Intimal Hyperplasia. Experimental and Clinical Studies

Abstract

Introduction: Intimal hyperplasia (IH) is recognized as the primary cause of reocclusion 2-24 months following vascular intervention and surgery, reportedly occurring in 30-50% of patients. It is an exaggerated form of the normal healing process of the iatrogenic trauma. The two main processes of IH are proliferation and migration of vascular smooth muscle cells (VSMCs) with a subsequent deposition of extracellular matrix. This accumulation of cells and matrix leads to a narrowing or, ultimately, occlusion of the lumen. IH usually affects arterial anastomoses, but a very distinct type of IH called pannus also exists. It occurs at the valvular annuli of the heart following implantation of valve prostheses and can obstruct valvular motion, thereby leading to acute heart failure. The treatment for IH is reintervention, resulting in major clinical issues for the patients and strain on the health care system. No prophylactic treatment currently exists. Heparins have demonstrated inhibitory effects in cell and animal experiments, but the results of clinical studies have been disappointing. There are no known predisposing factors enabling one to foresee who will develop clinically significant IH and who will not. This thesis sought to investigate the processes of IH in cell cultures, the effects of different heparins on these processes and the clinical manifestations in animals and patients. Paper I: The aim of this study was to investigate if heparin coated vascular grafts could improve patency and recruit less IH compared to uncoated grafts at a longer term in a sheep model. Twenty-eight common carotid arteries in fourteen sheep were bypassed with a luminal heparin-coated graft on one side and a control uncoated contralaterally. The grafts were explanted after six months. The thickness of IH in open grafts was measured with histomorphometrical methods. In conclusion, heparin coated grafts improved patency and recruited less IH compared to control uncoated grafts at six months in a sheep model. Paper II This study was designed to investigate the influence of heparin on the processes of IH of patient-derived vascular cell mono- and co-cultures. VSMCs and endothelial cells (ECs) were isolated from vessel wall biopsies from six patients. The cell cultures were treated with heparin and evaluated for effects on proliferation, migration and mitogen-activated protein kinase - extracellular signal-regulated kinase (MAPKERK) signal transduction using image-based cell enumeration, real-time migration monitoring and flow cytometry. Heparin inhibited proliferation and migration of patient-derived VSMC cultures, but only at concentrations exceeding clinical doses. The proliferative response of VSMCs to heparin was not affected by the presence of ECs. Heparin stimulated MAPK-ERK phosphorylation of VSMCs at lower concentrations in the presence of human fibroblast growth factor (hFGF). MAPK-ERK phosphorylation of unstimulated cultures was inhibited dose-dependently. Thus, heparin seems to influence patient-derived vascular cells through both a stimulatory hFGF-dependent and an inhibitory hFGF-independent pathway. These findings may explain the divergence of results between previous in vitro and clinical studies and provide a basis for new therapeutic strategies. Paper III This study was designed to investigate the effects of two low-molecular weight heparins (LMWHs), enoxaparin and dalteparin, compared to unfractioned heparin on proliferation, migration and MAPK-ERK signal transduction of patient-derived VSMCs. VSMCs were treated with the LMWHs in a range of concentrations and evaluated using image based cell enumeration, real time migration monitoring and flow cytometry. Series treated with unfractioned heparin were included as positive controls and untreated series as negative controls. This study demonstrated a difference in proliferative and migratory effects between the two LMWHs and unfractioned heparin in patient-derived VSMCs. The effects corresponded to the MAPK-ERK phosphorylation, suggesting different mechanisms of action. These results may explain why clinical trials using LMWHs to prevent IH have failed to observe a reduced incidence of restenosis and do not support prolonged therapeutic use to prevent it. Paper IV: The aim of this study was to analyse the incidence, treatment and results of acute prosthetic valve obstruction due to IH overgrowth originating from the periannular area (pannus), in a large series of patients with a Medtronic-Hall tilting disc aortic prosthesis. This was a retrospective study of patients seen at Haukeland University Hospital over the course of the last 30 years. Special attention was given potential predisposing factors of pannus development. This study demonstrated that females and younger patients are at higher risk for pannus development. When acute dysfunction of a mechanical aortic valve by pannus is suspected, an immediate echocardiogram and an emergency aortic valve replacement should be performed to prevent an otherwise fatal outcome.