Targeting of Scavenger Receptors Stabilin-1 and Stabilin-2 Ameliorates Atherosclerosis by a Plasma Proteome Switch Mediating Monocyte/Macrophage Suppression

Abstract

BACKGROUND: Scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are preferentially expressed by liver sinusoidal endothelial cells. They mediate the clearance of circulating plasma molecules controlling distant organ homeostasis. Studies suggest that Stab1 and Stab2 may affect atherosclerosis. Although subsets of tissue macrophages also express Stab1, hematopoietic Stab1 deficiency does not modulate atherogenesis. Here, we comprehensively studied how targeting Stab1 and Stab2 affects atherosclerosis. METHODS: ApoE-KO mice were interbred with Stab1-KO and Stab2-KO mice and fed a Western diet. For antibody targeting, Ldlr-KO mice were also used. Unbiased plasma proteomics were performed and independently confirmed. Ligand binding studies comprised glutathione-S-transferase–pulldown and endocytosis assays. Plasma proteome effects on monocytes were studied by single-cell RNA sequencing in vivo, and by gene expression analyses of Stabilin ligand–stimulated and plasma-stimulated bone marrow–derived monocytes/macrophages in vitro. RESULTS: Spontaneous and Western diet–associated atherogenesis was significantly reduced in ApoE-Stab1-KO and ApoE-Stab2-KO mice. Similarly, inhibition of Stab1 or Stab2 by monoclonal antibodies significantly reduced Western diet–associated atherosclerosis in ApoE-KO and Ldlr-KO mice. Although neither plasma lipid levels nor circulating immune cell numbers were decisively altered, plasma proteomics revealed a switch in the plasma proteome, consisting of 231 dysregulated proteins comparing wildtype with Stab1/2-single and Stab1/2-double KO, and of 41 proteins comparing ApoE-, ApoE-Stab1-, and ApoE-Stab2-KO. Among this broad spectrum of common, but also disparate scavenger receptor ligand candidates, periostin, reelin, and TGFBi (transforming growth factor, β-induced), known to modulate atherosclerosis, were independently confirmed as novel circulating ligands of Stab1/2. Single-cell RNA sequencing of circulating myeloid cells of ApoE-, ApoE-Stab1-, and ApoE-Stab2-KO mice showed transcriptomic alterations in patrolling (Ccr2 – /Cx3cr1 ++ /Ly6C lo ) and inflammatory (Ccr2 + /Cx3cr1 + /Ly6C hi ) monocytes, including downregulation of proatherogenic transcription factor Egr1. In wildtype bone marrow–derived monocytes/macrophages, ligand exposure alone did not alter Egr1 expression in vitro. However, exposure to plasma from ApoE-Stab1-KO and ApoE-Stab2-KO mice showed a reverted proatherogenic macrophage activation compared with ApoE-KO plasma, including downregulation of Egr1 in vitro. CONCLUSIONS: Inhibition of Stab1/Stab2 mediates an anti-inflammatory switch in the plasma proteome, including direct Stabilin ligands. The altered plasma proteome suppresses both patrolling and inflammatory monocytes and, thus, systemically protects against atherogenesis. Altogether, anti-Stab1– and anti-Stab2–targeted therapies provide a novel approach for the future treatment of atherosclerosis.

Deficiency for scavenger receptors Stabilin-1 and Stabilin-2 leads to age-dependent renal and hepatic depositions of fasciclin domain proteins TGFBI and Periostin in mice

“…We here show that scavenging by Stab1 and Stab2 is crucial not only for homeostasis of TGFBI and POSTN plasma levels in mice (Manta et al, 2022), but also to prevent age-dependent accumulation of TGFBI and POSTN in distant organs. We hypothesize that POSTN and TGFBI are not produced locally in Stab-DKO livers and kidneys, but are deposited in distant organs, possibly due to their high concentrations in Stab-DKO plasma, since their scavenger receptors are missing.…”

“…These findings bear consequences for the use of therapeutical inhibition of Stabilins, which for Stab1 is already in Phase 2 clinical trials in cancer (Virtakoivu et al, 2021) and might be a treatment option for atherosclerosis in future studies (Manta et al, 2022). Single deficiency for Stab1 or Stab2 alone did not seem to induce strong damage to kidney function, although a mild increase in TGFBI depositions was observed in young, but not aged Stab2-KO glomeruli.…”

“…Nevertheless, these findings point towards a slight amelioration of the kidney phenotype by deletion of POSTN. Whether TGFBI is solely responsible for the possibly fatal glomerulofibrosis in Stab-DKO and Stab-POSTN-TrKO is unclear, since we observed more than 100 dysregulated proteins comparing the plasma proteome of Stab-DKO mice compared to WT mice (Manta et al, 2022). Stab1 and Stab2 not only contain fasciclin domains, but also EGF-like domains and Laminin EGF-like domains beside the X-link domain (Politz et al, 2002).…”

“…Stab1 and Stab2 not only contain fasciclin domains, but also EGF-like domains and Laminin EGF-like domains beside the X-link domain (Politz et al, 2002). The EGF-like domain containing protein Reelin has previously been identified by us as a Stabilin-2 ligand (Manta et al, 2022). No Reelin depositions were found in kidneys and livers of our models (data not shown).…”

“…Previously, we showed that Stab1/Stab2-double-knockout mice (Stab-DKO) present an impaired kidney function and a decreased lifespan likely due to their impaired scavenging function through LSECs (Schledzewski et al, 2011). Only recently, we were able to show that single Stabilin inhibition reduces atherosclerosis development and that extracellular matrix proteins Transforming growth factor, beta-induced, 68kDa (TGFBI) and Periostin (POSTN) are ligands of Stab1 and Stab2, likely due to fasciclin domain interactions and their plasma concentration is strongly increased in Stab-DKO mice (Manta et al, 2022). Interestingly, Stab1 and Stab2 as well as POSTN and TGFBI are the only four fasciclin-domain containing proteins in mammals (Seifert, 2018).…”