α1-Proteinase Inhibitor, α1-Antichymotrypsin, and α2-Macroglobulin Are the Antiapoptotic Factors of Vascular Smooth Muscle Cells*

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors α1-proteinase inhibitor, α1-antichymotrypsin, and α2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.

Serum depletion has been demonstrated to induce apoptosis (1). Integrin-mediated adhesion to extracellular matrix is required for growth and survival of many cell types. This is due in part to the fact that adhesion to extracellular matrix is required for progression of cells through the cell cycle by regulating cyclin D1 and cyclin E-Cdk2 (2). Disruption of adhesion arrests cells in the G 1 phase and causes apoptosis (3)(4)(5)(6)(7)(8). Signaling mediated by ␤3-integrin stimulates NF-B, which results in cell survival (9). Therefore, extracellular matrix proteins are required in addition to growth factors for cell survival.
It is sometimes difficult to keep smooth muscle cells in culture without serum despite the presence of extracellular matrix proteins and growth factors. This observation suggests that serum contains unidentified survival factors.
In a previous study, we showed that ␣1-proteinase inhibitor (␣1PI), 1 ␣1-antichymotrypsin (␣1ACT), and ␣2-macroglobulin (␣2M) are necessary for cell spreading and adhesion in fibrin gels (10). These proteinase inhibitors protect degradation of extracellular matrix proteins by cell-derived proteinases. Thus, the plasma proteinase inhibitors are essential to ensure appropriate cell-matrix interactions. Analysis of the cells that detach in the absence of proteinase inhibitors showed a similarity to apoptotic cells induced by serum depletion. Based on these results, we hypothesized that the proteinase inhibitors function as critical survival factors in vascular smooth muscle cells.

EXPERIMENTAL PROCEDURES
Induction of Cell Death-Cultured human vascular smooth muscle cells (HNB18E6E7) were originally derived from the aorta of a newborn infant (2-day-old) autopsy (11,12). The cells were detached by brief exposure to 0.05% trypsin, 0.02% EDTA. Trypsin was inactivated by excess amounts of soybean trypsin inhibitor (Sigma). After washing with PBS, the cells were resuspended in serum-free DMEM containing 0.2% bovine serum albumin, at a concentration of 50,000 cells/50 l. A 96-well culture plate was previously coated with either fibronectin (10 g/ml) or vitronectin (10 g/ml) at room temperature for 1 h. The cell suspensions (50 l) were plated on the 96-well culture plate. 50 l of each sample were overlaid. Final concentration of the samples was 5% calf serum, PDGF-BB (20 ng/ml), ␣1PI (0.125 mg/ml), ␣1ACT (0.125 mg/ml), and ␣2M (0.125 mg/ml) in DMEM with 0.2% bovine serum albumin. The plates were incubated at 37°C with 5% CO 2 for 24 h. Attached cells were counted using phase contrast microscopy. Fibronectin, vitronectin, ␣1PI, ␣1ACT, and ␣2M were purchased from CalBiochem. PDGF-BB was from Life Technologies, Inc. The experiments were repeated three times.
Western Blot Analysis-After observing cell morphology, 25 l of 25% SDS was added to the wells and shaken for 1 h. The samples were run on an 8% SDS-polyacrylamide gel electrophoresis, and proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad). The membrane was blotted with polyclonal anti-fibronectin antibody (R790), a kind gift provided by Dr. William G. Carter (Fred Hutchinson Cancer Research Institute, Seattle, WA), at 1:10,000 dilution, and the antigen was visualized with Renaissance Western blot chemiluminescence reagent (PerkinElmer Life Sciences).
Trypan Blue Exclusion Assay-After collecting floating cells, attached cells were exposed to 0.05% trypsin, 0.02% EDTA. Trypsin was inactivated by soybean trypsin inhibitor (Sigma). All the attached and detached cell populations were combined to determine the proportion of dead cells. Trypan blue (Life Technologies, Inc.) was mixed with cells (1:1), and trypan blue exclusion by living cells was scored using phase contrast microscopy.
TUNEL Staining-TUNEL staining was performed using the in situ Cell Death Detection Kit, Fluorescein (Roche Molecular Biochemicals cat. No. 1 684 795). The nuclei were counterstained with Hoechst at 5 g/ml.
Inactivation of ␣1PI and ␣2M-␣1PI was inactivated according to Johnson and Travis (13); 0.1 mg of human ␣1PI (CalBiochem) in 40 l * This study was supported by National Institutes of Health Grants HL-03174 and ROI HL-61860 and the Mitsui Life Social Welfare Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Effect of z-VAD-A 96-well culture plate was previously coated with fibronectin. Cell suspension of 50,000 cells/94.5 l were plated. Each sample was overlaid; 0.5 l of 20 mM z-VAD in Me 2 SO plus 5 l of PBS, 0.5 l of Me 2 SO plus 5 l of 0.125 mg/ml ␣2M in PBS, or 0.5 l of Me 2 SO plus 5 l of PBS. Final concentration of z-VAD was 0.1 mM and that of ␣2M was 6.25 g/ml. Cell morphology and trypan blue exclusion were performed as noted above.
Assay for Caspase Activity-Activity of caspase was determined using fluorescent caspase-specific substrate PhiPhiLux-G1D2 (Oncoimmunin) of which the main peptide sequence is GDEVDGI. Human vascular smooth muscle cells were cultured on a plate previously coated with fibronectin for 24 h with ␣2M at 1.7 ϫ 10 Ϫ7 M, without ␣2M, with z-VAD at 1 ϫ 10 Ϫ4 M or with staurosporin at 1 ϫ 10 Ϫ6 M. Cells were collected, then washed in PBS and incubated at 37°C for 1 h with 50 l of 10 M substrate solution. Flow cytometry analysis was performed at ex ϭ 505 nm and em ϭ 530 nm. Statistics-All experiments were repeated three times. Analysis of variance test was performed using SAS software version 6 in order to compare mean and variance.

RESULTS
Human vascular smooth muscle cells (HNB18E6E7) were plated in DMEM medium containing either calf serum, PDGF-BB, ␣1PI, ␣1ACT, or ␣2M on 96-well culture plate previously coated with either fibronectin or vitronectin. PDGF-BB induces cell division of HNB18E6E7 cells as shown by increased thymidine incorporation (data not shown). Attached cells were counted using a phase contrast microscope. At 1 h, cell attachment on the surface was similar. However, at 24 h, the numbers of attached cells were significantly different (Fig. 1A). Fig.   1B shows photographs at 24 h, when cells had detached and looked round upon serum depletion or in the presence of PDGF-BB. In contrast; with serum, ␣1PI, ␣1ACT, or ␣2M; cells remained attached and spread. A number of cells with calf serum was about 10% more than ␣1PI, ␣1ACT, or ␣2M, probably because the growth factors in serum supported proliferation.
To determine whether the endogenous cellular proteinases disrupted the cell matrix, we examined the integrity of fibronectin. Specifically, fibronectin was extracted by 5% SDS, and then Western blot analysis was performed. Fibronectin was intact with serum, ␣1PI, ␣1ACT, or ␣2M; however, it was degraded with PDGF or no serum (Fig. 1C). Therefore, the morphology of the cells depended on the integrity of adhesion molecules.
To determine how many cells were dead, we collected all the detached and attached cells and then counted the total number of dead cells by trypan blue exclusion test ( Fig. 2A). Dead cell numbers were significantly higher with no serum or PDGF alone than with serum or proteinase inhibitors. Next, we confirmed apoptosis using TUNEL staining although TUNEL detects apoptosis only of attached cells (Fig. 2B). TUNEL positivity was significantly higher with no serum or PDGF than other groups. These data suggest that proteinase inhibitors prevent degradation of matrix, which supports cell attachment and may result in cell survival.
Next, we quantified the survival effect of the proteinase inhibitors. ␣1PI, ␣1ACT, and ␣2M induce cell survival in a dose-dependent manner by the trypan blue exclusion test (Fig.  3A). In this assay, 50% survival was achieved at 4.4 ϫ 10 Ϫ8 M in ␣2M, at 3.3 ϫ 10 Ϫ7 M in ␣1PI, and at 4.2 ϫ 10 Ϫ7 M in ␣1ACT. Thus, ␣2M has a 7.6 times higher ability than ␣1PI and 9.6 times higher than ␣1ACT. We next removed both ␣1PI and ␣2M from human serum by immunoprecipitation. We did not have an appropriate antibody against ␣1ACT and thus could not deplete it. Ninety-five percent of the proteinase inhibitory activity was lost by the simultaneous removal of both proteins. The remaining 5% activity was probably due to the presence of ␣1ACT. The immunoprecipitated serum lost ϳ95% of survival activity, assessed by trypan blue (Fig. 3B). Furthermore, we tried an admixture of three proteins at normal serum level. Survival rate with the admixture was not statistically different from that with serum in this assay (Fig. 3B).
Chemical inactivation was performed on ␣1PI and ␣2M. ␣1PI was inactivated with N-chlorosuccinimide. ␣2M was inactivated with methylamine. Both ␣1PI and ␣2M lost their survival effect (Fig. 3C). These results indicate that the ability of cell survival is attributable to proteinase inhibitory activity.
We compared the proteinase inhibitors with a caspase inhibitor as survival factors. Cells attached on a plastic surface previously coated with fibronectin at 24 h with ␣2M (Fig. 4A); however, cells detached with no serum (Fig. 4B) or with z-VAD (Fig. 4C). The level of attached cells was significantly higher with ␣2M than other groups (Fig. 4D). In contrast, with z-VAD, the cells are alive (assessed by trypan blue exclusion) although many cells have detached (Fig. 4E). z-VAD had no effect on attachment although detached cells survived with z-VAD.
Next, we determined caspase activity using caspase-specific fluorescent substrate, PhiPhiLux-G1D2. With ␣2M, caspase activity was as low as with z-VAD (Fig. 5). However, without ␣2M, caspase activity was significantly high, similar to that with staurosporin. This suggests that the proteinase inhibitor not only supports attachment but also prevents activation of caspases.
The data suggest a novel mechanism for cell survival induced by plasma proteinase inhibitors. Cell survival is dependent on the ability of proteinase inhibitor to protect adhesion matrix. If the proteinase inhibitors are not present, the matrix is degraded by proteinases, and the cells cannot maintain attachment.

DISCUSSION
It is well documented that extracellular matrix and integrin interactions generate intracellular signals that lead to caspase activation (14 -16). However, combinations of adhesion molecules and growth factors alone are often insufficient to keep cells in culture alive without serum. Serum therefore, has been assumed to include unidentified survival factor(s). We have demonstrated that the critical antiapoptotic factors in serum are ␣1PI, ␣1ACT, and ␣2M. We also show that the mechanism of survival involves proteinase inhibitors, protecting the extracellular matrix from being degraded by cellderived proteinase(s).
Cell migration is required during development, in tissue repair and in wound healing. Degradation of the extracellular PBSϩPI indicates an admixture of the three proteinase inhibitors at normal serum level. Final concentration was equal to 2.5% serum in each well. The error bars represent S.D.; asterisk indicates p Ͻ 0.01 compared with negative control. C, ␣1-PI inactivated by N-succinichloramide (PI(Ϫ)) and native ␣1-PI were compared at each concentration. ␣2-M inactivated by methylamine (M(Ϫ)) and native ␣2-M were compared at each concentration. Death was quantified by trypan blue exclusion. matrix is a prerequisite for cell migration into the three-dimensional matrix. As part of this homeostatic process, cells produce proteinases, including matrix metalloproteinases (17) and/or serine proteinases. Careful regulation of proteolytic activity is required, as too much degradation will induce detachment that may result in cell death. In many cases, matrix proteinase activity is immediately neutralized. In addition to plasma proteinase inhibitors, many proteinase inhibitors such as tissue inhibitor of matrix metalloproteinases (TIMPS) have activity against specific cell-derived proteinases (18). Because a broad spectrum of proteases have apoptotic activity, it is reasonable to assume that many proteinase inhibitors are potentially antiapoptotic factors, acting via a mechanism similar to what we have shown here.
There is some controversy over whether TIMPs are proapoptotic or antiapoptotic. TIMP-3 induces apoptosis (19 -22) via a death domain located within the N terminus (23). Alexander et al. (24) showed that overexpression of TIMP-1 prevents apoptosis of epithelial cells from stromelysin-1 transgenic mice. Several other studies have shown antiapoptotic effects of TIMP-1 (25,26) and TIMP-2 (27). The role of proteinases is not limited to matrix degradation in vivo. For example, extracellular proteinases can directly modify intracellular signals via proteinase-activated receptors (28). Thus, the interaction between proteinases and proteinase inhibitors may influence a cells response to its environment in several ways.
Compared with the abundance of albumin and globulin, proteinase inhibitors are the third most prevalent group of plasma proteins. In our assay, neither albumin nor globulin demonstrated any antiapoptotic effect whereas ␣2M, a very nonspecific protease inhibitor, was very cytoprotective. ␣2M is an ancient protein that is found in all vertebrates and in nonvertebrates such as eels and crabs (29). There is speculation that ␣2M is a carrier of transforming growth factor-␤ and other cytokines (30) or that it functions as an agonist of ␣2M receptors (31). No ␣2M genetic deficiency has been found in our species despite extensive screening of human plasma (32). Furthermore, ␣2M knock-out mice have no phenotype (33). ␣1PI is a universal inhibitor of serine proteinases, which when diminished or mutated causes pulmonary emphysema and liver fi- brosis. Physiologically, ␣1PI is considered to be an elastase inhibitor (32). ␣1ACT is a chymotrypsin type serine proteinase inhibitor. ␣1ACT is recognized as an acute phase protein. Serum concentrations of ␣1ACT increase rapidly and dramatically after a variety of events including surgery, burn injury, inflammatory bowel disease, and some types of cancer (32). This probably indicates that an increase in ␣1ACT supports the survival of resident cells in areas of inflammation. Although the actual roles of the plasma proteinase inhibitors are not completely clarified, our data show a novel biological activity of these proteinase inhibitors.
One obvious question is what proteinase(s) does the smooth muscle cell produce? Based on the specificity of proteinase inhibitors, chymotrypsin type serine proteinases are candidates. We observed that this cell line produced chymotryptic activity when co-cultured with a chymogenic substrate (data not shown). Additional experiments using degenerate PCR primers and screening of a large gene expression array also identified a trypsin-like protease (manuscript in preparation).
In conclusion, we have shown a novel anti-apoptotic activity of plasma proteinase inhibitors. Because of the high concentration of ␣1PI and ␣2M, the proteinase inhibitors may function as the major antiapoptotic proteins present in serum. The proteinase inhibitors prevent extracellular matrix from degradation by cell-derived proteinase(s). This novel activity may suggest that the proteinase inhibitors could play a role in vascular disease such as atherosclerosis or aortic aneurysm.