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ApoA-I nanotherapy rescues inadequate post-ischemic vascular adaptation in diabetic mice by modulating macrophage phenotype

ABSTRACT: Purpose: To clarify cellular and molecular mechanisms underlying impaired post-ischemic adaptive vascular responses and to evaluate reconstituted HDL (rHDL)-ApoA-I nanotherapy to rescue the defect in type 2 diabetic (T2DM) mouse model of hindlimb ischemia (HLI). Methods: Mice with beta-cell specific over-expression of insulin-like growth factor-2 in atherosclerotic background (IGF-II/ LDLR-/-ApoB100/100) with T2DM features were used in the study with C57BL/6J mice fed with a regular chow-diet (R36, Lactamin) serving as controls. Mice aged between 16 to 20 weeks were used in the study . Mice were anaesthetized with 1.5-2% isoflurane/air mixture during the surgical procedure. Hair was removed from the operation site and cleaned with sterile water. Single incision was made just above medial thigh and superficial femoral artery was separated from femoral vein and nerve. HLI was induced by unilateral ligation of femoral artery distal to the origin of the profunda femoral artery. The skin was closed with interrupted silk sutures. Mice were were administered with reconstituted ApoA-I nanoparticles with phosphatidyl serine core (rHDL) intravenously starting 2 days post-HLI with doses every 2 days until scarification with Saline injections serving as controls. Adductors muscle aortic endothelial cells (AECs) and Ischemic muscle Macrophages and Endothelial Cells (ECs) were isolated using a combination of magnetic assisted cell sorting and flow Cytometry using Macrophage (F4/80) and EC (CD-31) specific markers. Total RNA isolated from FACS sorted cells were used for RNA seq library preparation. Results: Cell-specific gene expression, flow cytometry and immunohistochemistry revealed a persistent Macrophage and Endothelial cell inflammation and a reduced anti-inflammatory M2-macrophage activation associated with impaired collateral remodelling and sprouting angiogenesis in T2DM mice. Furthermore, reconstituted HDL (rHDL)- ApoA-I was found to rescue impaired collateral remodelling and angiogenesis in T2DM mice through modulating EC and macrophage phenotypes. Conclusions: Our results suggest that an impaired collateral remodelling and sprouting angiogenesis in T2DM mice after HLI is associated with a persistent EC and macrophage inflammation and a reduced activation of anti-inflammatory M2-macrophage. Skewing the macrophage phenotype towards the pro-arteriogenic and pro-angiogenic anti-inflammatory M2-macrophages using reconstituted HDL (rHDL)-ApoA-I nanotherapy may serve as a potential therapeutic modality for T2DM PAD.

ORGANISM(S): Mus musculus

PROVIDER: GSE197392 | GEO | 2023/01/30

REPOSITORIES: GEO

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Project description:Protein lysine residue carbamylation is an irreversible post-translational modification, resulting in the generation of protein-bound homocitrulline (N--carbamyllysine). Carbamylation alters protein structure and can thus also alter protein function because of the loss of the charged -amino lysine moiety. Two distinct pathways can promote protein carbamylation. The first pathway occurs as a result of urea decomposition forming an equilibrium mixture of cyanate (CNO-) and the reactive electrophile isocyanate; the second (enzymatic) pathway occurs from myeloperoxidase (MPO)–catalyzed oxidation of thiocyanate (SCN-), yielding CNO- and isocyanate. We and others have previously shown that apolipoprotein A-I (apoA-I), the major protein constituent of high density lipoprotein (HDL), is a target for MPO-catalyzed modification in vivo altering its function, converting the traditionally cardio-protective lipoprotein into a pro-atherogenic and pro-apoptotic one. We hypothesized that insights into the chemical environment within the human artery wall could be gained by monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta. Toward testing this hypothesis, we first mapped and quantified carbamyllysine obtained from in vitro carbamylation of apoA-I by: (i) the urea driven (cyanate) vs. the inflammatory driven (MPO) pathways; as well as (ii) in lipid-poor vs. lipidated apoA-I (reconstituted nascent HDL). Our in vitro studies, interpreted in the context of existing structural models, suggest lysine residues within regional proximity of the known MPO binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the non-enzymatic (cyanate) pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I recovered from human aortic atheroma identified 16 of the 21 total lysine residues as carbamylated and suggests that the majority of apoA-I carbamylation in vivo occurs via the non-enzymatic cyanate pathway, and on ‘lipid-poor’ apoA-I forms. Monitoring the patterns of post translational modification of apoA-I lysine residues in vivo can provide insights into the chemical environment within human atheroma.

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ApoA-I nanotherapy rescues inadequate post-ischemic vascular adaptation in diabetic mice by modulating macrophage phenotype

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