CXCR7 Inhibits Fibrosis via Wnt/β-Catenin Pathways during the Process of Angiogenesis in Human Umbilical Vein Endothelial Cells

Although SDF-1/CXCR7 plays an important role in angiogenesis, the function and the pathway of the SDF-1/CXCR7 axis might depend on the cell type or tissue origin and not fully understood. In this study, we investigated the effect of CXCR7 in SDF-1-induced proliferation, migration, apoptosis, tube formation, and endothelial-to-mesenchymal transition (EndMT) of human umbilical vein endothelial cells (HUVECs), and the potential pathway of SDF-1/CXCR7. We confirmed that the silencing of CXCR7 inhibited the proliferation of HUVECs and contributed the apoptosis, while overexpressed CXCR7 increased SDF-1-induced HUVECs migration and tube formation. However, upregulated CXCR7 inhibited the expression of α-SMA, suggesting that CXCR7 might attenuate EndMT. In addition, overexpressed CXCR7 activated AKT and ERK signaling pathways but suppressed Wnt/β-catenin pathways in HUVECs. The inhibition of Wnt/β-catenin pathways decreased the expression of α-SMA. Altogether, these results suggest that CXCR7 might inhibit fibrosis via Wnt/β-catenin pathways during the process of angiogenesis.


Introduction
Angiogenesis is a key component of many pathological conditions, including corneal neovascularization (CorNV). Corneal neovascularization, resulting from a variety of etiologies, is a sight-threatening pathological change and part of a wound healing response culminating in fibrosis and scar formation [1,2]. Although the mechanism of CorNV has been unclear, the mounting evidence shows that lymphangiogenesis and angiogenesis are closely related to CorNV [3]. Chemokines, including stromal-derived factor 1 (SDF-1), are small molecules for leukocyte migration and recruitment [4] and have been reported involved with lymphangiogenesis and angiogenesis [5].
The cornea is transparent and devoid of blood and lymphatic vessels. In certain pathological conditions, the corneal host cells, including corneal epithelial, stromal, and endothelial cells can play intricate roles in angiogenesis via releasing series factors, which shift the balance toward CorNV [33]. Then, the process of neovascularization includes the activation, proliferation, and migration of endothelial cells, and the formation of vascular tubes and networks [34]. Therefore, endothelial cells are the effector cells. Human umbilical vein endothelial cells (HUVECs) exhibit key endothelial cell phenotypes [35] and are widely used in the in vitro research of CorNV. In the current study, we determined the role of CXCR7 in angiogenesis and fibrosis as well as the potential pathway of SDF-1/CXCR7 axis in HUVECs.
To upregulate the CXCR7 expression in HUVECs, the plasmid encoding overexpressed CXCR7 was constructed by cloning the CXCR7 gene into XhoI and EcoRI sites on pEX-1 vector (Shanghai GenePharma, Shanghai, China). The CXCR7 cDNA was amplified using the following primers: forward (CGCAAATGGGCGGTAGGCGTG) and reverse (TAGAAGGCACAGTCGAGG). HUVECs were transfected with recombinant CXCR7 or control vector using Lipofectamine 3000 overnight. After transfection for 48 h, the efficiency was determined by quantitative real-time PCR and western blot.

Western
Blot. The cells were lysed in RIPA Lysis Buffer (Beyotime, China) supplemented with protease inhibitors. The total protein concentration was measured using a BCA protein assay kit (Pierce, MA, USA) according to the manufacturer's instruction. A total of 20 μg protein was separated on 10% SDS-PAGE and transferred onto PVDF membranes (Beyotime). Blocking was performed in 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature for 2 hours. The membranes were incubated overnight at 4°C with primary antibodies, including the fol- . The membranes were then washed with TBST three times, incubated with HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 2 hours at room temperature, then detected with ECL detection reagents (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as a loading control.
2.5. Cell Proliferation Assay. After CXCR7 siRNA or overexpressed CXCR7 transfection for 48 h, the HUVECs were seeded into 96-well plate (3000 cells/well). Cell proliferation was detected after SDF-1 (100 ng/ml) treatment for 24 h with CCK-8 kit (Dojindo, Kumamoto, Japan) according to the manufacturer's instruction. The optical density was measured with a microplate reader at 450 nm.
2.6. Flow Cytometry Analysis of Cell Apoptosis. Apoptosis was detected by annexin V-FITC kit (Beyotime). In brief, HUVECs were transfected with CXCR7 siRNA or overexpressed CXCR7 for 48 h, then cultured with SDF-1 (100 ng/ml) for 24 h. After experimental treatment, HUVECs were collected and washed 2 times, then incubated with annexin V-FITC and propidium iodide (PI) for 15 min at room temperature in the dark. Cells were then immediately analyzed by flow cytometry.

Transwell Migration Assay.
Cell migration assay was performed in a 6-well plate with 8.0 μm pore-size transwell inserts (Corning, Corning, NY, USA). The upper surface chamber was pretreated with Matrigel (Corning) and serum-free DMEM/F12 (1 : 8) medium. After CXCR7 siRNA or overexpressed CXCR7 transfection for 48 h, a total of 1 × 10 5 HUVECs were seeded to the upper chamber with serum-free DMEM/F12 media. The lower chamber was filled with 500 μl DMEM/F12 medium containing SDF-1 (100 ng/ml) and 10% FBS. After incubation at 37°C for 24 h, the cells that migrated to the lower membrane surface were fixed with 4% paraformaldehyde and stained by 0.1% crystal 2 BioMed Research International violet. The number of cells in randomized 5 fields was counted under a microscope.

Scratch Wound Assay.
A total of 1:5 × 10 4 upregulated or downregulated CXCR7 HUVECs were seeded onto a 6-well plate for 24 h, and then exposed to SDF-1(100 ng/ml). A wound was created after manually scraping the cell monolayer with a p200 pipet tip. The initial wound quantification was performed on images collected at 0 hour after wounding, and further images were collected randomly in wound areas at 18 h after wounding. The wound width was measured, and the migration was represented as percentage migration considering migration in untreated control as 100%. Cells were rinsed in PBS and incubated with DAPI for 5 min. Images were captured with an inverted microscope (Leica, Wetzlar, Germany).
2.11. Statistical Analysis. The data were presented as the mean values of 3 or 4 independent experiments. Student's t test and one-way ANOVA analysis were performed using SPSS 20.0 (SPSS, Chicago, Ill, USA) for all statistical data. All analyses were carried out using GraphPad Prism (Graph-Pad Software, La Jolla, CA). Values were expressed as the mean ± SD, and statistical significance was set at p < 0:05.

The Downregulation and Upregulation of CXCR7 in
HUVECs. After selection with puromycin, the expression of CXCR7 in HUVECs was detected by qRT-PCR and western blotting. The level of CXCR7 mRNA and protein in HUVECs transfected with CXCR7-siRNA 3 was decreased (p < 0:001) (Figures 1(a) and 1(b)). On the contrary, the level of CXCR7 mRNA was significantly increased with overexpressed CXCR7 plasmid vector transfected (p < 0:001) (Figures 1(c) and 1(d)). These results indicated that CXCR7 knockdown and overexpressed HUVECs could be available to further researches.

CXCR7 Activates ERK and AKT Pathways but Suppresses the Wnt/β-Catenin Pathway in HUVECs.
To investigate the signaling pathway of CXCR7, we assessed total ERK1/2, phospho-ERK1/2, pan AKT, phospho-AKT, and β-catenin protein levels in the presence of SDF-1 by western blotting. The phosphorylation of ERK1/2 and AKT increased when CXCR7 was overexpressed, while the level of phosphorylated ERK1/2 and AKT was decreased in CXCR7 downregulated HUVECs (Figure 4(a)). In contrast, the protein level of βcatenin decreased when CXCR7 was overexpressed. In addition, the levels of Wnt/β-catenin downstream target proteins c-Myc, survivin, and cyclinD1 significantly decreased by upregulating CXCR7 (Figure 4(b)). These data suggest that overexpressed CXCR7 suppresses the Wnt/β-catenin pathway in HUVECs.

Discussion
Angiogenesis refers to many neovascular diseases when the balance is damaged between angiogenic factors and angiogenic inhibitors. In most pathological conditions, angiogenesis is part of a wound healing response culminating, via an angiofibrotic switch, in fibrosis, and scar formation [2]. Our pilot research showed that CXCR7 might be involved in CorNV [23]. However, the role and the mechanism of CXCR7 in CorNV were not fully understood. In this study, we determined the role of SDF-1/CXCR7 axis in angiogenesis in HUVECs.
CXCR7, one of the chemokine receptor for SDF-1, has been demonstrated to regulate the proliferation [11,37], migration [16,26,27,29,31,37], invasion [11,16,38], adhesion [11,21,37], and tube formation [11,15,24,26,29,30,37,39] in different cell lines. The function of the CXCR7 depends on the cell type or tissue origin [26]. In this study, we found CXCR7 enhanced the proliferation, migration, and tube formation of HUVECs and inhibited the apoptosis. Consistent with most of the previous researches, these results demonstrate that CXCR7 is essential to the angiogenesis induced by SDF-1. However, we also found that overexpressed CXCR7 reduced the expression of α-SMA, suggesting that CXCR7 might attenuate EndMT. Cao et al. [36] reported TC14012, a CXCR7 agonist, promoted lung alveolar repair and reduced fibrosis. Guan and Zhou [40] also proposed that CXCR7 upregulation during angiogenesis was a feedback mechanism to ameliorate pulmonary fibrosis. Together, these results suggest that CXCR7 might inhibit fibrosis during the process of angiogenesis. Besides, TC14012 seemed to have an inhibitory effect on angiogenesis at the late stage of CorNV [23]. Thus, upregulation of CXCR7 might not result in excessive neovascularization along with the inhibition of fibrosis.

Conclusion
In summary, overexpressed CXCR7 might inhibit fibrosis via Wnt/β-catenin pathways during the process of angiogenesis in HUVECs. CXCR7 could be a regulator for the pathophysiology of angiogenesis via an angiofibrotic switch.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors have no conflicts of interest to declare.