Interleukin-1ß suppression dampens inflammatory leucocyte production and uptake in atherosclerosis.

AIMS: Targeting vascular inflammation represents a novel therapeutic approach to reduce complications of atherosclerosis. Neutralizing the pro-inflammatory cytokine interleukin-1ß (IL-1ß) using canakinumab, a monoclonal antibody, reduces the incidence of cardiovascular events in patients after myocardial infarction (MI). The biological basis for these beneficial effects remains incompletely understood. We sought to explore the mechanisms of IL-1ß-targeted therapies. METHODS AND RESULTS: In mice with early atherosclerosis (ApoE-/- mice on a high-cholesterol diet for 6 weeks), we found that 3 weeks of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3)-inflammasome inhibition or anti-IL-1ß treatment (using either MCC950, an NLRP3-inflammasome inhibitor which blocks production and release of active IL-1ß, or a murine analogue of canakinumab) dampened accumulation of leucocytes in atherosclerotic aortas, which consequently resulted in slower progression of atherosclerosis. Causally, we found that endothelial cells from atherosclerotic aortas lowered expression of leucocyte chemoattractants and adhesion molecules upon NLRP3-inflammasome inhibition, indicating that NLRP3-inflammasome- and IL-1ß-targeted therapies reduced blood leucocyte recruitment to atherosclerotic aortas. In accord, adoptive transfer experiments revealed that anti-IL-1ß treatment mitigated blood myeloid cell uptake to atherosclerotic aortas. We further report that anti-IL-1ß treatment and NLRP3-inflammasome inhibition reduced inflammatory leucocyte supply by decreasing proliferation of bone marrow haematopoietic stem and progenitor cells, demonstrating that suppression of IL-1ß and the NLRP3-inflammasome lowered production of disease-propagating leucocytes. Using bone marrow reconstitution experiments, we observed that haematopoietic cell-specific NLRP3-inflammasome activity contributed to both enhanced recruitment and increased supply of blood inflammatory leucocytes. Further experiments that queried whether anti-IL-1ß treatment reduced vascular inflammation also in post-MI accelerated atherosclerosis documented the operation of convergent mechanisms (reduced supply and uptake of inflammatory leucocytes). In line with our pre-clinical findings, post-MI patients on canakinumab treatment showed reduced blood monocyte numbers. CONCLUSIONS: Our murine and human data reveal that anti-IL-1ß treatment and NLRP3-inflammasome inhibition dampened vascular inflammation and progression of atherosclerosis through reduced blood inflammatory leucocyte (i) supply and (ii) uptake into atherosclerotic aortas providing additional mechanistic insights into links between haematopoiesis and atherogenesis, and into the beneficial effects of NLRP3-inflammasome- and IL-1ß-targeted therapies.

Imaging atherosclerotic plaques by targeting Galectin-3 and activated macrophages using (89Zr)-DFO- Galectin3-F(ab')2 mAb.

Rationale: The high expression of Galectin-3 (Gal3) in macrophages of atherosclerotic plaques suggests its participation in atherosclerosis pathogenesis, and raises the possibility to use it as a target to image disease severity in vivo. Here, we explored the feasibility of tracking atherosclerosis by targeting Gal3 expression in plaques of apolipoprotein E knockout (ApoE-KO) mice via PET imaging. Methods: Targeting of Gal3 in M0-, M1- and M2 (M2a/M2c)-polarized macrophages was assessed in vitro using a Gal3-F(ab')2 mAb labeled with AlexaFluor®488 and 89Zr- desferrioxamine-thioureyl-phenyl-isothiocyanate (DFO). To visualize plaques in vivo, ApoE-KO mice were injected i.v. with 89Zr-DFO-Gal3-F(ab')2 mAb and imaged via PET/CT 48 h post injection. Whole length aortas harvested from euthanized mice were processed for Sudan-IV staining, autoradiography, and immunostaining for Gal3, CD68 and α-SMA expression. To confirm accumulation of the tracer in plaques, ApoE-KO mice were injected i.v. with Cy5.5-Gal3-F(ab')2 mAb, euthanized 48 h post injection, followed by cryosections of the body and acquisition of fluorescent images. To explore the clinical potential of this imaging modality, immunostaining for Gal3, CD68 and α-SMA expression were carried out in human plaques. Single cell RNA sequencing (scRNA-Seq) analyses were performed to measure LGALS3 (i.e. a synonym for Gal3) gene expression in each macrophage of several subtypes present in murine or human plaques. Results: Preferential binding to M2 macrophages was observed with both AlexaFluor®488-Gal3-F(ab')2 and 89Zr-DFO-Gal3-F(ab')2 mAbs. Focal and specific 89Zr-DFO-Gal3-F(ab')2 mAb uptake was detected in plaques of ApoE-KO mice by PET/CT. Autoradiography and immunohistochemical analyses of aortas confirmed the expression of Gal3 within plaques mainly in macrophages. Moreover, a specific fluorescent signal was visualized within the lesions of vascular structures burdened by plaques in mice. Gal3 expression in human plaques showed similar Gal3 expression patterns when compared to their murine counterparts. Conclusions: Our data reveal that 89Zr-DFO-Gal3-F(ab')2 mAb PET/CT is a potentially novel tool to image atherosclerotic plaques at different stages of development, allowing knowledge-based tailored individual intervention in clinically significant disease.

Functional investigation of the coronary artery disease gene SVEP1.

A missense variant of the sushi, von Willebrand factor type A, EGF and pentraxin domain containing protein 1 (SVEP1) is genome-wide significantly associated with coronary artery disease. The mechanisms how SVEP1 impacts atherosclerosis are not known. We found endothelial cells (EC) and vascular smooth muscle cells to represent the major cellular source of SVEP1 in plaques. Plaques were larger in atherosclerosis-prone Svep1 haploinsufficient (ApoE-/-Svep1+/-) compared to Svep1 wild-type mice (ApoE-/-Svep1+/+) and ApoE-/-Svep1+/- mice displayed elevated plaque neutrophil, Ly6Chigh monocyte, and macrophage numbers. We assessed how leukocytes accumulated more inside plaques in ApoE-/-Svep1+/- mice and found enhanced leukocyte recruitment from blood into plaques. In vitro, we examined how SVEP1 deficiency promotes leukocyte recruitment and found elevated expression of the leukocyte attractant chemokine (C-X-C motif) ligand 1 (CXCL1) in EC after incubation with missense compared to wild-type SVEP1. Increasing wild-type SVEP1 levels silenced endothelial CXCL1 release. In line, plasma Cxcl1 levels were elevated in ApoE-/-Svep1+/- mice. Our studies reveal an atheroprotective role of SVEP1. Deficiency of wild-type Svep1 increased endothelial CXCL1 expression leading to enhanced recruitment of proinflammatory leukocytes from blood to plaque. Consequently, elevated vascular inflammation resulted in enhanced plaque progression in Svep1 deficiency.