Endothelial Gata6 deletion reduces monocyte recruitment and proinflammatory macrophage formation and attenuates atherosclerosis through Cmpk2-Nlrp3 pathways

Abstract

Endothelial dysfunction results in chronic vascular inflammation, which is critical for the development of atherosclerotic diseases. Transcription factor Gata6 has been reported to regulate vascular endothelial cell activation and inflammation in vitro. Here, we aimed to explore the roles and mechanisms of endothelial Gata6 in atherogenesis. Endothelial cell (EC) specific Gata6 deletion was generated in the Apoe^KO hyperlipidemic atherosclerosis mouse model. Atherosclerotic lesion formation, endothelial inflammatory signaling, and endothelial-macrophage interaction were examined in vivo and in vitro by using cellular and molecular biological approaches. EC-GATA6 deletion mice exhibited a significant decrease in monocyte infiltration and atherosclerotic lesion compared to littermate control mice. Cytosine monophosphate kinase 2 (Cmpk2) was identified as a direct target gene of GATA6 and EC-GATA6 deletion decreased monocyte adherence, migration and pro-inflammatory macrophage foam cell formation through regulation of the CMPK2-Nlrp3 pathway. Endothelial target delivery of Cmpk2-shRNA by intercellular adhesion molecule 2 (Icam-2) promoter-driven AAV9 carrying the shRNA reversed the Gata6 upregulation mediated elevated Cmpk2 expression and further Nlrp3 activation and thus attenuated atherosclerosis. In addition, C-C motif chemokine ligand 5 (Ccl5) was also identified as a direct target gene of Gata6 to regulate monocyte adherence and migration influencing atherogenesis. This study provides direct in vivo evidence of EC-GATA6 involvement in the regulation of Cmpk2-Nlrp3, as well as Ccl5, on monocyte adherence and migration in atherosclerosis development and advances our understanding of the in vivo mechanisms of atherosclerotic lesion development, and meanwhile provides opportunities for future therapeutic interventions.

Keywords: Atherosclerosis; Cytosine monophosphate kinase 2 (Cmpk2); Endothelial cells; Gata6 transcription factors; Nlrp3 inflammasome.

Fig. 1

Elevated Gata6 expression in the endothelium of atherosclerotic lesions of human coronary artery and Apoe knockout hyperlipidemic mouse artery. (A) The representative images of immunostaining of GATA6 in the endothelium of human coronary artery with or without atherosclerotic lesions by co-staining with EC marker CD31 (n = 6), with quantification data on the right and the arrows indicating Gata6 positive endothelial cells (ECs) (n = 6). (B–D) Gata6 expression in aortic ECs of Apoe knockout (Apoe^KO) hyperlipidemic and Apoe^WT mice by co-staining with EC marker CD31 (B) (n = 6), quantitative reverse transcription PCR (RT-qPCR) (C) and Western blot (D) (n = 5). (E–G) GATA6 expression in human aortic ECs (HAECs) treated with oxidized low density lipoprotein (oxLDL, 50 μg/ml for 48hr) or vehicle control by RT-qPCR (E), Western blot (F) with quantification data on the right (n = 5) and digital PCR (G) (n = 3). (H) Gata6 expression in aortic arch or thoracic arch ECs by RT-qPCR (n = 5). (I) Schematic diagrams on design of in vitro shear stress flow system and cross sections of cell chamber. (J) GATA6 expression in HAECs treated with oscillatory shear stress (OSS) or laminar shear stress (LSS) by RT-qPCR (n = 5). Quantification of Western blot was carried out with Image J and normalized to loading control β-actin. Unpaired 2-tailed student t-test for A-F, H, J and nonparametric statistical test for G. Scale bars: A, B, 100 μm.

Fig. 2

Endothelial Gata6 deletion reduces atherosclerotic lesions and lesional macrophages due to decreased macrophage adhesion, migration and reduced pro-inflammatory macrophage foam cell formation. (A–B) Gata6 expression of Gata6^fl/fl;Cdh5-Cre^ERT2;Apoe^KO(Gata6^ECKO;Apoe^KO) mutant and Gata6^WT/WT;Cdh5-Cre^ERT2;Apoe^KO (WT;Apoe^KO) littermate wild-type mice by RT-qPCR (A) and Western blot (B) following tamoxifen treatment (100 mg/kg, i.p. every other day for total 4 times) (n = 5). (C–E) The representative images of Sudan IV staining of en face aorta (C), aortic root (D)and F4/80 immunostaining aortic root sections (E) in Gata6^ECKO;Apoe^KO and littermate wild-type mice following 8 weeks high-fat western diet with quantification data on the right (n = 12, 6 each for male and female mice per group). (F–G) GATA6 expression by RT-qPCR (F) and Western blot (G) with quantification data on the right in HAECs of GATA6-siRNA knockdown and scramble siRNA control(n = 5). (H–J) Adhesion of monocytes to GATA6-siRNA or scramble-siRNA ECs (H), monocyte migration in Boyden chamber assay (I) and macrophages engulf of Dil-labeled oxidized low-density lipoprotein (oxLDL) (J) after treatment of condition medium from GATA6-siRNA or scramble-siRNA ECs, with representative images on the left and quantitative data left (n = 5). (K) The expression of oxidized low density lipoprotein receptor 1 (OLR1), interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) in macrophages following treatment by oxLDL and condition medium from GATA6-siRNA or scramble-siRNA ECs. Quantification of Western blot was carried out with Image J and normalized to loading control β-actin. Unpaired 2-tailed student t-test for A, B and F–K and two-way ANOVA followed by Turkey post hoc test for C-E. Scale bars: C, 2 mm; D, 500 μm; E, H, I, 100 μm; J, 50 μm.

Fig. 3

Cmpk2 is identified as Gata6 direct downstream target gene in ECs for the regulation of monocyte adhesion, migration and pro-inflammatory macrophage formation. (A–C) The expression of Cytidine/uridine monophosphate kinase 2 (Cmpk2) by RT-qPCR (A) and Western blot (B) with quantification data on the right in aortic ECs of Gata6^ECKO;Apoe^KO hyperlipidemic and Apoe^WT mice (n = 5), and in oxLDL stimulated HAECs of GATA6-siRNA knockdown and scramble siRNA control by RT-qPCR (C) (n = 5). (D) Sequence analysis of GATA6 binding sites (red oval) in the proximal promoter region of CMPK2 gene. (E) Chromatin immunoprecipitation (ChIP) assay of GATA6 and the promoter of CMPK2 gene with gel images on the top. (F) The luciferase activity of proximal-2kb Cmpk2 promoter with Gata6 binding sites by Gata6 expression vector in 293T cells (n = 3). (G–H) The expression of NLRP3 inflammasome components, NLRP3 and IL-1β, in oxLDL-treated ECs by RT-qPCR (G) and Western blot (H) with quantification data on the right, and the effects of GATA6-siRNA, CMPK2 vector for overexpression or the combination on NLRP3 inflammasome activation. (I) Secreted IL-1β level in conditioned medium of GATA6-siRNA or scramble-siRNA ECs by ELISA (n = 5). (J–L) The quantitative data of monocyte adhesion to GATA6-siRNA or scramble-siRNA ECs (J), monocyte migration in Boyden chamber assay (K) and macrophages engulf of Dil-labeled oxLDL (L) after treatment of condition medium from GATA6-siRNA or scramble-siRNA ECs (n = 5). Quantification of Western blot was carried out with Image J and normalized to loading control β-actin. Unpaired 2-tailed student t-test for A-C, G-L and nonparametric statistical test for F.

Fig. 4

Ccl5 is identified as Gata6 direct downstream target gene in ECs for the regulation of monocyte adhesion and migration. (A–C) The expression of chemokine Ccl5 in ECs of Gata6^ECKO;Apoe^KO hyperlipidemic and Apoe^WT mice (A), in HAECs treated with oxLDL or vehicle (B) and HAECs with GATA6-siRNA and scramble siRNA (C) (n = 5). (D) The sequence analysis of GATA6 binding sites (red oval) in the proximal promoter region of CCL5 gene. (E) Chromatin immunoprecipitation (ChIP) assay of GATA6 and the promoter of CCL5 gene with gel images on the top. (F) The luciferase activity of proximal-2kb Ccl5 promoter with Gata6 binding sites by Gata6 expression vector (n = 3). (G–H) Adhesion of monocytes to GATA6-siRNA and scramble siRNA ECs (G) and monocyte migration in Boyden chamber assay after treatment with condition medium from GATA6-siRNA and scramble siRNA ECs (H), with representative image on the left and quantitative data right (n = 5). (I) Secreted CCL5 level in conditioned medium of GATA6-siRNA or scramble-siRNA ECs by ELISA (n = 5). Unpaired 2-tailed student t-test for A-C, E and G-I and nonparametric statistical test for F. Scale bars: G-H,100 μm

Fig. 5

Icam-2 promoter-driven adeno-associated virus 9 target delivery of Cmpk2-shRNA into ECs inhibits the elevated Gata6-Cmpk2 mediated Nlpr3 inflammasome activation and reduces atherosclerosis. (A) The schematic figure of administration of Icam-2 promoter-driven adeno-associated virus 9 (AAV9) carrying Cmpk2-shRNA or scramble-shRNA by tail vein injection for viral EC target delivery affecting atherosclerotic lesion formation in 8-week high-fat diet Apoe^KO mice. (B–E) Viral GFP fluorescence in aortic ECs co-staining with EC marker CD31 (B) and Cmpk2 expression in ECs by RT-qPCR (C), Western blot (D) with quantification data on the right and digital PCR (E) following tail vein injection of Icam-2 promoter-driven AAV9 carrying Cmpk2-shRNA or scramble-shRNA. (F–H) The representative images of Sudan IV staining of en face aorta (F), aortic root (G) and F4/80 immunostaining aortic root sections (H) following administration of Icam-2 AAV9 carrying Cmpk2-shRNA or scramble shRNA, with quantification on the right (n = 12, 6 each for male and female mice per group). (I–J) Gata6, Cmpk2 and in turn Nlrp3 and IL-1β expression by RT-qPCR (I) and Western blot (J) with quantification data on the right in ECs of ApoE^KO hyperlipidemic and wild-type mice following tail vein injection of Icam-2 AAV9 carrying Cmpk2-shRNA or scramble-shRNA. Quantification of Western blot was carried out with Image J and normalized to loading control β-actin. Unpaired 2-tailed student t-test for C-D and I-J, two-way ANOVA followed by Turkey post hoc test for F–H. Scale bars: B, 500 μm, 100 μm; F, 2 mm; G, 500 μm; H, 100 μm.

Fig. 6

Schematic figure of working model. Model of endothelial Gata6 exerts its atherogenic effects through regulation of vascular inflammation by Cmpk2-Nlrp3 inflammasome activation and chemokine Ccl5 signals that affect the monocyte adherence, migration and recruitment into the vessel wall, as well as the pro-inflammatory macrophage formation.