Vascular Endothelial Growth Factor Gene Expression Research Articles

Cytocompatibility of electrospun poly-L-lactic acid membranes for Bruch's membrane regeneration using human embryonic stem cell-derived retinal pigment epithelial cells.

Cell replacement therapy is under development for dry age-related macular degeneration (AMD). A thin membrane resembling the Bruch's membrane is required to form a cell-on-membrane construct with retinal pigment epithelial (RPE) cells. These cells have been differentiated from human embryonic stem cells (hESCs) in vitro. A carrier membrane is required for cell implantation, which is biocompatible for cell growth and has dimensions and physical properties resembling the Bruch's membrane. Here a nanofiber electrospun poly-L-lactic acid (PLLA) membrane is tested for capacity to support cell growth and maturation. The requirements for laminin coating of the membrane are identified here. A porous electrospun nanofibrous PLLA membrane of ∼50 nm fiber diameter was developed as a prototype support for functional RPE cells grown as a monolayer. The need for laminin coating applied to the membrane following treatment with poly-L-ornithine (PLO), was identified in terms of cell growth and survival. Test membranes were compared in terms of hydrophilicity after laminin coating, mechanical properties of surface roughness and Young's modulus, porosity and ability to promote the attachment and proliferation of hESC-RPE cells in culture for up to 8 weeks. Over this time, RPE cell proliferation, morphology, and marker and gene expression, were monitored. The functional capacity of cell monolayers was identified in terms of transepithelial electrical resistance (TEER), phagocytosis of cells, as well as expression of the cytokines, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PLLA polymer fibers are naturally hydrophobic, so their hydrophilicity was improved by pretreatment with PLO for subsequent coating with the bioactive protein laminin. They were then assessed for amount of laminin adsorbed, contact angle and uniformity of coating using scanning electron microscopy (SEM). Pretreatment with 100% PLO gave the best result over 10% PLO treatment or no treatment prior to laminin adsorption with significantly greater surface stiffness and modulus. By 6 weeks after cell plating, the coated membranes could support a mature RPE monolayer showing a dense apical microvillus structure and pigmented 3D polygonal cell morphology. After 8 weeks, PLO (100%)-Lam coated membranes exhibited the highest cell number, cell proliferation, and RPE barrier function measured as TEER. RPE cells showed the higher levels of specific surface marker and gene expression. Microphthalmia-associated transcription factor expression was highly upregulated indicating maturation of cells. Functionality of cells was indicated by expression of VEGF and PEDF genes as well as phagocytic capacity. In conclusion, electrospun PLLA membranes coated with PLO-Lam have the physical and biological properties to support the distribution and migration of hESC-RPE cells throughout the whole structure. They represent a good membrane candidate for preparation of hESC-RPE cells as a monolayer for implantation into the subretinal space of AMD patients.

Development of VEGF mimetic QK peptide on polycaprolactone nanofiber for vascular tissue engineering

Electrospinning allows promoting vascular tissue formation. Polycaprolactone (PCL) is a promising polymer for vascular tissue engineering because of its great mechanical strength and efficient fabrication. However, PCL shows limited biocompatibility owing to its hydrophobic structure. PCL nanofibers can be modified by peptides to improve biological activity. KLTWQELYQLKYKGI (QK) is a peptide that mimics the Vascular Endothelial Growth Factor (VEGF) by organizing endothelial cells. It is critically important to identify the optimum concentration of QK peptide conjugation onto PCL nanofiber for vascularization. Thus, PCL nanofibers were conjugated by different QK peptide concentrations (10 µM, 100 µM and 1000 µM). QK conjugated nanofibers were characterized by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) analysis. The vascularization was evaluated by Live and Dead staining assay, real time PCR of platelet/endothelial cell adhesion molecule (PECAM-1), VEGF and vascular endothelial-cadherin (VE-cadherin) genes and VEGF immunofluorescence analysis. The surfaces of PCL nanofibers were modified by different concentrations of QK peptide. The roughness of nanofibers increased depending on the increased concentration of QK peptide. The higher cell attachment and cell viability were observed in 1000 µM of QK peptide. 1000 µM of QK peptide showed higher vasculogenic potential in terms of PECAM, VEGF, and VE-cadherin gene expression than all the other experimental groups. In the same on gene expression analysis by qPCR, 1000 µM concentration of QK peptide showed the higher VEGF protein than the other experimental groups. 1000 µM concentration of QK peptide can be used to improve the vasculogenic property of PCL nanofibers.

Efficacy of Sailuotong on neurovascular unit in amyloid precursor protein/presenilin-1 transgenic mice with Alzheimer's disease.

To discuss the influence of Sailuotong (, SLT) on the Neurovascular Unit (NVUs) of amyloid precursor protein (APP)/presenilin-1(PS1) mice and evaluate the role of gas supplementation in activating blood circulation during the progression of Alzheimer's disease (AD). The mice were allocated into the following nine groups: (a) the C57 Black (C57BL) sham-operated group (control group), (b) ischaemic treatment in C57BL mice (the C57 ischaemic group), (c) the APP/PS1 sham surgery group (APP/PS1 model group), (d) ischaemic treatment in APP/PS1 mice (APP/PS1 ischaemic group), (e) C57BL mice treated with aspirin following ischaemic treatment (C57BL ischaemic + aspirin group), (f) C57BL mice treated with SLT following ischaemic treatment (C57BL ischaemic + SLT group), (g) APP/PS1 mice treated with SLT (APP/PS1 + SLT group), (h) APP/PS1 mice treated with donepezil hydrochloride following ischaemic treatment (APP/PS1 ischaemic + donepezil hydrochloride group) and (i) APP/PS1 mice treated with SLT following ischaemic treatment (APP/PS1 ischaemic + SLT group). The ischaemic model was established by operating on the bilateral common carotid arteries and creating a microembolism. The Morris water maze and step-down tests were used to detect the spatial behaviour and memory ability of mice. The hippocampus of each mouse was observed by haematoxylin and eosin (HE) and Congo red staining. The ultrastructure of NVUs in each group was observed by electron microscopy, and various biochemical indicators were detected by enzyme-linked immunosorbent assay (ELISA). The protein expression level was detected by Western blot. The mRNA expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The results of the Morris water maze and step-down tests showed that ischemia reduced learning and memory in the mice, which were restored by SLT. The results of HE staining showed that SLT restored the pathological changes of the NVUs. The Congo red staining results revealed that SLT also improved the scattered orange-red sediments in the upper cortex and hippocampus of the APP/PS1 and APP/PS1 ischaemic mice. Furthermore, SLT significantly reduced the content of Aβ, improved the vascular endothelium and repaired the mitochondrial structures. The ELISA detection, western blot detection and qRT-PCR showed that SLT significantly increased the vascular endothelial growth factor (VEGF), angiopoietin and basic fibroblast growth factor, as well as the levels of gene and protein expression of low-density lipoprotein receptor-related protein-1 (LRP-1) and VEGF in brain tissue. By increasing the expression of VEGF, SLT can promote vascular proliferation, up-regulate the expression of LRP-1, promote the clearance of Aβ and improve the cognitive impairment of APP/PS1 mice. These results confirm that SLT can improve AD by promoting vascular proliferation and Aβ clearance to protect the function of NVUs.

Mechanism underlying the anti-diabetic potential of bee venom as compared to bone marrow mesenchymal stem cells in the diabetic rat tongue.

Diabetes mellitus (DM) is a critical chronic metabolic disease. Several treatment modalities are currently under investigation. Both bee venom (BV) and bone marrow mesenchymal stem cells (BMSCs) can possibly offer an approach for treating type I diabetes. The aim of the present study was to investigate the mechanism underlying the anti-diabetic effect of BV as compared to BMSCs on the tongue mucosa of diabetic rats. A total of 52 male albino rats were used in the current study. The rats were randomly assigned into 4 groups: group 1 (control); group 2 (streptozocin (STZ)); group 3 (BV-treated); and group 4 (BMSC-treated). Diabetes mellitus was induced via an intraperitoneal (IP) injection of STZ in the rats from groups 2, 3 and 4. Following the diagnosis of DM, the rats in group 3 were injected with a daily dose of 0.5 mg/kg of BV, while the rats in group 4 were treated with a single injection of BMSCs. All rats were euthanized after 4 weeks, and their tongues were dissected and divided into halves. The right halves of the tongues were utilized for the histological examination, followed by morphometric analysis. In contrast, the left halves were used to detect the local gene expression of transforming growth factor beta 1 (TGF-β1) and vascular endothelial growth factor (VEGF). Group 2 revealed marked disruption in the morphology of the fungiform and filiform papillae, and atrophic epithelial changes in both dorsal and ventral surface epithelium as compared to other groups. Group 4 showed a significantly larger number of taste buds, and a higher gene expression of TGF-β1 and VEGF as compared to groups 2 and 3. Additionally, BV and BMSCs effectively increased the thickness of dorsal and ventral surface epithelium as compared to group 2. Treatment with BMSCs was associated with significant improvement in the morphology and number of lingual epithelial cells and taste buds in the tongues of diabetic rats as compared to BV-treated rats, which was due to the local upregulation of TGF-β1 and VEGF gene expression.

Impact of umbelliprenin-containing niosome nanoparticles on VEGF-A and CTGF genes expression in retinal pigment epithelium cells.

To investigate the impact of niosome nanoparticles carrying umbelliprenin (UMB), an anti-angiogenic and anti-inflammatory plant compound, on the expression of vascular endothelial growth factor (VEGF-A) and connective tissue growth factor (CTGF) genes in a human retinal pigment epithelium (RPE)-like retina-derived cell line. UMB-containing niosomes were created, optimized, and characterized. RPE-like cells were treated with free UMB and UMB-containing niosomes. The IC50 values of the treatments were determined using an MTT assay. Gene expression of VEGF-A and CTGF was evaluated using real-time polymerase chain reaction after RNA extraction and cDNA synthesis. Niosomes' characteristics, including drug entrapment efficiency, size, dispersion index, and zeta potential were assessed. Free UMB had an IC50 of 96.2 µg/mL, while UMB-containing niosomes had an IC50 of 25 µg/mL. Treatment with UMB-containing niosomes and free UMB resulted in a significant reduction in VEGF-A expression compared to control cells (P=0.001). Additionally, UMB-containing niosomes demonstrated a significant reduction in CTGF expression compared to control cells (P=0.05). However, there was no significant reduction in the expression of both genes in cells treated with free UMB. Both free UMB and niosome-encapsulated UMB inhibits VEGF-A and CTGF genes expression. However, the latter demonstrates significantly greater efficacy, potentially due to the lower UMB dosage and gradual delivery. These findings have implications for anti-angiogenesis therapeutic approaches targeting age-related macular degeneration.

An evaluation of photobiomodulation effects on human gingival fibroblast cells under hyperglycemic condition: an in vitro study.

An in vitro study was designed to evaluate the effects of photobiomodulation (PBM) with 915-nm diode laser on human gingival fibroblast (HGF) cells under hyperglycemic condition. The HGF cells were cultured in Dulbecco's modified eagle medium (DMEM) medium containing 30mM glucose concentration for 48h to mimic the hyperglycemic condition. Subsequently, the cells received three sessions of PBM (915nm, continuous emission mode, 200 mW, energy density values of 3.2, 6, and 9.2J/cm2). Twenty-four hours post-irradiation, cell proliferation, expression of interleukin 6 (IL-6), and vascular endothelial growth factor (VEGF) were assessed with MTT and real-time polymerase chain reaction (PCR) tests, respectively. Also, reactive oxygen species (ROS) production was measured using CM-H2DCFDA fluorimetry. No changes were detected in the cell proliferation rate between the high glucose control group and laser-treated cells, while VEGF and IL-6 gene expression levels increased significantly after PBM in the high glucose-treated cells group. ROS level was significantly decreased in the irradiated cells in high-glucose medium compared with the high glucose control group. Our study revealed the inductive role of 915-nm-mediated PBM on VEGF and the inflammatory response while concurrently reducing reactive oxygen species production in HGF cells in hyperglycemic conditions.

Investigating changes in the post-transcriptional pattern of VEGF and CD146 genes carrying miR-573 in breast cancer cells treated with tamoxifen.

Recent developments regarding the contribution of microRNAs (miRNAs) to tumor angiogenesis and the oncogenic effects of miRNAs point to their potential role in breast cancer angiogenesis. Tumor-derived exosomes are considered a rich source of miRNAs that can regulate the function of other cells in the tumor microenvironment, including vascular endothelial cells. This study analyzes the effect of tamoxifen chemotherapy on the expression of a key miRNA, miR-573, involved in the angiogenesis of the tumor exosomes and introduces a regulatory link between this miRNA and the CD146 gene associated with the vascular endothelial growth factor (VEGF) messaging pathway. MCF-7 breast cancer cells were purchased and cultured in a complete culture medium. These cells were treated with tamoxifen and then their exosomes were extracted from the culture medium. The RNAs of the exosomes were isolated and the expression of miR-573, VEGF, and CD146 genes in the exosomes was investigated using the real-time polymerase chain reaction (PCR) method. The results of this study showed that tamoxifen treatment increased the expression of miR-573 in exosomes derived from MCF-7 cancer cells. The expression of CD146 and VEGF genes in drug-treated cell exosomes had a downward pattern. The results of this experiment demonstrated that the treatment of breast cancer cells with tamoxifen reduces the expression of VEGF and CD146 by increasing miR-573. Thus, angiogenesis is reduced and, therefore, its anti-tumor effects are applied.

Phenobarbital ameliorates hyperglycemia-induced angiogenesis in diabetic nephropathy-possible intervention at the HIF-1α/VEGF axis

Hyperglycemia contributes to a cascade of inflammatory responses in kidneys that result in the development of renal hypoxia and angiogenesis with subsequent chronic renal failure. As the hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) axis is a key pathway for neovascularization, the inhibition of this axis is a target for renal angiogenesis therapy. We speculate that Phenobarbital (PB) which has a potential to reduce vascularization in clinical settings might have an influence on the development of angiogenesis in diabetic kidney. The aim of the study was to explore the effects of PB on the HIF-1α and VEGF expression and angiogenesis in renal tissue of rats with hyperglycemia and diabetic nephropathy. Sixty-four male Wistar rats were devided into 4 groups: control group received a single intraperitoneal saline injection; PB group received 0.05% PB orally in drinking water; diabetic group received a single intra-peritoneal STZ (65 mg/kg) injection; PB-STZ group received 0.05% PB orally two weeks before STZ administration. At the end of the experiment period (8 weeks), the kidneys were removed and used for biochemical analyses. Serum glucose, urea and creatinine levels, IL-6 levels in kidney homogenate and changes in HIF-1α and VEGF gene expression were estimated. Hematoxylin-eosin staining was performed for histopathological exa­mination. The results obtained showed that both HIF-1α and VEGF gene expression and IL6 level in diabetic rat group were significantly elevated compared to that in control group, whereas in PB and PB-STZ groups, these indices were significantly down-regulated compared to the diabetic group. Abundant glomerular congestion and mesangial proliferation were detected in diabetic rat renal tissues. However, in PB-treated diabetic group, newly formed vessels were significantly decreased. These findings confirmed that phenobarbital, affecting the HIF-1α/VEGF signaling pathway, ameliorates angiogenesis after hyperglycemic kidney injury. Keywords: angiogenesis, HIF-1α, hyperglycemia, IL6, kidney injury, VEGF

Comprehensive metabolite and biological profile of “Sulmona Red Garlic” ecotype’s aerial bulbils

“Sulmona Red Garlic” is a well-known Italian traditional product. Bulbs, used for culinary purposes, have been largely investigated for their medicinal properties whereas aerial bulbils are usually removed as waste material. Here, for the first time, chemical composition and biological properties of the hydroalcoholic extract from aerial bulbils were investigated. Complementary information on metabolite composition were obtained using both NMR based untargeted and HPLC-DAD targeted methodologies. The NMR analysis revealed the presence of sugars, organic acids, amino acids, organosulphur compounds (methiin, alliin, allicin and cycloalliin), and other secondary metabolites. In particular, methiin and alliin were identified for the first time in the NMR spectra of aerial bulbil garlic extracts. Polyphenol content was determined by HPLC-DAD analysis: catechin, chlorogenic acid, and gallic acid turned out to be the most abundant phenolics.Hydroalcoholic extract blocked cell proliferation of colon cancer cell line HCT116 with an IC50 of 352.07 µg/mL, while it was non-toxic to myoblast cell line C2C12. In addition, it caused seedling germination reduction of two edible and herbaceous dicotyledon species, namely Cichorium intybus and C. endivia. Moreover, the same extract reduced the gene expression of TNF-α (tumor necrosis factor), HIF1-α (hypoxia-inducible factor), VEGFA (vascular endothelial growth factor), and transient receptor potential (TRP) M8 (TRPM8) indicating the ability to contrast cancer development through the angiogenic pathway. Final, in silico experiments were also carried out supporting the biological effects of organosulphur compounds, particularly alliin, which may directly interact with TRPM8.The results here reported suggest the potential use of garlic aerial bulbils often considered a waste product as a source in phytotherapeutic remedies.

Secretome of stem cells from human exfoliated deciduous teeth (SHED) and its extracellular vesicles improves keratinocytes migration, viability, and attenuation of H2 O2 -induced cytotoxicity.

Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H2 O2 in vitro was performed with H2 O2 followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4-0.6 μg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H2 O2 -induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.

Enhanced Angiogenesis in HUVECs Preconditioned with Media from Adipocytes Differentiated from Lipedema Adipose Stem Cells In Vitro.

Lipedema is a connective tissue disorder characterized by increased dilated blood vessels (angiogenesis), inflammation, and fibrosis of the subcutaneous adipose tissue. This project aims to gain insights into the angiogenic processes in lipedema using human umbilical vein endothelial cells (HUVECs) as an in vitro model. HUVECs were cultured in conditioned media (CM) collected from healthy (non-lipedema, AQH) and lipedema adipocytes (AQL). The impacts on the expression levels of multiple endothelial and angiogenic markers [CD31, von Willebrand Factor (vWF), angiopoietin 2 (ANG2), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMPs), NOTCH and its ligands] in HUVECs were investigated. The data demonstrate an increased expression of CD31 and ANG2 at both the gene and protein levels in HUVECs treated with AQL CM in 2D monolayer and 3D cultures compared to untreated cells. Furthermore, the expression of the vWF, NOTCH 4, and DELTA-4 genes decreased. In contrast, increased VEGF, MMP9, and HGF gene expression was detected in HUVECs treated with AQL CM cultured in a 2D monolayer. In addition, the results of a tube formation assay indicate that the number of formed tubes increased in lipedema-treated HUVECs cultured in a 2D monolayer. Together, the data indicate that lipedema adipocyte-CM promotes angiogenesis through paracrine-driven mechanisms.

The promoting effect of modified Dioscorea pills on vascular remodeling in chronic cerebral hypoperfusion via the Ang/Tie signaling pathway.

The objective of this study was to investigate the effect of modified Dioscorea pills (MDP) on microcirculatory remodeling in the hippocampus of rats with chronic cerebral hypoperfusion (CCH) through the angiopoietin (Ang)/tyrosine kinase receptor tyrosine kinase with immunoglobulin-like and EGF-like domains (Ang receptor) 2 (Tie-2) signaling pathways, which may underlie the cognitive improvement observed in CCH rats. Forty male Sprague-Dawley rats raised under specific pathogen-free conditions were randomly divided into three groups: control group (10 rats), model group (15 rats), and MDP group (15 rats). The rats in the model group and MDP group underwent bilateral common carotid artery occlusion using the 2-vessel occlusion (2-VO) method to induce CCH. Rats in the control group underwent the same surgical procedures as those in the model group, except for ligation and occlusion of the carotid arteries. After 1 week of 2-VO, rats in the MDP group were administered MDP condensed decoction intragastrically at a dose of 1 ml/100 g body weight (prepared by the Preparation Room of Hubei Provincial Hospital of Traditional Chinese Medicine) for 45 days, while rats in the other two groups received normal saline intragastrically with the same dose and duration as the MDP group. After the intervention, all rats were euthanized, and brain perfusion was performed to obtain the hippocampal tissue for analysis. Immunohistochemical staining for CD43 was performed to assess microvessel density (MVD); western blot and the reverse transcription-polymerase chain reaction (RT-PCR) were used to analyze the expression of proteins and genes in angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-2, and vascular endothelial growth factor (VEGF) proteins and genes in the hippocampal tissue and compute the Ang-1/Ang-2 ratio. MDP treatment reduced neuronal loss and promoted restoration of the damaged hippocampal structure in CCH rats. The model group showed significantly higher MVD (14.93 ± 1.92) compared to the control group (5.78 ± 1.65) (P < 0.01), whereas MDP treatment further increased MVD (21.19 ± 2.62). Western blot and RT-PCR analysis revealed that CCH significantly increased the expression of Ang-1, Ang-2, Tie-2, and VEGF proteins and genes, while MDP treatment further significantly upregulated the expression of these proteins and genes. In addition, MDP significantly elevated the gene and protein expression of the Ang-1/Ang-2 ratio compared to the control group (P = 0.041, P = 0.029). CCH induces microvascular neogenesis in the hippocampus, and MDP promotes angiogenesis and microcirculation remodeling in CCH rats via the Ang/Tie signaling pathway, which may be an important mechanism for its restorative effects on hippocampal perfusion and improvement of cognitive function in CCH rats.

Fusobacterium nucleatum-Mediated Alteration in Expression of VEGF and CCL3 Genes and KRAS Mutation in Colorectal Cancer Patients

Background: Colorectal cancer (CRC) is the third most common cancer worldwide, and its development is influenced by genetic and environmental factors, including the gut microbiota. Recent studies have reported an association between Fusobacterium nucleatum abundance and CRC. Objectives: This study aimed to investigate the abundance of F. nucleatum in CRC and polyp patients and its association with the expression of chemokine ligand -3(CCL3), vascular endothelial growth factor (VEGF), and nuclear factor-kappa B (NF-KB11) genes and the presence of deoxyribonucleic acid (DNA) mutations and polymorphisms in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene. Methods: A total of 80 biopsy samples were collected from CRC, polyp, and colitis patients. Moreover, F. nucleatum abundance was measured by quantitative polymerase chain reaction (qPCR). The expression of CCL3, VEGF, and NF-KB11 genes was measured by reverse transcription polymerase chain reaction (RT-PCR). Additionally, KRAS gene mutations and polymorphisms were detected by the Mutation Surveyor software (V5.1.2). Results: The results showed that F. nucleatum abundance was significantly higher in CRC and polyp patients than in colitis patients (P &lt; 0.05). The expression of CCL3 and VEGF genes was also significantly higher in F. nucleatum-positive samples (P &lt; 0.05). However, NF-KB11 gene expression was non-significant. Fusobacterium nucleatum-positive biopsy samples had a higher frequency of KRAS gene mutations and polymorphisms than F. nucleatum-negative CRC patients (odds ratio = 3). Most of the mutations observed in the positive samples were (6144A&gt;AT,31E&gt;E) at exon 2 of the KRAS gene. Conclusions: The study findings suggest that F. nucleatum might play a role in CRC and polyp development and contribute to KRAS gene mutations. Therefore, targeting F. nucleatum in the gut microbiota could be a potential therapeutic strategy for preventing CRC and polyp development.

SALL4 promotes angiogenesis in gastric cancer by regulating VEGF expression and targeting SALL4/VEGF pathway inhibits cancer progression

BackgroundSpalt-like protein 4 (SALL4) is a stemness-related transcription factor whose abnormal re-expression contributes to cancer initiation and progression. However, the role of SALL4 in cancer angiogenesis remains unknown.MethodsAnalyses of clinical specimens via TCGA datasets were performed to determine the expression level and clinical significance of SALL4 in STAD (Stomach Adenocarcinoma). SALL4 knockdown, knockout, and overexpression were achieved by siRNA, CRISPR/Cas9, and plasmid transfection. The effects of conditioned medium (CM) from SALL4 knockdown or overexpression of gastric cancer cells on endothelial cell proliferation, migration, and tube formation were investigated by CCK-8 assay, transwell migration assay, and tube formation assay. The regulation of VEGF gene expression by SALL4 was studied by qRT-PCR, western blot, chromatin immunoprecipitation (ChIP) assay, and electrophoretic mobility shift assay (EMSA). Engineered exosomes from 293T cells loaded with si-SALL4-B and thalidomide were produced to test their therapeutic effect on gastric cancer progression.ResultsSALL4 expression was increased in STAD and positively correlated with tumor progression and poor prognosis. SALL4-B knockdown or knockout decreased while over-expression increased the promotion of human umbilical vein endothelial cells (HUVEC) cell proliferation, migration, and tube formation by gastric cancer cell-derived CM. Further investigation revealed a widespread association of SALL4 with angiogenic gene transcription through the TCGA datasets. Additionally, SALL4-B knockdown reduced, while over-expression enhanced the expression levels of VEGF-A, B, and C genes. The results of ChIP and EMSA assays indicated that SALL4 could directly bind to the promoters of VEGF-A, B, and C genes and activate their transcription, which may be associated with increased histone H3-K79 and H3-K4 modifications in their promoter regions. Furthermore, si-SALL4-B and thalidomide-loaded exosomes could be efficiently uptaken by gastric cancer cells and significantly reduced SALL4-B and Vascular Endothelial Growth Factor (VEGF) expression levels in gastric cancer cells, thus inhibiting the pro-angiogenic role of their derived CM.ConclusionThese findings suggest that SALL4 plays an important role in angiogenesis by transcriptionally regulating VEGF expression. Co-delivery of the functional siRNA and anticancer drug via exosomes represents a useful approach to inhibiting cancer angiogenesis by targeting SALL4/VEGF pathway.

Effect of amoxicillin and clindamycin on the gene expression of markers involved in osteoblast physiology

Background/purposeAmoxicillin and clindamycin are the most effective decontaminants for intraoral bone grafts before their application in bone regeneration without cytotoxic effects on osteoblasts, but their effects on the gene expression of markers involved in osteoblast growth and differentiation remain unclear. The study objective was to determine the effects of amoxicillin and clindamycin on the gene expression of markers involved in osteoblast growth and differentiation. Materials and methodsReal-time polymerase chain reaction (RT-PCR) was performed to explore the effect of 150 μg/mL clindamycin or 400 μg/mL amoxicillin on the gene expression by primary human osteoblasts (HOBs) of runt-related transcription factor 2 (Runx-2), osterix (OSX), alkaline phosphatase (ALP), osteocalcin (OSC), osteoprotegerin (OPG), receptor activator for nuclear factor κ B ligand (RANKL), type I collagen (Col-I), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), TGF-β1 and TGF-β receptors (TGF-βR1, TGF-βR2, and TGF-βR3), and vascular endothelial growth factor (VEGF). ResultsTreatment with 150 μg/mL clindamycin significantly increased the gene expression of TFG-β1, TGF-βR1, TGF-βR2, TGF-βR3, RUNX-2, Col-1, OSX, OSC, BMP-2, BMP-7, ALP, VEGF, and RANKL by HOBs. Treatment with 400 μg/mL amoxicillin significantly increased the gene expression of TGF-β R1, Col-I, OSC, RANKL, and OPG alone. ConclusionThese findings suggest that 150 μg/mL clindamycin is the decontaminant of choice to treat intraoral bone grafts before their application in bone regeneration. The osteogenic and antibacterial properties of clindamycin can favor and accelerate the integration of bone grafts in the oral cavity.

The Exercise Preconditioning Effect on Cardiac Tissue Injury following Induction of Myocardial Infarction in Male Rats.

Twenty-four male rats were divided into 4 groups including MI, Sham, HIIT, and HIIT+MI (N = 6). HIIT and HIIT+MI which underwent high-intensity interval training (HIIT) for 4 weeks (5 days a week). The training protocol included 10 intervals of 1-minute running, with 2 minutes rest between each interval. The training intensity was different every week according to the peak treadmill running speed (v peak) percentage of each rat. Isoproterenol injection was used to induce myocardial infarction (MI). Expressions of creatine kinase-MB (CK-MB), PGC-1α troponin-I, mitochondrial transcription factor A (TFAM), vascular endothelial growth factor (VEGF), and microRNA 126 (miR-126) genes were measured. The variables were measured using biochemical and RT-PCR methods. The significance level (P value≤0.05) was analyzed using ANOVA test. The results showed that 4 weeks of HIIT training led to a significant increase in PGC-1α, TFAm, and VEGF levels in the MI, HIIT, and HIIT+MI groups compared to the sham group (P = 0.001). HIIT exercises increased miR-126 in the different groups compared to the sham group; however, it was not significant. The results obtained showed that HIIT exercise exerts cardio-protective effects to reduce cardiac tissue injury and necrosis against MI. These effects increase mitochondrial biogenesis and angiogenesis by inducing the increased expression of VEGF, TFAM, PGC-1α, and miR-126 genes in the heart tissue. Therefore, HIIT training, as a preconditioning program, was able to protect the cardiac tissue against MI.

O-222 Functional characterization and clinical implications of patient-specific iPSC-derived endothelial cells to unravel the pathogenesis associated with androgen-mediated endothelial dysfunction in polycystic ovarian syndrome

Abstract Study question Whether and how the endothelial cell (EC) dysfunction is involved in the pathogenesis of PCOS revealed by iPSC-based disease modeling? Summary answer PCOS-specific iPSC-derived ECs were successfully established and the results showed that EC proliferation and function were impaired in PCOS through the androgen receptor(AR)-mediated signaling pathway. What is known already ECs have been shown to play critical biological roles in the regulation of vascular tone and inflammatory process and any injury to ECs or disturbance in their homeostasis is called EC dysfunction, which has been recognized as an early marker of atherosclerosis and cardiovascular diseases. Several studies have also revealed indirect evidence of EC dysfunction among women with PCOS, including elevated circulatory markers of endothelial damage and impaired vascular structure. But whether and how EC dysfunction is involved in the pathogenesis of PCOS is still unclear. Study design, size, duration This is an experimental study at tertiary university hospital. iPSCs were established from skin fibroblasts and peripheral blood mononuclear cells from two patients with PCOS and two control participants. Participants/materials, setting, methods iPSCs were differentiated into ECs through monoculture with chemically defined conditions. Single cell RNA sequencing (scRNA-seq) was performed on the iPSCs-derived ECs to compare the differences between PCOS and control. iPSCs-derived ECs were treated with different dosages (1, 10 and 100 nM) of dihydrotestosterone (DHT). Cell cycle phases were analyzed by flow cytometry with BrdU incorporation assay. The expression of AR, cyclin-dependent kinase 1 (CDK1) and vascular endothelial growth factor (VEGF) were analyzed using rtPCR. Main results and the role of chance Up to 90% of iPSC-derived ECs successfully expressed EC markers, including CD31, CD144 and von Willebrand Factor in both PCOS and control groups, showing a high differentiation efficiency in the present study. The pathway enrichment analysis of transcriptomic data of iPSC-derived ECs showed functional differences involving cell cycle process, VEGF signaling and apoptotic process between PCOS and control. Analysis using sc-RNA seq revealed decreased cellular proliferation in the PCOS iPSC-derived ECs compared to control group, which was due to cell cycle arrest revealed by flow cytometry. The modulating effects of androgen on the proliferation and gene expression of iPSC-derived ECs were further investigated. DHT at doses ranging from 1 to 100 nM stimulated iPSC-derived ECs proliferation in the control group through upregulation of AR, CDK1 and VEGF gene expression. In contrast, DHT did not promote the cellular proliferation of PCOS iPSC-derived ECs at physiological concentrations of 1 and 10 nM and the expression of AR, CDK1, and VEGF remained unchanged. These data suggest that DHT-induced cellular proliferation and the expression of associated genes were more sensitive and dose-dependent in the control iPSC-derived ECs and blunted in the PCOS iPSC-derived ECs. Limitations, reasons for caution One of the major limitations of our study was the limited number of cases during the establishment of iPSCs, which was unable to reflect the heterogeneity and complexity of PCOS. Wider implications of the findings Impaired cellular proliferation and differentially expressed genes involved in cell proliferation, immune responses, cardiovascular functions, and apoptosis were observed among PCOS iPSC-derived ECs. Additionally, androgen-induced ECs proliferation was blunted in PCOS through a diminished AR/VEGF/cyclin D1 signaling pathway, which might hinder VEGF-dependent vascular repair and probably increase cardiovascular risks. Trial registration number not applicable

Fenugreek Seed Extract Regulates Human Umbilical Vein Endothelial Cell Angiogenesis and Proliferation via the PI3K/Akt/Cyclin D1 Pathway.

The significance of angiogenesis in tumour progression has been widely documented. Hence, the identification of anti-angiogenic agents with fewer common side effects would be valuable in cancer therapy. In this study, we evaluated the anti-angiogenic and anti-proliferative effects of a hydro-alcoholic extract of fenugreek seed (HAEF) on human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells were treated with various concentrations of HAEF and the half-maximal inhibitory concentration (IC50) value was estimated by using the MTT assay. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase enzyme (MMP-2 and MMP-9) gene expression profiles were evaluated by using quantitative RT-PCR (qRT-PCR). Moreover, MMP activities and PI3K, Akt and cyclin D1 protein expression levels were evaluated by gel zymography and Western blotting, respectively. HAEF reduced HUVEC viability, with an IC50 value of 200μg/ml. The qRT-PCR results demonstrated that treatment with HAEF markedly reduced MMP-2/MMP-9, VEGF and bFGF gene expression, as compared to the control group. We also found that MMP-2/MMP-9 enzyme activity and PI3K/Akt/cyclin D1 protein expression were notably decreased in cells treated with HAEF. Our results suggest that HAEF can potentially inhibit angiogenesis, and also affect cellular proliferation by targeting the PI3K/Akt/cyclin D1 pathway. Thus, fenugreek seed extract merits further investigation as a source of compounds with anti-cancer properties.

1491-P: ER Stress Drives the VEGF Expression in Monocytic Cells by Mechanisms Involving CHOP/ROS/HIF-1alpha/NRF2 and NF-kappaB/p38 MAPK Pathways

Obesity is marked by metabolic inflammation and by metabolic impairment caused by increased endotoxin, free fatty acids, and vascular endothelial growth factor (VEGF) levels; and involves the endoplasmic reticulum (ER) stress as well. However, it remains unclear whether the ER stress can induce/amplify VEGF expression in metabolically-stressed monocytic cells; and if so, by which mechanism(s). To test this, metabolic stress was induced in THP-1 monocytic cells by treating cells separately with lipopolysaccharide (LPS), palmitic acid (PA), and oleic acid (OA), in presence/absence of ER stressor thapsigargin (TG). Gene expression of VEGF/ER stress markers was assessed by qRT-PCR, protein expression by ELISA, ROS by DCFH-DA assay, phosphorylation of HIF-1α, NF-κB, ERK1/2, and p38 MAPK by immunoblotting, and the insulin response in stressed cells by glucose-uptake assay. Regarding clinical analyses, adipose VEGF gene and protein expression was detected using qRT-PCR and IHC, respectively, while plasma hs-CRP, TNF-α, MDA, and OX-LDL levels were measured by ELISA. The experimental data show that a cooperative interaction between the metabolic and ER stresses led to the expression of VEGF, ROS, CHOP, ATF6, SOD2, and NRF2 (P˂0.05), as well as stimulated the CHOP and NRF2 promoter activities in reporter cells (P˂0.05). However, the glucose uptake was not impaired. The VEGF expression was dependent on phosphorylation of HIF-1α, NF-κB, and p38 MAPK; and the inhibitors of NF-κB/MAPK pathways as well as antioxidants or ROS scavengers suppressed the VEGF production. Further, individuals with obesity showed increased VEGF expression which associated positively with plasma levels of hs-CRP, TNF-α, MDA, and OX-LDL (P≤0.05). Overall, our findings support a cooperativity model in which the ER and metabolic stresses interact to augment VEGF expression in monocytic cells via the mechanisms involving CHOP/ROS/HIF-1α/NRF2 and NF-κB/p38 MAPK pathways. Disclosure S.A.K.Sindhu: None. F.Alzaid: None. F.Almulla: None. R.Ahmad: None. N.Akhter: None. A.Wilson: None. H.Arefanian: None. A.Al madhoun: None. R.S.Thomas: None. S.P.Kochumon: None. F.Alrashed: None. F.Bahman: None. Funding Kuwait Foundation for the Advancement of Sciences (RA2015-027, RA2010-003, RAHM-2019-022)

#6465 MIR-451B AND CHRONIC KIDNEY DISEASE: A NOVEL TARGET TO MODULATE RENAL VEGF SIGNALING

Abstract Background and Aims Chronic kidney disease (CKD) is a progressive disorder that affects over 15% of adults worldwide. A major component of CKD pathophysiology is renal microvascular rarefaction and loss that pairs the progression of the disease and complications. We developed a translational swine model of CKD and showed that loss of renal function associates with a significant decrease in renal vascular endothelial growth factor (VEGF) signaling and progressive microvascular (MV) rarefaction. VEGF is a key endogenous cytokine for MV proliferation and repair, but underlying mechanisms of renal VEGF downregulation in CKD are unknown. We hypothesized that micro-RNA (miRNA)-mediated processes contribute to the downregulation of renal VEGF signaling in CKD. Method Unbiased renal micro-RNA (miRNA)-seq was performed in a swine model of CKD stage 3 (14 weeks of bilateral renal artery stenosis + atherogenic diet) and normal controls (n = 5/group). Micro-RNAs up- (&amp;gt;1.4 FC, p&amp;lt;0.05) and down-regulated (&amp;lt;0.7 FC, p&amp;lt;0.05) were identified, followed by target prediction analysis (miRwalk 3.0, Target Scan 7.2). Renal gene and protein expression of VEGF was measured by qPCR and western blot, respectively, and renal MV architecture was studied by 3D micro-CT. Results Unbiased miRNA-seq and target prediction analysis identified four miRs upregulated in the kidney of CKD pigs vs. controls, of which miR-451b (validated by qPCR), broadly conserved among species, was capable of targeting VEGFA (FC = 4.46, p = 0.04). Renal gene and protein expression of VEGF was downregulated in CKD vs. controls, associated with significant cortical and medullary microvascular rarefaction (Figure 1). Conclusion Our data suggest that post-transcriptional modulation of renal VEGF signaling may serve as a prominent mechanism of VEGF downregulation, which leads to MV rarefaction and disease progression in CKD. Furthermore, this work in progress identified a novel candidate (miR-451b) and may set the stage for its targeted modulation to preserve VEGF signaling as a potential strategy to protect the renal microvasculature in CKD.