail1 and 2 were up-regulated, though insignificant. Our results provide novel evidence to support that even in semen washed twice, SP is still detectable. The changes in EMT/metaplasia markers and mediators give a new insight into a possible effect of SP on the pathogenesis of endometriosis.