CXCR4 blockade induces atherosclerosis by affecting neutrophil function

Abstract

Aims: The SDF-1α/CXCR4 dyad was previously shown by us and others to be instrumental in intimal hyperplasia as well as early stage atherosclerosis. We here sought to investigate its impact on clinically relevant stages of atherosclerosis in mouse and man.

Methods and results: Immunohistochemical analysis of CXCR4 expression in human atherosclerotic lesions revealed a progressive accumulation of CXCR4(+) cells during plaque progression. To address causal involvement of CXCR4 in advanced stages of atherosclerosis we reconstituted LDLr(-/-) mice with autologous bone marrow infected with lentivirus encoding SDF-1α antagonist or CXCR4 degrakine, which effects proteasomal degradation of CXCR4. Functional CXCR4 blockade led to progressive plaque expansion with disease progression, while also promoting intraplaque haemorrhage. Moreover, CXCR4 knockdown was seen to augment endothelial adhesion of neutrophils. Concordant with this finding, inhibition of CXCR4 function increased adhesive capacity and reduced apoptosis of neutrophils and resulted in hyperactivation of circulating neutrophils. Compatible with a role of the neutrophil CXCR4 in end-stage atherosclerosis, CXCR4 expression by circulating neutrophils was lowered in patients with acute cardiovascular syndromes.

Conclusion: In conclusion, CXCR4 contributes to later stages of plaque progression by perturbing neutrophil function.

Keywords: CXCR4; SDF-1α; atherosclerosis; neutrophils; senescence.

Fig. 1. Analysis of CXCR4 and SDF-1α expression in atherosclerotic human carotid artery plaques

A, Microarray analyses of CXCR4 and SDF-1α mRNA expression in human atherosclerosis. Four two-armed plaque cohorts were analyzed for mRNA expression patterns: advanced versus early stable carotid artery plaque cohorts obtained at autopsy (advanced:early cohort 1; n = 9 and 8, resp), advanced versus early stable carotid artery plaque cohort from GSE28829 which served as validation cohort (early/advanced cohort 2); advanced unstable versus stable plaque cohort (n = 12 and 3, respectively, unstable:stable cohort 1); a second advanced unstable versus stable plaque cohort from the human plaque transcriptomics study [16] (n = 22 and 22, stable/unstable cohort 2), both obtained by endarterectomy surgery. Values represent relative CXCR4 expression intensity in the first versus the second tissue component; all expression values were false-discovery-rate (FDR) corrected (*P < 0.05, **P < 0.01) B, CXCR4 and SDF-1α protein expression in non-diseased arteries and early lesions, as well as in advanced and ruptured plaques (magnification: 100×). Histological classification is described in the supplemental methods (* = lumen, I = intima and # = media). C, CXCR4 and SDF-1α expression by intimal cells (magnification: 400×). D, Mouse isotype IgG2b control (magnification: 100×).

Fig. 2. In vitro and in vivo analysis of functionality of lentiviral CXCR4 degrakine and SDF-1α antagonist

A, Quantification of migrated FDCP-MixA4 cells infected with LV.Empty, LV.SDF1-α(P2G), LV.CXCR4deg or a shRNA targeting CXCR4 (shX4HM) without stimulation (black bars) or in response to SDF-1α (white bars) in a slope well assay. (*P < 0.05 and **P < 0.01 compared to LV.Empty control, ^##P < 0.01 compared to LV.Empty + SDF-1α). B, Quantification of migrated neutrophils, that were isolated from either LV.Empty control transplanted mice, or LV.CXCR4deg chimeras in a transwell migration assay. LV.CXCR4 deg expressing neutrophils migrated significantly less towards SDF-1α (**P < 0.01 compared to LV.Empty + SDF-1α). C, Flow cytometry analysis of relative circulating CXCR4⁺ neutrophils in LV.CXCR4deg versus LV.Empty chimeras (***P < 0.005).

Fig. 3. CXCR4 and SDF-1α lentiviral blockade deteriorates atherosclerotic plaque progression

LDLr^−/− mice were transplanted with LV.empty, LV.CXCR4deg or LV.SDF1(P2G) bone marrow and placed on Western type diet for 6 (plaque initiation study, n = 8 per group) and 10 weeks (plaque progression study, n = 8–11 per group). A, Oil-Red-O staining of the aortic root. Blockade of CXCR4 by LV.CXCR4deg aggravated lesion progression after 10 weeks of diet feeding in LDLr^−/− mice (**P < 0.01 compared to LV.Empty, left graph). Similarly, hematopoietic overexpression of the SDF-1α(P2G) antagonist tended to induce lesion progression after 10 weeks of diet feeding (P = 0.06 compared to LV.Empty, left graph). Right panel: representative lesions of each group (50× magnification). Initial plaque development as measured after 6 weeks of western-type diet feeding was not affected by CXCR4 blockade (P = 0.25 compared to LV.Empty, right graph). B, Intraplaque hemorrhage in a LV.CXCR4deg plaque indicated by the arrow (upper left: 50× magnification, lower right: 200× magnification). C, Increased intraplaque hemorrhage was observed as measured by the relative erythrocyte surface area in plaques of LV.CXCR4deg chimeras (*P < 0.01). Data are represented for the 6 weeks group. D, Plaque macrophage (MOMA-2) content, represented as the percentage of MOMA2⁺ cells per plaque area. Data are represented for the 10 weeks group. E, Plasma cholesterol level distribution did not differ between the groups.

Fig. 4. CXCR4 blockage induces neutrophil adhesion

A, Flow cytometry analysis of the percentage of Ly6G^highCD11b⁺(CD71⁻) neutrophils in blood from LV.Empty (n = 4) versus LV.CXCR4deg (n = 4) bone marrow transplanted mice. Data are represented for the 6 weeks group. B, Napthol CAE staining showing adhering neutrophils to the plaque endothelium (left panel, upper picture: 400× magnification, lower picture: 1000× magnification). Represented in the graphs are the number of adhering neutrophils per endothelial cell length in LV.Empty versus LV.CXCR4deg transplanted LDLr^−/− bone marrow chimeras 6 and 10 weeks after Western type diet feeding (**P < 0.01 compared to LV.Empty). C, HL60 cells were treated with retinoic acid to induce neutrophil development. D, Adhesion of non-treated versus AMD3100 treated neutrophils in control, fibronectin (FN) and gelatin coated wells. *P < 0.05 compared to HL60 + RA, ^##P < 0.01 compared to uncoated controls, **P < 0.01 compared to HL60 + RA (FN). E, Relative gene expression of FAK in neutrophils differentiated in the presence or absence of AMD3100 (*P < 0.05). F, Adhesion of neutrophils isolated from bone marrow of LV.Empty versus LV.CXCR4deg transplanted mice to either control, fibronectin (FN) and gelatin coated wells. **P < 0.05 compared to LV.control, ^###P < 0.001 compared to uncoated controls. G, Adhesion of neutrophils isolated from bone marrow of LV.Empty, LV.SDF-1α(P2G), LV.CXCR4deg transplanted mice to mouse endothelial cells. *P < 0.05 compared to LV.Empty control.

Fig. 5. CXCR4 blockage does not alter neutrophil recruitment

In vivo chemokinesis assay in LDLr^−/− mice, transplanted with either bone marrow infected with LV.Empty or LV.CXCR4deg A, Percentage of peritoneal Ly6G^highCD11b⁺(CD71⁻) neutrophils at 2 h after KC injection. ***P < 0.001 compared to T = 0. B, Percentage of recruited neutrophils in response to SDF-1α. *P < 0.05 compared to T = 0, ^#P < 0.05 compared to LV.Empty. C, Percentage of peritoneal neutrophils in response to 3% Brewer’s Thioglycollate in control versus AMD3100 treated mice.

Fig. 6. Neutrophils lacking functional CXCR4 expression show increased cell survival

A, Treatment of HL60 cells with AMD3100 inhibits differentiation of HL60 cells into neutrophils as demonstrated by a reduction in the percentage of Ly6G^highCXCR4⁺ (left panel) and Ly6G^highCXCR4⁺ cells (right panel) (**P < 0.01 compared to control HL60 cells, ^##P < 0.01 compared to RA differentiated HL60 cells). B, AMD3100 reduced neutrophil precursor proliferation during differentiation (**P < 0.01). C, Myeloperoxidase activity (**P < 0.01). D, Percentage of cell survival (***P < 0.001). E, Relative gene expression of Akt in neutrophils differentiated in the presence or absence of AMD3100 (*P < 0.05).

Fig. 7. Increased numbers of senescent neutrophils in the absence of CXCR4

LDLr^−/− mice on western type diet were treated with AMD3100 for 3 weeks. Control mice received PBS injections. A, Circulating Ly6G^highCD11b⁺ granulocyte numbers (*P < 0.05). B, Mean fluorescence intensity for DHR123 (*P = 0.05). C, Normalized CXCR4 protein expression on circulating granulocytes isolated from healthy (control) persons versus carotid endarterectomy (CEA) and patients with unstable angina pectoris (UAP, defined in the online methods) (**P < 0.01, ***P < 0.001 compared to controls).