Human vascular smooth muscle cells of diabetic origin exhibit increased proliferation, adhesion, and migration.

PURPOSE: Patients with diabetes mellitus (DM) experience progressive macrovascular atherosclerosis and intimal hyperplastic restenosis with increased frequency as compared with nondiabetic patients. These observations suggest that vascular smooth muscle cells (VSMCs) behave in a phenotypically different and more aggressive manner in diabetic patients. In this study, we compared the in vitro rates of proliferation, adhesion, and migration of human VSMCs obtained from diabetic and nondiabetic patients. METHODS: Human VSMC cultures were isolated from 23 diabetic patients (9 artery, 14 vein) and 15 nondiabetic patients (9 artery, 6 vein) with extensive lower extremity atherosclerosis. All patients were between 61 and 78 years of age (average: 68.4 years [diabetic]; 67.3 years [nondiabetic]). All diabetic patients had type 2 DM. Vascular specimens were obtained at the time of amputation from infragenicular arteries and during arterial revascularization from saphenous veins. Cells from passages 2 and 3 were assayed for their proliferative capacity with total DNA fluorescence photometry and for adhesion and migration with a modified Boyden chamber. RESULTS: The average duration of diabetes was 11.6 +/- 4.1 years. The average number of diabetic complications (retinopathy, neuropathy, nephropathy, coronary artery disease) was 2.8 +/- 0.7 per patient. Diabetic VSMCs exhibited abnormal morphology in cell culture with loss of the normal hill and valley configuration. Proliferation was significantly increased in VSMCs of diabetic origin (156 +/- 57 absorption units) as compared with those of nondiabetic origin (116 +/- 42 absorption units) (P <.001). Diabetic VSMCs demonstrated significantly greater adhesion (63.6 +/- 24 per high-power field vs 37.9 +/- 13 per high-power field; P =.002) and migration (397 +/- 151 per low-power field vs 121 +/- 99 per low-power field; P =.001) rates. CONCLUSIONS: Diabetic VSMCs exhibit significantly increased rates of proliferation, adhesion, and migration as well as abnormal cell culture morphology suggestive of abnormal contact inhibition. These observations of human VSMCs in culture are consistent with the increased rate of infragenicular atherosclerosis and the increased rates of restenosis observed clinically in diabetic patients. The atherosclerosis- and intimal hyperplasia-promoting behavior exhibited appears to be intrinsic to the DM-VSMC phenotype and must be considered when designing methods to limit atherosclerosis and intimal hyperplasia in diabetic patients.

Relationship of the 20S proteasome and the proteasome activator PA28 to atherosclerosis and intimal hyperplasia in the human vascular system.

Down-regulation of the proteasome activator PA28 results in abnormal proteasome activation and has been implicated in the development of intimal hyperplasia (IH) in animal models. Demonstration of proteasome and PA28 expression has not yet been documented in the human vascular system. This study sought to define the distribution of the 20S proteasome and its activator PA28 in human vessels and determine the relationship between the expression of the proteasome and PA28 and the development of atherosclerosis and IH. Vascular biopsies were obtained from 70 patients at the time of surgery, were snap frozen and sectioned in 5-micron sections, and prepared using standard histological techniques. The immunoperoxidase technique was used to identify 20S proteasome and PA28 expression in diseased and normal human arteries and veins as well as in patent bypass grafts with and without IH. Expression was graded by a blinded pathologist (scale: 1-4). Repeat quantification of the immunopositive cells was also performed. Expression of 20S proteasome and PA28 was identified in all vascular tissues examined. The proteins were identified predominately within the cytoplasm of vascular smooth muscle cells and endothelial cells. PA28 was more intensely expressed in quiescent regions of the vessel wall as compared to areas undergoing active proliferation and remodeling. PA28-mediated activation of the proteasome may be necessary to maintain normal cellular homeostasis and prevent excessive cellular proliferation in the human vascular system. Abnormalities of proteasome activation may have a significant role in the development of atherosclerosis and IH.

In vivo assessment of a novel dacron surface with covalently bound recombinant hirudin.

Prosthetic arterial graft surfaces are relatively thrombogenic and fail to heal with a cellular neointima. The goal of this study was to characterize the in vivo antithrombin properties of a novel Dacron surface with covalently linked recombinant hirudin (rHir) implanted in a canine thoracic aorta with high flow and shear rates. rHir was bound to a knitted Dacron patch using crosslinker-modified bovine serum albumin (BSA) as a basecoat protein. BSA was first reacted with the heterobifunctional crosslinker, sulfo-SMCC. This BSA-SMCC complex was then bound to the carboxylic acid groups of hydrolyzed Dacron patches using the carbodiimide crosslinker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Iodinated, Traut's-modified rHir (125I-rHir-SH) was then reacted with the Dacron-BSA-SMCC surface, thereby covalently binding 125I-rHir. Graft segments were washed and sonicated to remove any nonspecifically bound 125I-rHir. Dacron-BSA-SMCC-S-125I-rHir patches (n = 5) and control Dacron-BSA patches (n = 5) were implanted in series in the thoracic aortas of canines. These patches were exposed to nonheparinized, arterial blood flow for 2 hours. Patches were explanted and assessed for 125I-rHir loss. Antithrombin activity of explanted 1-cm2 patch segments was evaluated using a chromogenic assay with 1, 5, 10, 15 units of added thrombin. Light microscopy was performed to qualitatively examine the pseudointima. Two animals were excluded from the study owing to excessive bleeding through the knitted 125I-rHir patch. Comparison of preoperative and postoperative 125I-rHir gamma counts revealed an overall decrease of 20+/-5.4% over the period studied. Explanted 125I-rHir patch segments were able to inhibit 1, 5, and 7 NIHU of thrombin, demonstrating retained antithrombin activity. Gross and microscopic examination of the control and test Dacron surfaces showed marked differences. Dacron surfaces with covalently bound 125I-rHir had no gross thrombus and a thin pseudointima of platelets and plasma proteins. In contrast, the control patches had a thick pseudointima composed of fibrin-rich thrombus. rHir, covalently bound to Dacron patches, maintains its biologic activity as well as prevents thrombus formation on the graft surface. This novel antithrombin coating, by modifying the blood/ graft interface, may improve both short- and long-term patency in small-diameter prosthetic arterial grafts and has applications with respect to other implantable or indwelling biomaterials.