Up-Regulated Expression of Matrix Metalloproteinases in Endothelial Cells Mediates Platelet Microvesicle-Induced Angiogenesis.

BACKGROUND/AIMS: Platelet microvesicles (PMVs) contribute to angiogenesis and vasculogenesis, but the mechanisms underlying these contributions have not been fully elucidated. In the present study, we investigated whether PMVs regulate the angiogenic properties of endothelial cells (ECs) via mechanisms extending beyond the transport of angiogenic regulators from platelets. METHODS: In vitro Matrigel tube formation assay and in vivo Matrigel plug assay were used to evaluate the pro-angiogenic activity of PMVs. The effects of PMVs on the migration of human umbilical vein endothelial cells (HUVECs) were detected by transwell assay and wound-healing assay. Real-time PCR and western blot were conducted to examine mRNA and protein expression of pro-angiogenic factors in HUVECs. Matrix metalloproteinase (MMP) activity was assayed by gelatin zymography. Moreover, the effects of specific MMP inhibitors were tested. RESULTS: PMVs promoted HUVEC capillary-like network formation in a dose-dependent manner. Meanwhile, PMVs dose-dependently facilitated HUVEC migration. Levels of MMP-2 and MMP-9 expression and activity were up-regulated in HUVECs stimulated with PMVs. Inhibition of MMPs decreased their pro-angiogenic and pro-migratory effects on HUVECs. Moreover, we confirmed the pro-angiogenic activity of PMVs in vivo in mice with subcutaneous implantation of Matrigel, and demonstrated that blockade of MMPs attenuated PMV-induced angiogenesis. CONCLUSION: The findings of our study indicate that PMVs promote angiogenesis by up-regulating MMP expression in ECs via mechanism extending beyond the direct delivery of angiogenic factors.

Deoxynivalenol Induces Intestinal Damage and Inflammatory Response through the Nuclear Factor-κB Signaling Pathway in Piglets.

Deoxynivalenol (DON) is highly toxic to animals and humans, but pigs are most sensitive to it. The porcine mucosal injury related mechanism of DON is not yet fully clarified. Here, we investigated DON-induced injury in the intestinal tissues of piglet. Thirty weanling piglets [(Duroc × Landrace) × Yorkshire] were randomly divided into three groups according to single factor experimental design (10 piglets each group). Piglets were fed a basal diet in the control group, while low and high dose groups were fed a DON diet (1300 and 2200 µg/kg, respectively) for 60 days. Scanning electron microscopy results indicated that the ultrastructure of intestinal epithelial cells in the DON-treated group was damaged. The distribution and optical density (OD) values of zonula occludens 1 (ZO-1) protein in the intestinal tissues of DON-treated groups were decreased. At higher DON dosage, interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α mRNA levels were elevated in the intestinal tissues. The mRNA and protein levels of NF-κB p65, IκB-α, IKKα/ß, iNOS, and COX-2 in the small intestinal mucosa were abnormally altered with an increase in DON concentration. These results indicate that DON can persuade intestinal damage and inflammatory responses in piglets via the nuclear factor-κB signaling pathway.

Celastrol alleviates angiotensin II­mediated vascular smooth muscle cell senescence via induction of autophagy.

Reactive oxygen species (ROS) production has been implicated in the promotion of cellular senescence. Celastrol, a quinone methide triterpenoid isolated from the Celastraceae family, exerts antioxidant effects and enhances autophagy in various cell types. Since autophagy serves an important role in regulating ROS, it was hypothesized that the antioxidant effect of celastrol is via enhanced autophagy, thus inhibiting cell senescence. Therefore, the present study used a Senescence ß­Galactosidase Staining kit, western blot analysis and cell cycle analysis to investigate whether celastrol alleviates angiotensin (Ang) II­induced cellular senescence by upregulating autophagy in vascular smooth muscle cells (VSMCs). The results demonstrated that celastrol reduced Ang II­induced senescence of VSMCs. Ang II­induced generation of ROS and the subsequent VSMC senescence were counteracted by pretreatment with celastrol, determined by a ROS assay kit. Celastrol significantly upregulated VSMC autophagy, which reduced intracellular ROS and the subsequent cellular senescence induced by Ang II. Furthermore, celastrol markedly suppressed activity of the mechanistic target of rapamycin signaling pathway in VSMCs. In conclusion, the present study demonstrated that celastrol counteracts VSMC senescence probably by reducing ROS production via activation of autophagy, which may hold promise for the prevention and treatment of aging­associated cardiovascular disorders such as atherosclerosis.

HMGB1 Induces Secretion of Matrix Vesicles by Macrophages to Enhance Ectopic Mineralization.

Numerous clinical conditions have been linked to ectopic mineralization (EM). This process of pathological biomineralization is complex and not fully elucidated, but thought to be started within matrix vesicles (MVs). We hypothesized that high mobility group box 1 (HMGB1), a cytokine associated with biomineralizing process under physiological and pathological conditions, induces EM via promoting MVs secretion from macrophages. In this study, we found that HMGB1 significantly promoted secretion of MVs from macrophages and subsequently led to mineral deposition in elevated Ca/Pi medium in vitro. Transmission electron microscopy of calcifying MVs showed formation of hydroxyapatite crystals in the vesicle interior. Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization. Mechanistic experiments revealed that HMGB1 activated neutral sphingomyelinase2 (nSMase2) that involved the receptor for advanced glycation end products (RAGE) and p38 MAPK (upstream of nSMase2). Inhibition of nSMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and mineral deposition. Collectively, HMGB1 induces MVs secretion from macrophages at least in part, via the RAGE/p38 MAPK/nSMase2 signaling pathway. Our findings thus reveal a novel mechanism by which HMGB1 induces ectopic mineralization.

A sensitive immunochromatographic assay using colloidal gold-antibody probe for rapid detection of fumonisin B1 in corn.

A sensitive immunochromatographic assay (ICA) using a colloidal gold-antibody probe for the rapid detection of fumonisin B1 (FB1) in corn samples was developed. The colour density of the test line correlated with the concentration of FB1 in the range 2-40 ng ml(-1) by the assay, and the detection limit for FB1 was 2 ng ml(-1). The linear range for FB1 was 50-1000 µg kg(-1), and the visual limit detection of the test was 1000 µg kg(-1) in corn samples. The ICA to detect FB1 is sensitive, specific and rapid. Specific anti-FB1 monoclonal antibody (mAb) and FB1-ovalbumin (FB1-OVA) conjugate antigen were prepared. FB1 mAb, labelled with colloidal gold, was used as the probe on the immunochromatographic strip. FB1-OVA and goat-anti-mouse IgG were coated onto a nitrocellulose (NC) membrane as test lines and control lines, respectively. FB1 in samples will competitively combines the FB1 mAb with the FB1-OVA in an NC membrane and the results are directly observed by the colour of the detection and quality control lines. The concentrations of FB1 mAb labelled with colloidal gold, detecting antigen and goat anti-mouse IgG, were optimised. The results indicate that the test strip is specific for FB1, with no cross-reactivity to other toxins. The strip assay for FB1 was simple, only needing one step without complicated assay performance and expensive equipment, and the total time for visual evaluation was less than 10 min. A survey of 24 corn samples from Hefei, China, was performed with the test strip and HPLC, and the detection results showed that the developed ICA and the HPLC were in excellent agreement. Hence, the developed ICA can be used as a method for rapid detection of FB1 in corn samples.