Quercetin Synergistically Potentiates the Anti-Angiogenic Effect of 5- Fluorouracil on HUVEC Cell Line

background: Since tumors need oxygen and nutrients to grow and spread, angiogenesis has an essential role in cancer growth and metastasis. So, inhibition of angiogenesis at the initial stage of cancer is a critical strategy in metastasis inhibition. 5-Fluorouracil (5-FU) is a chemotherapy drug that has also been shown to have anti-angiogenic effects. Quercetin, a natural polyphenolic compound, has anti-angiogenic effects. The present study was conducted to investigate the enhancement of the anti-angiogenic effect of 5-FU in combination with Quercetin (Que) and compare it with the application of 5-FU alone.Method and Results: Following the treatment of human umbilical vein endothelial cells (HUVECs) with Que, 5-FU and their combination, the cell viability, migration, and gene expression of VEGFR2 and VEGFR1 were assessed through MTT assay, wound healing assay, and real-time RT-PCR, respectively. In vivo angiogenesis was evaluated using chicken chorioallantoic membrane (CAM) assay. Our study showed that cell viability, migration, gene expression of VEGFR2 and VEGFR1, and angiogenesis significantly decreased following Que and 5-FU alone treatment, and the decrease in combination state was significant compare to 5-FU alone.Conclusions: In summary, the present study showed that the combination of Que with 5-FU improves its anti-angiogenic effects. Therefore, this combination can be suggested for future in vivo studies.

Vascular endothelium protective property of curcumin

Objectives: In the present study, we aimed to examine the efficacy of curcumin on endothelial vascular protection on human umbilical vein endothelial cells (HUVEC) cells in the in vitro setting and to determine effective doses. Materials and methods: Cytotoxic effect and wound healing activity of curcumin on the HUVEC cell line were evaluated and angiogenesis was studied by tube formation assay. The cytotoxic activity of 0.5 to 8 μM curcumin on HUVEC cell line was tested using the WST-1 cell proliferation assay. In wound healing determination, the scratched cells were incubated with the half maximal effective concentration (EC50) dose of curcumin and wounds were monitored by the JuLITM Br live cell movie analyzer. Wound gaps were measured using the ImageJ software. To determine the angiogenesis, tube formation assay was performed and the results were analyzed. Results: The 1.77 μM of curcumin increased the cell viability by 150% after 48 h of treatment. According to the wound healing results, after 48 h of incubation, the control group and 1.77 μM curcumin exhibited 47.1% and 43.8% closure, respectively. The mean maximum and minimum tube lengths were found to be 21,858±3,945 and 12,438±3,817 pixel for curcumin, respectively with low fetal bovine serum (FBS) and curcumin with high FBS, respectively. Conclusion: Our results show that curcumin is a promising endothelial protective agent for HUVEC cell line.

Doxorubicin Enhances Procoagulant Activity of Endothelial Cells after Exposure to Tumour Microparticles on Microfluidic Devices

The majority of cancer patients undergoing chemotherapy have a significantly increased risk of venous thromboembolism via a mechanism not yet fully elucidated but which most probably involves tumour microparticles (MP) combined with damaged/activated endothelium. Tumour cell lines (ES-2 and U87) were cultured as 3D spheroids and transferred to biochips connected through to a second chip precultured with an endothelial cell layer (human umbilical vein endothelial cells [HUVECs]). Media were introduced with and without doxorubicin (DOX) to the spheroids in parallel chips under constant flow conditions. Media samples collected pre- and post-flow through the biochip were analysed for tissue factor microparticles (TFMP) and procoagulant activity (PCA). HUVECs were also harvested and tested for PCA at a constant cell number. TFMP levels in media decreased after passing over HUVECs in both conditions over time and this was accompanied by a reduction in PCA (indicated by a slower coagulation time) of the media. The relationship between PCA and TFMP was correlated (r = −0.85) and consistent across experiments. Harvested HUVECs displayed increased PCA when exposed to tumour spheroid media containing TFMP, which was increased further after the addition of DOX, suggesting that the TFMP in the media had bound to HUVEC cell surfaces. The enhanced PCA of HUVECs associated with the DOX treatment was attributed to a loss of viability of these cells rather than additional MP binding. The data suggest that tumour MP interact with HUVECs through ligand-receptor binding. The model described is a robust and reproducible method to investigate cytotoxic agents on tumour spheroids and subsequent downstream interaction with endothelial cells.

Preclinical study of innovative peptides mimicking the space structure of the α-helix B of erythropoietin

Introduction: The aim of this study was to examine the effectiveness of innovative peptides obtained by addition of polypeptide motifs with antiaggregation activity (Arg-Gly-Asp, Lys-Gly-Asp and Pro-Gly-Pro) to a peptide mimicking the space structure of the α-helix B of erythropoietin pHBSP (Pyr-Glu-Gln-Leu-Glu-Arg-Ala-Leu-Asn-Ser-Ser). Materials and methods: The cytoprotective activity of innovative peptides mimicking the space structure of the α-helix B of erythropoietin at the doses of 5 μg/ml, 30 μg/ml and 50 μg/ml was studied on human endothelial cell culture in a simulated oxidative stress. An ADMA-like model of preeclampsia was simulated in the experiment. The study was conducted in 260 female Wistar rats, weighing 250–300 g. Results and discussion: Innovative peptides mimicking the space structure of the α-helix B of erythropoietin retain their cytoprotective activity in a simulated oxidative stress in HUVEC cell culture at the doses of 5 μg/ml, 30 μg/ml, and 50 μg/ml. The compounds with laboratory codes P-αB1 and P-αB3 had the most pronounced cytoprotective activity. Administration of N-nitro-L-arginine-methyl ether to pregnant females for 7 days causes the morphofunctional changes similar to clinical changes in preeclampsia. The innovative peptide under laboratory code P-αB4 at the dose of 50 μg/kg mimicking the space structure of the α-helix B of erythropoietin shows the most pronounced protective properties. Conclusion: Innovative peptides mimicking the space structure of the α-helix B of erythropoietin have a pronounced positive influence on the morphofunctional disorders in animals with ADMA-like preeclampsia.

Exosomes From 3d Culture of Marrow Stem Cells Enhances Endothelial Cell Proliferation, Migration, and Angiogenesis via Activation of the Hmgb1/akt Pathway

BackgroundAngiogenesis is an essential step in tissue engineering. MSCexosomes play an important role in angiogenesis. Functional biomolecules in exosomes vested by the culture microenvironment can be transferred to recipient cells and affects their effect. 3D culture can improve the proliferation and activity of MSCs. However, whether exosomes derived from 3D culture of MSCs have an enhanced effect on angiogenesis is unclear. MethodsHerein, we compared the bioactivity of exosomes produced by conventional 2D culture (2D-exos) and 3D culture (3D-exos) of bone marrow stem cells (BMSCs) in angiogenesis. ResultsA series of in vitro and in vivo experiments indicated that 3D-exos exhibited stronger effects on HUVEC cell proliferation, migration, tube formation, and in vivo angiogenesis compared with 2D-exos. Moreover, the superiority of 3D-exos might be attributed to the activation of HMGB1/AKT signaling. ConclusionsThese results indicate that exosomes from 3D culture of MSCs may serve as a potential therapeutic approach for pro-angiogenesis.

Vascular NRP2 triggers PNET angiogenesis via activating SSH1-cofilin axis

Background Angiogenesis is a critical step in pancreatic neuroendocrine tumour (PNET) growth and may be a selective target for PNET therapy. However, PNET is robustly resistant to current antiangiogenic therapies which primarily target the VEGFR pathway. Thus, PNET angiogenesis mechanism was urgently to be clarified. Methods Dataset analysis was used to identify PNET angiogenesis related genes. Immunohistochemistry was performed to determine relation among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assay were investigated to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2 inducing angiogenesis was detected by mutant construction, Western blot, and immunofluorescence assay. In vivo, mice model was performed to evaluate the effect of NRP2 antibody, and clinical data was recruited to verify association between NRP2 and patients prognosis. Results NRP2, a VEGFR2 co-receptor, positively correlated with vascularity rather than with VEGFR2 in PNET tissues. NRP2 promoted PNET cell medium-treated HUVEC cell migration via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Mechanistically, cofilin phosphatase slingshot-1 (SSH1) was highly expressed involved in NRP2-activating cofilin. Silencing SSH1 rescued NRP2-activated HUVEC cell migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumour growth. High NRP2 expression was associated with poor prognosis in PNET patients. Conclusion Vascular NRP2 promotes PNET angiogenesis via activating SSH1/cofilin/actin axis. Our findings demonstrate NRP2 to be an important regulator of angiogenesis and potential therapeutic target of anti-angiogenesis therapy for PNET.

All-Trans Retinoic Acid Grafted Poly Beta-Amino Ester Nanoparticles: A Novel Anti-angiogenic Drug Delivery System

Purpose: Developing chemotherapy with nanoplatforms offers a promising strategy for effective cancer treatment. In the present study, we propose a novel all-trans retinoic acid (ATRA) grafted poly beta-amino ester (PBAE) copolymer for preparing nanoparticles (NPs). Methods: ATRA grafted PBAE (ATRA-g-PBAE) copolymer was synthesized by grafting ATRA to PBAE; it was characterized by proton nuclear magnetic resonance, Fourier transform infrared, and thermogravimetric analysis. ATRA-g-PBAE NPs were prepared by the solvent displacement method. Design-Expert software was employed to optimize size of NPs. The morphology was evaluated by transmission electron microscope, and ultraviolet-visible spectroscopy was applied for drug release. Cytotoxicity was evaluated toward HUVEC cell line, and the 3D collagencytodex model was used to evaluate anti-angiogenic property of PBAE, ATRA, and NPs. Results: The optimum size of the NPs was 139.4 ± 1.41 nm. After 21 days, 66.09% ± 1.39 and 42.14% ± 1.07 of ATRA were released from NPs at pH 5.8 and 7.4, respectively. Cell culture studies demonstrated antiangiogenic effects of ATRA-g-PBAE NPs. Anti-angiogenesis IC50 was 0.007 mg/mL for NPs (equal to 0.002 mg/mL of ATRA) and 0.005 mg/mL for free ATRA. Conclusion: This study proposes the ATRA-g-PBAE NPs with inherent anti-angiogenic effects as promising carrier for anticancer drugs with purpose of dual drug delivery.