Dihydroartemisinin Inhibits Vascular Endothelial Growth Factor-induced Endothelial Cell Migration By A P38 Mitogen -Activated Protein Kinase-Independent Pathway

BackgroundThe herb Artemisia annua has been used for more than two thousands years in Chinese traditional medicine as a treatment for fever and malaria. In 1971, Chinese chemists isolated from the leafy portions of the plant the substance responsible for its reputed medicinal action. This compound, called artemisinin, is a sesquiterpene lactone that bears a peroxide grouping. The compound has been used successfully in several thousands malaria patients in the world, including those with both chloroquine-sensitive and chloroquine-resistant strains of plasmodium falciparum. Artemisinin and its derivatives, such as dihydroartemisinin(DHA), artemether, arteether and the water-soluble sodium artesunate, are effective antimalarial drugs, with fewer side effects compared with other drugs. They are recommended by the World Health Organization as the first-line antimalarial treatment for plasmodium falciparum malaria.Artemisinin and its derivatives have strong anti-inflammatory, anti-tumor, anti-fungaland anti-angiogenic effects in addition. They drew the researchers’ attention for its anti-angiogenic effect recently, but the exact mechanism is not clear.Atherosclerotic cardiovascular disease (Arteriosclerotic cardiovascular disease, ASCVD) is one of the highest mortality rates of human diseases. Approximately 75% of acute coronary events are associated with disruption of atherosclerotic plaques. Plaque angiogenesis may have an important role in the development of atherosclerosis. Vasavasorum angiogenesis and medial infiltration provides nutrients to the developing and expanding intima and therefore, may prevent cellular death and contribute to plaque growth and stabilizationin early lesions. However in more advanced plaques, inflammatory cell infiltration, and concomitant production of numerous pro-angiogenic cytokines may be responsible for induction of uncontrolled neointimal microvessel proliferation resulting in production of immature and fragile neovessels. These could contribute to development of an unstable haemorrhagic rupture-prone environment. Increasing evidence has suggested that the expression of intimal neovessels is directly related to the stage of plaque development, the risk of plaque rupture, and subsequently, the presence of symptomatic disease, the timing of ischemic neurological events and myocardial infarction. Ischemia and hypoxia are the basic causes of plaque angiogenesis. Density of angiogenesis is positively correlated with the thickness of endometrial and the degreement of arterial stenosis. Plaque angiogenesis is closely related to the degree of plaque development and vulnerability. Immature plaque neovascularization is directly related to the stage of plaque development, the risk of plaque rupture, thrombosis, and subsequently, the presence of acute event. Thus, it is very important to inhibit plaque angiogenesis.Atherosclerosis is a chronic inflammatory disorder, and angiogenesis plays a complex role in atherothrombosis. In addition it is recognized that during plaque development many pro-angiogenic pathways are re-activated and this leads to formation of immature blood vessels prone to rupture. Therefore, inhibition of angiogenesis might be an important target to prevent the development of active, unstable plaque lesions.Tyrosine kinase inhibitors are the most mature drugs in antiangiogenic drugs. However, most of the multiple tyrosine kinase inhibitors inhibit the tyrosine kinase targets, resulting in high blood pressure, proteinuria, poor wound healing, gastrointestinal perforation, hemorrhage, thrombosis, cardiotoxicity, endocrine dysfunction and other adverse reactions in the anti-tumor activity at the same time. Development of highly selective, well tolerated, with few side effects of angiogenesis inhibitors become a research trend.Endothelial cells migration is an essential component of angiogenesis. This motile process is regulated by chemotactic, haptotactic and mechanotactic stimuli.VEGF plays major roles in regulating the functions of endothelial cells migration. VEGF, is a highly controlled process requiring the co-operation of signaling pathways, involving the extracellular matrix, transmembrane receptors, such as integrins, and actin cytoskeleton-associated motile apparatus. p38 MAPK pathways are important components of the signaling network, transducing the migratory signals generated by VEGF and playing an important role in the regulation of angiogenesis. ation is an ideal target for anti-angiogenic therapy.Dihydroartemisinin (DHA) is the active metabolite of artemisinin compounds, and a semisynthetic derivative of artemisinin. DHA is an effective, water-soluble antimalarial drug, with fewer side-effects compared with other drugs. In addition, DHA exhibits strong antiangiogenesis effects.To the best of our knowledge, there isn’t any study to investigate the signaling transduction pathways mediating the effects of the artemisinin family of drugs on EC migration.ObjectiveDHA was hypothesized to inhibit VEGF-induced endothelial cells migration via the p38 MAPK pathway. We will discuss the mechanism of p38 MAPK signaling pathways in endothelial cell migration in DHA and provide the theoretical guidance for DHA inhibition of atherosclerotic vulnerable plaque angiogenesis.MethodsBoyden chamber migration assay.Cell migration assays were performed using modified 24-well Boyden chambers (Costar, Acton, MA, USA), containing a polycarbonate membrane with 8.0μm pores. HUVECs were starved in basic EGM-2 (serum/growth factor free) overnight at 37℃, prior to being harvested with trypsin and resuspended in basic EGM-2. The single cell suspensions with 20 μM DHA or 20 μM SB203850 were seeded at 1×105 cells/well in the upper chamber, while 0.5 ml EGM-2 with 20 ng/ml VEGF was added to the bottom chamber as chemoattractants. After 24 h incubation, the migrated cells on the bottom surface were stained with 0.1% crystal violet and counted under a microscope.Wound healing migration assay.HUVECs were grown to confluence in 24-well plates and starved for 2 h. The media were changed to basic endothelial growth basal medium (EBM-2), supplemented with 100 ng/ml VEGF, and a scratch (wound) was made across the monolayer using a sterile pipette tip. DHA was added to the culture medium with a final concentration of 20 μM. Images of the wells were captured at fixed points to record the area of clearing at time 0 and 8 h, and ImageJ software was used to quantitate the cleared area.Western blot analysis.Cell Iysates were prepared in radioimmunoprecipitation assay buffer [20 mM Tris (pH 7.5),150 mM NaCl,50 mM NaF,1% NP-40,0.1% deoxycholate,0.1% SDS and 1 mM EDTA], supplemented with 1 mM phenylmethylsulfonyl fluoride and 1 μg/ml leupeptin. Cleared cell Iysates were subjected to SDS-PAGE using 10% polyacrylamide gel and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 2.5% non-fat milk, and incubated with primary antibodies at 4℃ overnight in phosphate-buffered saline Tween-20. The primary antibodies used were total-p38, phospho-p38 (Cell Signaling Technology, Inc.) and P-actin (Sigma-Aldrich, St. Louis, MO, USA). Immunoreactivity was visualized with horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Inc.). The blots were analyzed using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA).ECIS migration analysis. Real-time EC migration was measured using the ECIS technique (ECIS model 1600; Applied BioPhysics, Troy, NY, USA). Briefly, eight well ECIS arrays (8W10E+) were coated with fibronectin (Invitrogen Life Technologies, Carlsbad, CA, USA). HUVECs were plated at a confluent density to form monolayers directly on top of the electrodes. Next, an elevated voltage pulse of 40 kHz was applied to the arrays for 30 sec, which resulted in the death and detachment of cells from the electrodes. As a result, the wound was healed by the surrounding cells. Following treatment with DHA or SB203850, an alternating current was applied to the cells across the electrodes and the electrical resistance was recorded. Data plots are representative of triplicate experiments, with each graph showing the resistance readings from a separate well, at 40 distinct electrodes per well.Statistical analysis.Statistically significant differences were assessed using a paired-samples t-test. P<0.05 was used to indicate a significant difference.ResultsDHA inhibits EC migration.Boyden chambertype cell migration assays were used to assess the effect of DHA on EC migration. A low concentration of 20 μM DHA was used, since it had been demonstrated to be sufficient to inhibit angiogenesis in vitro. The number of HUVECs migrating across the polycarbonate membrane was significantly reduced in the groups treated with 20 μM DHA (33.76%, P<0.01). Cell migration during in vitro wound healing mimics the process of EC motility in vivo. Thus, wound healing migration assays were also performed, and the migrated area of HUVECs was significantly reduced in the DHA-treated groups when compared with the groups treated with vehicle alone (40.97%, P<0.01).Activation of p38 MAPK is unaffected by DHA in ECs.In ECs, p38 MAPK activation by VEGF mediates actin reorganization and cell migration. To examine the effects of DHA on p38 MAPK activation, HUVECs cultured in EBM-2, containing 100 ng/ml VEGF, were treated with 20 μM DHA and the protein expression was analyzed at different time points. HUVECs treated with anisomycin, a known activator of p38 MAPK, were used as a positive control. Western blot analysis showed that the total p38 and phospho-p38 MAPK protein expression levels remained unchanged during DHA treatment at all the indicated time points. Densitometric analysis further confirmed that DHA did not affect p38 MAPK activation or expression.DHA-induced repression of EC migration is unaffected by SB203850.SB203850 is a pyridinyl imidazole derivative that inhibits p38 MAPK phosphorylation, but does not reduce p38 MAPK expression. The role of p38 MAPK in mediating the DHA-induced inhibition of EC migration was ascertained by examining the effect of SB203580 on the DHA-treated HUVECs. In the presence of SB203580, DHA also induced the reduction of EC migration in the Boyden chamber assay (30.38%, P<0.01) and wound healing assay (43.04%, P<0.05). The levels of reduction induced by DHA in the two assays were similar in the absence or presence of SB203850.To capture the subtle alterations of cell migration, an ECIS system was implemented, which allowed for real-time measurements of the resistance caused by the migration of ECs. The transendothelial resistance was significantly reduced when HUVECs were treated with 20 μM DHA (P<0.05). Treatment with 20 μM SB203580 also significantly decreased the transendothelial resis-tance (P<0.05); however, treatment with DHA and SB203850 induced an additional reduction in transendothelial resistance at 4,8 and 12 h. In the absence or presence of SB203850, the reduction of transendothelial resistance induced by DHA showed no statistically significant difference at any of the time points.ConclusionDHA inhibits VEGF-induced endothelial cell migration by a p38 mitogen-activated protein kinase-independent pathway.Significance1. To the best of our knowledge, the present study is the first to investigate the signaling transduction pathways mediating the effects of the artemisinin family of drugs on EC migration. The results confirmed that DHA inhibits VEGF-induced endothelial cell migration and by a p38 mitogen-activated protein kinase-independent pathway, which provide a theoretical basis for DHA inhibit atherosclerotic vulnerable plaque angiogenesis.2. To capture the subtle alterations of cell migration, electric cell-substrate impedance sensing system was implemented, which allowed for real-time measurements of the resistance caused by the migration of ECs. The experimental results with a high degree of repeatability, accuracy and advancement, These results have reached the leading level in the world.