α1-Proteinase Inhibitor, α1-Antichymotrypsin, or α2-Macroglobulin Is Required for Vascular Smooth Muscle Cell Spreading in Three-dimensional Fibrin Gel

Abstract

It is assumed that vitronectin and other adhesion molecules induce cell spreading. We found that vascular smooth muscle cells require unidentified plasma components besides adhesion molecules to spread in fibrin gel, a likely provisional matrix at wound sites. By purification, the plasma components were found to be ␣ 1 -proteinase inhibitor, ␣ 1 -antichymotrypsin, and ␣ 2 -macroglobulin. The chemically inactivated ␣ 1 -proteinase inhibitor and ␣ 2 -macroglobulin lose the spreading activity, indicating that these proteins function as proteinase inhibitors but not as adhesion molecules. Not only antiintegrin (␣ v ␤ 3 and ␣ 5 ␤ 1 ) antibodies but also anti-fibronectin antibodies inhibit the cell spreading. The spreading occurs without the addition of fibronectin and integrins, suggesting that cells produce these molecules. In the absence of the proteinase inhibitors, Western blot analysis shows that the fibronectin is degraded in fibrin gel, while it is intact in the presence of the inhibitors. Thus, the proteinase inhibitors prevent adhesion molecules such as fibronectin from being degraded by a cell-derived proteinase(s) and thus play a role in cell spreading.Under physiological states, especially in wound healing and vascular injury, cell spreading has been observed in threedimensional matrices. In most of the studies, however, spreading has been conducted on artificial two-dimensional surfaces rather than in three-dimensional matrices. Cell spreading relates to the cells' ability to migrate, proliferate (1), and resist apoptosis (2). While fibrin is known as a blood coagulant protein, it has also been implicated in several forms of tissue response to injuries including angiogenesis (3), vascular disease (4, 5), wound healing (6), and metastasis of cancer (7,8). During in vitro experiments, we unexpectedly found that human smooth muscle cells do not spread in fibrin gel unless serum or plasma was added. Vitronectin, which is identified as the serum-spreading factor (9, 10), surprisingly did not promote the spreading. We then tested other molecules such as fibronectin, osteopontin, thrombospondin, platelet-derived growth factor, and plasminogen activator inhibitor type-1 (PAI-1) 1 as possible spreading factors. However, none of these factors were able to promote the cell spreading. We then attempted to purify the cell spreading factor(s) from bovine plasma and found that these are serine proteinase inhibitors. MATERIALS AND METHODSCell Spreading in Fibrin Gel-Cultured human vascular smooth muscle cells (HNB18E6E7) were originally derived from the aorta of a newborn infant (2 days old) autopsy (11,12). The cells were detached by brief exposure to 0.05% trypsin (Life Technologies, Inc.), 0.02% EDTA. The remaining trypsin was inactivated by an excess of soybean trypsin inhibitor (Sigma). After washing with 20 mM phosphate, pH 7.4, containing 0.15 M NaCl, the cells were resuspended at 62,500 cells/49 l in serum-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 25 mM HEPES, pH 7.4. The cell ...

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