Smoking is a major risk factor for atherosclerosis. In this study, we evaluated the effects of benzo[a]pyrene (BaP, a prominent component of tobacco smoke) on the function and pro-inflammatory response of human endothelial progenitor cells (EPCs). EPCs were isolated from umbilical cord blood and treated with different concentrations (10,20 and 50 mu mol/l) of BaP. The proliferation, migration, adhesion and angiogenesis of BaP-treated EPCs were evaluated using the cell counting kit-8 (CCK-8), Transwell assay, adhesion assay and in vitro tube formation assay, respectively. The activation of nuclear factor-kappa B (NF-kappa B) was evaluated by measuring the mRNA expression of NF-kappa B p65 and p.50 by real-time RT-PCR and NF-kappa B translocation assay. Reactive oxygen species (ROS) production was determined by the reduction of fluorescent 2',7'-dichlorofluorescein diacetate (DCFH-DA). The results demonstrated that BaP treatment significantly inhibited the proliferation, migration, adhesion and angiogenesis of EPCs in vitro. In addition, BaP induced the release of interleukin (IL)-1 beta and tumor necrosis factor-a from these cells. Moreover, the exposure of EPCs to BaP induced ROS generation and the activation of NF-kappa B. Experiments with EPCs pre-treated with pyrrolidine dithiocarbamate, an inhibitor of NF-kappa B, revealed that the BaP-mediated inhibition of proliferation, migration, adhesion and angiogenesis of EPCs is mainly regulated by NF-kappa B. Thus, tobacco smoke may induce oxidant-mediated stress responses in EPCs and impair their function via the activation of the NF-kappa B pathway.

In this study, we examined the protective effects of Danshen both on endothelial progenitor cells (EPCs) in patients with hypercholesterolemia and on in-vitro EPCs of healthy volunteers. In the clinical study, we randomly divided 24 subjects with hypercholesterolemia into two groups (the control group and the Danshen-treated group). At the end of two weeks of treatment, the EPC cellular functions of both groups were tested. The results indicated that, compared to the control group, EPCs in the Danshen-treated group showed significantly better cellular functions, which was manifested in the cloning number, the proliferation capacity, the number of EPC adhesions, and cell migration. In the subsequent in-vitro experiments, EPCs were treated with vehicle, oxidized low-density lipoprotein (Ox-LDL, 100 µg/ml), or Ox-LDL (100 µg/ml) plus different concentrations of Danshen (Danshensu 2, 10, or 50 µg/ml, respectively) for 24 h. The results showed that Danshen treatments can prevent the detrimental effects of Ox-LDL on EPC cellular functions measured by proliferation capacity (0.24±0.08, 0.37±0.11, 0.30±0.04 vs. 0.13±0.02, P<0.05, P<0.01, and P<0.01, respectively), and adhesion ability (63.00±11.60, 70.00±10.80, 85.50±11.41 vs. 40.50±6.85, all P<0.01). Compared to the group treated with Ox-LDL alone, Danshen treatment significantly decreased the lipid peroxidation end product malondialdehyde (MDA) [(4.34±0.54), (3.98±0.47), (3.46±0.31) vs. (5.57±0.64) nmol/ml, all P<0.01], increased the production of superoxide dismutase (SOD) [(29.74±0.71), (31.09±0.83), (30.41±0.65) vs. (14.76±3.99) U/ml, all P<0.01], and lowered the expression of interleukin-6 (IL-6) [(24.62±7.69), (27.04±3.14), (33.38±18.86) vs. (230.67±33.53) pg/ml, all P<0.01] and tumor necrosis factor-α (TNF-α) [(41.72±6.10), (17.02±6.82), (3.73±2.26) vs. (228.71±41.53) pg/ml, all P<0.01] in Ox-LDL treated EPCs. These results suggest that Danshen may exert a protective effect through its antioxidant and anti-inflammatory features.

Objective To observe the effects of Danshensu on the functions of endothelial progenitor cells(EPCs)with inflammatory injuries.Methods Mononuclear cells(MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation,and were identified by demonstrating the expression of CD34,VEGFR-2 and AC133 with flow cytometry.The cells were then plated on fibronectin-coated culture dishes.After 7 days incubation,attached cells were collected and treated with ox-LDL(100 mg/L)alone,or ox-LDL(100 mg/L)as well as different concentrations of Danshensu(2,10 or 50 mg/L,respectively) for 24 hours.EPCs′ cellular functions were measured by proliferation capacity and adhesion ability after the intervention,and cell supernatant of each group was tested for IL-6 and TNF-α concentrations.Results Compared to the groups treated with ox-LDL alone,the groups treated with the concentrations of 2,10 or 50 mg/L.Danshensu had less detrimental effects of ox-LDL on EPCs′ cellular proliferation capacity(OD value:0.24±0.08,0.37±0.11,0.30±0.04 vs 0.13±0.02,P0.05),and adhesion ability(63.00±11.60,70.00±10.80,85.50±11.41 vs 50.50±10.12,P0.01).Danshensu also decreased the expression of IL-6(24.62±7.69,27.04±3.14,33.38±18.86 vs 230.67±33.53,P0.01)and TNF-alpha(41.72±6.10,17.02±6.82,3.73±2.26 vs 228.71±41.53,P0.01).Conclusion Danshensu had protective effect on EPCs with inflammatory injuries through its anti-inflammatory functions.