Endothelial Cell Insulin Signaling Regulates CXCR4 (C-X-C Motif Chemokine Receptor 4) and Limits Leukocyte Adhesion to Endothelium

Abstract

Background: An activated, proinflammatory endothelium is a key feature in the development of complications of obesity and type 2 diabetes and can be caused by insulin resistance in endothelial cells.

Methods: We analyzed primary human endothelial cells by RNA sequencing to discover novel insulin-regulated genes and used endothelial cell culture and animal models to characterize signaling through CXCR4 (C-X-C motif chemokine receptor 4) in endothelial cells.

Results: CXCR4 was one of the genes most potently regulated by insulin, and this was mediated by PI3K (phosphatidylinositol 3-kinase), likely through FoxO1, which bound to the CXCR4 promoter. CXCR4 mRNA in CD31+ cells was 77% higher in mice with diet-induced obesity compared with lean controls and 37% higher in db/db mice than db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. SDF-1 (stromal cell-derived factor-1)-the ligand for CXCR4-increased leukocyte adhesion to cultured endothelial cells. This effect was lost after deletion of CXCR4 by gene editing while 80% of the increase was prevented by treatment of endothelial cells with insulin. In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling after intravenous injection of SDF-1, but most of this response was prevented in transgenic mice with endothelial overexpression of IRS-1 (insulin receptor substrate-1).

Conclusions: Endothelial cell insulin signaling limits leukocyte/endothelial cell interaction induced by SDF-1 through downregulation of CXCR4. Improving insulin signaling in endothelial cells or inhibiting endothelial CXCR4 may reduce immune cell recruitment to the vascular wall or tissue parenchyma in insulin resistance and thereby help prevent several vascular complications.

Keywords: chemokines; diabetes; endothelial cells; insulin resistance; leucocyte rolling; mice; obesity.

Figure 1.. Identification of insulin-regulated genes in human endothelial cells.

A, Human umbilical vein endothelial cells (HUVEC) were treated with 100 nM insulin for 3 hours or left untreated. Cell lysate from 5 cultures treated with insulin and 5 cultures left untreated was analyzed by RNAseq. Volcano plot of differentially regulated genes is shown. B-C, HUVEC were treated with insulin or IGF-1 for 3 hours or left untreated. Cell lysate was analyzed by TaqMan real-time PCR. Results are from 6 independent experiments. C, *, p=0.03 for all (Wilcoxon signed-rank test). D, *, p≤0.0025.

Figure 2.. Insulin regulates CXCR4 in primary endothelial cells through PI3K.

A, mRNA expression in primary mouse lung endothelial cells (MLEC) after treatment with 10 nM insulin for the time indicated. B-E, MLEC were isolated from mice with floxed mutations in the Insr and Igf1r gene with (“DKO”) or without (“lox”) a cre transgene controlled by the Cdh5 promoter causing knockout of the insulin and IGF-1 receptors in vascular endothelial cells. Serum-starved cultures were treated with 10 nM insulin or 10 nM IGF-1 for 4 hours. B, Western blots of MLEC lysate. C, Normalized Cxcr4 mRNA expression analyzed by real-time PCR. Results from 3 independent experiments are shown. *, p≤0.001. D, Analysis of cell surface CXCR4 expression in MLEC from lox and DKO mice using flow cytometry, representative result shown. E, Flow cytometry results from 6 independent experiment. F, MLEC were treated with 1 μM wortmannin, a PI3K inhibitor, or 10 μM U0126, a MEK1/2 inhibitor or left untreated. After 30 minutes, cultures were treated with 100 nM insulin for 4 hours or left untreated. CXCR4 mRNA was measured by real-time PCR. Results from 6 independent experiments are shown. *, p≤0.004. G-H, MLEC were isolated from Foxo1/3/4^flox/flox mice and infected with adenovirus expressing GFP or cre. After 48 hours, cultures were lysed. G, Protein expression was measured by Western blotting. H, CXCR4 mRNA analyzed by real-time PCR. Results from 6 independent experiments are shown. *, p=0.007. I, MS1 endothelial cells were infected with an adenovirus expressing wild-type FOXO1 as described previously. Cells were serum-starved overnight and chromatin immunoprecipitation (ChIP) was performed using FOXO1 antibody or rabbit IgG followed by real-time PCR using primers amplifying a candidate FoxO1 binding motifs in the Cxcr4 promoter or a sequence without such a motif in the Gck promoter.

Figure 3.. Regulation of CXCR4 in mouse models of insulin resistance.

A-D, Myocardial cells were dissociated enzymatically and sorted by FACS. CD31 sorting was performed after gating on PI– CD45– cells. mRNA in sorted cell lysate was analyzed by real-time PCR. A-C, Analysis of cells sorted from mice fed a high-fat diet (Hi-fat) or control mice fed a a low-fat diet (Lo-fat). C, *, p=0.04. D, Cells sorted from db/db mice or their db/+ controls. *, p=0.05 (Mann-Whitney test).

Figure 4.. Endothelial insulin signaling regulates myeloid cell adhesion to endothelial cells induced by SDF-1.

A, Adhesion of THP-1 myeloid cells to MS1 endothelial cells. Endothelial cells were treated with 100 ng/ml SDF-1 for the time indicated or left untreated, then washed before adding THP-1 cells, labeled with Vybrant CFDA SE, for 30 minutes. Non-adhering cells were washed away. B-F, The Cxcr4 gene was targeted by CRISPR/Cas9 in MS1 cells using two independent gRNAs (Cxcr4Δ1 and Cxcr4Δ2, respectively, abbreviated Δ1 and Δ2) and compared to wildtype cells (wt) cells. B, Fraction of CXCR4+ cells, measured by flow cytometry. C-F, Cells were treated with 100 ng/ml SDF-1 for 4 hours. C, Representative Western blots. D, ICAM-1 relative to actin densitometry from 5 independent experiments. a, p=0.06; b, p=0.06 (Wilcoxon signed-rank test). E, Western blot results from 6 independent experiments. a, p=0.02 and b, p=0.04. F-G, Adhesion of THP-1 myeloid cells to MS1 endothelial cells as in A. F, Results from 5-6 independent experiments using the wt, Δ1 and Δ2 cell lines. *, p=0.01. G, Serum-starved, “wildtype” MS1 endothelial cells were treated with 100 nM insulin for 12 hours or left untreated, then treated with 100 ng/ml SDF-1 for 4 hours or left untreated, with or without AMD3100, an inhibitor of CXCR4. Results from 4 independent experiments. a, p=0.007; b, p=0.01.

Figure 5.. Endothelial insulin signaling regulates leukocyte-endothelial cell interaction in vivo

A-B, Mice with deletion of CXCR4 targeted to endothelial cells (cre Cxcr4^lox/lox mice abbreviated Cxcr4^iΔEC) were compared to Cxcr4lox/lox mice (flox). A, The fraction of CXCR4+ cells in primary cultures of lung endothelial cells was measured by flow cytometry. B, Intravital microscopy of leukocyte interaction with mesenteric venules was performed in 3-4 week old mice. The frequency of rolling leukocytes in 7 pair of animals was analyzed. C-D, Tg(Irs1) (“Tg”) mice, with overexpression of IRS-1 in endothelial cells, or their wildtype controls wildtype (“wt”) were studied. C, The fraction of CXCR4+ cells in primary cultures of lung endothelial cells was measured by flow cytometry. A representative result is shown. D, Flow cytometry was performed in 3 wt mice and 3 Tg mice. *, p=0.008. E-G, 3-4 week old wildtype (“wt”) or Tg(Irs1) (“Tg”) mice were studied 4 hours after intravenous injection of 1 μg SDF-1α or saline. E, Leukocyte interaction with mesenteric venules was observed during intravital microscopy. Arrows point to rolling leukocytes. Scalebar, 50 μm. F, Quantification of leukocyte rolling. *, p=0.03 (Mann-Whitney test).