Time and flow-dependent changes in the p27 gene network drive maladaptive vascular remodeling

Abstract

Objectives Although clinical studies have identified that a single nucleotide polymorphism in the p27kip1 gene is associated with success or failure following vein bypass grafting, the underlying mechanisms for this difference are not well defined. Using a high-throughput approach in a flow-dependent vein graft model, we explored the differences in p27kip1 -related genes that drive the enhanced hyperplastic response under low flow conditions. Methods Bilateral rabbit carotid artery interposition grafts with jugular vein were placed with unilateral distal outflow branch ligation to create differential flow states. Grafts were harvested at 2hr, 1d, 3d, 7d, 14d, and 28 days after implantation, measured for neointimal area, and assayed for cell proliferation. Whole-vessel mRNA was isolated and analyzed using an Affymetrix gene array platform. Ingenuity Pathway Analysis (IPA) was utilized to identify the gene networks surrounding p27kip1. This gene set was then analyzed for temporal expression changes following graft placement and for differential expression in the alternate flow conditions. Results Outflow branch ligation resulted in an 8-fold difference in mean flow rates throughout the 28d perfusion period (P<.001). Flow reduction led to a robust hyperplastic response, resulting in a significant increase in intimal area by 7d (0.13±0.04 vs. 0.014±0.006mm2, P<.005) and progressive growth to 28d (1.37±0.05 vs. 0.39±0.06mm2, P<.001). At 7 days, low flow grafts demonstrated a burst of actively dividing intimal cells (36.4 cells/mm2 ± 9.4 vs. 11.5 ± 1.9, P=.04). 65 unique genes within the microarray were identified as components of the p27kip1 network. At a false discovery rate of 0.05, 26 genes demonstrated significant temporal changes and two dominant patterns of expression was identified. Class comparison analysis identified differential expression of 11 genes at 2h and 7 genes and 14d between the high and low flow grafts (P<.05). At 2h, Oncostatin M and cadherin 1 were the most differentially expressed. Cadherin 1 and PKB exhibited the greatest differential expression at 14d. Conclusions Alterations in flow and shear stress result in divergent patterns of vein graft remodeling. Associated with the dramatic increase in neointimal expansion observed in low flow versus high flow grafts is a subset of differentially expressed p27kip1-associated genes that correlate with critical stages in the adaptive response. These represent potential biologic targets whose activity may be altered to augment maladaptive vascular remodeling.

Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia

“…Anesthesia was maintained with endotracheal intubation and continuous isoflurane inhalant anesthesia (2% during painful stimuli, and 1% at latent periods). Vein bypass grafts were constructed with an anastomotic cuff technique as previously as described [ 11 , 12 ]. Bilateral carotid artery interposition grafting with jugular vein and unilateral distal carotid artery branch ligation were performed to create two distinct flow states.…”

“…Bilateral carotid artery interposition grafting with jugular vein and unilateral distal carotid artery branch ligation were performed to create two distinct flow states. Briefly, differential flow states between the right and left vein grafts were accomplished by ligation of the internal carotid artery and three of the four primary branches of the external carotid artery, resulting in an immediate six fold difference in mean shear stress [ 11 , 12 ]. Routine daily postoperative care and analgesia was provided, including daily neurologic assessment up to day 5 post surgery to identify any or all of head tilt, ear droop, or extremity weakness.…”

“…To determine the contribution of hemodynamic forces in the development of IH, we previously developed a model of bilateral rabbit carotid artery interposition grafts plus a unilateral distal outflow ligation to model differential flow states [ 11 , 12 ]. In this setting, we identified divergent patterns of vein graft remodeling under low versus high flow and shear stress [ 12 ].…”

“…To determine the contribution of hemodynamic forces in the development of IH, we previously developed a model of bilateral rabbit carotid artery interposition grafts plus a unilateral distal outflow ligation to model differential flow states [ 11 , 12 ]. In this setting, we identified divergent patterns of vein graft remodeling under low versus high flow and shear stress [ 12 ]. To further study the impact of hemodynamic forces after human vein arterial engraftment, we developed an ex-vivo human vein perfusion system (EVPS) [ 13 , 14 ] and observed a 7-day high-pressure perfusion (mean = 100 mmHg) to be sufficient for IH formation.…”

Multi-scale Modeling of the Cardiovascular System: Disease Development, Progression, and Clinical Intervention

“…The mechanisms behind how genes affect biological systems are currently being studied in vascular biology. For example, clinical studies have shown that a “single nucleotide in the p27 kip1 gene” affects the likelihood of success versus failure following vein bypass grafting 35 . The mechanical response of the graft to the high arterial pressure – or of medial-layer smooth muscle to loading in the case of arterial remodeling – should also be considered, so it is imperative that one understands the mechanical properties of the tissue at the continuum scale and also at the microscopic scale.…”