G protein-coupled receptor GPR182 negatively regulates sprouting angiogenesis via modulating CXCL12-CXCR4 axis signaling

Abstract

Angiogenesis is a critical process for tumor progression, regulated by various signaling pathways. Although antiangiogenic therapies targeting the VEGF pathway have shown potential, their effectiveness is inconsistent across different tumor types. GPR182, an endothelial cell-specific G protein-coupled receptor, is frequently downregulated in hypervascular tumors, but its specific role in angiogenesis has not been well defined. Our study reveals that GPR182 expression is markedly reduced in hepatocellular carcinoma (HCC) and inversely correlates with CD31, a pan-endothelial marker. In zebrafish embryos, Gpr182 deficiency resulted in enhanced angiogenic sprouting and hypervascularization, and GPR182-deficient human umbilical vein endothelial cells (HUVECs) showed increased migration and proliferation. At the molecular level, GPR182 acts as a decoy receptor, binding CXCL12 and regulating its gradient, which in turn suppresses CXCR4-mediated angiogenesis. The pharmacological blockade of CXCR4 with AMD3100 corrected the abnormal angiogenic phenotype in Gpr182-deficient zebrafish embryos and in the livers of a zebrafish HCC model. This work uncovers GPR182 as a negative regulator of angiogenesis, a key process in tumor growth and metastasis, and proposes that targeting GPR182 may offer a novel therapeutic approach for antiangiogenic strategies in cancer treatment.

Fig. 1

Downregulation of GPR182 in HCC correlates with poor prognosis. (A) Comparative expression analysis of GPR182 in normal liver versus HCC tissues within the TCGA-LIHC dataset. (B and C) Kaplan-Meier survival curves for overall survival (OS) and disease-free survival (DFS) of HCC patients with high and low expression levels of GPR182 in TCGA-LIHC dataset. (D) Representative IHC staining for GPR182 in peritumoral and tumoral regions of HCC tissues. (E) Whole-mount in situ hybridization analysis of gpr182 expression in zebrafish HCC models at various stages of tumor progression and in controls during development

Fig. 2

Comparative expression of GPR182 across species. (A, D, G) UMAP visualizations depict the distribution of GPR182 in the Human, Mouse, and Zebrafish cell atlases, respectively. (B, E, H) Relative distribution patterns of GPR182 across various cell clusters in Human, Mouse, and Zebrafish. (C, F, I) Mean expression levels of GPR182 within distinct cell clusters for each species. (J) Immunofluorescence staining of peritumoral and tumoral regions in HCC sections showing expressions of CD31 (a pan-endothelial marker) and GPR182. (K and L) Quantitative analysis of GPR182 expression intensity and microvessel density in peritumoral and tumoral regions of HCC sections (n = 10). Data are presented as mean ± SD, with statistical significance determined by Student’s t-test. ****, p < 0.0001. (M) Expression profiling of LSEC-specific markers in liver cell populations. (N-R) Whole-mount in situ hybridization of embryos reveals high gpr182 expression in the zebrafish vascular system and liver. The hybridization signals in developing ISVs, PCV, DA, and liver are indicated by green, blue, red, and magenta arrowheads, respectively

Fig. 3

Gpr182 deficiency promotes sprouting angiogenesis in zebrafish embryos. (A) Trunk vascular morphology in control MO and gpr182-MO-injected Tg(fli1ep: EGFP-CAAX)^ntu666 embryos at 32, 48, and 72 hpf. Gpr182 deficiency results in increased ISV sprouting. Dashed rectangles highlight aberrant vascular phenotypes, and red arrowheads point to the sprouts. Scale bar, 100 μm. (B-D) Quantitative analysis of ectopic sprouts at 32 hpf and total ISV length at 48 and 72 hpf. (E) Confocal microscopy analysis of ISV tip cell filopodia in 24-hpf Tg(fli1ep: EGFP-CAAX)^ntu666 embryos and ISV endothelial cells in 32- and 72-hpf Tg(fli1a: nEGFP) embryos. (F) Number of ISV tip cell filopodia per ISV in control and gpr182 morphant embryos at 24 hpf. (G and H) Number of ECs per ISV in control and gpr182 morphant embryos at 32 and 72 hpf, respectively. Data are presented as mean ± SD, with each data point representing an individual fish. A total of 10 fish were analyzed per group. Statistical significance was determined using Student’s t-test. ****, p < 0.0001

Fig. 4

Loss of GPR182 promotes EC migration and tube formation. (A and B) EC migration is assessed using the wound closure assay in HUVECs. Scale bar, 100 μm. (C) Representative microscopic images display tube formation by HUVECs. Scale bar, 100 μm. (D-G) Quantitative analysis of the number of junctions, meshes, total mesh area, and total tube length in HUVECs. Data are presented as mean ± SD. Statistical significance was determined using Student’s t-test. ****, p < 0.0001

Fig. 5

Whole-genome transcriptomic profiling of control embryos and gpr182 morphants. (A) Heatmaps based on bulk RNA-seq data represent the expressions of cardiovascular-related genes in 36 and 48 hpf embryos. Zebrafish cxcr4a, vegfaa, and esm1 are upregulated in gpr182 morphants. (B and C) Expression levels of cxcr4a, vegfaa, and esm1 are validated by qPCR and WISH. Data are presented as mean ± SD, with statistical significance determined by Student’s t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Fig. 6

GPR182 does not trigger downstream signaling in response to CXCL12. (A and G) and (B and H), HeLa/cAMP/Ca2 + cells expressing GPR182 or an empty vector were treated with CXCL12. (C and D) and (E and F), HeLa/RhoA/ERK cells expressing GPR182 or an empty vector were treated with CXCL12. The addition CXCL12 starts at 5 min. Representative images of different biosensors before (left) and after (right) the stimulation of CXCL12. The response of the biosensors for cAMP levels (I and M), RhoA activity (J and N), ERK activity (K and O), and Ca2 + levels (L and P) is normalized by dividing by the averaged value before stimulation and plotted as a function of elapsed time after stimulation. The red and gray lines represent the time-course for the average and individual cells, respectively

Fig. 7

GPR182 modulates CXCR4 signaling through internalizing CXCL12. (A and D) FRET analyses via acceptor photobleaching assess the formation of CXCR4 homodimers (positive control) and CXCR4/GPR182 heterodimers. Representative images display CFP and YFP fluorescent signals before and after photobleaching. White rectangles indicate the regions of interest (ROI) for photobleaching; negative controls lack photobleaching. (B, C, E, and F) Relative intensities of CFP and YFP in the corresponding ROIs for panels A (B and C) and D (E and F). (G) Confocal microscopy images depict CXCL12 internalization mediated by GPR182. HEK293T cells expressing YFP-tagged GPR182 are incubated with or without CXCL12, and endocytosis is assessed using an anti-CXCL12 antibody. (H) Quantification of intracellular GPR182 localization as shown in panel G. (I) Intracellular colocalization of GPR182 and Rab5 upon CXCL12 addition. HEK293T cells expressing YFP-tagged GPR182 are incubated with or without CXCL12, and cells are stained with an anti-Rab5 antibody

Fig. 8

Inhibition of CXCR4 signaling normalizes the vasculature in gpr182 morphants and the liver of HCC model. (A) Confocal microscopy images of trunk vessels in 48-hpf Tg(fli1ep: EGFPCAAX)^ntu666 control embryos, embryos injected with gpr182 MO, embryos injected with gpr182 MO and gpr182 mRNA, and embryos injected with gpr182 MO and AMD3100. (B) Quantitative analysis of total ISV length. (C-E) Confocal images of liver vasculature in control zebrafish, HCC model zebrafish, and AMD3100-treated HCC zebrafish at 7 dpf. (F-L) Three-dimensional reconstructions of liver vasculature in controls, HCC zebrafish, and AMD3100-treated HCC zebrafish. Vascular vessels and branching points are indicated by green and orange, respectively. (M-O) Quantifications of total hepatic vessel length, number of vascular branching points, and vessel diameters of liver vasculature. Data are presented as mean ± SD. One-way ANOVA analysis was applied. Not significant (ns); ****, p < 0.0001. (P) Vessel diameter distribution in controls, HCC zebrafish, and AMD3100-treated HCC zebrafish. (Q) Survival rates of controls, HCC zebrafish, and AMD3100-treated HCC zebrafish until 10 dpf

Fig. 9

Schematic overview of GPR182’s role in regulating sprouting angiogenesis. This illustration depicts how GPR182 functions as a chemokine scavenger to modulate the CXCL12/CXCR4 signaling axis, thereby controlling the process of sprouting angiogenesis