Phentolamine inhibits angiogenesis in vitro : Suppression of proliferation migration and differentiation of human endothelial cells

Abstract

It is widely known that the β-adrenergic receptor (AR) blocker (propranolol) inhibits human endothelial cell (EC) angiogenesis in vitro, but how the α-AR antagonist (phentolamine) affects human EC angiogenesis has not yet been studied. Here, we show for the first time that both human dermal microvascular ECs (HDMECs) and human brain microvascular ECs (HBMECs) express α-ARs. Moreover, our results indicate that phentolamine inhibits the proliferation, migration, and tubulogenesis of HDMECs and HBMECs. Finally, VEGFR-2 and Ang1/2 expression of HDMECs was suppressed by phentolamine. Together, these results indicate that phentolamine impairs several critical events of neovascularization, and α-ARs, as well as the VEGF/VEGFR-2 and Ang/Tie-2 signaling pathways, may be involved in these processes. Our results suggest a novel therapeutic strategy for the use of α-blockers in the treatment of human angiogenesis-dependent diseases.

Keywords

Phentolamine endothelial cell angiogenesis angiogenesis-dependent disease

1 Introduction

Angiogenesis is a complex, multistage process important for both physiological (e.g. wound healing, embryogenesis and reproduction) and pathological processes (e.g. autoimmune diseases, atherosclerosis, tumors and age-related macular degeneration) [3]. Endothelial cells (ECs) play a key role in new vessel formation by proliferating, migrating, and subsequently form lumen-containing, tube-like structures to initiate angiogenesis [23]. Moreover, vascular endothelial growth factor (VEGF) is the most important pro-angiogenic factor [16]. VEGF is a permeability-enhancing factor for vessels [24] and mitogen for ECs, and exerts its biological effects mainly via the receptor, VEGFR-2. In particular, VEGF initiates EC proliferation, elongation, and reorientation; transforms EC morphology into a more highly ordered state; and elongates the phenotype of cells lining the inner surface of blood vessels [31]. Furthermore, VEGF is an EC survival factor [13], and can promote EC migration [12]. Indeed, the VEGF/VEGFR-2 system is the most studied signaling pathway involved in angiogenesis [4 , 21].

Besides VEGF/VEGFR-2, the angiopoietins (Ang) and their receptors, Tie-1 and Tie-2, represent another major tyrosine kinase-ligand system critical for the regulation of angiogenesis [4, 21]. Mediated by Tie-2, angiopoietin 1 (Ang1) can regulate many aspects of angiogenesis [29], including stimulation of EC sprouting, migration, and tube formation [18]. Acting through PI3K/Akt pathway, Ang1 has also been shown to inhibit serum deprivation-induced apoptosis of human umbilical vein endothelial cells (HUVECs). On the other hand, Ang2, an antagonist of the Ang1/Tie2 pathway, can promote EC survival, migration, and tube formation [30]. In addition, Ang2/Tie2 signaling has been found to facilitate vascular instability and increase VEGF-induced angiogenesis [21].

More recently, multiple researchers have focused on the biological roles of adrenergic receptors (ARs) in angiogenesis. In animal models of breast cancer and lung cancer, direct activation of β-ARs was shown to significantly increase tumor vascularization and, in turn, significantly decrease blood vessel density. As the β-ARs present on many tumor cells become activated, the synthesis and release of pro-angiogenic factors (VEGF, IL-8, and IL-6) increases, leading to accelerated tumor growth [26, 27]. Moreover, the non-selective β-AR agonist, isoproterenol, has been found to decrease angiogenesis by reducing secretion of the pro-angiogenic fibroblast growth factor (FGF)-2 from human dermal microvascular ECs (HDMECs) [14]. On the other hand, in infant hemangioma ECs (IHECs), isoproterenol has been reported to stimulate cell proliferation [5, 14]. Furthermore, several researches indicate that the non-selective β-AR antagonist, propranolol, inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells(HUVECs) [10]. Propranolol also suppresses proliferation and induces apoptosis of IHECs in vitro [6, 28]. Compared with β-ARs, there have been few studies investigating α-ARs in angiogenesis, especially on how α-AR antagonists regulate EC angiogenesis in vitro.

Phentolamine is a non-selective α-AR blocker, which expands blood vessels to reduce peripheral vascular resistance, and is used in the treatment of pheochromocytoma, Raynaud’s syndrome, heart failure, erectile dysfunction, and other diseases. Recently, a few studies have investigated the role of phentolamine in angiogenesis. Administration of phentolamine (p.o.) was found to have no impact on blood vessel density in the cutaneous wound of a normal blood glucose rat [20]. On the other hand, in an assay of human corpus cavernosal ECs in vitro, the experimental groups contaning phentolamine(phentolamine+ papaverine or phentolamine+ papaverine+ prostaglandin E1) were shown to cause a marked reduction in the cell number and metabolic activity of the cells [17]; however, its mechanism has not been further elucidated. Therefore, whether phentolamine influences angiogenesis in vitro requires further exploration. This study aimed to observe the effect of phentolamine on proliferation, migration, and tube formation of HDMECs and human brain microvascular ECs (HBMECs) in vitro, and explore its mechanism of action. The results of this study will contribute to our understanding of angiogenesis.

2 Materials and methods

2.1 Cell culture

Primary HDMECs were purchased from Sciencell Research Laboratories (CA, USA). The HBMEC line was a gift from the Pathogenic Microbiology Laboratory of Southern Medical University (Guangzhou, China). HDMECs were maintained in endothelial cell medium (ECM, Sciencell Research Laboratories, CA, USA), which contains 1% endothelial cell growth supplements (ECGs, Sciencell Research Laboratories) (which contains VEGF), 5% FBS (Sciencell Research Laboratories) (which contains no VEGF), 100 U/ml penicillin coupled with 100 mg/ml streptomycin (Sciencell Research Laboratories), and cultured in bottles or plates coated with 30 mg/ml Bovine Plasma Fibronectin Purified Protein (Sciencell Research Laboratories). HBMECs were maintained in DMEM (Gibco, Thermo Fisher Scientific, Waltham, USA) supplemented with 10% FBS (Life, Thermo Fisher Scientific, Waltham, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (Sciencell Research Laboratories). HDMECs were restricted for use between passages 3 and 5. Cells were cultured at 37 °C under a humidified 95% /5% (v/v) mixture of air and CO₂.

2.2 Western blot analyzes

Based on the methods described in O’Leary [14] and Jiang [7], HDMECs and HBMECs were washed twice with PBS (Hyclone, Utah, USA) at room temperature, and then lysed with lysis buffer (Beyotime Biotechnology, Jiangsu, China) containing 1 mM PMSF (Beyotime Biotechnology) on ice. The lysates were centrifuged at 10,000g for 15 min at 4 °C. The protein concentrations of the samples were detected with the Bradford Protein Assay Kit (Beyotime Biotechnology). Next, loading buffer was added, and the samples were boiled for 5 min, before being electrophoretically separated on 10% Bis-tris gels (Beyotime Biotechnology) and electro-transferred onto a polyvinylidene fluoride membrane (Millipore, Massachusetts, USA). The membrane was blocked with 5% skim milk, and then incubated with the following primary antibodies in Tris-Buffered Saline with Tween 20 (TBS-T) overnight at 4 °C: Rabbit anti-α1-AR (ab3462, Abcam, Cambridge, UK), rabbit anti-α2-AR (orb10054, Biorbyt, Cambridge, UK), rabbit anti- β-actin (ab129348, Abcam). Following overnight incubation, HRP-conjugated anti-rabbit antibodies were added and incubated for 1 h in TBS-T at 37 °C. For angiogenesis-associated proteins, HDMECs were treated with complete medium containing 50μg/mL phentolamine. As described above, proteins were incubated with the primary antibody, i.e., rabbit polyclonal anti-Ang1/2 antibodies (ab8451/ab155106, Abcam), mouse monoclonal anti-Tie-2 antibody (ab24859, Abcam), and rabbit polyclonal anti-GADPH antibody (ab37168, Abcam), followed by incubation with the corresponding secondary antibodies (Abcam). Western blots were developed with electro-chemiluminescence detection agents (Millipore).

2.3 Cell proliferation assays

HDMECs (the third passage) and HBMECs were seeded into 96-well plates at a density of 4×10³ cells/well in 100μL complete medium, and incubated at 37 °C under a humidified atmosphere containing 5% CO₂. After 24 h, the old medium was removed and replaced by 100μL of fresh complete medium, containing the indicated concentrations (0, 10, 30, 50, 70μg/ml) of phentolamine (Sigma, CA, USA) based on the methods described in Schultheiss et al. [22]. After 48 h incubation, the cell counting kit-8 (CCK-8, Dojindo, Japan) was used to detect cell quantity. Briefly, 10μl of CCK-8 dye was added to each well, and the cells were incubated at 37 °C under a humidified atmosphere containing 5% CO₂ for 2 h. Absorbance at 450 nm was detected using the Multiskan GO microplate reader (Thermo Fisher Scientific).

2.4 Cytotoxicity assays

To assess the effect of phentolamine on cell toxicity, the release of lactate dehydrogenase (LDH) upon damage of the plasma membrane was analyzed in the culture medium of HDMECs and HBMECs. Samples from the cell medium were harvested from cells treated with phentolamine for 48 h. LDH activity was measured at 30 °C by a continuous optical test based on the extinction change of pyridine nucleotide at 340 nm, as described by the manufacturer’s instructions (Promega, Madison, Wisconsin, USA).

2.5 Scratch assay

HDMECs and HBMECs were plated in triplicate on 24-well plates at a density of 3×10⁴ cells/well. When the cells reached a density of 100%, HDMECs and HBMECs were starved with new medium (i.e., ECM containing 1% FBS and 0.2% ECGs for HDMECs; or DMEM containing 0.5% FBS for HBMECs) for 24 h. In order to create two wound edges, a 1 mm-wide gap in the well center was scratched by a 20μl pipette tip. Dead cells were then washed away with PBS, and new medium (i.e., the incomplete medium described in the cell starvation assay, which suppresses cell proliferation but maintains cell survival) was added containing differing concentrations of phentolamine (0, 10, or 20μg/ml for HBMECs; and 0, 20, or 40μg/ml for HDMECs). The demarcated areas of each well were then photographed (40×) at time 0, 12, 24 and 48 h under a Nikon Eclipse phase contrast microscope, with a Hamamatsu digital camera, and Improvision software (Perkin Elmer, Waltham, Massachusetts, USA). The areas of wound healing in the pictures were measured with ImageJ Software.

2.6 Endothelial cell tube formation assays

Matrigel (BD Biosciences, Franklin lakes, New Jersey, USA) was thawed at 4°C, and 80μL was quickly added to each well of a 48-well plate and allowed to solidify for 30 min at 37 °C. Once solidified, the wells were incubated for 30 min with 300μL of cells (6×10⁴ cells/well) in complete medium. After adhesion of the cells, complete medium (containing 50μg/ml phentolamine for HDMECs and 30μg/ml phentolamine for HBMECs) was added, and cells were incubated at 37 °C for 12 h. The formation of tube-like structures was observed microscopically (Nikon Eclipse phase contrast microscope), and photographs (40×) were taken with a Hamamatsu digital camera with Improvision software at time 0, 4, 8 and 12 h (Perkin Elmer). The extent of capillary-like structures formed in the gel was quantified by analysis of digitized images to determine the thread length of the capillary-like network, using a commercially available image analysis program (Northern Eclipse, SAN jose, CA, USA).

2.7 VEGF-A and VEGFR-2 ELISAs

HDMECs were seeded into 6-well plates at a density of 10×10⁴ cells/well (day 0), and cultured with 2 mL complete medium at 37 °C under a humidified atmosphere containing 5% CO₂. When cells reached a density of 70% at the third day, old medium was replaced with 2 mL of fresh complete medium containing 50μg/mL phentolamine. After cells were incubated at 37 °C under a humidified atmosphere containing 5% CO₂ for 48 h, VEGFR-2 levels were detected using the human VEGFR-2 ELISA kit, according to manufacturer’s instructions (CST, Boston, USA). To detect intracellular VEGF-A levels, and VEGF-A levels in the cell supernatants, cells were grown to a density of 70% (at the third day) and treated with incomplete medium (ECM with 8% FBS, and 0% ECGs) containing 50μg/mL phentolamine. The incomplete medium was used in order to exclude interference of VEGF-A from the ECGs, and in this condition, the concentration of FBS (which contains no VEGF) must be raised to 8% for the survival of HDMECs. Levels of intracellular VEGF-A and VEGF-A in the cell supernatants were using the human VEGF-A ELISA kit, according to the manufacturer’s instructions (Abcam).

2.8 Statistical analysis

To compare one experimental group with the control group, statistical analysis was performed with the Student’s t-test. When two or more experimental groups were compared with the control group, one-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used. All statistical analyzes were performed using IBM SPSS v19 software. Differences with P < 0.05 were considered significant.

3 Results

3.1 HDMECs and HBMECs both express α-ARs

Western blot analysis showed that HBMECs expressed the α1-AR (both the α1A-AR (50 kDa) and α1D-AR isoforms (60 kDa)) and the α2-AR (Fig. 1A). In addition, HDMECs expressed α1A-AR (three bands from 34 kDa to 43 kDa represent three isoforms of α1A-AR (35kDa, 37kDa, and 40kDa)) and α2-AR (Fig. 1B). This is the first time that human microvascular ECs have been shown to express α-ARs.

3.2 Phentolamine inhibits proliferation of HDMECs and HBMECs

Next we determined the effects of phentolamine on cell proliferation in HDMECs and HBMECs. As there is an obvious influence of the passage number on the proliferation of primary ECs, e.g. cells of lower passage have stronger ability of proliferation [9], we used the third passage of the primary HDMECs for proliferation assay. The primary HDMECs of the first 3 passages grow fast and have a good morphology. Using the CCK-8 cell proliferation assay, we found that phentolamine significantly inhibited proliferation of HDMECs and HBMECs in a dose-dependent manner, with a half maximal inhibitory concentration (IC50) of 30μg/ml for HBMECs (Fig. 2A) and 50μg/ml for HDMECs (Fig. 2C) without toxicity (Fig. 2B, D). These IC50 concentrations were used in the subsequent tube formation assay, ELISA, and western blot experiments.

3.3 Phentolamine delays scratch wound closure of HDMECs and HBMECs

The scratch wound assay was used to detect the effects of phentolamine on the migration of HDMECs and HBMECs from a monolayer wound edge. It was obvious that phentolamine inhibited migration of the two ECs in a dose-dependent way (Fig. 3). Low concentrations of phentolamine also reduced cell migration, but not so significantly (data not shown).

3.4 Phentolamine inhibits tube formation of HDMECs and HBMECs

We next investigated the effect of phentolamine on the morphological differentiation of ECs into capillary-like structures using the in vitro tube formation assay on the matrigel. As shown in Fig. 4, phentolamine inhibited HBMECs tube formation by 30.67%, and HDMECs tube formation by34.45%.

3.5 Phentolamine reduces VEGFR-2 expression of HDMECs, but not VEGF-A expression

In order to determine whether phentolamine regulates the VEGF/VEGFR pathway in HDMECs, we detected expression levels of membrane-bound VEGFR-2, intracellular VEGF-A, and VEGF-A in the cell supernatants using ELISAs. As shown in Fig. 5A, phentolamine reduced VEGFR-2 expression of HDMECs by 32.7%. However, there was no significant change in VEGF-A levels in the cell supernatant (Fig. 5B) or intracellularly (Fig. 5C) compared with the control group. For a secreted protein, the level of VEGF-A in the cell supernatants was very low (13.9 pg/mL for control group, 0 pg/mL for phentolamine group). However, the intracellular VEGF-A levels in HDMECs were very high (226.3 pg/mL for control group, 234.5 pg/mL for phentolamine group).

3.6 Phentolamine inhibits Ang1/2 expression, but promotes Tie-2 expression of HDMECs

Finally, we examined Ang1/2 and Tie-2 expression of HDMECs after treatment with phentolamine for 48 h by western blot. As shown in Fig. 6, phentolamine inhibited Ang1 and Ang2 expression, but unexpectedly, promoted Tie-2 expression of HDMECs.

4 Discussion

In this study, we selected the most common human microvascular ECs (HBMECs and HDMECs) for the study. In order to make the phenomenon more persuasive, we used phentolamine to act on primary HDMECs and HBMECs line respectively. As primary cells have high specificity, and better reflect the state of the body, we detected total VEGF-A, VEGFR-2 and Ang/Tie-2 protein expression following incubation of primary HDMECs with phentolamine. We found that both HDMECs and HBMECs expressed α-ARs, and that the α-AR blocker, phentolamine, inhibited the proliferation, migration, and tubulogenesis of these two EC types. In addition, our results show that phentolamine suppresses the expression of VEGFR-2 and Ang1/2 in HDMECs, but promotes the expression of the Ang receptor, Tie-2.

Previous studies have shown that the non-selective β-AR antagonist (propranolol) can inhibit the proliferation, migration, and tube formation of ECs in vitro [10]. Similarly, we find that phentolamine inhibits the proliferation, migration, and tube formation of primary HDMECs and HBMECs, which are critical processes for new vessel sprouting. As the inhibition of cell proliferation is correlated with reduced chemotactic motility, as well as reduced EC ability to differentiate into capillary-like structures [11, 19], it is not difficult to deduce that the reduced chemotactic motility may also contribute to impaired tubulogenesis.

Indeed, many prior in vitro and in vivo studies have investigated the role of β-ARs on ECs angiogenesis. However, relatively few studies have evaluated the relationship between α-AR and ECs angiogenesis, and there have been contradictory results. For example, in a proliferation assay using pregnant ewe uterine artery ECs, activation of β2-ARs and β3-ARs was found to promote cell division, whereas α-ARs activation played no role [8]. On the other hand, a nonvasoconstrictive α-AR agonist (Phenylepinephrine) has been shown to augment the proliferation and migration of HUVECs, and promote capillary generation [27]. Conversely, an α1-AR antagonist (doxazosin) was also shown to promote aortic EC proliferation, migration, and tube formation, and enhance neo-angiogenesis in the ischemic hindlimb in Wistar-Kyoto rats. While α1-AR agonist phenylephrine had the contrary effects, which indicated that the α1-AR in ECs may be involved in the negative regulation of angiogenesis [2]. Moreover, blockade of α-ARs has no effect on blood vessel density during cutaneous wound healing in rats [20]. Our results indicating that the α-ARs antagonist phentolamine inhibits angiogenesis in vitro are different from the above studies. These differences may have been due to the differing conditions (e.g., species, in vitro/in vivo, cell sources, and so on) used in previous studies compared to in the present study. In addition, the differences may also be related to the fact that the function and potential for angiogenesis of microvascular ECs may differ based on the different donors (who are in the different states of health) of the cells [25]. Thus, Our study riches the theories of α-ARs and ECs angiogenesis.

The VEGF/VEGFR-2 system is the most studied pathway for angiogenesis [4, 21]. Mediated by VEGFR-2, VEGF promotes EC proliferation, migration [12], and prolongs EC survival [13], contributing to new vessel formation. Here, we found that phentolamine significantly inhibited VEGFR-2 expression of HDMECs, but had no significant effect on the total VEGF-A in the cell supernatant or intracellularly. The intracellular VEGF-A levels in HDMECs were very high (>200 pg/mL), while the level of VEGF-A in the cell supernatant was very low (<20 pg/mL). In the phentolamine group, there was almost no VEGF-A in the cell supernatant. As the receptors were significantly decreased, the VEGF/VEGFR-2 pathway might be suppressed, leading to impaired EC proliferation, migration, and tube formation. However, we only detected the changes in total VEGF-A and VEGFR-2 levels, and in the future, it would be useful to simultaneously examine VEGFR-2 tyrosine phosphorylation levels.

The Ang/Tie-2 system is another critical pathway involved in angiogenesis [1]. Mediated by Tie-2, Ang regulates many aspects of angiogenesis [29]. In the present research, we found that phentolamine suppressed Ang1/2 expression accordingly, but promoted Tie-2 expression. The upregulation of Tie-2 expression might result from negative feedback. However, as the Ang ligands decrease, the increase in Tie-2 receptors may not be able to compensate, leading to decreased signaling, and thus, delayed scratch healing and impaired tube formation. Similar to the experiments on the VEGF/VEGFR-2 pathway, here we detected the total synthesis of Ang1, Ang2, and Tie-2, and the Tie-2 phosphorylation level could be further investigated in future experiments.

Finally, as angiogenesis plays an important role in pathological diseases [15], inhibiting this process may be useful in a clinical setting. For example, propranolol inhibits EC angiogenesis in vitro [6 , 28], which may have some relation to its being used in the treatment of infant hemangiomas. Here, we found that phentolamine had the same effects as propranolol on normal EC angiogenesis in vitro. Therefore, phentolamine may also be able to used to inhibit EC angiogenesis in infant hemangiomas, leading to new therapeutic strategies. Besides infant hemangiomas, phentolamine may be a valuable factor for studying tissue repair, tissue regeneration, tumorigenesis, angiogenesis-dependent diseases, and so on. However, these potential uses for phentolamine require further study.

5 Conclusions

This is the first study to verify that human microvascular ECs can express α-ARs. In addition, we found that the non-selective α-AR antagonist phentolamine inhibits EC angiogenesis in vitro, similar to propranolol. However, the molecular mechanisms for this phenomenon, and for the previously identified role of α-AR agonists on ECs angiogenesis in vitro and in vivo, requires further investigation. On the other hand, our results show that phentolamine impairs several critical events of neovascularization, indicating a novel therapeutic strategy for the use of α-blockers in the treatment of human angiogenesis-dependent diseases.

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Acknowledgments

This work was suported by the Guangzhou City Science and Technology Project (201508020253), the Guangzhou Province Science and Technology Project (2014B020212010), the National Natural Science Foundation of China (81171812 and 81272105), and the National Basic Science and Development Program (2012CB518105).

We thank Prof. Wei Zhao from the Southern Medical University for the gift of HBMECs line.

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