scholarly journals Pravastatin Promotes Endothelial Colony-Forming Cell Function, Angiogenic Signaling and Protein Expression In Vitro

2021 ◽  
Vol 10 (2) ◽  
pp. 183
Author(s):  
Nadia Meyer ◽  
Lars Brodowski ◽  
Katja Richter ◽  
Constantin S. von Kaisenberg ◽  
Bianca Schröder-Heurich ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Growth Factor ◽  
Endothelial Dysfunction ◽  
Protein Expression ◽  
Tube Formation ◽  
In Vitro Assays ◽  
Heme Oxygenase 1 ◽  
Expression Levels ◽  
Functional Capacities

Endothelial dysfunction is a primary feature of several cardiovascular diseases. Endothelial colony-forming cells (ECFCs) represent a highly proliferative subtype of endothelial progenitor cells (EPCs), which are involved in neovascularization and vascular repair. Statins are known to improve the outcome of cardiovascular diseases via pleiotropic effects. We hypothesized that treatment with the 3-hydroxy-3-methyl-glutaryl–coenzyme A (HMG-CoA) reductase inhibitor pravastatin increases ECFCs’ functional capacities and regulates the expression of proteins which modulate endothelial health in a favourable manner. Umbilical cord blood derived ECFCs were incubated with different concentrations of pravastatin with or without mevalonate, a key intermediate in cholesterol synthesis. Functional capacities such as migration, proliferation and tube formation were addressed in corresponding in vitro assays. mRNA and protein levels or phosphorylation of protein kinase B (AKT), endothelial nitric oxide synthase (eNOS), heme oxygenase-1 (HO-1), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and endoglin (Eng) were analyzed by real time PCR or immunoblot, respectively. Proliferation, migration and tube formation of ECFCs were enhanced after pravastatin treatment, and AKT- and eNOS-phosphorylation were augmented. Further, expression levels of HO-1, VEGF-A and PlGF were increased, whereas expression levels of sFlt-1 and Eng were decreased. Pravastatin induced effects were reversible by the addition of mevalonate. Pravastatin induces beneficial effects on ECFC function, angiogenic signaling and protein expression. These effects may contribute to understand the pleiotropic function of statins as well as to provide a promising option to improve ECFCs’ condition in cell therapy in order to ameliorate endothelial dysfunction.

scholarly journals Upregulation of ETV2 Expression Promotes Endothelial Differentiation of Human Dental Pulp Stem Cells

2021 ◽  
Vol 30 ◽  
pp. 096368972097873
Author(s):  
Jing Li ◽  
Youming Zhu ◽  
Na Li ◽  
Tao Wu ◽  
Xianyu Zheng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Dental Pulp ◽  
Tube Formation ◽  
Endothelial Differentiation ◽  
Cell Source ◽  
Lentiviral Infection ◽  
Mrna And Protein Expression ◽  
Human Dental Pulp

The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.

scholarly journals Hyperglycaemia up-regulates placental growth factor (PlGF) expression and secretion in endothelial cells via suppression of PI3 kinase-Akt signalling and activation of FOXO1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samir Sissaoui ◽  
Stuart Egginton ◽  
Ling Ting ◽  
Asif Ahmed ◽  
Peter W. Hewett
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Endothelial Dysfunction ◽  
Akt Activation ◽  
Forkhead Box ◽  
Pi3k Activity ◽  
Binding Sequence

AbstractPlacenta growth factor (PlGF) is a pro-inflammatory angiogenic mediator that promotes many pathologies including diabetic complications and atherosclerosis. Widespread endothelial dysfunction precedes the onset of these conditions. As very little is known of the mechanism(s) controlling PlGF expression in pathology we investigated the role of hyperglycaemia in the regulation of PlGF production in endothelial cells. Hyperglycaemia stimulated PlGF secretion in cultured primary endothelial cells, which was suppressed by IGF-1-mediated PI3K/Akt activation. Inhibition of PI3K activity resulted in significant PlGF mRNA up-regulation and protein secretion. Similarly, loss or inhibition of Akt activity significantly increased basal PlGF expression and prevented any further PlGF secretion in hyperglycaemia. Conversely, constitutive Akt activation blocked PlGF secretion irrespective of upstream PI3K activity demonstrating that Akt is a central regulator of PlGF expression. Knock-down of the Forkhead box O-1 (FOXO1) transcription factor, which is negatively regulated by Akt, suppressed both basal and hyperglycaemia-induced PlGF secretion, whilst FOXO1 gain-of-function up-regulated PlGF in vitro and in vivo. FOXO1 association to a FOXO binding sequence identified in the PlGF promoter also increased in hyperglycaemia. This study identifies the PI3K/Akt/FOXO1 signalling axis as a key regulator of PlGF expression and unifying pathway by which PlGF may contribute to common disorders characterised by endothelial dysfunction, providing a target for therapy.

Hydroxysafflor yellow A promotes angiogenesis in rat brain microvascular endothelial cells injured by oxygen-glucose deprivation/reoxygenation(OGD/R) through SIRT1-HIF-1α-VEGFA signaling pathway

2021 ◽  
Vol 18 ◽  
Author(s):  
Juxuan Ruan ◽  
Lei Wang ◽  
Jiheng Dai ◽  
Jing Li ◽  
Ning Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Signaling Pathway ◽  
Ischemia Reperfusion ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Hydroxysafflor Yellow A ◽  
Microvascular Endothelial

Objective: Angiogenesis led by brain microvascular endothelial cells (BMECs) contributes to the remission of brain injury after brain ischemia reperfusion. In this study, we investigated the effects of hydroxysafflor yellow A(HSYA) on angiogenesis of BMECs injured by OGD/R via SIRT1-HIF-1α-VEGFA signaling pathway. Methods: The OGD/R model of BMECs was established in vitro by OGD for 2h and reoxygenation for 24h. At first, the concentrations of vascular endothelial growth factor (VEGF), Angiopoietin (ang) and platelet-derived growth factor (PDGF) in supernatant were detected by ELISA, and the proteins expression of VEGFA, Ang-2 and PDGFB in BMECs were tested by western blot; the proliferation, adhesion, migration (scratch healing and transwell) and tube formation experiment of BMECs; the expression of CD31 and CD34 were tested by immunofluorescence staining. The levels of sirtuin1(SIRT1), hypoxia-inducible factor-1α (HIF-1α), VEGFA mRNA and protein were tested. Results: HSYA up-regulated the levels of VEGF, Ang and PDGF in the supernatant of BMECs under OGD/R, and the protein expression of VEGFA, Ang-2 and PDGFB were increased; HSYA could significantly alleviate the decrease of cell proliferation, adhesion, migration and tube formation ability of BMECs during OGD/R; HSYA enhanced the fluorescence intensity of CD31 and CD34 of BMECs during OGD/R; HSYA remarkably up-regulated the expression of SIRT1, HIF-1α, VEGFA mRNA and protein after OGD/R, and these increase decreased after SIRT1 was inhibited. Conclusion: SIRT1-HIF-1α-VEGFA signaling pathway is involved in HSYA improves angiogenesis of BMECs injured by OGD/R.

Effects of Hypocrellin A on Expression of Vascular Endothelial Growth Factor and Endothelin-1 in Human Umbilical Endothelial Cells

2007 ◽  
Vol 35 (04) ◽  
pp. 713-723 ◽  
Author(s):  
Lei Dang ◽  
J. Paul Seale ◽  
Xianqin Qu
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Endothelial Dysfunction ◽  
Protein Expression ◽  
Vascular Endothelial ◽  
Naturally Occurring ◽  
Hypocrellin A ◽  
Endothelial Growth Factor ◽  
Vegf Protein

Increased endothelin-1 (ET-1), vascular endothelial growth factor (VEGF) and activation of protein kinase C (PKC) are co-contributors to endothelial hyperpermeability in diabetes. Several lines of evidence have suggested a hypothesis that activation of specific PKC isoforms are the causative factor in ET-1 and VEGF mediated endothelial dysfunction. In the present study, we tested this hypothesis with hypocrellin A, a naturally occurring PKC inhibitor from a Chinese plant. Human umbilical vein endothelial cells (HUVECs) were incubated with 20 mM glucose in both the presence and absence of hypocrellin A, after which, the protein expression and release of VEGF and mRNA expression and release of ET-1 were measured. VEGF and ET-1 were released into the medium and expressions of VEGF protein and ET-1 mRNA were significantly increased in HUVECs incubated with 20 mM glucose. Hypocrellin A (150 nM) significantly decreased VEGF release (117 ± 3 vs. 180 ± 11 pg/mg, p < 0.05) and VEGF protein expression (from 130 ± 14% to 88 ± 18.5%, p < 0.05). ET-1 release was also reduced in hypocrellin A treated HUVECs (63.3 ± 9.9 vs. 75.2 ± 12.6 ng/mg). Hypocrellin A significantly reversed the effect of high glucose on ET-1 mRNA expression ( p < 0.05). The results revealed that PKC activation plays a pivotal role in VEGF and ET-1 mediated endothelial permeability. The naturally occurring compound hypocrellin A may be a potentially novel treatment for endothelial dysfunction in diabetes.

The Effect of High CDC6 Levels on Predicting Poor Prognosis in Colorectal Cancer

Chemotherapy ◽  
2022 ◽  
pp. 1-10
Author(s):  
Cheng Yang ◽  
Na Xie ◽  
Zhifei Luo ◽  
Xiling Ruan ◽  
Yixin Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Protein Expression ◽  
Poor Prognosis ◽  
Cancer Metastasis ◽  
Mrna Level ◽  
Normal Mucosa ◽  
Tnm Stage ◽  
Expression Levels ◽  
Normal Tissues

<b><i>Introduction:</i></b> We investigated the function of cell division cycle 6 (CDC6) on the prognosis in colorectal carcinoma (CRC). <b><i>Methods:</i></b> CDC6 protein expression levels in 121 patients with colorectal cancer and adjacent normal mucosa were detected by immunohistochemistry. <b><i>Results:</i></b> Compared to adjacent normal tissues, CDC6 mRNA level was overexpressed in CRC tissues. Moreover, CDC6 protein levels were expressed up to 93.39% (113/121) in CRC tissues in the cell nucleus or cytoplasm. However, there were only 5.79% (7/121) in normal mucosal tissues with nuclear expression. CDC6 expression was significantly correlated with TNM stage and tumor metastasis. The 5-year survival rate was lower in the high CDC6 expression group than the low group. After silencing of CDC6 expression in SW620 cells, cell proliferation was slowed, the tumor clones were decreased, and the cell cycle was arrested in G1 phase. In multivariate analysis, increased CDC6 protein expression levels in colon cancer tissues were associated with cancer metastasis, TNM stage, and patient survival time. <b><i>Conclusion:</i></b> CDC6 is highly expressed in CRC, and downregulation of CDC6 can slow the growth of CRC cells in vitro. It is also an independent predictor for poor prognosis and may be a useful biomarker for targeted therapy and prognostic evaluation.

scholarly journals Impaired Hematopoiesis in Paroxysmal Nocturnal Hemoglobinuria/Aplastic Anemia Is Not Associated With a Selective Proliferative Defect in the Glycosylphosphatidylinositol-Anchored Protein-Deficient Clone

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1173-1181 ◽  
Author(s):  
Jaroslaw P. Maciejewski ◽  
Elaine M. Sloand ◽  
Tadatsugu Sato ◽  
Stacie Anderson ◽  
Neal S. Young
Keyword(s):  
Protein Expression ◽  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Surface Proteins ◽  
In Vitro Assays ◽  
Normal Numbers ◽  
Normal Volunteers ◽  
Cd34 Cells ◽  
Cd59 Expression

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) results from somatic mutations in the PIG-A gene, leading to poor presentation of glycosylphosphatidylinositol (GPI)-anchored surface proteins. PNH frequently occurs in association with suppressed hematopoiesis, including frank aplastic anemia (AA). The relationship between GPI-anchored protein expression and bone marrow (BM) failure is unknown. To assess the hematopoietic defect in PNH, the numbers of CD34+ cells, committed progenitors (primary colony-forming cells [CFCs]), and long-term culture-initiating cells (LTC-ICs; a stem cell surrogate) were measured in BM and peripheral blood (PB) of patients with PNH/AA syndrome or patients with predominantly hemolytic PNH. LTC-IC numbers were extrapolated from secondary CFC numbers after 5 weeks of culture, and clonogenicity of LTC-ICs was determined by limiting dilution assays. When compared with normal volunteers (n = 13), PNH patients (n = 14) showed a 4.7-fold decrease in CD34+ cells and an 8.2-fold decrease in CFCs. LTC-ICs in BM and in PB were decreased 7.3-fold and 50-fold, respectively. Purified CD34+ cells from PNH patients had markedly lower clonogenicity in both primary colony cultures and in the LTC-IC assays. As expected, GPI-anchored proteins were decreased on PB cells of PNH patients. On average, 23% of monocytes were deficient in CD14, and 47% of granulocytes and 58% of platelets lacked CD16 and CD55, respectively. In PNH BM, 27% of CD34+ cells showed abnormal GPI-anchored protein expression when assessed by CD59 expression. To directly measure the colony-forming ability of GPI-anchored protein-deficient CD34+ cells, we separated CD34+ cells from PNH patients for the GPI+ and GPI− phenotype; CD59 expression was chosen as a marker of the PNH phenotype based on high and homogeneous expression on fluorescent staining. CD34+CD59+ and CD34+CD59− cells from PNH/AA patients showed similarly impaired primary and secondary clonogeneic efficiency. The progeny derived from CD34+CD59− cells were both CD59− and CD55−. A very small population of CD34+CD59− cells was also detected in some normal volunteers; after sorting, these CD34+CD59− cells formed normal numbers of colonies, but their progeny showed lower CD59 levels. Our results are consistent with the existence of PIG-A–deficient clones in some normal individuals. In PNH/AA, progenitor and stem cells are decreased in number and function, but the proliferation in vitro is affected similarly in GPI-protein–deficient clones and in phenotypically normal cells. As measured in the in vitro assays, expansion of PIG-A– clones appears not be caused by an intrinsic growth advantage of cells with the PNH phenotype.

scholarly journals Single-Cell Receptor Quantification of an In Vitro Coculture Angiogenesis Model Reveals VEGFR, NRP1, Tie2, and PDGFR Regulation and Endothelial Heterogeneity

Processes ◽  
2019 ◽  
Vol 7 (6) ◽  
pp. 356 ◽  
Author(s):  
Si Chen ◽  
P. I. Imoukhuede
Keyword(s):  
Systems Biology ◽  
Growth Factor ◽  
Plasma Membranes ◽  
Temporal Dynamics ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Dermal Fibroblasts ◽  
Steady Increase

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for both normal development and numerous pathologies. Systems biology has offered a unique approach to study angiogenesis by profiling tyrosine kinase receptors (RTKs) that regulate angiogenic processes and computationally modeling RTK signaling pathways. Historically, this systems biology approach has been applied on ex vivo angiogenesis assays, however, these assays are difficult to quantify and limited in their potential of temporal analysis. In this study, we adopted a simple two-dimensional angiogenesis assay comprised of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) and examined temporal dynamics of a panel of six RTKs and cell heterogeneity up to 17 days. We observed ~2700 VEGFR1 (vascular endothelial growth factor receptor 1) per cell on 24-h-old cocultured HDF plasma membranes, which do not express VEGFR when cultured alone. We observed 4000–8100 VEGFR2 per cell on cocultured HUVEC plasma membranes throughout endothelial tube formation. We showed steady increase of platelet-derived growth factor receptors (PDGFRs) on cocultured HDF plasma membranes, and more interestingly, 1900–2900 PDGFRβ per plasma membrane were found on HUVECs within the first six hours of coculturing. These quantitative findings will offer us insights into molecular regulation during angiogenesis and help assess in vitro tube formation models and their physiological relevance.

5 PROPYLENE GLYCOL FEEDING SUPPLEMENTATION MODIFIES INSULIN-LIKE GROWTH FACTOR SYSTEM GENE EXPRESSION IN CUMULUS–OOCYTE COMPLEXES AND THE EXPRESSION OF SELECTED CANDIDATE GENES IN EMBRYOS PRODUCED IN VITRO IN FEED-RESTRICTED HEIFERS

2015 ◽  
Vol 27 (1) ◽  
pp. 95
Author(s):  
G. Gamarra ◽  
C. Ponsart ◽  
S. Lacaze ◽  
F. Nuttinck ◽  
P. Mermillod ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Rate ◽  
Growth Factor ◽  
Propylene Glycol ◽  
Dietary Supplementation ◽  
Cumulus Cells ◽  
Oestrous Cycle ◽  
Expression Levels ◽  
Igf System

Dietary supplementation with propylene glycol (PG) increases the rate of grade 1 embryos produced from feed restricted females (Gamarra et al. 2014 Reprod. Fertil. Dev.). The aim of this study was to evaluate if a PG feeding supplement could modify the expression profile of selected candidate genes that are important for in vitro embryo development and the gene expression patterns of the insulin-like growth factor (IGF) system in oocytes and cumulus cells in feed-restricted heifers. Feed-restricted heifers (n = 16, growth rate of 600 g day–1) received a single daily drench of 400 mL of water (group restricted, R) from Day 1 to Day 9 of a first synchronized oestrous cycle followed by 400 mL of PG from Day 1 to Day 9 of the second synchronized oestrous cycle (group restricted + PG, RPG). Ovum pick-up (OPU) was performed following superovulation, on Day 5 of the oestrous cycle to produce embryos in vitro and on Day 9 without superovulation to obtain oocytes and cumulus cells. The same protocol was used in control animals (n = 6, growth rate of 800 g day–1). Real-time PCR was used to determine the relative abundance of genes involved in lipid metabolism and storage (PLIN2, SCD), energy metabolism (ATP5A1, GLUT1), membrane permeability (AQP3), epigenetic marks (DNMT3a), apoptosis (BAX, TP53), and protein processing (HSPA9B) in grade 1 blastocysts, IGF1, IGF1R, IGFBP2, IGFBP4 in cumulus cells, and IGF1R and IGFBP2 in oocytes. Mann-Whitney nonparametric tests were performed to analyse gene expression results. The expression of PLIN2, ATP5A1, GLUT1, AQP3, DNMT3a, BAX, and HSPA9B were decreased in embryos collected from restricted compared with control animals. The expression levels of these genes were restored when females were supplemented with PG. The expression of TP53 and SCD were not affected. In cumulus cells, the expression levels of IGF1, IGF1R, and IGFBP4 were decreased in restricted compared with control animals. The expression levels of IGF1 and IGF1R were restored with PG supplementation. No differences were observed for the IGFBP2 gene. In the oocytes, no differences were observed for the expression levels of IGF1R and IGFBP2 genes. In conclusion, this work shows for the first time that feed restriction and dietary supplementation by PG in heifers produced changes in gene expression in blastocysts and modified the pattern of the IGF system in cumulus cells. These results suggest the existence of an epigenetic regulation induced by PG during follicular growth, which can regulate the level of gene expression up to the blastocyst stage. In general, PG supplementation of feed-restricted donors restored gene expression at the levels observed after normal feeding.

scholarly journals MicroRNA-204-5p regulates apoptosis by targeting Bcl2 in rat ovarian granulosa cells exposed to cadmium†

2020 ◽  
Vol 103 (3) ◽  
pp. 608-619
Author(s):  
Ping Zhong ◽  
Jin Liu ◽  
Hong Li ◽  
Senbin Lin ◽  
Lingfeng Zeng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Granulosa Cells ◽  
B Cell Lymphoma ◽  
Experimental Conditions ◽  
Expression Levels ◽  
Ovarian Granulosa Cells ◽  
Mrna And Protein Expression ◽  
Induced Apoptosis ◽  
Apoptosis Regulation

Abstract This study aimed to investigate whether cadmium (Cd) cytotoxicity in rat ovarian granulosa cells (OGCs) is mediated through apoptosis or autophagy and to determine the role of microRNAs (miRNAs) in Cd cytotoxicity. To test this hypothesis, rat OGCs were exposed to 0, 10, and 20 μM CdCl2 in vitro. As the Cd concentration increased, OGC apoptosis increased. In addition, Cd promoted apoptosis by decreasing the mRNA and protein expression levels of inhibition of B-cell lymphoma 2 (Bcl2). However, under our experimental conditions, no autophagic changes in rat OGCs were observed, and the mRNA and protein expression levels of the autophagic markers microtubule-associated protein 1 light chain 3 alpha (Map1lc3b) and Beclin1 (Becn1) were not changed. Microarray chip analysis, miRNA screening, and bioinformatics approaches were used to further explore the roles of apoptosis regulation-related miRNAs. In total, 19 miRNAs putatively related to Cd-induced apoptosis in rat OGCs were identified. Notably, miR-204-5p, which may target Bcl2, was identified. Then, rat OGCs were cultured in vitro and used to construct the miR-204-5p-knockdown cell line LV2-short hairpin RNA (shRNA). LV2-shRNA cells were exposed to 20 μM Cd for 12 h, and the mRNA and protein expression levels of Bcl2 were increased. Our findings suggest that Cd is cytotoxic to rat OGCs, and mitochondrial apoptosis rather than autophagy mediates Cd-induced damage to OGCs. Cd also affects apoptosis-related miRNAs, and the underlying apoptotic mechanism may involve the Bcl2 gene.

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