[Impacts of microparticles on vascular endothelial cells].

OBJECTIVE: To investigate the effects of microparticles (Mps) from different people on the vascular endothelial cell function. METHODS: Third generation human umbilical vein endothelial cells (HUVECs) were cultured with Mps-containing plasma samples from 5 systemic lupus erythematosus (SLE) patients undergoing heavy steroid treatment, 5 patients with steroid-induced avascular necrosis of femoral head (ANFH), 4 patients with alcohol induced ANFH, and 4 healthy persons for 12 h, 24 h, and 48 h respectively. Plasma samples from the above persons with the Mps filtered were used as experimental controls. HUVECs cultured with blank culture fluid were used as blank controls. Inverse phase contrast microscopy was used to observe the morphology of the HUVECs. PE-CD31 and PEcy5-CD62E were added into the flow cytometric test tubes and flow cytometry (FC) was used to count the number of CD62E +/ CD31 + Mps in the medium. RT-PCR was used to measure the mRNA expression of the apoptotic gene fas (fas/beta-actin). RESULTS: Microscopy showed no distinct difference between the morphology of the HUVECs among the different groups. FC showed that the number of CD62E +/CD31 + Mps 48 hours after the 20% Mp stimulation of the steroid-treated SLE group was significantly lower than that of the control group (P = 0.035). RT-PCR showed that 48 hours after the stimulation the levels of mRNA fas/beta-actin of the steroid-treated SLE group and steroid induced ANFH group were 0.914 +/- 0.226 and 0.776 +/- 0.230 respectively, both significantly higher than those of the control groups (0.832 +/- 0. 200 and 0.669 +/- 0.148 respectively, P = 0.005 and P = 0.006). CONCLUSION: The Mps from the steroid treated patients aggravate the apoptosis of HUVECs, and the Mps from the steroid induced ANFH patients augment the production of apoptosis gene in HUVECs. The Mps from healthy people and alcohol induced ANFH patients have no relationship with HUVEC apoptosis.

Platelet releasates promote the proliferation of hepatocellular carcinoma cells by suppressing the expression of KLF6.

Platelets in the primary tumor microenvironment play crucial roles in the regulation of tumor progression, but the mechanisms underlying are poorly understood. Here, we report that platelet releasates exerted a proliferative effect on hepatocellular carcinoma (HCC) cells both in vitro and in vivo. This effect depended on a reduction of KLF6 expression in HCC cells. After incubation with either platelets or platelet granule contents, SMMC.7721 and HepG2 cells exhibited significant increases in proliferation and decreases in apoptosis. However, no effect was observed when incubating cancer cells with resuspended activated platelet pellet which exhausted of releasates. Platelet releasates also increased the population of HCC cells in the S and G2/M phases of the cell cycle and reduced the cell population in the G0/G1 phase. Moreover, knocking down KLF6 expression significantly diminished the platelet-mediated enhancement of HCC growth. In addition, blocking TGF-ß signaling with the TGF-ß receptor inhibitor SB431542 counteracted the effect of platelets on KLF6 expression and proliferation of HCC cells. Based on these findings, we conclude that platelet releasates, especially TGF-ß, promote the proliferation of SMMC.7721 and HepG2 cells by decreasing expression of KLF6. This discovery identifies a potential new therapeutic target for the prevention and treatment of hepatocellular carcinoma.

Antiplatelet activity of chrysin via inhibiting platelet αIIbß3-mediated signaling pathway.

SCOPE: Propolis is thought to help prevent thrombotic and related cardiovascular diseases in humans. Chrysin, a bioflavonoids compound found in high levels in propolis and in honey, has been reported to possess antiplatelet activity. However, the mechanism by which it inhibits platelet function is unclear. METHODS AND RESULTS: The effects of chrysin on agonist-activated platelet-aggregation, granule-secretion, and integrin αIIbß3 activation were examined. Its effects on the phosphorylation of Akt, GSK3ß, MAPKs, and several proteins of the glycoprotein VI (GPVI) signaling pathway were also studied on collaged-activated platelets. In addition, human platelet spreading on immobilized fibrinogen was also tested. We found that chrysin dose dependently inhibited platelet aggregation and granule secretion induced by collagen, as well as platelet aggregation induced by ADP, thrombin, and U46619. Chrysin also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen. Biochemical analysis revealed that chrysin inhibited collagen-induced activation of Syk, PLCγ2, PKC, as well as the phosphorylation of Akt and ERK1/2. Additionally, chrysin attenuated phosphorylation of molecules such as FcγRIIa, FAK, Akt, and GSK3ß in platelet spreading on immobilized fibrinogen. CONCLUSIONS: Our findings indicate that chrysin suppresses not only integrin αIIbß3-mediated "inside-out" signaling, but also the "outside-in" signal transmission. This implies that chrysin may represent a potential candidate for an antiplatelet agent.

Expression of E-selectin in Endothelial Cells Effects of Wild Type p53 Gene.

To investigate expression of E-selectin in endothelial cells(EC)and effect of wild type p53 gene transduction on its expression, flow cytometry and RT-PCR were employed to measure the E-selectin protein and mRNA, respectively. The effects of TNFalpha stimulation and p53 gene transduction on the expression of E-selectin was studied. Flow cytometric analysis revealed that there was no E-selectin expression on the surface of resting EC, although its mRNA was detectable by RT-PCR. TNFalpha (10-1 000 ku/L)induced E-selectin expression on the surface of EC in a concentration-dependent manner. The level of E-selectin mRNA was also increased with the increasing concentrations of TNFalpha. The expression of E-selectin protein was notable after 2 h of TNFalpha stimulation and reached a peak at 4-6 h, then rapidly declined to around the basic level by 12 h. Transduction of p53 gene inhibited TNFalpha-induced EC expression of E-selectin. The E-selectin positive cell proportion was reduced from(21.31+/-1.06)% to(11.83+/-0.98)%, (n = 3, P < 0.001). The E-selectin mRNA level was also reduced correspondingly. In resting EC, neither E-selectin nor its mRNA level was affected by the transduction of p53 gene. The results indicated that p53 gene inhibited TNFalpha-induced expression of E-selectin on the surface of EC, at least partially, by reducing its mRNA level.