Prevention of neointimal hyperplasia after coronary artery bypass graft via local delivery of sirolimus and rosuvastatin: network pharmacology and in vivo validation

Abstract

Background: Coronary artery bypass graft (CABG) is generally used to treat complex coronary artery disease. Treatment success is affected by neointimal hyperplasia (NIH) of graft and anastomotic sites. Although sirolimus and rosuvastatin individually inhibit NIH progression, the efficacy of combination treatment remains unknown.

Methods: We identified cross-targets associated with CABG, sirolimus, and rosuvastatin by using databases including DisGeNET and GeneCards. GO and KEGG pathway enrichment analyses were conducted using R studio, and target proteins were mapped in PPI networks using Metascape and Cytoscape. For in vivo validation, we established a balloon-injured rabbit model by inducing NIH and applied a localized perivascular drug delivery device containing sirolimus and rosuvastatin. The outcomes were evaluated at 1, 2, and 4 weeks post-surgery.

Results: We identified 115 shared targets between sirolimus and CABG among databases, 23 between rosuvastatin and CABG, and 96 among all three. TNF, AKT1, and MMP9 were identified as shared targets. Network pharmacology predicted the stages of NIH progression and the corresponding signaling pathways linked to sirolimus (acute stage, IL6/STAT3 signaling) and rosuvastatin (chronic stage, Akt/MMP9 signaling). In vivo experiments demonstrated that the combination of sirolimus and rosuvastatin significantly suppressed NIH progression. This combination treatment also markedly decreased the expression of inflammation and Akt signaling pathway-related proteins, which was consistent with the predictions from network pharmacology analysis.

Conclusions: Sirolimus and rosuvastatin inhibited pro-inflammatory cytokine production during the acute stage and regulated Akt/mTOR/NF-κB/STAT3 signaling in the chronic stage of NIH progression. These potential synergistic mechanisms may optimize treatment strategies to improve long-term patency after CABG.

Keywords: Inflammation; Intimal hyperplasia; Network pharmacology; Rosuvastatin; Sirolimus.

Fig. 1

Workflow of the network pharmacology approach for identifying the mechanism underlying the effects of sirolimus and rosuvastatin in treatment coupled with CABG. PPI protein–protein interaction, CABG coronary artery bypass graft

Fig. 2

Intersection of potential targets of CABG, sirolimus, and rosuvastatin along with overlapping targets in a drug-disease network. A Venn diagram showing the potential targets of CABG, sirolimus, and rosuvastatin. Yellow section represents the potential targets of CABG, blue section represents the potential targets of sirolimus, and pink section represents the potential targets of rosuvastatin. B Overlapping targets in the drug-disease network. CABG, coronary artery bypass graft

Fig. 3

GO and KEGG pathway analysis of overlapping targets between CABG and sirolimus. A Bar graph presents the top 10 significant GO terms related to vascular disease among overlapping targets. X-axis presents the top 10 BPs, CCs, and MFs identified via GO enrichment analysis. Y-axis presents the − log10 (adjusted p-value) identified within each GO term. Dot plots illustrating the enrichment of B BP and C KEGG pathways. X-axis presents the enrichment gene ratio, while y-axis presents the BP and KEGG pathways. Dot size indicates the number of genes, and color indicates the enriched − log10 (adjusted p-value). GO gene ontology, KEGG Kyoto encyclopedia of genes and genomes, CABG coronary artery bypass graft, BP biological process, CC cellular component, MF molecular function

Fig. 4

GO and KEGG pathway analysis of overlapping targets between CABG and rosuvastatin. A Bar graph presents the top 10 significant GO terms related to vascular disease among overlapping targets. X-axis presents the top 10 BPs, CCs, and MFs identified via GO enrichment analysis. Y-axis presents the − log10 (adjusted p-value) identified within each GO term. Dot plots illustrating the enrichment of B BP and C KEGG pathways. X-axis presents the enrichment gene ratio, while y-axis presents BP and KEGG pathway. Dot size indicates the number of genes, and color indicates the enriched − log10 (adjusted p-value). GO gene ontology, KEGG Kyoto encyclopedia of genes and genomes, CABG coronary artery bypass graft, BP biological process, CC cellular component, MF molecular function

Fig. 5

GO and KEGG pathway analysis of overlapping targets among CABG, sirolimus, and rosuvastatin. A Bar graph presents the top 10 significant GO terms related to vascular disease among overlapping targets. X-axis presents the top 10 BPs, CCs, and MFs identified via GO enrichment analysis. Y-axis presents the − log10 (adjusted p-value) identified within each GO term. Dot plots illustrating the enrichment of B BP and C KEGG pathways. X-axis presents the enrichment gene ratio, while y-axis presents BP and KEGG pathways. Dot size indicates the number of genes, and color represents the enriched − log10 (adjusted p-value). GO gene ontology, KEGG Kyoto encyclopedia of genes and genomes, CABG coronary artery bypass graft, BP biological process, CC cellular component, MF molecular function

Fig. 6

Prediction of the drug combination of sirolimus and rosuvastatin for CABG via PPI network analysis. A–C The interactive PPI network of A CABG and sirolimus, B CABG and rosuvastatin, and C CABG and both drugs. The top 10 hub and bottleneck genes identified using CytoHubba are shown. Dark red color indicates a relatively higher maximal clique centrality or bottleneck score. The figures were created using BioRender. CABG coronary artery bypass graft, PPI protein–protein interaction

Fig. 7

Sirolimus and rosuvastatin co-treatment alleviate balloon injury-induced intimal hyperplasia in an experimental model. A Schematic illustration of the injury and application protocol. The rabbit abdominal aortas were injured using a balloon catheter and treated using a localized perivascular drug delivery device containing either a control or SIR + RSV. Blood vessels were harvested after 1, 2, and 4 weeks. The figure was created using BioRender. B Histological changes in the aortas were observed using hematoxylin and eosin staining after control or SIR + RSV treatment (scale bars = 50 μm). C Quantification of intimal and medial thickness, the ratio of intima/media, and neointima formation. C–E Transmission electron microscopy images of vascular smooth muscle cells in the treated aorta after 1 and 2 weeks and in the native aorta (Normal). Statistical significance was determined using Fisher’s least significant difference test (*p < 0.05, **p < 0.01, ***p < 0.001). SIR + RSV combination of sirolimus and rosuvastatin

Fig. 8

Sirolimus and rosuvastatin inhibit pro-inflammatory cytokine production in the acute stage. A–D Protein levels of inflammatory cytokines IL-6, TNF-α, and IL-1β were detected via western blot analysis. Data are presented as the mean ± standard error of mean. Statistical significance was determined using Fisher’s least significant difference test (* p < 0.05, ** p < 0.01, ***p < 0.001). SIR + RSV combination of sirolimus and rosuvastatin, IL-6 interleukin 6, TNF-α tumor necrosis factor alpha, IL-1β interleukin 1 beta

Fig. 9

Sirolimus and rosuvastatin attenuate STAT3 phosphorylation in the chronic stage. A–C Protein levels of STAT3 and p-STAT3 were determined via western blot analysis. Data are presented as the mean ± standard error of mean. Statistical significance was determined using Fisher’s least significant difference test (*p < 0.05, **p < 0.01, ***p < 0.001). SIR + RSV combination of sirolimus and rosuvastatin, STAT3 signal transducer and activator of transcription 3, p-STAT3 phosphorylated STAT3

Fig. 10

Sirolimus and rosuvastatin regulate MMP expression in the chronic stages via Akt/mTOR/NF-kB signaling. A–F Protein levels of Akt1, mTOR, p-mTOR, NF-κB, and MMP9 were detected via western blot analysis. Data are presented as the mean ± standard error of mean. Statistical significance was determined using Fisher’s least significant difference test (*p < 0.05, **p < 0.01, ***p < 0.001). SIR + RSV combination of sirolimus and rosuvastatin, Akt a protein cloned from the v-akt oncogene of retrovirus AKT8, mTOR mammalian target of rapamycin, p-mTOR phosphorylated mTOR, NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells, MMP matrix metalloproteinase

Fig. 11

A schematic representation illustrating the role of sirolimus and rosuvastatin in preventing neointimal hyperplasia (created using BioRender). IL-6 interleukin 6, IL-1β interleukin 1 beta, TNF-α tumor necrosis factor alpha, Akt a protein cloned from the v-akt oncogene of retrovirus AKT8, mTOR mammalian target of rapamycin, STAT3 signal transducer and activator of transcription 3, NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells