Effect Of Hemin On The Phosphorylation Of Erk1/2 In Human Umbilical Vascular Endothelial Cells

Background and AimIt is well known that hemoglobin is the most important part of erythrocyte. Hemoglobin released from senescent erythrocytes is separated to the heme and globin moieties. Heme rapidly intercalates into the plasma membrane of endothelial cells (ECs), where it can catalyze oxidative injury and cause cell death which are proved to be involved in the pathogenesis of atherogenesis, reperfusion injury, and the organ injury accompanying hemoglobinemia or myoglobinemia. In contrast, Balla et al found that more prolonged contact of ECs with heme renders them remarkably resistant to subsequent oxidant challenge caused by exposure to hydrogen peroxide (H₂O₂), activated neutrophils, or oxidized low density lipoprotein. Incubation of ECs with heme induces the production of hemedegrading enzyme, heme oxygenase-1 (HO-1). HO-1 is the key enzyme in heme catabolism, which degrades heme to CO, Fe, and biliverdin. CO is known as a gaseous messenger in the vascular and nervous systems. Biliverdin is rapidly converted tobilirubin, whose antioxidative effect is proposed to protect cells against reactive oxygen species. So HO-1 plays an important role in heme protection in the vasculature.Hemin, a kind of heme, has been reported to be protective. Some investigators have reported that hemin lowered blood pressure in spontaneously hypertensive rats and prevented hypoxia-induced pulmonary hypertension. And hemin also has been reported to prevent vascular smooth muscle cells against Balloon injury and to prevent rat carotid artery against Balloon injury. But at present it is not very clearly about its protective mechanisms, which may be partly provided by the induction of HO-1.Erkl/2 is belonged to the mitogen-activated protein kinases (MAPK) cascade family, which is a major signalling pathway that is shared by various types of cells. In mammalian cells, three important groups of kinases compose the MAPK family, including Erkl/2, c-jun NH2-terminal kinase (JNK), and p38 MAPK. Erkl/2 cascade appears to mediate signals promoting cell proliferation, differentiation, or survival, whereas JNK and p38 MAPK cascades appear to be involved in the cell responses to stress.Whether hemin can induce the phosphorylation of Erkl/2 in vascular endothelial cells has not been reported. So in this present study, we aim to elucidate the relationship between hemin and Erkl/2 phosphorylation in human umbilical vascular endothelial cells (HUVECs) and how phosphorylated Erkl/2 is induced by hemin in HUVECs. Since treatment of HUVECs with H2C>2 can induce the phosphorylation of Erkl/2. The one point of this study is to make clear the exact role of H2O2 in the induction of Erkl/2 phosphorylation by hemin in HUVECs. And another point of this study is to make sure whether HO-1 or its three metabolites (CO, Fe2+ and biliverdin) are involved in the hemin-induced Erkl/2 phosphorylation in HUVECs.Methods1. Cell culture and treatmentAfter serum starvation for 16 h, HUVECs were incubated with hemin (1, 5, 10, 25 or 50u.mol/L) or 100 umol/L of H2O2 in a 37°C humidified environment containing 5% CO2, and the phosphorylation of Erkl/2 in HUVECs was detected by Western blot assay.After being separately pretreating with HO-1 inhibitor ZnPP, CO scavenger Hb or iron chelator DFOM for 2 h, HUVECs were incubated with hemin for 2 h. Then the phosphorylation of Erkl/2 in HUVECs was detected by Western blot assay.HUVECs were separately incubated with 14.29 umol/L of Tricarbonyldichlororuthenium (II) dimer (CORM, a kind of exterior carbon monoxide-releasing molecule), 10 umol/L of FAC or 10 Hmol/L biliverdin for 2 h to investigate the induction of Erkl/2 phosphorylation.2. Western blot assayHUVECs were harvested at different times after treatment. Cell lysates were subjected to a sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and the resolved proteins were transferred to a nitrocellulose membrane. After blocked, nitrocellulose membranes were incubated with primary antibodies against total-Erkl/2 and phospho-Erkl/2. Membranes were then washed and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies. Immune complexes were detected using the enhanced chemiluminescence detection (ECL) system.3. Cell cycle and apoptosis analysisHUVECs were collected after trypsinization and then fixed in 70% ethanol overnight. After PI staining, phase distribution of cell cycle and cell apoptosis was analyzed by flow cytometry.4. MTT assayHUVECs were seeded into 96-well plates and were incubated in medium containing 10 umol/L of hemin or biliverdin for 24 hours at 37°C. MTT was dissolved in DMSO at 5 mg/ml. The stock solution was added to the culture medium at a dilution of 1:10. The plates were incubated at 37°C for 4 h. The dark brown formazan crystals formed after MTT metabolizationwere dissolved in DMSO, and after centrifiigation, the optical density of the supernatant was read at 570 ran.Results1. Hemin induced and sustaind the phosphorylation of Erkl 12 in HUVECs(1) Phosphorylation of Erkl 12 was induced after incubating with 25 umol/L of hemin or with 100umol/L of H2O2 in HUVECs. Phosphorylation of Erkl 12 induced by hemin was less than that induced by H2O2.(2) HUVECs were incubated with hemin of different concentrations (1, 5, 10, 25 or 50 umol/L) for 24 h. And Western blot assay found that hemin at the concentration of 1-10 umol/L induced the Erkl/2 phosphorylation in HUVECs, but with no change at the concentration of 25 or 50 umol/L compared with blank group.(3) HUVECs were treated with 10 umol/L of hemin for some times. Then the phosphorylation of Erkl/2 was found to be obviously induced in HUVECs after incubating with hemin for 30 min, and be continuously increased until 2 h. The phosphorylation of Erkl/2 sustained on a high level for 3 h.2. Mechanism of the induction of Erkl/2 phosphorylation in HUVECs by hemin(1) HUVECs were incubated with 100 umol/L of H2O2 for some times. And the results showed that the phosphorylation of Erkl/2 was obviously induced in HUVECs after incubating with H2O2 for 10 min, and sustained for 30 min. When HUVECs incubating with H2O2for 1 h, H2O2 didn't induced the phosphorylation of Erkl/2 any more. The time course was absolutely different from that of the phosphorylation of Erkl/2 induced by hemin.(2) After HUVECs preincubating with HO-1 inhibitor ZnPP, CO scavenger Hb or iron chelator DFOM separately for 2 h, the phosphorylation of Erkl/2 induced by hemin in HUVECs was mostly prohibited by HO-1 inhibitor ZnPP. But Hb or DFOM also induced thephosphorylation of Erkl/2 after incubating with HUVECs alone. And there was no difference between administration Hb or DFOM alone and combination with hemin. (3) We investigated the effect of three metabolites of hemin (CO, iron and biliverdin), on the phosphorylation of Erkl/2 in HUVECs. And the results showed that the phosphorylation of Erkl/2 was not induced by CO releasing molecule CORM nor iron donor FAC, but was significantly induced by biliverdin.3. Effect of hemin on HUVEC cells growth(1) Cell cycle and cell apoptosis of HUVECs were analysised by Flow cytometry. And the dates showed that 10 umol/L of hemin had no effect on the cell apoptosis, but G2/M phase ratio of HUVECs was increased by hemin.(2) Comparing with the control, the proliferation of HUVECs was obviously promoted by hemin or biliverdin administration.Conclusions10 umol/L of hemin induced and sustaind the phosphorylation of Erkl/2 in HUVECs. Both HO-1 induced by hemin or biliverdin produced by hemin metabolism might be involved in the phosphorylation of Erkl/2 induced by hemin in HUVECs. Hemin at the concentration of 10 umol/L not only has no cytotoxicity to HUVECs, but also promotes HUVEC cells growth.