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Pericellular plasmin induces smooth muscle cell anoikis¹

INSERM U460, CHU Bichat-Claude Bernard, 75018 Paris; and
^* INSERM U36, Collège de France 11, 75231 Paris Cedex 05, France

²Correspondence: INSERM U460, CHU Bichat-Claude Bernard, 46, rue Henri Huchard, 75877-Cdx, Paris 18, France. E-mail: u460{at}bichat.inserm.fr

SPECIFIC AIMS

Anoikis is a subtype of apoptosis induced by detachment of adherent cells from the extracellular matrix that could be involved in the smooth muscle cell disappearance observed in atherosclerotic plaque rupture and aneurysm formation. We sought to demonstrate that plasminogen activator-induced proteolysis is an intermediary mechanism of anoikis.

PRINCIPAL FINDINGS

1. Plasmin induces anoikis of VSMCs
In situ TUNEL reaction provided evidence of DNA fragmentation in adherent and floating plasmin-treated cells (Fig. 1 A2, A3, respectively) compared with untreated control cells (Fig. 1A1 ). After cytospinning the floating cells, hematoxylin/eosin (H/E) staining showed morphological changes such as nuclear condensation (black arrows), fragmentation (white arrow), and membrane blebbing (circled, Fig. 1A4 ). VSMCs treated with 0.25 µM plasmin for 16 h exhibited a DNA ladder profile characteristic of apoptosis (Fig. 1B ) as well as 40% cell detachment, assessed with the MTT test that quantifies the viable adherent cells remaining after plasmin stimulation (Fig. 1C ). TUNEL reaction performed on aortic segments treated ex vivo with 0.25 µM plasmin for 16 h showed DNA fragmentation: 40.5 ± 3% positive nuclei vs. 11 ± 2% in control (untreated aortas) (Fig. 1 D4 vs. D2). Analysis of the corresponding conditioned medium shows bands representing cellular (240 kDa) and remnant plasma fibronectin (70 kDa) fragments.

View larger version (88K): [in this window] [in a new window]   Figure 1. A) 1-3 TUNEL reaction performed on control (1) or plasmin-treated VSMCs (2: remaining adherent cells, 3: cytospun floating cells); 4: floating plasmin-treated VSMCs stained by H/E (4: nuclear condensation, : nuclear fragmentation, O: membrane blebbing). B) Internucleosomal DNA fragmentation. C)MTT quantification of viable adherent VSMCs after treatment with plasmin. D) TUNEL reaction (2, 4) or DAPI staining (1, 3) on adventitia-free rat abdominal aorta segments treated with 0.25 µM of plasmin for 16 h (3, 4) compared with untreated segments (1, 2). E) Fibronectin fragmentation detected by Western blot (0: control untreated aorta, Pn 0.25: aorta treated with 0.25 µM plasmin; : Fn: 0.5 ng of purified cellular fibronectin, Fn+Pn: 1 ng of cellular fibronectin incubated with 10 µl of 0.25 µM plasmin for 1 h at 37°C, Pn: 0.25 µM plasmin).

2. VSMC-tPA converts plasminogen to plasmin
Cultured VSMCs are able to convert plasminogen into plasmin in a time and dose-dependent manner. The cell extract but not the conditioned medium of VSMCs displays fibrinolytic activity corresponding to a free form of t-PA as indicated by its electrophoretic migration and inhibition in gels supplemented with 100 µg/mL purified anti-t-PA IgG. The conversion of plasminogen into plasmin by VSMCs was selectively inhibited by a t-PA inhibitor (dGGACK) but not by amiloride, a u-PA inhibitor. Altogether, these data indicate that in the cell system used in our experiments, constitutively expressed t-PA appears to be responsible for pericellular plasminogen activation and the subsequent generation of plasmin.

3. Deleterious effects of plasminogen activation: pericellular proteolysis of extracellular matrix-mediated anoikis
Plasmin generated from plasminogen by VSMCs induced cell retraction and death by anoikis. Similar results were obtained using human VSMCs. The pericellular generation of plasmin induced the degradation of extracellular matrix molecules such as fibronectin, which was prevented by ValPheLysCH₂Cl, a peptide that blocks the plasmin catalytic site. We also observed the 66 kDa active form of matrix metalloproteinase 2 (MMP2) in the culture medium of cells incubated with increasing concentrations of plasminogen. The use of GM6001, an MMP inhibitor, did not prevent fibronectin degradation, cell retraction and detachment, nor did it modify plasmin generation in our cell culture system, at concentrations known to inhibit MMPs (from 10 µM and up to 0.5 mM).

4. VSMC-mediated plasminogen activation: a pericellular process
Plasmin was detected by using a selective substrate and by Western blot. The activation of plasminogen was prevented by -amino-caproic-acid (EACA) (IC₅₀=0.9±0.2 mM), a competitor for plasminogen binding to the cell surface via its lysine binding sites. VSMCs were incubated with a saturating plasminogen solution (0.5 µM) and plasmin generation kinetics was monitored for 16 h in the presence of increasing concentrations of₂-antiplasmin (₂AP), the main physiological inhibitor of plasmin (Fig. 2A ). At the end of the kinetics, both cell-bound and released plasmin activity were measured in the cell extract and cell-conditioned medium (Fig. 2A , inset). ₂AP caused a concentration-dependent decrease in plasmin generation with an IC₅₀ = 64 nM. At a concentration of 31 nM, ₂AP totally quenched the plasmin present in the supernatant (Fig. 2A , inset , ) but failed to prevent cell retraction and detachment (Fig. 2C , ). Inactive plasmin/₂AP complexes were detected at 140 kDa by Western blot in the supernatant (Fig. 2B ). No trace of such complexes could be detected at the cell surface. These results indicate that plasmin released from the surface was immediately quenched by its specific inhibitor whereas cell-bound plasmin was still active (Fig. 2A , inset ). Anoikis of VSMCs could thus be attributed to pericellular proteolysis induced by plasmin generated at the surface of the cells by t-PA constitutively expressed by SMCs.

View larger version (28K): [in this window] [in a new window]   Figure 2. Activation of 0.5 µM plasminogen by VSMCs. A) Inhibition of plasmin generation by 2-antiplasmin (2AP) (•). Quantification of plasmin accumulated in the supernatant and membrane-bound plasmin (inset, and ). B) Western blot detecting 2AP in its free form () or as a complex with plasmin () in VSMC-conditioned medium. Complexes of 2AP:plasmin used as positive control. C) Viable adherent cells were quantified using the MTT test. indicates the 2AP concentration at which, despite the lack of free plasmin (A), there is marked cell detachment (C).

CONCLUSIONS

Using in vivo experimental models, it has been shown that plasmin generation is involved in such pathological processes as aneurysm formation and rupture. In the present study, we show that plasmin is indeed able to induce VSMCs detachment in vitro and ex vivo, leading to morphological and biochemical changes characteristic of anoikis. Our hypothesis is that under pathological conditions, plasmin could be generated in situ at the surface of the cells and be responsible for inducing anoikis by degrading pericellular adhesive glycoproteins such as fibronectin, vitronectin, or laminin, which participate in cell anchorage and survival signaling. Incubation of VSMCs with various concentrations of plasminogen induced a dose-dependent generation of plasmin at the surface of the cells, leading to VSMC anoikis. No addition of exogenous plasminogen activators was required to obtain these effects. Expression of t-PA and u-PA by VSMCs depends on various factors such as phenotype, potential extracellular stimulation, or a pathological context such as atherosclerosis. The cell fraction of VSMCs that we used for our study exhibited tPA activity. The presence of t-PA/PAI-1 complexes and the absence of t-PA activity in the supernatant suggest that PAI-1 is secreted in excess compared with t-PA. Excess of PAI-1 in the supernatant did not affect the activity of membrane-bound t-PA, confirming previously reported binding experiments.

Activation of plasminogen at the cell surface and anoikis were also inhibited by EACA, a competitor of plasminogen binding, suggesting that specific binding to cellular acceptors via carboxy-terminal lysine residues is necessary to convert plasminogen into plasmin. In our cell system, assembly of plasminogen and t-PA on the cell membrane is therefore required for the generation of plasmin in situ. Plasmin released into the conditioned medium could be trapped by addition of ₂AP, the physiological circulating inhibitor of plasmin.

The observation of anoikis in the absence of active plasmin in the supernatant adds further support to the concept of pericellular proteolysis as a surface-controlled process. Fibronectin is implicated in cell survival through activation of the focal adhesion kinase signaling pathway and could regulate expression of anti-apoptotic genes such as bcl-2. Disruption of fibronectin/integrin signaling has been reported to be the cause of serum-deprived thyroid cell anoikis and serum starvation-induced apoptosis of SMCs has been associated with cellular fibronectin degradation. Intracellular signaling linking fibronectin degradation to anoikis is not the focus of the present work and remains to be investigated in comparison with the well-explored intracellular signaling leading to spontaneous physiological anoikis. Plasmin is a wide-spectrum protease also able to cleave and activate directly or indirectly the matrix metalloproteinases. Despite the activation of MMP2 by plasmin, GM6001 (a broad-range inhibitor of MMPs) did not prevent plasmin-induced anoikis of VSMCs, in agreement with a recent report. This result suggests that plasmin might act as a pericellular proteolytic factor independent of the MMP activation system.

In conclusion, we provide evidence that constitutively expressed t-PA can generate plasmin at the surface of VSMCs in primary culture at concentrations sufficient to induce pericellular proteolysis and subsequent anoikis (Fig. 3 ). Pathological factors influencing expression of plasminogen activators by VSMCs and conditions favoring the bioavailability of plasminogen as a pericellular substrate remain to be further investigated. Such a cell-dependent phenomenon could be involved as an extracellular trigger capable of inducing cell disappearance in various vascular pathologies.

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic diagram. Plasmin-induced anoikis: a pericellular process. VSMCs can, under certain conditions, express plasminogen activators such as t-PA. Cell-associated t-PA converts plasminogen into plasmin, which may proteolyze the pericellular matrix leading to cell detachment and subsequent apoptosis, called anoikis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0687fje; doi: 10.1096/fj.02-0687fje

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