Macrophagic sclerostin loop2-ApoER2 interaction participates in the suppressive effect of sclerostin on atherosclerosis

Sclerostin inhibition by therapeutic sclerostin antibody promoted bone formation whereas imposed atherosclerosis-related severe cardiac ischemic events in clinical trials. Sclerostin protects cardiovascular system by inhibiting inflammatory responses, atherosclerosis, and aortic aneurysm (AA) in ApoE^-/- mice. The central residues of sclerostin form three loops. Sclerostin antibody targeted sclerostin loop2 and loop3. Our published data indicated that specific blockade of the interaction between sclerostin antibody and sclerostin loop2 could attenuate the sclerostin antibody-induced aggravation of inflammatory responses, atherosclerosis and AA in ApoE^-/- mice, whereas the protective effect of sclerostin on cardiovascular system was independent of sclerostin loop3. Now, it is necessary to investigate how sclerostin participates in protecting the cardiovascular system.
Our GWAS analysis of the SOST variants in U.K. Biobank indicated the association between sclerostin loop2-specific mutations and cardiovascular abnormalities. In single-cell RNA sequencing, we found that the proportion of the aortic anti-inflammatory macrophage cluster was dramatically elevated in ApoE^-/- mice after sclerostin knock-in (SOSTki.ApoE^-/- ), with Lrp8 (encoding ApoE receptor 2 - ApoER2) being among the top 5 genes upregulated. It was known that the absence of ApoER2 could lead to vascular inflammation and lesion progression in atherosclerosis. Here, the suppressive effects of sclerostin on inflammatory responses in macrophages were found to be dramatically attenuated upon ApoER2 silencing, indicating the suppressive effects of sclerostin on inflammatory responses could be dependent on macrophagic ApoER2 in vitro.
In our pull-down and SPR assays, it was further found that sclerostin loop2 bound to ApoER2 in macrophages. After identification of the interaction residues within ApoER2 to sclerostin loop2 in macrophages, K749A mutation within Lrp8 (Lrp8m) was designed to genetically block the interaction between sclerostin loop2 and ApoER2 in vitro. The suppressive effects of sclerostin on inflammatory responses in macrophages were attenuated upon Lrp8-K749A mutation in vitro. Based on the interaction, a validated peptide tool ApoER2-XY (L725-M764) was utilized to pharmacologically block the interaction between sclerostin loop2 and ApoER2 in vitro. The suppressive effects of sclerostin on inflammatory responses in macrophages were attenuated after pretreatment of the exogenous ApoER2-XY in vitro. The above genetic and pharmacologic approaches suggested that macrophagic sclerostin loop2-ApoER2 interaction participated in the suppressive effects of sclerostin on inflammatory responses in vitro.
After either AngII infusion or western-diet (WD) induction, ApoE^-/- mice with macrophagic Lrp8- K749A mutation (ApoE^-/- .Mac-Lrp8m mice) exhibited aggravated inflammatory responses and atherosclerosis than ApoE^-/- mice, which mirrored the phenotype of sclerostin knock-out ApoE^-/- mice (sost^-/- .ApoE^-/- ). To shield the effect of endogenous sclerostin, we constructed sost^-/- .ApoE^-/- .MacLrp8m mouse model. No significant differences were found in inflammatory responses and atherosclerosis between sost^-/- .ApoE^-/- .Mac-Lrp8m mice and sost^-/- .ApoE^-/- mice. Genetically, the sost^{- /-} .ApoE^-/- .Mac-Lrp8m mice could be used for treatment with recombinant sclerostin in the following Aim 1. As expected, the suppressive effects of sclerostin on expression of inflammatory cytokines and chemokines in SOSTki .ApoE^-/- mice with either AngII infusion or WD induction were remarkably attenuated after the treatment of the exogenous ApoER2-XY. Pharmacologically, the SOSTki .ApoE^-/- mice could be used for treatment with the exogenous ApoER2-XY in the following Aim 2. Together, it was hypothesized that macrophagic sclerostin loop2-ApoER2 interaction could participate in the suppressive effect of sclerostin on atherosclerosis. To test the hypothesis using the above mouse models with either AngII infusion or WD induction, we have the following two specific aims:
Aim 1. To genetically determine the role of macrophagic sclerostin loop2-ApoER2 interaction in the suppressive effect of sclerostin on atherosclerosis by comparing the inflammatory responses and atherosclerosis progression between sost ^-/- .ApoE^-/- .Mac-Lrp8m mice and sost ^-/- .ApoE^-/- mice, with and without treatment of recombinant sclerostin.
Aim 2. To pharmacologically determine the role of sclerostin loop2-ApoER2 interaction in the suppressive effect of sclerostin on atherosclerosis by comparing the inflammatory responses and atherosclerosis progression between SOSTki .ApoE^-/- mice with and without treatment of exogenous ApoER2-XY.
If the hypothesis could be supported, it would facilitate understanding how sclerostin could participate in protecting the cardiovascular system. Targeting sclerostin while preserving macrophagic sclerostin loop2-ApoER2 interaction would offer the first generation of precise sclerostin inhibition strategy to promote bone formation without increasing cardiovascular risk.