Expression of tissue inhibitor of metalloproteinase-1, -2, and -3 during neointima formation in organ cultures of human saphenous vein

Degradation of the extracellular basement membrane is implicated in atherosclerosis, restenosis after angioplasty, and intimal thickening of vein grafts. Upregulation of metalloproteinase (MMP)-2 and MMP-9 accompanies neointima formation in cholesterol-fed rabbits, in rat and pig models of angioplasty, and in organ cultures of human saphenous veins. MMPs are inhibited by binding to tissue inhibitors of MMPs (TIMPs). Relatively little is known about their regulation in relationship to neointima formation; thus, we investigated TIMP expression in the organ culture model. Qualitative reverse transcriptase-polymerase chain reaction of mRNA extracted from veins showed that TIMP-1, TIMP-2, and TIMP-3 are each expressed before rind after culture. Zymography revealed that TIMP-1 was the most abundant TIMP secreted and that its secretion increased dramatically between 0 to 2 and 12 to 14 days of culture. An enzyme-linked immunosorbent assay showed that TIMP-1 secretion increased from 3.2+/-1.5 (mean+/-SE) to 32+/-6 ng/mg wet weight per day (n=5, P<0.01). Immunocytochemical testing localized the increased expression of TIMP-1 to neointimal smooth muscle cells. Although less abundant, TIMP-2 secretion also increased from 0.8+/-0.3 to 4.7+/-0.2 ng/mg wet weight per day (n=5, P<0.001), and tissue levels increased from 33+/-7 to 150+/-70 ng/mg wet weight (P<0.05). TIMP-2 was also immunolocalized to neointimal smooth muscle cells and their surrounding matrix. TIMP-3 was not secreted but was detected variably and constitutively in tissue extracts (160+/-120 and 170+/-100 ng/mg wet weight [n=9] on days 2 and 14, respectively). TIMP-3 was found in the cells and extracellular matrix of the media and adventitia before culture and to a lesser extent in the neointima after 14 days of culture. Rates of total TIMP secretion on day 14 exceeded those of MMP-2 and MMP-9 (10.6+/-1.9 and 15.6+/-2.3 ng/mg wet weight per day, respectively). Consistent with this, in situ zymography showed that MMP gelatinase activity was highly localized to cell bodies in the media and neointima. Secretion of TIMP-1 and TIMP-2 is greatly increased during neointima formation in human saphenous veins. TIMP-1 is readily released, whereas TIMP-2 remains partially attached and TIMP-3 exclusively attached to the extracellular matrix. Regulation of TIMP expression is therefore an important determinant of net MMP activity during neointima formation, restricting it to the pericellular environment.