Effect of Arthrospira powders from different producers on the formation of endothelial cell monolayers

Arthrospira platensis (AP) and some of its derived products have well-established biological activities as antioxidants or as agents to reduce cardiovascular disease risk factors. Furthermore, AP products have gained increasing importance as potential anti-cancer agents. However, the ingredients of the available products vary greatly with the origin, the type of production and processing, which could have significant consequences for their biological effects. Therefore, the composition and biological influence of five distinct AP powders, which were acquired commercially or produced at a public biotechnology institute, were investigated in regard to their endothelialization capacity using a cell impedance- (CI) based measurement method. The study revealed that the AP composition and especially the influence on HUVEC proliferation differed significantly between the five AP powders up to 109%. Thus, it could be shown that the method used allows the reliable detection of quantitative differences in biological effects of different AP preparations.

miR-210 regulates vascular endothelial cell apoptosis in arteriosclerosis obliterans through the JAK1-STAT3 pathway

IntroductionAtherosclerosis is a continuously worsening chronic condition that starts in the arteries and may then affect other blood vessels. There is increasing evidence linking microRNAs to the development of arteriosclerosis obliterans (ASO). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied in this study to detect miR-210-5p (miR-210) expression, and significant upregulation was observed in human arterial walls where ASO was prevalent.Material and methodsThe proliferation of human umbilical vascular endothelial cells (HUVECs) with various levels of miR-210 expression was assessed via MTT and colony-formation assays. Cell proliferation was significantly promoted in HUVECs with upregulated miR-210 levels and reduced in HUVECs with downregulated miR-210 levels.ResultsFlow cytometric analysis of cells stained with annexin V-FITC and propidium iodide data demonstrated that miR-210 inhibited apoptosis, while miR-210 inhibition promoted apoptosis by mediating pro-apoptotic protein expression levels. These results were verified using a dual-luciferase reporter gene assay system, which showed that Janus kinase 1 (JAK1) was directly targeted by miR-210, while an miR-210 mimic significantly decreased downstream JAK1 and signal transducer and activator of transcription 3 (STAT3) activation at a post-transcriptional level in HUVECs, as detected by western blotting and qRT-PCR. Further inhibition of either JAK1 or STAT3 counteracted the effect of miR-210 on HUVEC proliferation and apoptosis. These findings suggest that miR-210 promotes HUVEC proliferation, at least in part, by targeting the JAK1-STAT3 signaling axis.ConclusionsThis study provides insights into the contribution of the miR-210-JAK1-STAT3 axis and its underlying mechanisms to ASO pathology.

NDRG2 inhibition facilitates angiogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an angiogenesis-dependent tumor, and angiogenesis plays pivotal roles in progression and hematogenous metastasis. Upregulating NDRG2 expression could inhibit endothelial cell proliferation and tumor angiogenesis. However, the development of angiogenesis is a complicated and dynamic process, and the specific mechanisms that NDRG2 influences its progression are largely unknown. Conditioned media (CM) was collected from HCC cells. Cell viability, migration assay, tube formation, and western blot were used to evaluate the effect of NDRG2 on angiogenesis in HCC cells. ELISA assay was used to measure the level of VEGFA in CM. CM from NDRG2 knockdown cells significantly promoted HUVECs proliferation, migration, and tube formation compared with control cells. The level of VEGFA in CM was increased by NDRG2 knockdown relative to the control group. The expression of VEGFA, HIF-1α, and p-Akt was significantly increased in NDRG2 knockdown cells. CM from NDRG2 knockdown cells with VEGFA antibody failed to induce HUVEC proliferation, migration, and tube formation. YC-1 significantly inhibited the level of VEGFA in CM from NDRG2 knockdown cells. YC-1 also inhibited the expression of VEGFA and HIF-1α. Therefore, NDRG2 inhibition promoted the angiogenesis of HCC via VEGFA and may be used to be an anti-angiogenesis target.

Structural Insight into Integrin Recognition and Anticancer Activity of Echistatin

Echistatin (Ech) is a short disintegrin with a long 42NPHKGPAT C-terminal tail. We determined the 3-D structure of Ech by X-ray crystallography. Superimposition of the structures of chains A and B showed conformational differences in their RGD loops and C-termini. The chain A structure is consistent with our NMR analysis that the GPAT residues of the C-terminus cannot be observed due to high flexibility. The hydrogen bond patterns of the RGD loop and between the RGD loop and C-terminus in Ech were the same as those of the corresponding residues in medium disintegrins. The mutant with C-terminal HKGPAT truncation caused 6.4-, 7.0-, 11.7-, and 18.6-fold decreases in inhibiting integrins αvβ3, αIIbβ3, αvβ5, and α5β1. Mutagenesis of the C-terminus showed that the H44A mutant caused 2.5- and 4.4-fold increases in inhibiting αIIbβ3 and α5β1, and the K45A mutant caused a 2.6-fold decrease in inhibiting αIIbβ3. We found that Ech inhibited VEGF-induced HUVEC proliferation with an IC50 value of 103.2 nM and inhibited the migration of A375, U373MG, and Panc-1 tumor cells with IC50 values of 1.5, 5.7, and 154.5 nM. These findings suggest that Ech is a potential anticancer agent, and its C-terminal region can be optimized to improve its anticancer activity.

Pigment Epithelium-Derived Factor Promotes the Angiogenic Activity of VEGF

Introduction: We successfully identified a subset of CD11b+ monocytes expressing CX3CR1, the only receptor of fractalkine (Fkn; CX3CL1), in G-CSF mobilized peripheral blood stem cell collection (PBSC). We found that Fkn-treated CD11b+CX3CR1+ monocytes express pigment epithelium-derived factor (PEDF) once Fkn activates Fkn/CX3CR1 signaling. PEDF is a glycoprotein, which belongs to the serpin family and involves various physiologic processes such as angiogenesis, cell proliferation, and survival. In this study, we investigate the role of PEDF and its mechanism of action in promoting angiogenesis. Materials andmethods: To mobilize mononuclear cells (MNCs) including CD11b+CX3CR1+ monocytes into peripheral blood (PB), heathy donors were subcutaneously injected with G-CSF (10μg/kg) for 5 days. Apheresis MNCs were collected from donor PB using a COBE spectra cell separator (COBE, Lakewood, CO) 5 days after daily G-CSF injection. Then, CD11b+CX3CR1+ cells were isolated with a MoFlo™ XDP Cell Sorter (Beckman Coulter, Brea, CA). Human umbilical vein endothelial cells (HUVECs) were isolated from human cords and cultured at 37 °C in 5% CO2 on top of collagen in four different settings: (1) untreated (HUVECs alone) (2) VEGF (1ng/mL) treated HUVECs; (3) PEDF (3,30 and 300 ng/mL) treated HUVECs; (4) VEGF (1ng/mL)+PEDF (3,30 and 300 ng/mL) treated HUVECs. To assess dose-dependency, PEDF was administered at different dosages (0, 3, 30 and 300 ng/mL) for 7 days in either the presence or absence of VEGF (1ng/mL). HUVEC growth areas were measured by image J. Results: CD11b+CX3CR1+ monocytes were found at 19.6±3.58% of total MNCs in human G-CSF mobilized PBSC. Fractalkine treatment (50ng/mL for 30 minutes) of these monocytes promoted endothelial cell proliferation of HUVECs and increased PEDF expression according to the angiogenic protein array results. Administration of PEDF inhibitor significantly decreased HUVEC proliferation and vascular structure formation compared to the control group (p <0.001). We further investigated pro-angiogenic effects of PEDF on HUVEC proliferation. In PEDF (3,30 and 300 ng/mL) treated HUVECs, PEDF itself has minimal effect on HUVEC proliferation. However, EC sprouting at the edge of vessel-like structures was greater in the 3 and 30 ng/mL. Interestingly, there was no EC sprouting with 300 ng/mL PEDF. PEDF (3 and 30 ng/mL) itself showed little effect on HUVEC proliferation. However, co-culturing with VEGF (1ng/mL) and low PEDF concentrations (3 and 30 ng/mL) significantly increased EC proliferation (EC proliferation area (mm2), 1.7±0.4 (VEGF only) vs 3.0±0.6 (VEGF+3 ng/mL PEDF) and 2.5±1.1 (VEGF+30 ng/mL PEDF); p=0.002 and 0.048, respectively). Higher concentration of PEDF did not induced EC proliferation even in the presence VEGF (1.7±0.4 (VEGF only) vs 1.5±0.9 (VEGF+300 ng/mL PEDF); p=0.192). The results suggest that low concentrations of PEDF (3 and 30 ng/mL) has no effect on HUVEC proliferation, but has a potent effect on HUVEC migration regardless of VEGF activity. PEDF is not pro-angiogenic by itself, but can markedly enhance HUVEC proliferation in PEDF (3 and 30 ng/mL) treated HUVECs with VEGF. Conclusion: Fractalkine-treated CD11b+CX3CR1+ monocytes promote angiogenesis by increasing PEDF expression, which acts as a pro-angiogenic factor only under conditions where VEGF is present. Disclosures No relevant conflicts of interest to declare.

Serum of coronary atherosclerotic heart disease patients induces oxidative stress injury on endothelial cells

Endothelial cell (EC) dysfunction has a fundamental role in the development of atherosclerosis, which leads to myocardial infarction and stroke. The aim of this study is to investigate the effect of serum from patients with coronary atherosclerotic heart disease (CAD) on endothelial cells and investigate the possible mechanism underlying these effects. Serum from 35 patients with CAD and 35 healthy volunteers was collected. Human umbilical vein endothelial cell (HUVEC) proliferation and apoptosis were assessed by a CCK‑8 assay and a flow cytometry assay, respectively. The synthesis of nitric oxide (NO) and reactive oxygen species (ROS) was measured using the nitrate reduction method and DCFH2-DA staining, respectively. The proliferation of HUVECs was inhibited by treatment with serum from CAD patients (P<0.05). Suppression of HUVEC proliferation by CAD serum occurred in a concentration-dependent manner. The synthesis of NO was also reduced in the CAD serum-treated group. Furthermore, the serum from CAD patients increased both apoptosis and intracellular ROS production in HUVECs. Moreover, treatment with tempol antagonized CAD serum-meditated HUVEC injuries. Taken together, these results suggest that HUVEC injury via CAD serum treatment is mediated by ROS production. Tempol may partly reverse this effect by abolishing HUVEC apoptosis.

Enhanced vascularization of PCL porous scaffolds through VEGF-Fc modification

VEGF-Fc interface improves the vascularization of PCL scaffolds by enhancing HUVEC proliferation and migration through activating VEGFR/Pi3k and VEGFR/MAPK pathways.