N. Momi2,6, P. Liang2,6, S. Bhasin2,5, F. W. LoGerfo2,6, C. Ferran2,4, L. Pradhan-Nabzdyk2,6, M. Bhasin2,5 2Harvard School Of Medicine,Brookline, MA, USA 4Beth Israel Deaconess Medical Center,Center For Vascular Biology Research And Division Of Nephrology, Department Of Medicine,Boston, MA, USA 5Beth Israel Deaconess Medical Center,Genomics And Proteomics Center, Division Of Interdisciplinary Medicine And Biotechnology,Boston, MA, USA 6Beth Israel Deaconess Medical Center,Division Of Vascular And Endovascular Surgery, Department Of Surgery,Boston, MA, USA
Introduction: Bypass grafting using autologous vein conduits is the cornerstone therapy for arterial occlusive disease. However, 30–50% of lower extremity vein grafts (VG) fail within 5 years from surgery. We hypothesize that VG implantation injury causes spatial and temporal genetic changes in the VG, triggering a cascade of interrelated molecular events starting with inflammation and culminating in vessel wall remodeling eventually leading to Intimal Hyperplasia (IH). This study aims to investigate the genomic contribution of individual cells including smooth muscle cells (SMC), endothelial cells (EC), adipocytes (Adipo), fibroblasts (FB), immune cells (T-Cells, Macrophages, NK etc.) towards VG failure and IH.
Methods: Canines underwent cephalic vein to common carotid artery interposition surgery. Cephalic VG and contralateral vein (CV) were harvested 24hrs post-surgery. Upon collagenase-I digestion, samples were subjected to 10X Single Cell (sc) genomics-based droplet sequencing to quantify and compare cell enrichment by ImmuneQuant software-based annotation and t-SNE clustering. Further, individual cell pools were validated by IHC. Additionally, bulk RNA-seq and ingenuity pathway analysis (IPA) was performed to elucidate key mediators/pathways.
Results: Our results show a dramatic difference in the cell types present in VG vs. CV 24hrs post-surgery. CV predominantly had non-immune cells (93% of total distributed in 9 clusters), including EC, SMC, FB and Adipo, with fewer immune (7%, T-Cells). In striking contrast, VG demonstrated partial loss of ECs, Adipo and FB with simultaneous infiltration of immune cells, accounting for 89% of total cells, with 4 T-cell clusters (52%) (CD3D+PTPRC+CD19+IL3RA+), primarily Th1 cells (67% of T-Cells), 4 monocyte/macrophages clusters (34%) (CD68+TGFB1_CANFA+IL1R1+) and 1 B-cell cluster (3%). Further, supervised analysis of individual clusters highlighted differential expression of several genes including, chemokines (IL-8, CCL4/2), TFs (FOS) and extracellular matrix/vascular components (collagen/COL1A1, endothelin/EDN). Intriguingly, these findings were corroborated by bulk-RNA-seq indicating significant activation of inflammatory pathway, Th1 pathway, CD28 signaling in Th1 cells, PI3K signaling in B-cells, IL-8 and chemokine signaling. Finally, IHC analysis also validated the higher number of CD3+ T-cells in the VG.
Conclusion: Significant monocytes/macrophages and T-cell infiltrates in VG vs CV reveal an early contribution of both innate and adaptive immune response towards the pathophysiology of VG remodeling. Sc-genomics provide a better understanding of the complex ecosystem that governs implantation injury and unsuccessful adaptations of the VG leading to graft failure, thereby opening avenues for plausible effective preventive measures and early therapeutic targets.