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Caspases from apoptotic myocytes degrade extracellular matrix: a novel remodeling paradigm

Division of Cardiovascular Research, Departments of Pediatrics, Laboratory Medicine and Pathobiology, and Department of Medicine, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

² Correspondence: Wall Center for Pulmonary Vascular Disease and the Departments of Pediatrics, and Developmental Biology, Stanford University School of Medicine, Stanford University, CCSR-Room 2245B, 269 Campus Dr., Stanford 94305-5162, California, USA. E-mail: marlener{at}stanford.edu

SPECIFIC AIMS

The goals of this study were to: 1) determine whether vascular smooth muscle cell (SMC) apoptosis is coordinated with degradation of elastin in collagen gels; 2) determine the nature of the elastin degrading enzyme released by apoptotic SMCs; 3) determine whether caspases were exteriorized or released by apoptotic SMCs that can degrade elastin at the cell surface; 4) use relatively selective substrates and inhibitors to better define the caspases that could degrade elastin upon release from apoptotic cells; 5) determine which candidate elastin-degrading caspases specifically localized to smooth muscle cell surfaces in apoptotic cells; and 6) determine whether caspase inhibitors can prevent elastolysis by apoptotic SMCs.

PRINCIPAL FINDINGS

1. Apoptosis in vascular SMCs is coordinated with ECM/elastin degradation
We first documented that SMCs derived from a rat aorta cell line (A10) undergo apoptosis when placed on floating vs. attached collagen gels for 48 h, consistent with our studies using pulmonary artery SMCs. Apoptosis was assessed by DNA fragmentation, using propidium iodide flow cytometry (Fig. 1 A–C), and was correlated with electron microscopic evidence of apoptosis (not shown).

View larger version (27K): [in this window] [in a new window]   Figure 1. A10 SMCs induced to apoptose on floating collagen gels exhibit enhanced elastin degradation related to caspase activity. A–E)SMC apoptosis in floating vs. attached cultures. Representative flow cytometry plots of SMC cultures are shown in panels A (attached) and B (floating), where the "M1" region represents SMCs with fragmented or hypodiploid DNA, and plots are summarized in panel C. D) Changes in mitochondrial membrane potential. E) Corresponding changes in cell number. F) Representative viable SMC, indicated by the red MitoTracker mitochondrial stain, on an attached collagen gel impregnated with fluoresce in-conjugated elastin. G)Representative SMC on a floating collagen gel which is apoptotic, indicated by loss of the normal red mitochondrial stain, and associated with green fluorescence indicative of gel impregnated fluorogenic elastin degradation. H) Fluorogenic elastin in plate reader assays, normalized to human leukocyte elastase (HLE), with SMC culture membrane fractions alone or spiked with GM-6001 (0.8µM) (GM), a general MMP inhibitor, ZD0892 (2mM) (ZD), a serine elastase inhibitor, or the general caspase inhibitor Boc-D-FMK (100µM) (Boc). Bars = mean ± SEof n= 4 (C, D,E); n = 3 (H); scale bar = 10 µm; *P< 0.05 vs. the attached condition and P < 0.05 compared to the floating condition in the absence of inhibitors.

Next, we used the mitochondrial membrane dye DePsipher to identify individual apoptotic cells in culture and observed loss of mitochondrial membrane potential in association with SMC apoptosis on floating collagen gels (Fig. 1D ), related to a corresponding loss in SMC number (Fig. 1E ).

We used a different mitochondrial membrane potential dye, MitoTracker, in which apoptotic SMCs are nonfluorogenic and nonapoptotic cells exhibit a red fluorescence as observed on an attached collagen gel impregnated with an elastin substrate, which fluoresces green upon degradation (Fig. 1F ). Nonfluorogenic apoptotic SMCs on floating gels acquired a green fluorescence, indicative of elastin degradation (Fig. 1G ).

2. Caspases, and not classical matrix-degrading enzymes, mediate apoptosis-associated ECM degradation
Membranes from SMCs on floating collagen gels exhibited a major increase in elastolytic activity equivalent to 0.0045 units of human leukocyte elastase (HLE) activity (Fig. 1H ) using a fluorogenic elastin substrate in plate-reader assays. This activity could not be inhibited by spiking the floating culture SMC membrane fractions with either serine or metalloproteinase inhibitors in concentrations shown to be effective in previous studies. However, a broad spectrum caspase inhibitor (Boc-D-FMK) attenuated the elastolytic activity of the membrane fractions (Fig. 1H ).

3. Caspases-2, -3, and -7 are elastolytic
Using fluorogenic caspase substrates relatively selective toward various caspases, we documented heightened activity consistent with caspase-2, -3, -6, and -7 on membrane fractions from SMCs in floating vs. attached cultures (7.1-, 3.4-, 4.1-, and 5.1-fold, respectively). No activity was found using substrates with relative selectivity toward caspase-1, -4, -5, and -9. In addition, there was a 4.8-fold increase in activity consistent with caspase-2 and -6 detected in the conditioned media of floating vs. attached SMC cultures. While caspase-6 did not degrade elastin, caspase-2, -3, and -7 were potently elastolytic, exhibiting 0.1–0.2 HLE equivalent units of activity per µg of enzyme. This activity, which rose progressively at higher doses, as shown for caspase-3, was inhibited with increasing concentrations of the general caspase inhibitor (Boc-D-FMK) (0.50-fold suppression at 1µM concentration). Indeed, a more selective caspase-3 inhibitor (Ac-DMQD-CHO) showed enhanced inhibition of this activity (0.86-fold suppression at 1µM concentration).

4. Elastolytic caspases (2, 3, and 7) are active on the apoptotic SMC membrane
We confirmed that the elastolytic caspases-2, -3, and -7 are localized to the cell surface of apoptotic SMCs by immunocytochemistry on nonpermeabilized SMCs using an antibody recognizing the active and latent form of caspase-2 and antibodies to the active forms of caspase-3 and -7 (Fig. 2 A–F). Viable SMCs on attached collagen showed minimal or no binding of antibodies to caspase-2, -3, or -7 (Fig. 2A, B, C , respectively). In contrast, caspase-2, -3, and -7 (Fig. 2D, E, F , respectively) were detected on the surface of apoptotic SMCs both diffusely and in foci. The most pronounced caspase immunoreactivity appeared to be related to caspase-3. Since the immunostaining was carried out on nonpermeabilized cells, we presumed that these enzymes were on the cell surface, but to further document this, at least for caspase-3, Western immunoblot analysis for active caspase-3 was performed on cytosolic and membrane components following cellular fractionation (Fig. 2G and quantified in Fig. 2H ).Immunoreactivity indicated that minimal active caspase-3 is present on the membrane or in the cytosol of viable cells at the time of cell harvest. However, SMC apoptosis is associated with activation of caspase-3 both in the cytosol (13.7-fold increase) and on the membrane (16.2-fold increase) (Fig. 2G, H ).

View larger version (25K): [in this window] [in a new window]   Figure 2. Activated elastolytic caspases are targeted to the plasma membrane of apoptotic SMCs. A–F) Representative immunocytochemistry for caspase-2 (active and latent form) and for the active form of caspase-3 and -7, co labeled with MitoTracker (red mitochondria indicate a nonapoptotic cell) and DAPI, a nuclear counter stain. A–C) No cell surface staining with antibodies to caspase-2, -3, and -7, respectively, in nonapoptotic SMCs on attached collagen. D–F) Apoptotic SMCs on floating collagen stain positive with active caspase-2, -3, and -7 antibodies, respectively. G–H) Representative western immunoblot analyses for active caspase-3 and the plasma membrane marker, PMCA, in cytosolic and membrane fractions from viable (–) and apoptotic (+) SMCs are shown in G and quantified by densitometry in H. I) Flow cytometry-sorted apoptotic vs. nonapoptotic SMCs incubated with fluorogenic elastin in plate-reader assays in the presence and absence of either Boc-D-FMK (1 and 100µM) (Boc) or Ac-DMQD-CHO (100µM) (DMQD). Bars = mean ± SEof n = 3; scale bar = 10 µm for immunocytochemistry; *P< 0.05 vs. viable condition; P < 0.05 vs. apoptotic condition in the absence of inhibitors.

5. Elastin degradation associated with vascular SMC apoptosis is largely accounted for by caspase-3 activity
DePsipher fluorescence was used to sort, by flow cytometry, SMCs cultured on attached and floating collagen into viable and apoptotic populations. A 10-fold induction of elastolytic activity (0.0027 units of HLE) was observed in apoptotic vs. viable SMCs using the fluorogenic elastin assay (Fig. 2I ). This heightened activity could be progressively inhibited by addition of increasing concentrations of the general caspase inhibitor (Boc-D-FMK), such that the observed increase in elastolytic activity was entirely attenuated at 100µM (Fig. 2I ). This inhibition of elastolysis could be only partially reproduced using the more specific caspase-3 inhibitor (Ac-DMQD-CHO) (100µM) (Fig. 2I ), suggesting that caspase-2 and -7 might also contribute, consistent with the interdependent nature of caspases.

CONCLUSIONS AND SIGNIFICANCE

The present studies introduce a novel paradigm related to remodeling of blood vessels and perhaps other tissues by providing evidence that active caspases localized to the surfaces of apoptotic cells are released and are functional as matrix degrading enzymes. While it is not surprising that caspase-3, with a number of known substrates, could degrade ECM proteins including elastin, our data suggest that caspase-2 and -7 can also degrade elastin. Given that only one intracellular target has been described to date for caspase-2, its role as a cell surface or secreted matrix degrading enzyme may be its primary function.

Localization of caspases to the cell surface may be a function of binding following release, similar to the binding of MMPs-2 and -9 to the cell surface receptors _vß₃ integrin and CD44, respectively, or there may be a more elaborate mechanism of intracellular targeting to a membrane associated protein or receptor, or selective flipping as has been described for phosphatidyl serine. Nevertheless, the biochemical data coupled with the immunohistochemistry in nonpermeabilized cells suggest that caspase-2, -3, and -7 are present on the plasma membrane of apoptotic SMCs, and caspase-2 only is released into the conditioned medium.

Observations in disease and development are consistent with a highly orchestrated process of apoptosis and ECM degradation. Resorption of angiogenic vessels following _vß₃ integrin blockade is associated with apoptosis and ECM degradation. In the formation of the preamniotic cavity, apoptosis of ectodermal cells is accompanied by ECM resorption. When apoptosis related to the formation of digits was prevented, the requisite loss of ECM did not occur. Thus, our report is relevant to processes of tissue remodeling in development and disease as it links coordinated cell and extracellular matrix loss to the action of caspases.

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic representation showing ECM degradation orchestrated together with vascular SMC apoptosis to mediate regression of advanced vascular disease. Vascular SMCs, which participate in the progression of vascular disease through proliferation and abundant production of ECM, including elastin, can be induced to undergo apoptosis. Initiation of apoptosis is associated with caspase activation and leads not only to cellular loss but, also, to coordinated resorption of ECM through the exteriorization of ECM-degrading caspases. SMC, smooth muscle cell; ECM, extra cellular matrix; C-2, caspase-2; C-3, caspase-3; C-7, caspase-7.

FOOTNOTES

¹ Current address:Faculty of Medicine and Dentistry, University of Western Ontario, 339-80F11 Windermere Rd., London, Ontario N6A 5A5, Canada

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3706fje;

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