Hsa-miR-134-5p predicts cardiovascular risk in circulating mononuclear cells and improves angiogenic action of senescent endothelial progenitor cells.

This research explores the role of microRNA in senescence of human endothelial progenitor cells (EPCs) induced by replication. Hsa-miR-134-5p was found up-regulated in senescent EPCs where overexpression improved angiogenic activity. Hsa-miR-134-5p, which targeted transforming growth factor ß-activated kinase 1-binding protein 1 (TAB1) gene, down-regulated TAB1 protein, and inhibited phosphorylation of p38 mitogen-activated protein kinase (p38) in hsa-miR-134-5p-overexpressed senescent EPCs. Treatment with siRNA specific to TAB1 (TAB1si) down-regulated TAB1 protein and subsequently inhibited p38 activation in senescent EPCs. Treatment with TAB1si and p38 inhibitor, respectively, showed angiogenic improvement. In parallel, transforming growth factor Beta 1 (TGF-ß1) was down-regulated in hsa-miR-134-5p-overexpressed senescent EPCs and addition of TGF-ß1 suppressed the angiogenic improvement. Analysis of peripheral blood mononuclear cells (PBMCs) disclosed expression levels of hsa-miR-134-5p altered in adult life, reaching a peak before 65 years, and then falling in advanced age. Calculation of the Framingham risk score showed the score inversely correlates with the hsa-miR-134-5p expression level. In summary, hsa-miR-134-5p is involved in the regulation of senescence-related change of angiogenic activity via TAB1-p38 signalling and via TGF-ß1 reduction. Hsa-miR-134-5p has a potential cellular rejuvenation effect in human senescent EPCs. Detection of human PBMC-derived hsa-miR-134-5p predicts cardiovascular risk.

Hsa-miR-409-3p regulates endothelial progenitor senescence via PP2A-P38 and is a potential ageing marker in humans.

We explored the roles of hsa-microRNA (miR)-409-3p in senescence and signalling mechanism of human endothelial progenitor cells (EPCs). Hsa-miR-409-3p was found upregulated in senescent EPCs. Overexpression of miRNA mimics in young EPCs inhibited angiogenesis. In senescent EPCs, compared to young EPCs, protein phosphatase 2A (PP2A) was downregulated, with activation of p38/JNK by phosphorylation. Young EPCs treated with siPP2A caused inhibited angiogenesis with activation of p38/JNK, similar to findings in senescent EPCs. Time series analysis showed, in young EPCs treated with hsa-miR-409-3p mimics, PP2A was steadily downregulated for 72 h, while p38/JNK was activated with a peak at 48 hours. The inhibited angiogenesis of young EPCs after miRNA-409-3p mimics treatment was reversed by the p38 inhibitor. The effect of hsa-miR-409-3p on PP2A signalling was attenuated by exogenous VEGF. Analysis of human peripheral blood mononuclear cells (PBMCs) obtained from healthy people revealed hsa-miR-409-3p expression was higher in those older than 65 years, compared to those younger than 30 years, regardless of gender. In summary, hsa-miR-409-3p was upregulated in senescent EPCs and acted as a negative modulator of angiogenesis via targeting protein phosphatase 2 catalytic subunit alpha (PPP2CA) gene and regulating PP2A/p38 signalling. Data from human PBMCs suggested hsa-miR-409-3p a potential biomarker for human ageing.

Ultrasonic microbubble VEGF gene delivery improves angiogenesis of senescent endothelial progenitor cells.

The therapeutic effects of ultrasonic microbubble transfection (UMT)-based vascular endothelial growth factor 165 (VEGF165) gene delivery on young and senescent endothelial progenitor cells (EPCs) were investigated. By UMT, plasmid DNA (pDNA) can be delivered into both young EPCs and senescent EPCs. In the UMT groups, higher pDNA-derived protein expression was found in senescent EPCs than in young EPCs. Consistent with this finding, a higher intracellular level of pDNA copy number was detected in senescent EPCs, with a peak at the 2-h time point post UMT. Ultrasonic microbubble delivery with or without VEGF improved the angiogenic properties, including the proliferation and/or migration activities, of senescent EPCs. Supernatants from young and senescent EPCs subjected to UMT-mediated VEGF transfection enhanced the proliferation and migration of human aortic endothelial cells (HAECs), and the supernatant of senescent EPCs enhanced proliferation more strongly than the supernatant from young EPCs. In the UMT groups, the stronger enhancing effect of the supernatant from senescent cells on HAEC proliferation was consistent with the higher intracellular VEGF pDNA copy number and level of protein production per cell in the supernatant from senescent cells in comparison to the supernatant from young EPCs. Given that limitations for cell therapies are the inadequate number of transplanted cells and/or insufficient cell angiogenesis, these findings provide a foundation for enhancing the therapeutic angiogenic effect of cell therapy with senescent EPCs in ischaemic cardiovascular diseases.

Enhanced Proliferation of Endothelial Progenitor Cells Post-Ultrasonic Microbubble Transfection Is Plasmid DNA Size Dependent and Contributed by Interleukin-6 Generation.

We investigated whether ultrasonic microbubble transfection (UMT) would enhance the transfection of large-sized luciferase plasmids (5.6, 9.2 and 33 kb) and biological impacts. Porcine venous blood endothelial progenitor cells (EPCs) were cultured in a medium containing plasmid DNA (pDNA) of different sizes followed by UMT and functional assays. Real-time polymerase chain reaction was conducted to investigate the effects of transfection of pDNA on multiple molecules central to endothelial function. The results indicated enhanced luciferase expression after UMT but the enhancement declined with increase in the size of the plasmid. UMT of pDNAs sized 5.6 and 9.2 kb into EPCs led to significant enhancement of proliferation. The interleukin-6 (IL-6) secreted from UMT of EPCs also increased in the 5.6- and 9.2-kb pDNA groups. Treatment of the transfected EPCs with anti-IL-6 antibody neutralized the proliferation. In conclusion, UMT of pDNAs sized 5.6 and 9.2 kb into EPCs increased the secretion of IL-6, which in turn enhanced cell proliferation.

The increase of VEGF secretion from endothelial progenitor cells post ultrasonic VEGF gene delivery enhances the proliferation and migration of endothelial cells.

We investigated the feasibility of exogenous gene expression in endothelial progenitor cells (EPCs) through the use of ultrasonic microbubble transfection (UMT). EPCs originating from porcine peripheral blood were cultured in a medium containing constructed vascular endothelial growth factor (VEGF) pDNA followed by UMT. Simultaneously, comprehensive functional evaluations were conducted to investigate the effects of UMT of the VEGF gene on the EPCs. The results showed that UMT yielded significant VEGF protein expression. VEGF-containing supernatant originating from EPCs post UMT led to significantly enhanced activities of proliferation by more than 20% and migration by approximately 30% in human aortic endothelial cells. The duration of additional secretion of VEGF protein attributable to the exogenous VEGF gene in the EPCs post UMT lasted more than 96 hours. In conclusion, UMT successfully delivers the VEGF gene into porcine EPCs, and VEGF-containing supernatant derived from EPCs post UMT enhances the proliferation and migration of human aortic endothelial cells.

Ultrasonic microbubble-mediated gene delivery causes phenotypic changes of human aortic endothelial cells.

Ultrasound, in combination with microbubbles, serves as a feasible nonviral method in vascular gene delivery. However, the effects of ultrasonic microbubble transfection (UMT) on vascular endothelial cells remained unclear. We therefore investigated whether UMT itself causes phenotypic changes of the human aortic endothelial cells (HAEC) in vitro. HAEC were cultured with solution containing luciferase reporter gene and microbubbles followed by exposure to ultrasound of selected parameters. Thereafter, the proliferation and migration activities of HAEC were investigated. Real-time RT-PCR and/or western blotting were performed to assess expression profile of HAEC, including growth-related factors (vascular endothelial growth factor, fins-like tyrosine kinase-1 [Flt-1] and kinase insert domain-containing receptor [KDR]), coagulatory factor (von Willebrand factor), vasodilatory enzyme (endothelial nitric oxide synthase), gap junctional protein connexin43 and adhesion molecules (P-selectin, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1). The results showed that in conditions where UMT lead to expression of luciferase, proliferation capacity is enhanced (p<0.001), partly attributable to the effect of ultrasound (p<0.05), after excluding the effect of contact inhibition. In addition, the expression of KDR and Flt-1 were found increased at either the mRNA level, protein level, or both (p<0.05). Other markers did not have significant changes (all p>0.2). Similarly, the migration capacity was minimally changed (p>0.3). In conclusion, UMT causes phenotypic changes of HAEC by enhancing proliferation and upregulating KDR and Flt-1, while possesses no obvious adverse effect on viable transfected cells. Further investigation is required to clarify the impact of these changes by UMT in vivo.

Immune response against grouper nervous necrosis virus by vaccination of virus-like particles.

The grouper is a high-value fish in the seafood market. Grouper nervous necrosis virus (GNNV) causes mass mortality, near 100% in larvae and juveniles, which has great economic impact on the aquaculture of the marine fish. Since vaccination is one of the best methods against viral diseases, grouper Epinephelus lanceolatus was injected with virus-like particles (VLPs) of GNNV at different dosages and injection frequencies. The anti-sera of vaccinated fish were analyzed with antigen-capture ELISA to quantify immunization titer. The antibody titers in the vaccinated fish increased remarkably within 4 weeks, during which time the antibody was definitely capable to neutralize the native virus. With one shot of 10-250 microg VLPs, the stimulated antibody titer reached a steady saturation level in 1 month, among which the titers by one shot of 100 and 250 microg VLPs were 13% higher than by 10 microg. Two shots of 10 and 100 microg VLPs increased to maximum titer, which was 29% higher than one shot, whereas two shots of 250 microg VLPs and four shots of 100 microg VLPs dramatically downgraded the titers by -23% and -44%, respectively. These results imply that the overdose effects occurred in total dosages higher than 200 microg VLPs. The experiments of VLP vaccine with adjuvant revealed that the adjuvant is not required for increasing the efficacy of the VLP vaccine. Immunization with the VLPs can also stimulate fish to produce high antibody titer for more than 5 months, which can be correlated to long-term protection. When VLPs are used as vaccine agent, a dosage at 1 microg/g of fish body weight is enough to stimulate a full-scale immune response.