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Recently, microRNA-297 (miR-297) and signal transducer and activator of transcription 3 (STAT3) have been demonstrated to be involved in dysfunction of vascular endothelial cells and inflammatory conditions, such as sepsis. The present study aimed to investigate the role of miR-297 and STAT3 in lipopolysaccharide (LPS)-induced inflammatory human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated by different concentrations of LPS. miR-297 mimics were transfected into HUVECs to overexpress miR-297. The qRT-PCR was used to measure the expression level of miR-297. Western blot was used to detect the expressions of STAT3, inflammatory cytokines, adhesion molecules and apoptosis-related proteins. Cell apoptosis was determined by flow cytometry. Compared with parental HUVECs, the expression of miR-297 was significantly down-regulated, while the expression of STAT3 was obviously up-regulated in LPS-induced HUVECs. The expressions of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin were also increased in LPS-induced HUVECs than those in parental HUVECs. In addition, LPS induced apoptosis of HUVECs through up-regulation of Bax and cleaved caspase 3 expressions. Conversely, miR-297 mimics inhibited LPS-activated expressions of STAT3, inflammatory cytokines, and adhesion molecules, and protected HUVECs against LPS-induced apoptosis through inhibition of Bax and cleaved caspase 3 expressions. Mechanistically, the 3'-untranslated region (3'-UTR) of STAT3 mRNA was validated as a direct target of miR-297. Over-expression of STAT3 partially abrogated protective effects of miR-297, whereas silencin g of STAT3 contributed to miR-297-mediated biological effects. miR-297 protects HUVECs against LPS-induced inflammatory response and apoptosis by targeting STAT3 pathway. Thus, miR-297 may be a promising therapeutic target for patients with sepsis.

Cardiovascular disease (CVD) is the leading cause of death worldwide, and extensive research has been performed to understand this disease better, using various experimental models. The endothelium plays a crucial role in the development of CVD, since it is an interface between bloodstream components, such as monocytes and platelets, and other arterial wall components. Human umbilical vein endothelial cell (HUVEC) isolation from umbilical cord was first described in 1973. To date, this model is still widely used because of the high HUVEC isolation success rate, and because HUVEC are an excellent model to study a broad array of diseases, including cardiovascular and metabolic diseases. We here review the history of HUVEC isolation, the HUVEC model over time, HUVEC culture characteristics and conditions, advantages and disadvantages of this model and finally, its applications in the area of cardiovascular diseases.

Boron nitride nanotubes (BNNTs) are tubular nanoparticles with a structure analogous to that of carbon nanotubes, but with B and N atoms that completely replace the C atoms. Many favorable results indicate BNNTs as safe nanomaterials; however, important concerns have recently been raised about ultra-pure, long (˜10 µm) BNNTs tested on several cell types. Here, we propose additional experiments with the same BNNTs, but shortened (˜1.5 µm) with a homogenization/sonication treatment that allows for their dispersion in gum Arabic aqueous solutions. Obtained BNNTs are tested on human endothelial and neuron-like cells with several independent biocompatibility assays. Moreover, for the first time, their strong sum-frequency generation signal is exploited to assess the cellular uptake. Our data demonstrate no toxic effects up to concentrations of 20 µg/ml, once more confirming biosafety of BNNTs, and again highlighting that nanoparticle aspect ratio plays a key role in the biocompatibility evaluation. Original submitted 3 December 2013; Revised submitted 28 January 2014; Published online 6 February 2014

Extracellular vesicles (EVs) such as exosomes are nano-sized vesicles that carry proteins and miRNAs and can transmit signals between cells. We hypothesized that exosomes from endothelial cells can transmit protective signals to cardiomyocytes. Co-culture of primary adult rat cardiomyocytes with normoxic HUVEC cells separated by a cell-impermeable membrane reduced the percentage of cardiomyocyte death following simulated ischaemia and reperfusion (sIR) from 80 ± 11% to 51 ± 4% (P < 0.05; N = 5). When EVs were removed from the HUVEC-conditioned medium it was no longer protective. Exosomes were purified from HUVEC-conditioned medium using differential centrifugation and characterized by nanoparticle tracking analysis, electron microscopy, and flow cytometry. Pre-incubation of cardiomyocytes with HUVEC exosomes reduced the percentage of cell death after sIR from 88 ± 4% to 55 ± 3% (P < 0.05; N = 3). This protection required ERK1/2 activity as it was prevented by inhibitors PD98059 and U0126. Ischaemic preconditioning caused about ~3-fold higher rate of exosome production from HUVEC and from isolated, perfused rat hearts. This increase resulted in significantly greater protection against sIR in cardiomyocytes. In conclusion, exosomes released from endothelial cells can confer resistance to sIR injury in cardiomyocytes via the activation of the ERK1/2 MAPK signalling pathway, and may contribute to IPC.

Background IL-6 classic signaling is linked to anti-inflammatory functions while the trans-signaling is associated with pro-inflammatory responses. Classic signaling is induced via membrane-bound IL-6 receptor (IL-6R) whereas trans-signaling requires prior binding of IL-6 to the soluble IL-6R. In both cases, association with the signal transducing gp130 receptor is compulsory. However, differences in the downstream signaling mechanisms of IL-6 classic- versus trans-signaling remains largely elusive. Methods In this study, we used flow cytometry, quantitative PCR, ELISA and immuno-blotting techniques to investigate IL-6 classic and trans-signaling mechanisms in Human Umbilical Vein Endothelial Cells (HUVECs). Results We show that both IL-6R and gp130 are expressed on the surface of human vascular endothelial cells, and that the expression is affected by pro-inflammatory stimuli. In contrast to IL-6 classic signaling, IL-6 trans-signaling induces the release of the pro-inflammatory chemokine Monocyte Chemoattractant Protein-1 (MCP-1) from human vascular endothelial cells. In addition, we reveal that the classic signaling induces activation of the JAK/STAT3 pathway while trans-signaling also activates the PI3K/AKT and the MEK/ERK pathways. Furthermore, we demonstrate that MCP-1 induction by IL-6 trans-signaling requires simultaneous activation of the JAK/STAT3 and PI3K/AKT pathways. Conclusions Collectively, our study reports molecular differences in IL-6 classic- and trans-signaling in human vascular endothelial cells; and elucidates the pathways which mediate MCP-1 induction by IL-6 trans-signaling.

PRP-Exos are nanoscale cup-shaped vesicles that carry a variety of proteins, mRNAs, microRNAs, and other bioactive substances. PRP-Exos can be formed through several induction pathways, which determine their molecular profiles and facilitate their tailormade participation in intercellular communication. Currently, little is known on how the PRP-Exos activation method influences the quality and quantity of PRP-Exos. The present study aims to observe and analyze the number, profile, and growth factors of PRP-Exos through TEM, Nanoflow, and WB after PRP activation and compare the difference in function of PRP-Exos on HUVECs, with different stimuli (calcium gluconate, thrombin, or both). We found that PRP activated with both thrombin and calcium gluconate harvested the highest concentration of exosomes [(7.16 ± 0.46) × 1010 particles/ml], compared to thrombin group [(4.87 ± 0.15) × 1010 particles/ml], calcium gluconate group [(5.85 ± 0.43) × 1010 particles/ml], or saline group [(7.52 ± 0.19) × 109 particles/ml], respectively (P < 0.05) via Nanoflow analysis. The WB analysis showed that cytokines (VEGF, PDGFBB, bFGF, TGF-β) are differentially encapsulated in PRP-Exos, depending on the PRP stimulus, in which the mixture-PRP-Exos yielded the highest concentration of cytokines. In the function assay of PRP-Exos on HUVECs, the mixture-PRP-Exos promoted HUVECs proliferation, increased HUVECs migration, promoted the formation of vessel-like by HUVECs via the AKT ERK signal pathway more dramatically, compared with other groups. In summary, our studies showed that PRP activated by the mixture of calcium gluconate and thrombin harvested the best quality of exosomes which had the top biological functions. This study provides a protocol for selecting appropriate PRP activators to obtain high-quality exosomes for future applications.

It remains a clinical challenge for cutaneous wound healing and skin regeneration. Endothelial cells participate in the formation of blood vessels and play an important role in the whole process of wound healing. Recent studies suggested that exosomes contribute to the intercellular communication through paracrine pathways, and sustained release of exosomes from hydrogel-based materials provide a promising strategy for curing wound defects. In this study, we isolated exosomes derived from human umbilical vein endothelial cells (HUVECs) and found that HUVECs derived exosomes (HUVECs-Exos) could promote the proliferation and migration activities of keratinocytes and fibroblasts, which are two important effector cells for skin regeneration. Then we developed gelatin methacryloyl (GelMA) hydrogel as the wound dressing to incorporate HUVECs-Exos and applied it to the full-thickness cutaneous wounds. It demonstrated that GelMA scaffold could not only repair the wound defect, but also achieve sustained release of exosomes. The in vivo results showed accelerated re-epithelialization, promotion of collagen maturity and improvement of angiogenesis. Collectively, our findings suggested that HUVECs-Exos could accelerate wound healing and GelMA mediated controlled release of HUVECs-Exos might offer a new method for repairing cutaneous wound defects.

Physicochemical properties play a crucial role in determining the toxicity of multi-walled carbon nanotubes (MWCNTs). Recently we found that MWCNTs with longer length and smaller diameters could induce toxicity to human umbilical vein endothelial cells (HUVECs) through the activation of endoplasmic reticulum (ER) stress. In this study, we further investigated the possible contribution of hydroxylation and carboxylation to the cytotoxicity of MWCNTs. The HUVECs were exposed to pristine (code XFM19), hydroxylated (code XFM20; content of hydroxyl groups 1.76 wt%) and carboxylated (code XFM21; content of carboxyl groups 1.23 wt%) MWCNTs, respectively. Then, the internalization, cytotoxicity, oxidative stress and activation of apoptosis-ER stress pathway were measured. In consequence, all types of MWCNTs could be internalized into the HUVECs, and the cellular viability was significantly reduced to a similar level. Moreover, the MWCNTs increased intracellular reactive oxygen species (ROS) and decreased glutathione (GSH) to similar levels, indicating their capacity of inducing oxidative stress. The Western blot results showed that all types of MWCNTs reduced BCL-2 and increased caspase-3, caspase-8, cleaved caspase-3 and cleaved caspase-8. The expression of ER stress gene DNA damage-inducible transcript 3 ( ) and protein level of chop were only significantly induced by XFM20 and XFM21, whereas protein level of p-chop was promoted by XFM19 and XFM21. In addition, the pro-survival gene was significantly down-regulated by all types of MWCNTs. These results suggested that MWCNTs could induce cytotoxicity to HUVECs via the induction of oxidative stress and apoptosis-ER stress, whereas a low degree of hydroxylation or carboxylation did not affect the toxicity of MWCNTs to HUVECs.

Meteorin-like (Metrnl) is a novel secreted protein with various biological activities. In this study, we investigated whether and how Metrnl regulated skin wound healing in mice. Global Metrnl gene knockout mice (Metrnl −/− ) and endothelial cell-specific Metrnl gene knockout mice (EC-Metrnl −/− ) were generated. Eight-mm-diameter full-thickness excisional wound was made on the dorsum of each mouse. The skin wounds were photographed and analyzed. In C57BL/6 mice, we observed that Metrnl expression levels were markedly increased in skin wound tissues. We found that both global and endothelial cell-specific Metrnl gene knockout significantly retarded mouse skin wound healing, and endothelial Metrnl was the key factor affecting wound healing and angiogenesis. The proliferation, migration and tube formation ability of primary human umbilical vein endothelial cells (HUVECs) were inhibited by Metrnl knockdown, but significantly promoted by addition of recombinant Metrnl (10 ng/mL). Metrnl knockdown abolished the proliferation of endothelial cells stimulated by recombinant VEGFA (10 ng/mL) but not by recombinant bFGF (10 ng/mL). We further revealed that Metrnl deficiency impaired VEGFA downstream AKT/eNOS activation in vitro and in vivo. The damaged angiogenetic activity in Metrnl knockdown HUVECs was partly rescued by addition of AKT activator SC79 (10 μM). In conclusion, Metrnl deficiency retards skin wound healing in mice, which is related to impaired endothelial Metrnl-mediated angiogenesis. Metrnl deficiency impairs angiogenesis by inhibiting AKT/eNOS signaling pathway.

Anti-angiogenesis is one of the most general clinical obstacles in cancer chemotherapy. Kaempferol is a flavonoid phytochemical found in many fruits and vegetables. Our previous study revealed that kaempferol triggered apoptosis in human umbilical vein endothelial cells (HUVECs) by ROS-mediated p53/ATM/death receptor signaling. However, the anti-angiogenic potential of kaempferol remains unclear and its underlying mechanism warranted further exploration in VEGF-stimulated HUVECs. In the present study, kaempferol significantly reduced VEGF-stimulated HUVEC viability. Kaempferol treatment also inhibited cell migration, invasion, and tube formation in VEGF-stimulated HUVECs. VEGF receptor-2 (VEGFR-2), and its downstream signaling cascades (such as AKT, mTOR and MEK1/2-ERK1/2) were reduced as determined by western blotting and kinase activity assay in VEGF-stimulated HUVECs after treatment with kaempferol. The present study revealed that kaempferol may possess angiogenic inhibition through regulation of VEGF/VEGFR-2 and its downstream signaling cascades (PI3K/AKT, MEK and ERK) in VEGF-stimulated endothelial cells.