Growth Factor In Epithelial Cells Research Articles

The Preventive Effect of Endostar on Radiation-induced Pulmonary Fibrosis.

Radiation-induced pulmonary fibrosis (RIPF) is a long-term complication of thoracic radiotherapy without effective treatment available. This study aimed to establish a RIPF mouse model and explore the therapeutic effects and mechanisms of recombinant human endostatin (Endostar). C57BL/6 mice received a 16-Gy dose of X-rays to the whole thorax with or without the administration of Endostar for 24 weeks. Radiation-induced body weight loss was partially attenuated by Endostar (P<0.05). Endostar significantly reduced alveolar inflammation (P<0.05) and pulmonary fibrosis (P<0.001), as indicated by a decrease in the expression levels of collagen I and collagen IV in lung tissue (both P<0.001). Angiogenesis (as shown by CD31 immunohistochemistry) was also decreased (P<0.01). In irradiated mice, Endostar inhibited the transforming growth factor-β1 (TGF-β1)/drosophila mothers against the decapentaplegic 3 (Smad3)/extracellular regulated protein kinases (ERK) signaling pathway (all P<0.05). In vitro, Endostar treatment decreased the radiation-induced expression of TGF-β1, vascular endothelial growth factor (VEGF), p-Smad3, and p-ERK in alveolar epithelial cells and vascular endothelial cells (all P<0.05). Endostar could alleviate RIPF through decreased antiangiogenic activity and inhibition of the TGF-β1/Smad3/ERK pathway.

Human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium.

To investigate human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium (CM). A human corneal epithelial cell line (HCE-T), human corneal fibroblasts (HCF) and keratoconus fibroblasts (KC-HCF) have been used. Twenty-four hours after RB-PDT (0.001% RB concentration, 565 nm wavelength illumination, 0.17 J/cm2 fluence) cell migration rate using scratch assay and growth factor concentrations in the cell culture supernatant using ELISA have been determined. In addition, the effect of CM has been observed. RB-PDT significantly reduced migration rate in all cell types, compared to controls (p≤0.02). Migration rate of HCE-T cultures without RB-PDT (untreated) was significantly higher using HCF CM after RB-PDT, than using HCF CM without RB-PDT (p<0.01). Similarly, untreated HCF displayed a significantly increased migration rate with HCE-T CM after RB-PDT, compared to HCE-T CM without treatment (p<0.01). Furthermore, illumination alone and RB-PDT significantly decreased keratinocyte growth factor (KGF) concentration in HCF and KC-HCF supernatant, and RB-PDT significantly decreased soluble N-Cadherin (SN-Cad) concentration in HCF supernatant, compared to controls (p<0.01 for all). In HCE-T CM, RB-PDT increased hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGFb) concentration (p≤0.02), while decreasing transforming growth factor β (TGF-β) concentration (p<0.01). FGFb concentration increased (p<0.0001) and TGF-β concentration decreased (p<0.0001) in HCF CM, by RB-PDT. Epidermal growth factor (EGF), HGF, and TGF-β concentration decreased (p≤0.03) and FGFb concentration increased (p<0.01) in KC-HCF CM, using RB-PDT. HCE-T, HCF and KC-HCF migration rate is reduced 24 hours after RB-PDT. In contrast, HCE-T migration is enhanced using HCF CM after RB-PDT, and HCF migration rate is increased through HCE-T CM following RB-PDT. Modulation of EGF, KGF, HGF, FGFb, TGF-β and N-Cadherin secretion through RB-PDT may play an important role in corneal wound healing.

Role of non-canonical T cells in homeostasis and pathology

In addition to the subsets of T lymphocytes and innate lymphocytes (innate lymphoid cells), the well-known players in adaptive immunity, there is an intermediate group of lymphocytes (innate-like cells) that already possess the T cell receptor, but with a restricted repertoire. This group includes γδT cells, subsets of type I and II NKT cells carrying both T cell receptor and NK-cell receptors, and mucosal-associated invariant T (MAIT) cells. The development of innate-like cells occurs in the thymus, but their positive and negative selection takes place without the participation of thymic epithelial cells. A distinctive feature is that innate-like cells acquire an effector phenotype already in the thymus, and therefore do not require complex activation reactions during antigen recognition. Upon exit from the thymus, noncanonical T cells express chemokine receptors, allowing them to migrate into barrier tissues at an early age. A characteristic feature of the T cell receptor innate-like cells is the recognition of non-peptide antigens presented in non-polymorphic histocompatibility molecules (MHC-Ib). This type of molecule includes the CD1 a/b/c/d/e molecule and the MR1 molecule. These molecules present lipid, glycolipid antigens and metabolites of B vitamins, synthesized by various representatives of the microbiota. The presence of functionally different subpopulations of innate-like cells with an activated phenotype allows them to quickly respond to the antigen by producing cytokines typical of Th1, Th2, Th17. They also exhibit cytotoxic and immunoregulatory activity. These cells are actively involved in regulation of barrier tissue homeostasis and interaction with microbiota. They synthesize growth factors for epithelial cells, fibroblasts, and vascular endothelium, which are required for regeneration of damaged tissues. They also participate in anti-infectious defense, directing the development of the immune response. Moreover, they have been found to be involved in many autoimmune diseases. The special functions of innate-like cells make them a promising target for therapeutic interventions. It has been shown that antibiotics, salicylates and some other well-known drugs exert certain effects on the innate-like cells. Different dietary options also affect the activity of these cells.

Hsa-miR-379 down-regulates Rac1/MLK3/JNK/AP-1/Collagen I cascade reaction by targeting connective tissue growth factor in human alveolar basal epithelial A549 cells

ObjectiveThis study was aimed to screen and identify miRNAs that could regulate human CTGF gene and downstream cascade reaction Rac1/MLK3/JNK/AP-1/Collagen I by bioinformatics and experimental means. MethodsTargetScan and Tarbase were used to predict miRNAs that may have regulatory effects on human CTGF gene. The dual-luciferase reporter gene assay was employed to verify the results obtained in bioinformatics. Human alveolar basal epithelial A549 cells were exposed to silica (SiO2) culture medium for 24 h to establish an in vitro model of pulmonary fibrosis, and bleomycin (BLM) of 100 ng/mL was used as a positive control. The miRNA and mRNA expression levels were determined by RT-qPCR, and the protein levels were measured by western blot in hsa-miR-379-3p overexpression group or not. ResultsA total of 9 differentially expressed miRNAs that might regulate the human CTGF gene were predicted. Hsa-miR-379-3p and hsa-miR-411-3p were selected for the subsequent experiments. The results of the dual-luciferase reporter assay showed that hsa-miR-379-3p could bind to CTGF, but hsa-miR-411-3p could not. Compared with the control group, SiO2 exposure (25 and 50 μg/mL) could significantly reduce the expression level of hsa-miR-379-3p in A549 cells. SiO2 exposure (50 μg/mL) could significantly increase the mRNA expression levels of CTGF, Collagen I, Rac1, MLK3, JNK, AP1, and VIM in A549 cells, while CDH1 level was significantly decreased. Compared with SiO2 + NC group, the mRNA expression levels of CTGF, Collagen I, Rac1, MLK3, JNK, AP1, and VIM were significantly decreased, and CDH1 level was significantly higher when hsa-miR-379-3p was overexpressed. At the same time, overexpression of hsa-miR-379-3p improved the protein levels of CTGF, Collagen I, c-Jun and phospho-c-Jun, JNK1 and phospho-JNK1 significantly compared with SiO2 + NC group. ConclusionHsa-miR-379-3p was demonstrated for the first time that could directly target and down-regulate human CTGF gene, and further affect the expression levels of key genes and proteins in Rac1/MLK3/JNK/AP-1/Collagen I cascade reaction.

Proanthocyanidins Promote Endothelial Cell Viability and Angiogenesis.

Botanic drugs are reportedly effective in treating ischemic conditions by improving vascular circulation. However, it has been very rare for biomaterial researchers to look into the possibility of using such products in the context of tissue regeneration. This work studied 4 botanic drugs to explore their effects on vascular endothelial cell growth. Human umbilical endothelial cells were cultured in the presence of different doses of astragalus powder extract, astragalus injection, puerarin injection, and proanthocyanidin (PAC). Among the 4 drugs, PAC showed a potent effect on cell viability and stimulated cell growth in a dose-dependent manner. In particular, the PAC under test was able to maintain a high level of cell viability/proliferation comparable with the cells supplemented with the endothelial cell growth medium, at both low and normal serum conditions. Blocking either endothelial cell growth factor receptors or epithelial cell growth factor receptors was ineffective in reducing the stimulatory effect. The PAC released from polyvinyl alcohol cryogels stimulated HUVECs proliferation. The chick embryo chorioallantoic membrane model was further used to test the angiogenicity of PAC, showing that this botanic drug was potent in stimulating vasculature development. This work therefore demonstrates for the first time that PAC is capable of upregulating endothelial cell activity and growth in vitro in the absence of growth factors and that PAC can be loaded and released from drug carriers and can stimulate angiogenesis. These findings suggest the application of PAC in angiogenesis and tissue regeneration.

Effects of Chitosan Drug Loaded Nanoparticles on Migration Activity of Retinal Pigment Epithelial Cells

ABSTRACTMethazolamide (MET) was used as a model drug. Chitosan (Chi), which had the advantages of biodegradation, non-toxicity and biocompatibility, was used to modify the surface of solid lipid nanoparticles (SLNs) to construct a stable and targeted drug carrier for eyes. Chi modified MET SLNs were prepared by emulsion evaporation low-temperature curing method. Then, the samples were characterized by infrared spectroscopy and X-ray diffraction. Its effect on hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells was analyzed. Meanwhile, the effect of the prepared nanoparticles (NPs) on the migration of retinal microvascular endothelial cells was analyzed. The results showed that the optimal formulation of Chi modified MET SLNs was as follows: 35 mg MET, 80 mg glyceryl monostearate (GMS), 15 mg phospholipid, and 10 ml anhydrous ethanol, which were heated and dissolved at 80 °C to form an organic phase. Besides, 20 ml solution composed of 1% Tween80 and 2% Polyethylene glycogen (PEG) 400 was used as the internal aqueous phase. Under hypoxia condition, the up-regulation of HIF-1α and VEGF expression was significantly inhibited; the RPE cells treated with prepared NPs migrated to the center, so as to inhibit the migration of vascular endothelial cells. The average inhibition rate was 44%.

A novel method for isolation and culture of primary swine gastric epithelial cells

BackgroundCulturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach.ResultsMechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37 °C, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively.ConclusionsA new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

β-elemene down-regulates HIF-lα, VEGF and iNOS in human retinal pigment epithelial cells under high glucose conditions

To investigate the effects and mechanism of β-elemene on the expressions of hypoxia-inducible factor-1α (HIF-lα), vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) in human retinal pigment epithelial (RPE) cells under high glucose conditions. ARPE-19 cell line was cultured under eight conditions: 1) low glucose (LG; 5.5 mmol/L); 2) high glucose (HG; 33 mmol/L); 3) high glucose with 20 µg/mL β-elemene (HG+20E); 4) high glucose with 40 µg/mL β-elemene (HG+40E); 5) high glucose with SB203590 [HG+SB203590, p38-mitogen-activated protein kinase (p38-MAPK) pathway inhibitor]; 6) high glucose with LY294002 [HG+LY294002, phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway inhibitor]; 7) high glucose with 40 µg/mL β-elemene and SB203590 (HG+40E+SB203590); and 8) high glucose with 40 µg/mL β-elemene and LY294002 (HG+40E+LY294002). Cells were treated in conditions 1-4 for 24 and 48h, while for 48h in conditions 5-8. Then mRNA and protein levels of HIF-1α, VEGF and iNOS in cells were measured by real-time polymerase chain reaction (qPCR), immunofluorescence and Western blotting, respectively. Furthermore, protein levels of total p38-MAPK, phosphorylated p38-MAPK (p38-MAPK-P), total Akt and phosphorylated Akt (Akt-P) in cells of conditions 2 and 4 which treated for 48h were measured by Western blotting. The mRNA levels and protein levels of HIF-1α, VEGF and iNOS in cells were significantly reduced in conditions 3-8 when compared with those in condition 2 (P<0.05). These reductions were more obvious in conditions treated for 48h than in conditions treated for 24h. The protein levels of p38-MAPK-P and Akt-P in cells of condition 4 were significantly lower than in condition 2 (P<0.01). β-elemene down-regulates HIF-1α, VEGF and iNOS in ARPE-19 cells under a high glucose condition. The inhibitory effect of β-elemene is more significant when its concentration and treatment time are increased, as well as it is combined with SB203590 or LY294002 treatment. P38-MAPK and PI3K/Akt signaling pathways may play a role in this inhibitory effect.

Tangeretin Inhibition of High-Glucose-Induced IL-1β, IL-6, TGF-β1, and VEGF Expression in Human RPE Cells.

Tangeretin, a natural compound extracted from citrus plants, has been reported to have antiproliferative, antidiabetic, anti-invasive, and antioxidant properties. However, the role of tangeretin in diabetic retinopathy (DR) is unknown. In the present study, we investigated whether tangeretin had any effect on the expression of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), transforming growth factor beta 1 (TGF-β1), and vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (RPE) cells under high-glucose (HG) conditions. Our results illustrated that HG levels induced IL-1β, IL-6, TGF-β1, and VEGF expression and that tangeretin significantly reduced HG-induced IL-1β, IL-6, TGF-β1, and VEGF expression in human RPE cells. Moreover, tangeretin efficiently inhibited the activation of the protein kinase B (Akt) signalling pathway in HG-stimulated RPE cells. Therefore, tangeretin may serve a role in the treatment of DR.

Factores de crecimiento en el plasma: herramienta terapéutica en dermatología

Las plaquetas contienen una gran cantidad de factores de crecimiento que participan en los procesos de cicatrización tisular. Entre ellos, el factor de crecimiento derivado de las plaquetas (PDGF), el factor de crecimiento transformante (TGF), el factor plaquetario 4 (PF4), la interleucina (IL)-1, el factor angiogénico derivado de las plaquetas (PDAF), el factor de crecimiento endotelial (VEGF), el factor de crecimiento epidérmico (EGF), el factor de crecimiento endotelial derivado de las plaquetas (PDEGF), el factor de crecimiento de células epiteliales (ECGF) y el factor de crecimiento similar a la insulina (IGF). El plasma rico en plaquetas (PRP) es un derivado sanguíneo concentrado de la sangre total con una alta concentración de plaquetas. Otro componente esencial del PRP son las proteínas que actúan a nivel de la adhesión celular (fibrina, fibronectina y vitronectina), que proporcionan el soporte estructural necesario para la migración celular y para la proliferación y crecimiento tridimensional de los tejidos sobre los que actúa. La fibrina es la forma activada del fibrinógeno, sustrato final de todas las reacciones de coagulación, se transforma en fibrina insoluble por acción de la trombina. El gel de fibrina polimerizado constituye la primera matriz cicatricial de las heridas. Tanto el plasma rico en plaquetas como las mallas de fibrina varían en la composición y concentración de factores de crecimiento, proteínas y citocinas. En este trabajo se revisan las características de estos productos biológicos, su aplicación en dermatología así como los principales requisitos para su preparación

Long non-coding RNA366.2 controls endometrial epithelial cell proliferation and migration by upregulating WNT6 as a ceRNA of miR-1576 in sheep uterus

Long non-coding RNAs (lncRNAs) play an important regulatory role in mammalian fecundity. Currently, most studies are primarily concentrated on ovarian lncRNAs, ignoring the influence of uterine lncRNAs on the fecundity of female sheep. In this study, we found a higher density of uterine glands and endometrial microvessel density (MVD) in high prolificacy group of Hu sheep compared to low prolificacy groups (p < 0.05) as well as an increased level of serum placental growth factor (PLGF). Hundreds of differentially expressed (DE) lncRNAs were identified in Hu sheep with different fecundity by RNA sequencing (RNA-seq), and their targets were enriched in some signaling pathways involved in endometrial functions, such as the estrogen signaling pathway, nuclear factor kappa B (NF-κB) signaling pathway, oxytocin signaling pathway, and Wnt signaling pathway. Furthermore, the underlying mechanisms of competitive endogenous RNA (ceRNA) of lncRNA366.2-miR-1576- WNT6 were determined by bioinformatics analysis. Functionally, our results indicated that lncRNA366.2 promoted endometrial epithelial cell (EEC) proliferation, migration, and growth factor expression by sponging miR-1576 to upregulate WNT6 expression and activate the Wnt/β-catenin pathway. Taken together, our research indicated the regulatory mechanism of the lncRNA366.2-miR-1576-WNT6 in EEC proliferation and migration. Furthermore, this study provides a new theoretical reference for the identification of candidate genes related to fecundity.

HGF/Met Axis Protects Human Retinal Pigment Epithelial Cells from Cell Death

Demise of the retinal pigment epithelium (RPE) caused by etiologies such as age‐related macular degeneration is a major underlying factor leading to vision loss. Cell death can occur through two major pathways: apoptosis and necrosis/necroptosis. In the former, Fas/FasL death receptor signaling induces apoptosis, but its role in human RPE cell death is not thoroughly elucidated. In the latter, reactive oxygen species (ROS) can induce necrosis/necroptosis. Additionally, Hepatocyte Growth Factor (HGF) is a potent growth and survival factor for epithelial cells, but its functional role in RPE is not well characterized, especially its ability to protect RPE cells from death and to induce their growth. Accordingly, we hypothesized that HGF/Met protects human RPE cells from Fas‐promoted apoptosis and H2O2‐induced necrosis/necroptosis.We analyzed whether the HGF receptor (Met) is expressed in human and non‐human primate (NHP) eye tissue via western blot analysis and immunohistochemistry. To determine whether HGF/Met protects RPE cells against death induced by Fas/FasL and H2O2, we utilized gain‐ and loss‐of‐function models, cell survival assays and gene expression analysis (RNAseq). Human ARPE‐19 cell line was treated with various combinations of HGF, Met kinase inhibitor SU11274 (SU), FasL or H2O2. Cell growth and survival was determined via MTT assay. Met, Akt, and Erk activation were analyzed by western blot.Western blot analysis and immunohistochemistry confirmed Met activity and signaling in human and NHP eye tissue, including in the RPE cell layer. We found that HGF elicited Met stimulation and signaling culminating in activation of its downstream effectors Akt and Erk, proteins involved in two major pathways controlling cell survival and proliferation. We also uncovered that HGF inhibited FasL‐ and ROS‐induced killing of RPE. Conversely, inhibiting Met signaling rendered RPE susceptible to cell death induced by FasL and H2O2. Global gene expression analysis using an RNAseq approach revealed that HGF controls expression of numerous genes governing RPE cell growth, metabolism, and survival to name a few. In particular, HGF significantly upregulated 400 and repressed expression of 420 genes six hours post‐treatment; at 24 hours post‐treatment, HGF upregulated 291 and downregulated expression of 260 genes. Expression of 11,000 genes was unchanged.Our studies establish that HGF and Met play a major role in promoting the survival and homeostasis of RPE cells by inhibiting two major pathways of RPE cell death. Our findings suggest HGF/Met is a potential druggable target for therapeutic management of RPE‐related human ocular diseases.

Early Events in Actin Cytoskeleton Dynamics and E-Cadherin-Mediated Cell-Cell Adhesion during Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) plays an important role in development and also in initiation of metastasis during cancer. Disruption of cell-cell contacts during EMT allowing cells to detach from and migrate away from their neighbors remains poorly understood. Using immunofluorescent staining and live-cell imaging, we analyzed early events during EMT induced by epidermal growth factor (EGF) in IAR-20 normal epithelial cells. Control cells demonstrated stable adherens junctions (AJs) and robust contact paralysis, whereas addition of EGF caused rapid dynamic changes at the cell-cell boundaries: fragmentation of the circumferential actin bundle, assembly of actin network in lamellipodia, and retrograde flow. Simultaneously, an actin-binding protein EPLIN was phosphorylated, which may have decreased the stability of the circumferential actin bundle. Addition of EGF caused gradual replacement of linear E-cadherin–based AJs with dynamic and unstable punctate AJs, which, unlike linear AJs, colocalized with the mechanosensitive protein zyxin, confirming generation of centripetal force at the sites of cell-cell contacts during EMT. Our data show that early EMT promotes heightened dynamics at the cell-cell boundaries—replacement of stable AJs and actin structures with dynamic ones—which results in overall weakening of cell-cell adhesion, thus priming the cells for front-rear polarization and eventual migration.

A composite Gelatin/hyaluronic acid hydrogel as an ECM mimic for developing mesenchymal stem cell-derived epithelial tissue patches.

Here we report fabrication of Gelatin-based biocomposite films and their application in developing epithelial patches. The films were loaded with an epithelial cell growth factor cocktail and used as an extracellular matrix mimic for in vitro regeneration of organized respiratory epithelium using Calu-3 cell line and mesenchymal stem cells (MSCs). Our data show differentiation of Calu-3 cells on composite films as evidenced by tight junction protein expression and barrier formation. The films also supported attachment, migration, and proliferation of alveolar basal epithelial cell line A549. We also show the suitability of the composite films as a biomimetic scaffold and growth factor delivery platform for differentiation of human MSCs to epithelial cells. MSCs differentiation to the epithelial lineage was confirmed by staining for epithelial and stem cell specific markers. Our data show that the MSCs acquire the epithelial characteristics after 2 weeks with significant reduction in vimentin, increase in pan cytokeratin expression, and morphological changes. However, despite the expression of epithelial lineage markers, these cells did not form fully functional tight junctions as evidenced by low expression of junctional protein ZO1. Further optimisation of culture conditions and growth factor cocktail is required to enhance tight junction formation in MSCs-derived epithelial cells on the composite hydrogels. Nevertheless, our data clearly highlight the possibility of using MSCs in epithelial tissue engineering and the applicability of the composite hydrogels as transferrable extracellular matrix mimics and delivery platforms with potential applications in regenerative medicine and in vitro modelling of barrier tissues.

6-sialyllactose ameliorates dihydrotestosterone-induced benign prostatic hyperplasia through suppressing VEGF-mediated angiogenesis

Benign prostatic hyperplasia (BPH), a common disease in elderly males, is accompanied by non-malignant growth of prostate tissues, subsequently causing hypoxia and angiogenesis. Although VEGF-related angiogenesis is one of the therapeutic targets of prostate cancer, there is no previous study targeting angiogenesis for treatment of BPH. Dihydrotestosterone (DHT)-induced expressions of vascular endothelial growth factor (VEGF) in prostate epithelial RWPE-1 cells and human umbilical vascular endothelial cells (HUVECs). Conditioned media (CM) from DHT-treated RWPE-1 cells were transferred to HUVECs. Then, 6SL inhibited proliferation, VEGFR-2 activation, and tube formation of HUVECs transferred with CM from DHT-treated RWPE-1 cells. In the rat BPH model, 6SL reduced prostate weight, size, and thickness of the prostate tissue. Formation of vessels in prostatic tissues were also reduced with 6SL treatment. We found that 6SL has an ameliorative effect on in vitro and in vivo the BPH model via inhibition of VEGFR-2 activation and subsequent angiogenesis. These results suggest that 6SL might be a candidate for development of novel BPH drugs.

Knockdown of dickkopf2 inhibits vascular endothelia growth factor expression through the Wnt/β-catenin signaling pathway in human retinal pigment epithelial cells under hypoxic conditions

Hypoxia has been demonstrated to be a proangiogenic factor that induces vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. Dickkopf2 (DKK2), originally known as Wnt antagonist, has recently been demonstrated to have an important regulatory role in angiogenesis; however, the specific role of DKK2 in RPE cells is not known. In the present study, the effects of DKK2 on VEGF expression under hypoxic conditions were investigated, as well as the molecular mechanisms involved. The results demonstrated that the expression of DKK2 was markedly increased under hypoxic conditions compared with normoxic conditions. Knockdown of DKK2 markedly attenuated the CoCl2-induced expression of hypoxia-inducible factor (HIF)-1α and VEGF in RPE cells. Furthermore, knockdown of DKK2 markedly inhibited the expression of β-catenin induced by hypoxia. In conclusion, the findings of the present study demonstrate that knockdown of DKK2 inhibits the hypoxia-induced production of VEGF by suppressing the activation of the Wnt/β-catenin signaling pathway.

Inhibitory effect of 1‑tetradecanol on Helicobacterpylori‑induced production of interleukin‑8 and vascular endothelial growth factor in gastric epithelial cells.

Helicobacterpylori (H.pylori) infection activates pro‑inflammatory mediators, including interleukin (IL)‑8 and vascular endothelial growth factor (VEGF) in gastric epithelial cells. 1‑Tetradecanol (1‑TD) has been purified from Dendropanaxmorbifera Leveille; its physiological activities are poorly understood. The present study assessed whether 1‑TD has an effect on H.pylori‑mediated inflammation in AGS gastric epithelial cells. 1‑TD reduced IL‑8 production by AGS cells in response to H.pylori in a significant and dose‑dependent manner, as measured by ELISA. Western blot analysis demonstrated that 1‑TD also suppressed the activation of nuclear factor‑κB, and two mitogen activated protein kinase species (p38 and extracellular signal‑regulated kinase 1/2), but not c‑Jun N‑terminal kinase in H.pylori‑infected AGS cells. As predicted, VEGF expression and hypoxia inducible factor‑1α stabilization induced by H.pylori in AGS cells were inhibited by 1‑TD. In addition, 1‑TD directly inhibited the growth of H.pylori in a dose‑dependent manner, as investigated by measuring the optical density. These findings indicated that 1‑TD may be a potential preventive or therapeutic agent for H.pylori‑induced gastric inflammation.

EGF regulation of proximal tubule cell proliferation and VEGF-A secretion

Proximal tubule cell (PTC) proliferation is critical for tubular regeneration and recovery from acute kidney injury. Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF‐A) are important for the maintenance of tubulointerstitial integrity and can stimulate PTC proliferation. We utilized HK‐2 cells, an immortalized human PTC line, to characterize the EGF‐dependent regulation of VEGF‐A secretion and proliferation in PTCs. We demonstrate that EGF stimulates VEGF‐A secretion via the EGF receptor (EGFR) and stimulates cell proliferation via activation of the VEGF receptor, VEGFR‐2. EGFR activation promotes MAPK (ERK1/2) activation and HIF‐1α expression, which are required for basal and EGF‐stimulated VEGF‐A secretion. EGF also stimulates the phosphorylation of P70S6 kinase (P70S6K), the downstream target of mTORC1. Rapamycin decreased basal and EGF stimulated HIF‐1α and enhanced MAPK (ERK1/2) activation, while MAPK (ERK/12) inhibition downregulated HIF‐1α expression and the phosphorylation of p70S6K. EGF stimulation of p70S6K was also independent of p‐AKT. Inhibition of the mTORC1 pathway with rapamycin abolished phosphorylation of p70S6K but had no effect on VEGF‐A secretion, indicating that EGF‐stimulated VEGF‐A secretion did not require mTORC1 pathway activation. We demonstrate evidence of a complex crosstalk between the MAPK/ERK and mTORC1 pathways, wherein MAPK (ERK1/2) activation stimulates p‐P70S6K, while p‐P70S6K activation seems to inhibit MAPK (ERK1/2) in EGF‐treated HK‐2 cells. Our results suggest that EGF stimulates MAPK (ERK1/2) in HK‐2 cells, which in turn increases HIF‐1α expression and VEGF‐A secretion, indicating that VEGF‐A mediates EGF‐stimulated cell proliferation as an autocrine proximal tubular epithelial cell growth factor.

Expressions of TGF-β2, bFGF and ICAM-1 in lens epithelial cells of complicated cataract with silicone oil tamponade.

To investigate the expression differences of transforming growth factor-β2 (TGF-β2), basic fibroblast growth factor (bFGF) and intercellular cell-adhesion molecule-1 (ICAM-1) in lens epithelial cells (LECs) of complicated cataract with silicone oil tamponade and age-related cataract. Totally 150 eyes of 150 patients (aged 35 to 77y) were investigated, including 75 patients with complicated cataract after silicone oil tamponade and 75 patients with age-related cataract. The central piece of anterior capsules was collected during cataract surgery. TGF-β2, bFGF and ICAM-1 were detected in the 60 specimens of the two groups by immunohistochemistry. The expression levels of the three kinds of messenger ribonucleic acid (mRNA) were determined by real-time quantitative reverse transcription-polymerase chain reaction in the 90 specimens of the two groups. TGF-β2 was detected in the cytomembrane and cytoplasm of the LECs and bFGF was detected in the nucleus. ICAM-1 was positive in the cytomembrane of the LECs and the distribution of positive cells was uneven. The mRNA genes expression of the TGF-β2, bFGF and ICAM-1 was significant differences between the two groups and markedly increased in complicated cataract group (P<0.05). The up-regulated TGF-β2, bFGF and ICAM-1 maybe associate with the occurrence and development of complicated cataract with silicone oil tamponade.

Up-Regulation of Transient Receptor Potential Melastatin 6 Channel Expression by Tumor Necrosis Factor-α in the Presence of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor.

Anti-epidermal growth factor receptor (EGFR) drugs such as erlotinib and gefitinib cause a side effect of hypomagnesemia, but chemotherapy to treat this has not yet been developed. The transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of Mg2+ in the renal tubule. We reported previously that the expression of TRPM6 is up-regulated by epidermal growth factor (EGF) in renal tubular epithelial NRK-52E and HEK293 cells. EGF-induced elevation of TRPM6 expression was inhibited by erlotinib, gefitinib, and lapatinib. We found that tumor necrosis factor-α (TNF-α) increases TRPM6 expression in the presence of erlotinib. Therefore, we investigated what molecules are involved in the up-regulation of TRPM6 expression by TNF-α. EGF increased the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2), which were inhibited by erlotinib. TNF-α did not change p-ERK1/2 levels, but increased the phosphorylation and nuclear localization of nuclear factor-κB (NF-κB), which were blocked by the NF-κB inhibitors BAY 11-7082 and pyrrolidinedithiocarbamate ammonium. Similarly, luciferase reporter activity of human TRPM6 was increased by TNF-α, which was blocked by NF-κB inhibitors, and was inhibited by a mutation in the κB-binding site in the proximal region of the TRPM6 promoter. A chromatin immunoprecipitation assay revealed that NF-κB binds to the κB-binding site, which was blocked by NF-κB inhibitors. In the presence of erlotinib, TNF-α increased Mg2+ influx, which was blocked by NF-κB inhibitors. These results suggest that TNF-α reverses the reduction in Mg2+ reabsorption caused by anti-EGFR drugs. J. Cell. Physiol. 232: 2841-2850, 2017. © 2016 Wiley Periodicals, Inc.