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Systems Approach to Discovery of Therapeutic Targets for Vein Graft Disease PPARα Pivotally Regulates Metabolism, Activation, and Heterogeneity of Macrophages and Lesion Development

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Background: Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discovering therapeutic targets for vein graft failure. Methods: Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The peroxisome proliferator-activated receptors (PPARs) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα siRNA and the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, qPCR, flow cytometry, metabolic assays, and single-cell RNA-sequencing on primary human and mouse macrophages. Results: We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, while gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity. Conclusions: This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacologic treatment for this unmet medical need.
Vein graft target discovery. A, Mouse model: donor's suprahepatic inferior vena cava (IVC) transplanted into the recipient's left midcommon carotid artery. B, Normal chow (NC)-fed wild-type (WT) C57BL6 12-week-old male mice and fat-fed (2 weeks prefed) low-density lipoprotein receptor (Ldlr -/-) 12-week-old male mice (C57BL6 background) vein grafts in ultrasound imaging (long-axis view) at week 4 after operation. (n=6 versus n=6). Near wall (ventral) = NW, far wall (dorsal) = FW. Scale =1 mm. C, Long axis view (scale bar 1 mm) of a representative vein graft (VG) lesion showing an increase of lesion size (blue arrowheads) from 1 week to 3 weeks after operation. Luminal stenosis evident at 3 weeks (yellow arrows) D, Immunofluorescence of vein graft at 4 weeks after operation using AF488-anti-CD68 (macrophages, green color), Cy3-α-smooth muscle actin, SMA (vascular smooth muscle cells, VSMCs, red color), and DAPI (4′,6-diamidino-2-phenylindole, nucleus, blue color). Co-loc. indicates co-localization. Scale=100 µm. E, VG tissue layer dissection of neointimal (NEO) and adventitial (ADV) layers of Ldlr -/-VG samples. WT VG samples were not microdissected. IVC was used as controls. Representative immunofluorescence staining of Ldlr -/-versus WT VG tissue (n=2 biological replicates, n=2 technical replicates). F, Tissue layer proteomics (n=2 mice, 2 technical replicates per tissue) by multigroup comparison, false discovery rate ≤0.05. Proteins that are relatively increased in IVC samples and VG of 1 WT animal are also relatively decreased in Ldlr -/-VG tissues in both NEO and ADV layers and in 1 VG of another WT animal, and vice versa. G, Time course proteomics of Ldlr -/-VGs: 1 day (D1), 3 days (D3), 14 days (W2), and 28 days (W4) after VG surgery (n=12 VG donors, 3 biological replicates per time point). Vein grafts were processed as a whole (no layer dissection) and paired with matching IVC controls from the same animals. H, Multiplexed analysis of proteome across time points showing "coabundance" proteins in time (see Expanded Methods in the Data Supplement).
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In vivo PPARα loss-of-function and gain-of-function studies. A, Study design. Upper, Loss-of-function. Twenty-two low-density lipoprotein receptor (Ldlr -/-) male mice used, aged 12 weeks, prefed with high-fat diet 2 weeks before operation. Randomization in 2 treatment groups: (1) set 1: siRNA control conjugated to C12-200 lipid nanoparticles (LNP); (2) set 2: siRNA PPARα (small interfering RNA or siRNA of PPAR alpha, 1:1 mixture of oligonucleotide 1 and 2 conjugated to C12-200 LNP. A 0.5 mg siRNA-LNP/kg body weight (BW)/dose given intravenously 11 times throughout the study. Dosage schedule: 2 days before operation, right after surgery, then 2 times per week (every 3 days) after surgery for 4 weeks with 1 extra dose 2 to 3 days after day 28. Lower, Pemafibrate (PPARα gain-of-function) study (n=11 versus n=11). B, Three-dimensional ultrasound rendered wall volume comparison of siControl (siControl= non-specific small interfering RNA, siRNA), and siRNA PPARα silenced group at 4 weeks after surgery. C, Vessel wall thickness, long-axis view. siRNA PPARα group had thicker vessel walls than siRNA control group (n=11 versus n=11). Scale=1 mm. D, Glucose uptake by RediJect assay (Perkin Elmer) and intravital fluorescence in the VG. Representative image. E, Gain-of-function study using the highly selective PPARα activator pemafibrate (drug) (n=11 versus n=11, control versus drug-treated) resulted in lesser neointimal plaque up to 3 weeks after surgery. F, Immunofluorescence histology of the midgraft transverse sections using CD68 (green) and α-SMA (red) antibodies (blue=DAPI [4′,6-diamidino-2-phenylindole], nuclear stain). Pemafibratetreated group had less macrophage accumulation at the lesion than control group. There was no difference in vascular smooth muscle cell content. G, Fluorescence-activated cell sorting analysis showing pemafibrate decreased circulating Ly6C ++ monocytes. ELISA of blood plasma shows pemafibrate decreases plasma recruitment chemokine CXCL11 levels. Corresponding normality tests. H, In situ transmitted light and intravital near-infrared fluorescence (NIRF) imaging of vein grafts 12 hours after MMPSense 680 intravenous injection. Red fluorescence indicates intensity of proteases MMP-2, 3, 9, and 13 (activity and relative abundance). I, Picrosirius red staining (PSR) of 4-week vein grafts midcross-section with quantification of red and green birefringence of collagen fibrils. *P<0.05; **P<0.01; ***P<0.001; n.s., P≥0.05. Co-loc indicates colocalization; Ctrl, control; FC, fragmented collagen; HFD, high-fat diet; LNP, lipid nanoparticle; Pema, pemafibrate; post-op, after operation; PPARα, peroxisome proliferator-activated receptor α; QQ or Q-Q, quantile-quantile; SC, stable collagen; siRNA, small interfering RNA; SMA, smooth muscle actin; and VG, vein graft.
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... For example, control centrality identified the statistical significance of 66 pathways in type 2 diabetes (102). This approach also identified the peroxisome proliferator-activated receptor alpha (PPARα) pathway as a potential causal factor, and thus a drug target in vein graft disease (103). ...
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