Markus J. Leskinen

Mast Cell Chymase Induces Smooth Muscle Cell Apoptosis by a Mechanism Involving Fibronectin Degradation and Disruption of Focal Adhesions

Objective—Chymase released from activated mast cells has been shown to induce apoptosis of vascular smooth muscle cells (SMCs) in vitro. The proteolytic activity of chymase is essential for the proapoptotic effect, but the mechanism of chymase-induced apoptosis has remained unknown. Methods and Results—Here we show by means of FACS analysis, immunohistochemistry, and Western blotting that mast cell–derived chymase induces SMC apoptosis by a mechanism involving degradation of an extracellular matrix component, fibronectin (FN), with subsequent disruption of focal adhesions. The FN degradation products induced SMC apoptosis of similar magnitude and with similar changes in outside-in signaling, as did chymase. Sodium orthovanadate, an inhibitor of tyrosine phosphatases, inhibited the chymase-induced SMC apoptosis. Focal adhesion kinase (FAK), one of the key mediators of integrin–extracellular matrix interactions and cell survival, was rapidly degraded in the presence of chymase or FN degradation products. Loss of phosphorylated FAK (p-FAK) resulted in a rapid dephosphorylation of the p-FAK–dependent downstream mediator Akt. Conclusions—The results suggest that chymase-secreting mast cells can mediate apoptosis of neighboring SMCs through a mechanism involving degradation of pericellular FN and disruption of the p-FAK–dependent cell-survival signaling cascade.

Regulation of smooth muscle cell growth, function and death in vitro by activated mast cells--a potential mechanism for the weakening and rupture of atherosclerotic plaques.

The fibrous cap of a lipid-containing atherosclerotic plaque consists of collagen produced by arterial smooth muscle cells (SMCs) of synthetic phenotype. A thick cap protects the lipid-rich core, whereas a thin cap predisposes it to rupture, with ensuing acute clinical complications, such as myocardial infarction. Among the pathological mechanisms leading to plaque weakening and rupture, one possibility is loss of the matrix-synthesizing SMCs. Indeed, caps of ruptured coronary plaques contain a reduced number of SMCs. In contrast, in such lesions, the number of activated inflammatory cells, such as mast cells, is increased, suggesting that they may regulate the SMC number. We have shown that heparin proteoglycans secreted by activated mast cells can efficiently inhibit proliferation of SMCs in vitro and reduce their ability to produce collagen. Chymase, a neutral serine protease secreted by activated mast cells, can also inhibit SMC-mediated collagen synthesis by a transforming growth factor-beta-dependent and -independent mechanism, and moreover, cause degradation of the collagen matrix by activating latent interstitial collagenase (MMP-1). Furthermore, chymase can induce SMC apoptosis by degrading the extracellular matrix component fibronectin necessary for SMC adhesion, with subsequent disruption of focal adhesions and loss of outside-in survival signaling. Thus, activated mast cells may participate in the weakening and rupture of atherosclerotic plaques by secreting mediators, such as heparin proteoglycans and chymase, which affect the growth, function and death of arterial SMCs.

Mast Cell Chymase Induces Apoptosis of Vascular Smooth Muscle Cells

In human coronary atheromas, the numbers of degranulated mast cells and of apoptotic smooth muscle cells (SMCs) are increased. Accordingly, the possibility exists that mast cells participate in the regulation of SMC apoptosis in the lesions. Mast cells isolated from the serosal cavities of rats were stimulated to release their secretory granules. The neutral protease chymase, present in the exocytosed granules, was found to induce apoptosis when added to rat aortic SMCs in culture. The chymase-induced apoptosis of SMCs was detected by flow cytometry, microscopic analysis of cellular morphology, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), and electrophoretic demonstration of DNA laddering. Chymase induced SMC apoptosis in a dose- and time- dependent manner, and its proteolytic activity was essential for the proapoptotic effect. In addition to rat chymase, recombinant human chymase was also found to induce apoptosis of human coronary artery SMCs in culture. These results suggest that mast cells may participate in the apoptotic regulation of SMCs in atherosclerotic lesions.

Proteolysis of the Pericellular Matrix: A Novel Element Determining Cell Survival and Death in the Pathogenesis of Plaque Erosion and Rupture

The 2 major general concepts about the cell biology of atherogenesis, growth of smooth muscle cells, and lipid accumulation in macrophages, ie, foam cell formation, have not been able to satisfactorily explain the genesis of acute coronary syndromes. Rather, the basic pathology behind the acute atherothrombotic events relates to erosion and rupture of unstable coronary plaques. At the cellular level, we now understand that a switch from cellular growth to cellular death, notably apoptosis, could be involved in turning at least some types of atherosclerotic plaques unstable. Because intimal cells require a proper matrix environment for normal function and survival, the vulnerability of an atherosclerotic plaque may critically depend on the integrity of the pericellular matrix of the plaque cells. In vitro studies have revealed that plaque-infiltrating inflammatory cells, such as macrophages, T-lymphocytes, and mast cells, by secreting a variety of proteases capable of degrading pericellular matrix components, induce death of endothelial cells and smooth muscle cells, and so provide a mechanistic explanation for inflammation-dependent plaque erosion and rupture. Thus, a novel link between inflammation and acute coronary syndromes is emerging. For a more explicit understanding of the role of proteases released by inflammatory cells in the conversion of a clinically silent plaque into a dangerous and potentially killing plaque, animal models of plaque erosion and rupture need to be established.

Activated Mast Cells Induce Endothelial Cell Apoptosis by a Combined Action of Chymase and Tumor Necrosis Factor-α

Objective—Activated mast cells (MCs) induce endothelial cell (EC) apoptosis in vitro and are present at sites of plaque erosions in vivo. To further elucidate the role of MCs in endothelial apoptosis and consequently in plaque erosion, we have studied the molecular mechanisms involved in MC-induced EC apoptosis. Methods and Results—Primary cultures of rat cardiac microvascular ECs (RCMECs) and human coronary artery ECs (HCAECs) were treated either with rat MC releasate (ie, mediators released on MC activation), rat chymase and tumor necrosis factor-&agr; (TNF-&agr;), or with human chymase and TNF-&agr;, respectively. MC releasate induced RCMEC apoptosis by inactivating the focal adhesion kinase (FAK) and Akt-dependent survival signaling pathway, and apoptosis was partially inhibited by chymase and TNF-&agr; inhibitors. Chymase avidly degraded both vitronectin (VN) and fibronectin (FN) produced by the cultured RCMECs. In addition, MC releasate inhibited the activation of NF-&kgr;B (p65) and activated caspase-8 and -9. Moreover, in HCAECs, human chymase and TNF-&agr; induced additive levels of apoptosis. Conclusions—Activated MCs induce EC apoptosis by multiple mechanisms: chymase inactivates the FAK-mediated cell survival signaling, and TNF-&agr; triggers apoptosis. Thus, by inducing EC apoptosis, MCs may contribute to plaque erosion and complications of atherosclerosis.

Mast cell-mediated apoptosis of endothelial cells in vitro: A paracrine mechanism involving TNF-α-mediated down-regulation of bcl-2 expression

Degranulated mast cells are present in the subendothelial space of eroded (de‐endothelialized) coronary atheromas. Upon degranulation, mast cells secrete into the surrounding tissue an array of preformed and newly synthesized mediators, including proapoptotic molecules, such as chymase and TNF‐α. In a co‐culture system involving rat serosal mast cells and rat cardiac (microvascular) endothelial cells, we could show, by means of competitive RT‐PCR, immunoblotting, immunocytochemistry, annexin staining, flow cytometry, and DNA‐laddering, that stimulation of mast cells with ensuing degranulation rapidly (within 30 min) down‐regulated the expression of both bcl‐2 mRNA and protein, with subsequent induction of apoptosis in the endothelial cells. The major effect of bcl‐2 down‐regulation resided in the exocytosed granule remnants, a minor effect also being present in the granule remnant‐free supernatant. No significant changes were observed in the expression levels of the pro‐apoptotic protein, bax. The mast cell‐mediated apoptotic effect was partially (70%) dependent on the presence of TNF‐α and involved the translocation of cytochrome C from mitochondria into cytoplasm. These results are the first to show that one of the cell types present in the atherosclerotic plaques, namely the mast cell, by releasing both granule‐remnant‐bound and soluble TNF‐α, may contribute to the erosion of atherosclerotic plaques by inducing apoptosis in adjacent endothelial cells. Published 2003 Wiley‐Liss, Inc.

Inhibition of smooth muscle cell proliferation by a chemically modified tetracycline.

IntroductionChemically modified tetracyclines (CMTs) are a group of nonantimicrobial derivatives of tetracycline, which exert antiproliferative and anticollagenolytic properties. The molecular mechanisms, however, are poorly understood. Materials and methodsThe effect of CMT-3 on cultured, subconfluent rat aortic smooth muscle cells (SMCs) was analyzed by [3H]-thymidine incorporation, counting cell numbers, and flow cytometry analysis. ResultsCMT-3 inhibited the incorporation of [3H]-thymidine and reduced the cell number dose-dependently, with ∼60% inhibition at the maximal CMT-3 concentration used (20 μmol/l). CMT-3 decreased the SMC proportion in S-phase and gradually increased the proportion at G2/M. Initially, the proportion of cells in G1-phase increased and then gradually decreased back to baseline as the CMT-3 concentration increased. CMT-3 treatment of confluent SMCs for 24 h did not induce apoptosis. ConclusionsCMT-3 inhibited SMC proliferation by inducing cell cycle arrest at the G2/M restriction point. Nonetheless, CMT-3 did not induce SMC apoptosis.

Mast cell-mediated apoptosis of endothelial cells in vitro: A paracrine mechanism involving TNF-α-mediated down-regulation of bcl-2 expression: MAST CELLS INDUCE ENDOTHELIAL CELL APOPTOSIS

Degranulated mast cells are present in the subendothelial space of eroded (de‐endothelialized) coronary atheromas. Upon degranulation, mast cells secrete into the surrounding tissue an array of preformed and newly synthesized mediators, including proapoptotic molecules, such as chymase and TNF‐α. In a co‐culture system involving rat serosal mast cells and rat cardiac (microvascular) endothelial cells, we could show, by means of competitive RT‐PCR, immunoblotting, immunocytochemistry, annexin staining, flow cytometry, and DNA‐laddering, that stimulation of mast cells with ensuing degranulation rapidly (within 30 min) down‐regulated the expression of both bcl‐2 mRNA and protein, with subsequent induction of apoptosis in the endothelial cells. The major effect of bcl‐2 down‐regulation resided in the exocytosed granule remnants, a minor effect also being present in the granule remnant‐free supernatant. No significant changes were observed in the expression levels of the pro‐apoptotic protein, bax. The mast cell‐mediated apoptotic effect was partially (70%) dependent on the presence of TNF‐α and involved the translocation of cytochrome C from mitochondria into cytoplasm. These results are the first to show that one of the cell types present in the atherosclerotic plaques, namely the mast cell, by releasing both granule‐remnant‐bound and soluble TNF‐α, may contribute to the erosion of atherosclerotic plaques by inducing apoptosis in adjacent endothelial cells. Published 2003 Wiley‐Liss, Inc.

Mast cell-mediated apoptosis of endothelial cells in vitro

Degranulated mast cells are present in the subendothelial space of eroded (de‐endothelialized) coronary atheromas. Upon degranulation, mast cells secrete into the surrounding tissue an array of preformed and newly synthesized mediators, including proapoptotic molecules, such as chymase and TNF‐α. In a co‐culture system involving rat serosal mast cells and rat cardiac (microvascular) endothelial cells, we could show, by means of competitive RT‐PCR, immunoblotting, immunocytochemistry, annexin staining, flow cytometry, and DNA‐laddering, that stimulation of mast cells with ensuing degranulation rapidly (within 30 min) down‐regulated the expression of both bcl‐2 mRNA and protein, with subsequent induction of apoptosis in the endothelial cells. The major effect of bcl‐2 down‐regulation resided in the exocytosed granule remnants, a minor effect also being present in the granule remnant‐free supernatant. No significant changes were observed in the expression levels of the pro‐apoptotic protein, bax. The mast cell‐mediated apoptotic effect was partially (70%) dependent on the presence of TNF‐α and involved the translocation of cytochrome C from mitochondria into cytoplasm. These results are the first to show that one of the cell types present in the atherosclerotic plaques, namely the mast cell, by releasing both granule‐remnant‐bound and soluble TNF‐α, may contribute to the erosion of atherosclerotic plaques by inducing apoptosis in adjacent endothelial cells. Published 2003 Wiley‐Liss, Inc.