Immunocytochemical determination of cell type and proliferation rate in human vein graft stenoses

Purpose: Vascular reconstructions are prone to fail as a result of the development of stenotic lesions, which have historically been attributed to myointimal hyperplasia. In animal models, these lesions are associated with marked proliferative smooth muscle cell (SMC) response to vascular injury. However, recent studies using sensitive immunocytochemical techniques in human lesions have generally failed to detect significant cellular proliferation. To clarify the role of cellular proliferation in humans, we characterized the cellular composition and proliferative index of 14 early infrainguinal vein graft stenoses. Methods: All infrainguinal vein grafts at our institution are prospectively enrolled in a duplex surveillance protocol, the details of which have been previously reported. Among 98 grafts placed within the last year, 11 patients were identified with 14 progressive, focal, high-grade lesions that met previously established threshold criteria for prophylactic revision to prevent graft thrombosis. Lesions were first detected from 1 week to 7 months after surgery and were removed and replaced with segmental interposition grafts (1.5 to 10 months). Freshly excised lesions were placed in Methyl Carnoy's fixative, paraffin embedded, and serially sectioned. The cellular composition of each lesion was determined with cell-specific immunochemical reagents: α SMC actin, von Willebrand factor (endothelial cell), CD 68 (macrophage), and CD 45RB (monocyte). Actively proliferating cells were identified using antibody to proliferating cell nuclear antigen (PCNA). The identity of PCNA-positive cells was determined by double-label immunocytochemical staining, and the proliferative index (PCNA-positive cells/total cells × 100) was calculated by computer-assisted counts of representative gridded cross-sections of each lesion. Results: All excised lesions demonstrated marked thickening with severe luminal encroachment and were highly cellular, with a predominance of α SMC actin+. Endothelial cells on the blood flow surface were present to a variable degree, and seven lesions exhibited striking numbers of macrophages and monocytes. The latter cell types were most abundant near microvessels in the deep neointima and adventitia. Active cellular proliferation was identified primarily in SMCs, with a mean PCNA index of 1.34%. However, significant PCNA reactivity was not limited to SMCs, but was also identified in macrophages and monocytes, particularly in lesions greater