Roles Of Blockade Of Adrenergic Receptor On The Angiogenesis Of Human Endothelial Cells In Vitro

BackgroundAngiogenesis is a complicated and multistage process. It plays an important part in physiologic processes (e.g., wound healing,embryogenesis, reproduction) and pathologic processes (e.g.,age-related macular degeneration,atherosclerosis, cancer, autoimmune diseases). Endothelial cells (ECs) are the essential parts during angiogenesis. They sprout out, proliferate, migrate and subsequently form tube-like structures. Furthermore, together with pericytes, they determine and regulate the stability of vessel walls. Recently, more and more researchers have started to concern on the biological roles of the sympathetic nervous system (SNS) during the process of new vessel formation. Many studies suggest that the SNS (Sympathetic neurotransmitters and sympathetic adrenoceptors (alpha and beta adrenoceptors, α-AR and β-AR)) has an important role in angiogenesis. Multiple findings suggest that blockade of β-AR inhibits the proliferation, migration and tube-formation of ECs in vitro. But how blockade of α-AR and simultaneous blockade of α-AR and β-AR affect angiogenesis of ECs in vitro remains unclear.Part I Roles of propranolol and phentolamine in the proliferation, migration, tube-formation and the expression of related proteins of human microvascular ECs respectivelyObjectiveTo study the roles of β-AR blocker (propranolol) and a-AR blocker (phentolamine) in the proliferation, migration, tube-formation and the expression of VEGF、VEGFR-2、Ang1、Ang2、Tie-2 respectively.Methods1. ImmunocytochemistryImmunocytochemical staining of Willebrand factor was used to identify ECs. Human dermal microvascular ECs (HDMECs) and human brain microvascular ECs (HBMECs) were planted on the glass slides in the 24-wellplates. After the density of the cells got to 80%-90%, cells were fixed with 4% cold paraformaldehyde for 20 min, then incubated in 0.2% Triton X-100 for 10 min and goat serum for 30 min to permeabilise the cells and block nonspecific protein-protein interactions.Then cells were incubated with rabbit polyclonal anti-von Willebrand factor antibodyovernight at 4℃, followed by incubation with Goat Anti-Rabbit IgG H&L (TRITC) at 37 ℃ for 2 h in the dark. Hochest was used to stain the cell nuclei for 15 min at a concentration of 1μg/ml. Lastly,10% glycerol was used to seal up the slides. Observed and photographed (200×) under the fluorescent microscopy.2. Western blot analysis for expression of a-AR in HDMECs and HBMECsUn-pretreated HDMECs and HBMECs were scraped in lysis buffer. The protein concentration of samples was detected by the Bradford Protein Assay Kit. Samples were boiledafter loading buffer was added into the container, then 40μg proteins were electrophoretically separated on 10% Bis-tris gels. The proteins were blocked with 5% BSA and incubated withrabbit anti-al-AR, anti-a2-AR and rabbit anti-β-actin overnight at 4 ℃, then followed by a 1 h incubation with HRP-conjugated anti-rabbit secondary antibodies. Blots were developed using electro-chemiluminescence detection agents in the dark.3. Proliferation assayHDMECs were seeded into the 96-wellplates. Cell were respectivelytreated with the indicated concentrations of propranolol (0,25,50,75, 100μM) and phentolamine(0,10,30,50,70 μg/ml) for 48h. Then,10 μl of CCK-8 dye was added into each well, and the cells were incubated at 37 ℃ under a humidified atmosphere containing 5% CO2 for 2 h. Absorbance at 450 nm was detected using the Multiskan GO microplate reader.4. Cytotoxicity assayHDMECs were seeded into the 96-wellplates. Cell were respectivelytreated with the indicated concentrations of propranolol (0,25,50,75, 100μM) and phentolamine(0,10,30,50,70 μg/ml) for 48h. Samples from the cell medium were harvested and LDH activity was measured at 30 ℃ by a continuous optical test based on the extinction change of pyridine nucleotide at 340 nm, as described by the manufacturer’s instructions.5. Scratch assayHDMECs were seeded into the 24-wellplates. After the density of the cells got to 100%, cells were starved with uncompleted medium (i.e., ECM containing 1% FBS and 0.2% ECGs for HDMECs) for 24 h. A 1 mm-wide gap in the well center was scratched by a 20 μpipette tip. Dead cells were then washed away with PBS, and new medium (i.e., the incomplete medium described in the cell starvation assay) were added containing differing concentrations of propranolol (0,0.1,1,10,20, 30μM) and phentolamine (0,0.1,1,10,20,40μg/ml). The demarcated areas of each well were then observed and photographed (40*) at time 0,12,24 and 48 h under the inverted fluorescence microscope.6. Tube-formation assayMatrigel (12.5 mg/ml) was thawed at 4℃. The 24-well plates and tips were both precooled, and the process was performed on ice. 100μl matrigel was quickly added to each well of the24-well plate and allowed to solidify for 30 min at 37℃ . Once solidified,the wells were incubated for 30 min with 500 μlcomplete medium which containedl.5×105 HDMECs or HBMECs. After adhesion of the cells, old media were removed, and complete media which contained 0,50 μM propranolol or 0,50μg/ml phentolamine were added, and cells were incubated at 37 ℃ for 24 h. The formation of tube-like structures was observed and photographed (40×) under an inverted fluorescence microscopeat time 0,4,8,12 and 24 h.7. VEGF ELISAHDMECs were seeded into 6-well plates. When the density of the cells got to 70%, old medium was replaced with fresh uncomplete medium (i.e., ECM containing 8% FBS and 0% ECGs) containing 0,50 μM propranolol or 0,50μg/ml phentolamine. After cells were incubated at 37 ℃ under a humidified atmosphere containing 5% CO2 for 48 h, cell proteins and the supernatants were obtained, and levels of intracellular VEGF and VEGF in the cell supernatantswere detected using the human VEGF ELISA kit, according to manufacturer’s instructions.8. VEGFR-2 ELISAHDMECs were seeded into 6-well plates. When the density of the cells got to 70%, old medium was replaced with fresh complete mediumcontaining 0,50 μM propranolol or 0,50μg/ml phentolamine. After cells were incubated at 37℃under a humidified atmosphere containing 5% CO2 for 48 h, cell proteins were obtained, and levels of VEGFR-2 were detected using the human VEGFR-2 ELISA kit, according to manufacturer’s instructions.9. Western blot analysis for expression of Ang1. Ang2% Tie-2HDMECs were treated with complete medium containing 0,50 μM propranolol or 0,50μg/ml phentolamine respectively for 48 h. Then cells were were scraped in lysis buffer. The protein concentration of samples was detected by the Bradford Protein Assay Kit. Samples were boiledafter loading buffer was added into the container, then 40 μg proteins were electrophoretically separated on 10% Bis-tris gels. The proteins were blocked with 5% BSA for 1 hand incubated with rabbit polyclonal anti-Angl/2 antibodies, mouse monoclonal anti-Tie-2 antibody, and rabbit polyclonal anti-GADPH antibody overnight at 4 ℃, followed by incubation with the corresponding secondary antibodies at 37 ℃ for 1 h. Blots were developed using electro-chemiluminescence detection agents in the dark.Results1. Both of the two kinds of cells expressed Willebrand factor, indicating that the cells were ECs.2. Western blot analysis showed that HBMECs expressed the al-AR (both the alA-AR (50 kDa) and α1D-AR (60 kDa) isoforms) and the a2-AR (50 kDa). In addition, HDMECs expressed al-AR (three bands from 34 kDa to 43 kDa represent three isoforms of al A-AR (35kDa,37kDa, and 40kDa)) and a2-AR (50 kDa). This is the first time that human microvascular ECs have been shown to express a-AR.3. Propranolol and phentolamine both significantly inhibited proliferation of HDMECsin a dose-dependent manner, with a half maximal inhibitory concentration (IC50) of about50μM,50μg/ml respectively.3. The drugs within the above concentration gradient had no obvious toxic effect on HDMECs. This suggested that it was the inhibited proliferation by propranolol and phentolamine contributed to the reduced cells, not the cytotoxicity of the drugs.4. Propranolol and phentolamine both significantly inhibited migration of HDMECs. After 48 h, compared to the control group, the wound closure of the drug treatment groups (propranolol 20,30 μM, phentolamine 20,40 μg/ml) was significantly decreased. Low concentrations of propranolol and phentolamine also reduced cell migration, but not so significantly.6. Propranolol and phentolamine both significantly inhibited tube-formation of HDMECs and HBMECs. Propranolol inhibited HDMECs tube-formationby 40.3%, and HBMECs 72.7%. Phentolamine inhibited HDMECs tube-formationby 65.7%, and HBMECs 77.7%.7. Propranolol and phentolamine inhibited VEGFR-2 expression of HDMECs, but had no effects on intracellular VEGF, and VEGF in the cell supernatants. Propranolol suppressed VEGFR-2 expression of HDMECs by 22.4%, and phentolamine 24.4%. The level of VEGF in the cell supernatant in the control group was very low (<13.9 pg/ml), and no VEGF was detected in the cell supernatant in the drug-treatment group. But both had no significance (p>0.05).8. Propranolol and phentolamine inhibited Angl, Ang2 expression of HDMECs, but promoted Tie-2 expression.ConclusionsPropranolol and phentolamine respectively inhibited proliferation, migration and tube-formation of ECs in vitro. This maybe relate to the drugs’inhibition of VEGFR-2, Angl and Ang2 expression of ECs.Part II Simultaneous blockade of α-AR andp-AR synergisticallyimpairs human microvascular ECs angiogenesis in vitroObjectiveTo observe wheather simultaneous blockade of a-AR and β-AR of human microvascular ECs synergisticallyimpairs angiogenesis in vitro.Methods1. The study was divided into 3 groups. Group 1, cells were treated with 50ug/ml phentolamine. Group 2, cells were treated with 50μM propranolol. Group 3, cells were treated with 50μg/ml phentolamine plus 50μM propranolol. Proliferation, tube-formation, and the expression of VEGF, VEGFR-2, Angl, Ang2, Tie-2 were detected. In the scratch assay, as the nutritional condition was worse, the concentration of the drugs were changed into 20μg/ml for phentolamine and 20μM for propranolol.2. The specific operation methods of proliferation, migration, tube-formation assay and ELISA, western blot analysis refered to part I.Results1. Simultaneous blockade of a-AR and β-AR synergistically impairs HDMECs proliferation. After 48 h, compared to the phentolamine group and the propranolol group, cell number of the phentolamine pluspropranolol group decresed by 43.5% and 45.5% respectively.2. Simultaneous blockade of a-AR and P-AR synergistically impairs HDMECs migration. After 48 h, wound closure of the phentolamine pluspropranolol group was significantly less than the phentolamine group and the propranolol group.3. Simultaneous blockade of a-AR and β-AR synergistically impairs tube-formation of HDMECs and HBMECs. At 4 h, intact capillary-like structures could be detected in the phentolamine group and the propranolol group, but were rare in the phentolamine pluspropranolol group. At 8 h, capillary-like structures in the phentolamine pluspropranolol group were obviously fewer than the phentolamine group and the propranolol group.4. Simultaneous blockade of a-AR and P-AR synergistically impairs VEGFR-2 expression of HDMECs. At 48 h, the OD value of the phentolamine pluspropranolol group was decreased by 33.3% compared to the phentolamine group, and 35% compared to the propranolol group. Furthermore, levels of intracellular VEGF of the three groups remained no significance. There was no VEGF in the cell supernatants of the three groups, with no significance.5. Simultaneous blockade of a-AR and β-AR synergistically suppressed the expression of Angl, Ang2 and Tie-2. After treatment of the drugs, the expression of Angl, Ang2 and Tie-2 of the phentolamine pluspropranolol group obviously down-regulated.ConclusionsSimultaneous blockade of a-AR and β-AR synergistically impairs proliferation, migration, tube-formation and the expression of VEGFR-2, Angl, Ang2 and Tie-2 of ECs. The inhibited VEGFR-2, Angl, Ang2 and Tie-2 maybe relate to the impairment of ECs angiogenesis in vitro. On the other hand, the results suggest that the inhibition of phentolamine andpropranolol in ECs angiogenesis in vitro maybe result from the impaired VEGF/VEGFR-2 and Ang/Tie-2 pathways.