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Apoptosis of endothelial cells triggers a caspase-dependent anti-apoptotic paracrine loop active on VSMC

MARC-ANDRÉ RAYMOND, ANIK DÉSORMEAUX, PATRICK LAPLANTE, NORMAND VIGNEAULT, JANOS G. FILEP, KARINE LANDRY^*, ALEXEY V. PSHEZHETSKY^* and MARIE-JOSÉE HÉBERT²

Research Centre CHUM, University of Montreal, East, Montreal, Canada;
^* Ste-Justine Hospital Research Centre, University of Montreal, Montreal, QC, Canada; and
Guy-Bernier Research Centre, Montreal, Canada

² Correspondence: CRCHUM, 1560 Sherbrooke East, Montreal, QC, H2L 4M1, Canada. E-mail: marie-josee.hebert.chum{at}ssss.gouv.qc.ca

SPECIFIC AIMS

Increased endothelial apoptosis and decreased apoptosis of vascular smooth muscle cells (VSMC) are pivotal events for initiation of myo-intimal thickening. Yet mechanisms responsible for opposite regulations of apoptosis in endothelial and smooth muscle cells at sites of vascular injury are still largely undefined. We hypothesize that apoptosis of endothelial cells (EC) triggers the release of mediators, which in turn inhibit the apoptotic machinery of VSMC.

PRINCIPAL FINDINGS

1. Apoptosis of EC is associated with caspase-dependent release of paracrine anti-apoptotic factors for VSMC
Apoptosis of human umbilical vein endothelial cells (HUVEC) was induced with serum starvation for 4 h. Then, serum-free medium conditioned by endothelial cells (SSC) was collected, centrifuged and used to serum starve rat transformed aortic VSMC (rVSMC) for 24 h. Development of apoptosis was significantly decreased in rVSMC exposed to SSC compared with fresh serum-free medium (SS) (Fig. 1 A, B). We then evaluated whether apoptosis of EC is a prerequisite for release of this/these anti-apoptotic factors. HUVEC were exposed to a pan-caspase inhibitor (ZVAD-FMK, 100 µM) or vehicle (DMSO) for 2 h. ZVAD-FMK and vehicle were then removed and HUVEC were serum starved for 4 h, followed by harvesting of the conditioned media and evaluation of apoptosis in HUVEC. Preincubation with ZVAD-FMK significantly inhibited development of apoptosis in HUVEC whereas preincubation with DMSO did not (Fig. 1C ). These serum-free conditioned media were then used to serum-starve rVSMC and human aortic VSMC (AOSMC) for 24 h. Serum-free medium conditioned by EC in which development of apoptosis was inhibited (preincubation with ZVAD-FMK) failed to inhibit apoptosis of rVSMC and AOSMC (Fig. 1D ) whereas serum-free medium conditioned by apoptotic EC did. These results suggest that apoptotic EC release paracrine anti-apoptotic mediators downstream of caspase activation.

View larger version (48K): [in this window] [in a new window]   Figure 1. A) Left panel: % of apoptotic cells in rVSMC exposed for 24 h to normal medium (N), serum starvation (SS), or serum-free medium conditioned by HUVEC (SSC). *P < 0.01 vs. N, n 4. Right panel: representative micrographs (Magnification 400 X). Arrows point to apoptotic cells. B) Upper panel: cell cycle analysis of rVSMC exposed for 16 h to N, SS or SSC. Lower panel: % of rVSM in the different phases of the cell cycle * P < 0.006 vs. N, n = 6. C) % of apoptotic cells in normal HUVEC (N), HUVEC serum-starved for 4 h (SS), HUVEC preincubated with normal medium containing either ZVAD-FMK or DMSO for 2 h and then exposed for 4 h to serum-free medium (respectively SS+ZVAD and SS+DMSO). Preincubation with ZVAD-FMK significantly attenuated development of apoptosis in HUVEC compared with preincubation with DMSO. SS+ ZVAD vs. SS+ DMSO:P=0.02, n = 12. D) Left panel: % of apoptotic cells in rVSMC exposed for 24 h to serum-free medium (SS), medium conditioned by apoptotic HUVEC (SSC), medium conditioned by apoptotic HUVEC preincubated with DMSO (SSC+DMSO) or medium conditioned by HUVEC in which apoptosis was inhibited (SSC+ZVAD). SSC + ZVAD vs. SSC + DMSO:P=0.0025, n = 6. Right panel: % of apoptosis in AOSMC exposed for 48 h to SS, SSC, SSC+DMSO or SSC+ZVAD. SSC + ZVAD vs. SSC + DMSO: P < 0.05, n 9. E) Upper panel: Northern analysis. Increased expression of Bcl-xl and decreased expression of p53 in rVSMC exposed to SSC for 24 h compared with N and SS. Lower panel: immunoblotting analysis. Increased protein level of Bcl-xl in rVSMC exposed to SSC for 24 h compared with SS. The results are representative of 3 independent experiments. F) Immunoblotting analysis. Upper panel: increased protein level of Bcl-xl in AOSMC exposed to SSC for 48 h compared with N and SS. Lower panel: decreased protein level of p53 in AOSMC exposed to SSC for 48 h compared with SS. The results are representative of 3 independent experiments. G) Immunoblotting analysis. Increased levels of phosphorylated ERK 1/2 in rVSMC exposed to SSC for 15 min, 30 min and 4 h compared with SS. The results are representative of 3 independent experiments.

2. Mediators released by apoptotic EC increase ERK 1/2 phosphorylation, enhance Bcl-xl expression, and inhibit p53 expression in VSMC
Exposure of rVSMC and AOSMC to SSC for 24 h significantly decreased p53 mRNA and protein levels and significantly increased Bcl-xl mRNA and protein levels compared with SS (Fig. 1E, F ). rVSMC exposed to SSC for 15 min to 4 h showed increased phosphorylation of ERK1/2 (Fig. 1G ). Blockade of ERK1/2 activity in rVSMC and AOSMC with PD98059 resulted in a significant inhibition of the anti-apoptotic activity of SSC (data not shown). In keeping with an ERK 1/2 dependent anti-apoptotic phenotype, PD98059 was found to prevent the induction of Bcl-xl at the protein level in rVSMC (data not shown).

3. Identification of a carboxyl-terminal fragment of perlecan as a bioactive mediator present in medium conditioned by apoptotic endothelial cells
Characterization of the paracrine mediator(s) released by apoptotic EC was achieved with concentration and fractionation of medium conditioned by apoptotic EC using ultrafiltration followed by FPLC ion-exchange chromatography. Of the 46 fractions, only one showed a significant prosurvival activity on rVSMC. An 23 kDa protein band present exclusively in the bioactive fraction was identified by tandem mass spectrometry (MS/MS) as a fragment of the carboxyl-terminal domain V of perlecan.

Perlecan is a 467 kDa modular proteoglycan composed of five distinct domains. Since a protein of 23 kDa was found in the bioactive fraction, this suggested that proteolysis of perlecan was needed for liberation of a cryptic carboxyl-terminal fragment. Sequences identified by mass spectral analysis contained the carboxyl-terminal end of the domain V but did not allow precise identification of the amino-terminal start of the fragment (data not shown). Using ExPASy Molecular Biology Server we deduced an approximative amino-terminal cleavage site based on a molecular weight of 23 ± 2 kDa and determined that the chondroitin 4-sulfate anchoring site and one EGF motif were part of the fragment we isolated in SSC. Supplementation of serum-free medium with either chondroitin 4-sulfate or a synthetic peptide containing the last EGF motif (CQLREPCLHGGTCQGTRCLCLPGFSGPRCQQGSGHG) significantly inhibited apoptosis of rVSMC (Fig. 2 A) but did not increase their proliferative rate (Fig. 2B ). Supplementation of serum-free medium with chondroitin 4-sulfate or the synthesized peptide led to increased Bcl-xl protein levels in rVSMC and AOSMC (Fig. 2D ) and development of an anti-apoptotic phenotype in rVSMC and AOSMC dependent on sustained ERK 1/2 phosphorylation (Fig. 2C, E ). Hence, these results suggest that fragments of the domain V of perlecan mimic the anti-apoptotic activity of SSC on VSMC whereas other mediators, yet to be identified, regulate p53 protein level (Fig. 2F ) and cell cycle reentry.

View larger version (44K): [in this window] [in a new window]   Figure 2. A) % of apoptotic cells in rVSMC exposed for 24 h to serum-free medium supplemented with chondroitin 4-sulfate (CS, 60 µg/mL, 125 µg/mL or 250 µg/mL), or synthetic peptide (Pep, 1 µg/mL). * P < 0.05 compared with SS. n 4. B) BrdU incorporation in rVSMC exposed to SS, SSC, CS (60 µg/mL, 125 µg/mL or 250 µg/mL), or Pep (1 µg/mL) for 8 or 24 h. Only SSC was associated with a significant proliferative response. * P < 0.0001 compared with SS. n = 12. C) Immunoblotting analysis for phosphorylated and nonphosphorylated forms of ERK 1/2 in rVSMC exposed for 4 h to N, SS, SSC, CS (125 µg/mL), or Pep (1 µg/mL) compared with SS. * P < 0.05 vs. SS. D) Immunoblotting for Bcl-xl. Upper panel: increased protein levels of Bcl-xl were found in rVSMC exposed to SSC, CS (125 µg/mL), and Pep (1 µg/mL) for 24 h compared with SS. * P < 0.05 vs. SS. Lower panel: increased protein levels of Bcl-xl were found in AOSMC exposed to SSC, CS (125 µg/mL), and Pep (1 µg/mL) for 48 h compared with SS. * P < 0.05 vs. SS. E) Upper panel: % of apoptotic cells in rVSMC exposed for 24 h to N, SS, SSC, CS (125 µg/mL), or Pep (1 µg/mL) alone or with PD98059 (50 µM) or vehicle (DMSO). * P < 0.05 vs. SS, n 9. Lower panel: % of apoptotic cells in AOSMC exposed for 48 h to N, SS, SSC, CS (125 µg/mL), or Pep (1 µg/mL), either alone or with PD98059 (50 µM) or vehicle (DMSO). * P < 0.05 vs. SS, n 6. F) Immunoblot analysis. Decreased protein level of p53 was found in AOSMC exposed to SSC for 48 h compared with SS. * P < 0.05 vs. SS.

CONCLUSIONS AND SIGNIFICANCE

Our results demonstrate that apoptosis of EC triggers a paracrine anti-apoptotic loop active on VSMC. We show that apoptotic EC release mediator(s) that inhibit apoptosis and increase proliferation of VSMC whereas normal EC do not. Release of anti-apoptotic mediator(s) by apoptotic EC occurs downstream of caspase activation, as preincubation of EC with ZVAD-FMK blocks the development of this paracrine activity.

Initiation of myo-intimal thickening is known to be associated with increased phosphorylation of ERK 1/2, increased expression of Bcl-xl and decreased expression of p53 within myo-intimal cells. The paracrine activity induced by apoptosis of EC modulates precisely the expression and activation of these genes and pathways in VSMC. Hence, the mediator(s) produced by apoptotic EC induce, at the molecular level, an anti-apoptotic phenotype in VSMC that has been linked to neointima formation.

We identified a fragment of the carboxyl-terminal domain of perlecan (domain V) as a mediator of this anti-apoptotic paracrine activity. The domain V of perlecan contains an anchoring site for chondroitin sulfate and three laminin-type G modules separated by four EGF-like modules. Our results suggest that proteolytic enzymes activated during apoptosis of EC are responsible for liberation of a truncated form of perlecan, active on VSMC. Consistent with this contention, our results showed that native perlecan does not inhibit apoptosis of VSMC whereas both chondroitin 4-sulfate and a synthetic peptide containing the EGF motif present in the domain V of perlecan inhibit VSMC apoptosis and increased ERK 1/2 phosphorylation and Bcl-xl protein levels in VSMC.

The enzymes responsible for the proteolysis of perlecan during apoptosis of EC have yet to be characterized. We showed that this paracrine anti-apoptotic activity does not develop in EC in presence of pan-caspase inhibition, yet evaluation of the amino acid sequence of perlecan did not reveal any caspase cleavage site. We propose that an endoprotease(s) yet uncharacterized, activated downstream of caspases in apoptotic EC, is responsible for the proteolysis of perlecan (Fig. 3 ). Sustained production of a carboxyl-terminal fragment of perlecan could form the basis of a maladaptive response that may ultimately foster pathological remodeling at sites of chronic vascular injury.

View larger version (66K): [in this window] [in a new window]   Figure 3. Schematic overview. Apoptosis of EC leads to activation of caspases and downstream activation of an uncharacterized protease, which in turn cleaves the domain V of perlecan. VSMC exposed to this fragment of perlecan develop increased ERK 1/2 phosphorylation, which leads to increased Bcl-xL expression and resistance to apoptosis. Inhibition of VSMC apoptosis initiates myo-intimal thickening.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0573fje; doi: 10.1096/fj.03-0573fje

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