Abstract
BACKGROUND:
The source of Ang1 is controversial. Although some people think that human endothelial cells can produce Ang1, more believe that endothelial cells produce Ang2 but not Ang1. However, in our recent study on endothelial cells and angiogenesis, we find that endothelial cells do produce Ang1.
OBJECTIVE:
This study aims to prove that endothelial cells can produce Ang1 and explore what manner does Ang1 act in.
METHODS:
Immunohistochemistry, western blotting and reverse transcription PCR were used to prove if human dermal microvascular endothelial cells, human brain microvascular endothelial cells and human umbilical vein endothelial cells produce Ang1. In order to explore Ang1’s act manner, Ang1 expression of human dermal microvascular endothelial cells pre-treated by propranolol IC50 was detected by western blotting.
RESULTS:
Immunohistochemistry, western blotting and reverse transcription PCR showed that human dermal microvascular endothelial cells, human brain microvascular endothelial cells and human umbilical vein endothelial cells all expressed Ang1, and propranolol significantly inhibited Ang1 expression of human dermal microvascular endothelial cells.
CONCLUSIONS:
Endothelial cells can also produce Ang1. In addition, endogenous Ang1 may be an autocrine agonistic regulator, participating in endothelial cells angiogenesis process.
Introduction
Ang1/2 and their receptor Tie2 represent an major tyrosine kinase-ligand system that is critical for the regulation of angiogenesis [1, 3]. Activation of Ang1/Tie2 signaling promotes interaction between endothelial cells (ECs) and peri-EC support cells to stabilize vessels [4]. In addition, Ang1 stimulates ECs migration, promotes sprouting of ECs [5, 6] and tube-formation [7]. Ang1 also dose-dependently inhibits apoptosis of human umbilical vein endothelial cells (HUVECs) [8, 9], and but don’t promote EC proliferation and DNA synthetize [10]. By now, the biological role of Ang1 is relatively clear. But about the source of Ang1, there are two contradictory viewpoints. Fan F et al. [11] think that human ECs produce Ang1, and find that interleukin-1β (IL-1β) significantly downregulates Angiopoietin-1 expression in HUVECs in vitro, which further proves their opinion. But many other reaserchers still believe that ECs don’t produce Ang1 [12–14] and Ang1 acts as a paracrine agonistic regulator [12]. The same as Fan F et al., in our recent study on ECs and angiogenesis, we find that ECs do produce Ang1, and Ang1 may act in an autocrine way. This study aims to prove that ECs can produce Ang1 and explore the manner Ang1 act in.
Materials and methods
Cell culture
Primary human dermal microvascular endothelial cells (HDMECs) were purchased from Sciencell Company (USA), HUVECs line was a present from Sun Yat-Sen University (China) and human brain microvascular endothelial cells (HBMECs) line was gifted by Southern Medical University (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 and HUVECs 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). Though the passage of primary human ECs significantly affected the endothelialization in vitro, and cell densities of human ECs of the first passage were comparable to cell densities in vivo [15], primary HDMECs were restricted for use between passages 3 and 5, as cells started losing their morphological characteristic from the 6th passage. Cells were cultured at 37°C under a humidified 95% /5% (v/v) mixture of air and CO2.
Immunohistochemistry
ECs were incubated with rabbit polyclonal anti-von Willebrand factor antibody (1 : 100, biorbyt), rabbit polyclonal anti-Ang1 antibody (1 : 200, Abcam) and Goat Anti-Rabbit IgG H&L (TRITC) (1 : 500, Abcam) to identify ECs themselves and detect Ang1 expression. Hoechst (Abcam) was used to stain the cell nuclei (blue) at a concentration of 1 ug/ml. Observed and photographed under fluorescent Microscopy (Olympus).
RT-PCR
The three ECs were analyzed by reverse transcription PCR (RT-PCR) using 4 ug total cellular RNA for reverse transcription. Primers: Ang-1 forward: 5′-cagcgccgaagtccagaaaac-3′; Ang-1 reverse: 5′-cacatgttccagatgttgaag-3′ [16]; GAPDH forward: 5′-atcccatcaccatcttccagg-3′; GAPDH reverse:5′-tgatgacccttttggctccc-3′.
Western blotting
Un-pretreated HDMECs, HBMECs,HUVECs and HDMECs which were pretreated with propranolol (Sigma) IC50 (150 uM) for 48 h were washed twice with room temperature PBS, then scraped in lysis buffer (CST) with 1 mM PMSF (Beyotime) on ice. The lysates were centrifuged at 10,000 g for 15 min at 4°C. The protein concentration of samples was detected by the Bradford Protein Assay Kit (Beyotime). Samples were boiled for 5 min after loading buffer was added into the container, then electrophoretically separated on 6% Bis-tris gels (Beyotime) and electro-transferred onto a polyvinylidene fluoride membrane (Millipore). The membrane was inhibited with 5% BSA (MPbio). The membranes were incubated with 1 : 1500 rabbit anti-Ang1 (Abcam) and 1 : 1000 rabbit anti-GAPDH (CST) overnight at 4°C, then 1 : 5000 HRP-conjugated anti-rabbit secondary antibodies (Abcam). Blots were developed using electro-chemiluminescence detection agents (Millipore).
Results
ECs produce Ang1
Immunohistochemistry, RT-PCR and Western blotting showed that all of the three kinds of ECs expressed Ang1 (Fig. 1A, B, C). From Fig. 1A, we could deduce that Ang1 was mainly produced in the cell nucleus, and cytoplasmic mildly expressed Ang1.
All of the three kinds of ECs produced Ang1, and propranolol significantly inhibited Ang1 expression. From (A) we could see that Ang1 was mainly produced in the cell nucleus, and cytoplasmic mildly expressed Ang1 (red represents vWF or Ang1, blue represents cell nucleus, 200×). Western blotting showed the three kinds of ECs expressed Ang1(B) and RT-PCR indicated Ang1 mRNA(C). HDMECs were pre-treated with propranolol IC50 for 48 h, then Ang1 expression was detected with western blotting. As (D) shown, propranolol sisnificantly inhibited Ang1 expression, indicating that reduced Ang1 may involve in propranolol’s suppression on ECs angiogenesis.
In order to observe Ang1’s pharmacodynamic responses to propranolol, HDMECs were pretreated with propranolol IC50 for 48 h. Ang1 level of HDMECs was detected by western blotting. As Fig. 1D shown, propranolol significantly inhibited Ang1 expression of HDMECs.
Discussion
Ang1 is an important part of the Ang/Tie-2 pathway. It stimulates ECs migration, promotes sprouting of ECs, facilitates the assembly of ECs into more complex networks in vitro models [5] and tube-formation [7]. As researchers have different opinions on if ECs produce Ang1, it is important to solve this problem. Our study directly proves that HDMECs, HBMECs and HUVECs produce Ang1 from cell phenotype, protein to mRNA level, and Ang1 is mainly produced in the cell nucleus. On the other hand, similar with Lamy et al. [17], we also find propranolol inhibits proliferation, migration and tube-formation of primary HDMECs as well, with a half-maximal inhibition (IC50) of 150 uM (data can be shown if necessary). Furthermore, propranolol has been found in our study to suppress HDMECs Ang1 expression significantly. Considering Ang1’s functions during angiogenesis, reduced Ang1 probably contribute to propranolol’s impairment of ECs proliferation, migration and tube-formation. This indicates that the endogenous Ang1 may be an autocrine agonistic regulator.
In conclusion, ECs (HDMECs, HBMECs and HUVECs) produce Ang1 and the endogenous Ang1 maybe participate in ECs proliferation, migration and tube-formation, all critical events of angiogenesis, in an autocrine agonistic manner. If all ECs of the body produce Ang1 and the action manner of Ang1 need further clarification. Meanwhile, this finding will contribute to new perspectives about Ang1, endothelial cells and angiogenesis.
Footnotes
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81671924 and 81272105), the National Key Research and Development Plan of China (2017YFC1103301), Health and Medical Treatment Collaborative Innovation Major Special Projects of Guangzhou (No.201508020253) and Science and Technology key Project of Guangdong province (2014B020212010). All of the authors have contributed significantly and declare no funding or conflicts of interest.
We thank Dr. Wei Zhao from Southern Medical University for the donation of HBMECs line, and Dr. Guoquan Gao from Sun Yat-Sen University, who presented us HUVECs line.