alpha 1-Proteinase Inhibitor, alpha 1-Antichymotrypsin, and alpha 2-Macroglobulin Are the Antiapoptotic Factors of Vascular Smooth Muscle Cells*

Yuji IkariDagger §, Eileen MulvihillDagger , and Stephen M. SchwartzDagger

From the Dagger  Department of Pathology, University of Washington, Seattle, Washington 98195 and the § Division of Cardiology, Mitsui Memorial Hospital, Tokyo 101-8643, Japan

Received for publication, September 18, 2000, and in revised form, November 10, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 G1 phase and causes apoptosis (3-8). Signaling mediated by beta 3-integrin stimulates NF-kappa 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 alpha 1-proteinase inhibitor (alpha 1PI),1 alpha 1-antichymotrypsin (alpha 1ACT), and alpha 2-macroglobulin (alpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 µl. A 96-well culture plate was previously coated with either fibronectin (10 µg/ml) or vitronectin (10 µg/ml) at room temperature for 1 h. The cell suspensions (50 µl) were plated on the 96-well culture plate. 50 µl of each sample were overlaid. Final concentration of the samples was 5% calf serum, PDGF-BB (20 ng/ml), alpha 1PI (0.125 mg/ml), alpha 1ACT (0.125 mg/ml), and alpha 2M (0.125 mg/ml) in DMEM with 0.2% bovine serum albumin. The plates were incubated at 37 °C with 5% CO2 for 24 h. Attached cells were counted using phase contrast microscopy. Fibronectin, vitronectin, alpha 1PI, alpha 1ACT, and alpha 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 µl 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 µg/ml.

Inactivation of alpha 1PI and alpha 2M-- alpha 1PI was inactivated according to Johnson and Travis (13); 0.1 mg of human alpha 1PI (CalBiochem) in 40 µl of 0.1 M Tris buffer, pH 8.8 was mixed with 10 µl of 80 mM N-chlorosuccinimide (Sigma) and incubated overnight. The excess of the reagent was removed by dialysis. The remaining antitrypsin activity was less than 1 × 10-4 of the original activity. alpha 2M (CalBiochem) was inactivated by treating with 0.2 M methylamine (Sigma) in 50 mM Tris, pH 8.0 for 6 h at room temperature. After dialysis, the remaining activity was less than 1 × 10-4.

Effect of z-VAD-- A 96-well culture plate was previously coated with fibronectin. Cell suspension of 50,000 cells/94.5 µl were plated. Each sample was overlaid; 0.5 µl of 20 mM z-VAD in Me2SO plus 5 µl of PBS, 0.5 µl of Me2SO plus 5 µl of 0.125 mg/ml alpha 2M in PBS, or 0.5 µl of Me2SO plus 5 µl of PBS. Final concentration of z-VAD was 0.1 mM and that of alpha 2M was 6.25 µg/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 alpha 2M at 1.7 × 10-7 M, without alpha 2M, with z-VAD at 1 × 10-4 M or with staurosporin at 1 × 10-6 M. Cells were collected, then washed in PBS and incubated at 37 °C for 1 h with 50 µl of 10 µM substrate solution. Flow cytometry analysis was performed at lambda ex = 505 nm and lambda em = 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human vascular smooth muscle cells (HNB18E6E7) were plated in DMEM medium containing either calf serum, PDGF-BB, alpha 1PI, alpha 1ACT, or alpha 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, alpha 1PI, alpha 1ACT, or alpha 2M; cells remained attached and spread. A number of cells with calf serum was about 10% more than alpha 1PI, alpha 1ACT, or alpha 2M, probably because the growth factors in serum supported proliferation.



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Fig. 1.   Cell attachment on fibronectin. A, vascular smooth muscle cells were cultured on a plastic surface previously coated with fibronectin. Culture medium was DMEM with 0.2% bovine serum albumin, pH 7.4, containing either 5% calf serum (CS), no serum (N), platelet-derived growth factor (PDGF) at 20 ng/ml, alpha 1-proteinase inhibitor (PI), alpha 1-antichymotrypsin (ACT), and alpha 2-macroglobulin (M) at 0.125 mg/ml. Attached cell numbers were counted using a phase contrast microscope at 1 h and 24 h. N and PDGF were significantly lower than the other four groups, p < 0.001. B, photographs were taken at 24 h with a phase contrast microscope. Previous coat on plastic surface was either fibronectin (FN) or vitronectin (VN) (original magnification, × 100). C, Western blot analysis for fibronectin. After observation of cell morphology as shown in B, the coated fibronectin was solubilized with 5% SDS. Each sample was electrophoresed on 8% SDS-polyacrylamide gels, transferred to a polyvinylidene difluoride membrane, and blotted with a polyclonal antibody against fibronectin.

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, alpha 1PI, alpha 1ACT, or alpha 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.



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Fig. 2.   Quantification of dead cells. A, all the detached and attached cells were collected after observation of cell morphology as shown in Fig. 1. Dead cells were counted using trypan blue exclusion. The error bars represent S.D.; asterisk represents p < 0.05 compared with the no serum group. B, TUNEL staining for the attached cells shown in Fig. 1. The attached cells were double stained with TUNEL and Hoechst. Positivity was counted using a fluorescent microscope. The error bars represent S.D.; asterisk represents p < 0.05 compared with the no serum group.

Next, we quantified the survival effect of the proteinase inhibitors. alpha 1PI, alpha 1ACT, and alpha 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 × 10-8 M in alpha 2M, at 3.3 × 10-7 M in alpha 1PI, and at 4.2 × 10-7 M in alpha 1ACT. Thus, alpha 2M has a 7.6 times higher ability than alpha 1PI and 9.6 times higher than alpha 1ACT. We next removed both alpha 1PI and alpha 2M from human serum by immunoprecipitation. We did not have an appropriate antibody against alpha 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 alpha 1ACT. The immunoprecipitated serum lost ~95% 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).



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Fig. 3.   Survival effect of proteinase inhibitors. A, trypan blue exclusion was performed for all the attached and detached cells. Each proteinase inhibitor worked as a survival factor in a dose-dependent manner. B, alpha 1PI and alpha 2M were subtracted from normal human serum by immunoprecipitation (serum-PI). PBS indicates a negative control. PBS+PI 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 < 0.01 compared with negative control. C, alpha 1-PI inactivated by N-succinichloramide (PI(-)) and native alpha 1-PI were compared at each concentration. alpha 2-M inactivated by methylamine (M(-)) and native alpha 2-M were compared at each concentration. Death was quantified by trypan blue exclusion.

Chemical inactivation was performed on alpha 1PI and alpha 2M. alpha 1PI was inactivated with N-chlorosuccinimide. alpha 2M was inactivated with methylamine. Both alpha 1PI and alpha 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 alpha 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 alpha 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.



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Fig. 4.   Smooth muscle cells were cultured on a plastic surface coated with fibronectin. The following samples were added to culture medium; alpha 2M at 0.125 mg/ml plus Me2SO (A, alpha 2M); Me2SO (B, N, negative control); or z-VAD at 50 µM in Me2SO (C, zVAD). Photographs were taken with a phase contrast microscope (original magnification, × 100). D, attached cells were counted using a phase contrast microscope. The error bars represent S.D.; asterisk indicates p < 0.01 compared with the negative control. E, all the detached and attached cells were collected, and death was quantified by trypan blue exclusion. Asterisk indicates p < 0.01 compared with negative control. With z-VAD, cells were alive even though many cells were detached.

Next, we determined caspase activity using caspase-specific fluorescent substrate, PhiPhiLux-G1D2. With alpha 2M, caspase activity was as low as with z-VAD (Fig. 5). However, without alpha 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.



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Fig. 5.   Caspase activity was determined using fluorescent substrate PhiPhiLux-G1D2 (see "Experimental Procedures"). Human vascular smooth muscle cells were cultured on a plate previously coated with fibronectin with alpha 2M at 1.7 × 10-7 M (A), without alpha 2M (B), with z-VAD at 1 × 10-4 M (C) or with staurosporin at 1 × 10-6 M (D). Intensity of fluorescence was measured using cell sorter. Mean of PhiPhiLux activity (E) and percent of cell numbers in a gait (M1) were plotted (F). St represents staurosporin. Each experiment was repeated three times. Asterisk represents p < 0.05 compared with alpha 2M(-).

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 1PI, alpha 1ACT, and alpha 2M. We also show that the mechanism of survival involves proteinase inhibitors, protecting the extracellular matrix from being degraded by cell-derived proteinase(s).

Cell migration is required during development, in tissue repair and in wound healing. Degradation of the extracellular 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 alpha 2M, a very nonspecific protease inhibitor, was very cytoprotective. alpha 2M is an ancient protein that is found in all vertebrates and in non-vertebrates such as eels and crabs (29). There is speculation that alpha 2M is a carrier of transforming growth factor-beta and other cytokines (30) or that it functions as an agonist of alpha 2M receptors (31). No alpha 2M genetic deficiency has been found in our species despite extensive screening of human plasma (32). Furthermore, alpha 2M knock-out mice have no phenotype (33). alpha 1PI is a universal inhibitor of serine proteinases, which when diminished or mutated causes pulmonary emphysema and liver fibrosis. Physiologically, alpha 1PI is considered to be an elastase inhibitor (32). alpha 1ACT is a chymotrypsin type serine proteinase inhibitor. alpha 1ACT is recognized as an acute phase protein. Serum concentrations of alpha 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 alpha 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 alpha 1PI and alpha 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.


    ACKNOWLEDGEMENT

This study was supported by NIH HL-03174, ROI HL-61860 and Mitsui Life Social Welfare Foundation.


    FOOTNOTES

* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Mitsui Memorial Hospital, 1, Kanda-Izumi-cho, Chiyoda-ku, Tokyo 101-8643, Japan. Fax: 81-3-5687-9765; E-mail: ikari-tky@umin.ac.jp.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008503200


    ABBREVIATIONS

The abbreviations used are: alpha 1PI, alpha 1-proteinase inhibitor; alpha 1ACT, alpha 1-antichymotrypsin; alpha 2M, alpha 2-macroglobulin; PDGF, platelet-derived growth factor; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; z-VAD, benzyloxycarbonyl-VAD; TUNEL, terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labeling.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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