Yenfeng Wang

Activation of paracrine TGF-β1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase

As a source of transforming growth factor β1 (TGF‐β1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF‐β1 have remained vague. We show here by means of RT‐PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF‐β1 in their chymase 1‐containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF‐β1, which is converted by chymase 1 into an active form recognized by the type II TGF‐β serine/ threonine kinase receptor (TβRII). Thus, mast cells secrete active TGF‐β1 by a unique secretory mechanism in which latent TGF‐β1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF‐β1 specifically by chymase was verified using recombinant human latent TGF‐β1 and recombinant human chymase. In isolated TβRI‐ and TβRII‐expressing peritoneal macrophages, the activated TGF‐β1 induces the expression of the plasminogen activator inhibitor 1 (PAI‐1), whereas in the mast cells, the levels of TβRI, TβRII, and PAI‐1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF‐β1 in peritoneal mast cells and of TβRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF‐β1 and by enhancing its response in target cells.—Lindstedt, K. A., Wang, Y., Shiota, N., Saarinen, J., Hyytiäinen, M., Kokkonen, J. O., Keski‐Oja, J., Kovanen, P. T. Activation of paracrine TGF‐β1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase. FASEB J. 15, 1377–1388 (2001)

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.

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.

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.

Heparin Proteoglycans Released From Rat Serosal Mast Cells Inhibit Proliferation of Rat Aortic Smooth Muscle Cells in Culture

-Mast cells are present in the human arterial intima. To study whether mast-cell degranulation influences the rate of proliferation of smooth muscle cells, we cocultured sensitized (IgE-bearing) rat serosal mast cells and rat aortic smooth muscle cells (SMCs). When sensitized mast cells were stimulated to degranulate with antigen, the rate of proliferation of the cocultured SMCs decreased sharply. This inhibitory effect was found to be due mainly to the very high molecular weight (Mr) heparin proteoglycans (average Mr 750 000) released from the stimulated mast cells. When the heparin proteoglycans were purified from mast-cell granule remnants and added to the SMC culture, they were found to block the cell cycle at the G0-->S transition and the exit from the G2/M phase, their inhibitory effect resembling that of commercial heparin. However, in contrast to the reported dependence of the inhibitory effect of commercial heparin on the release of transforming growth factor-beta from serum, the inhibitory effect of the mast cell-derived heparin proteoglycans in the presence of serum was not transforming growth factor-beta dependent. Moreover, the effect of the mast cell-derived heparin proteoglycans was more efficient than that of commercial heparins of high (average Mr 15 000) and low (average Mr 5000) molecular weight. We also purified heparin glycosaminoglycans (average Mr 75 000) from the mast cell-derived heparin proteoglycans and found that they also inhibited SMC growth efficiently, although less strongly than their parent heparin proteoglycans. These results reveal, for the first time, that mast cells are able to regulate SMC growth. Thus, activated mast cells, by releasing heparin proteoglycans, possibly participate in the regulation of SMC growth in the human arterial intima, the site of atherogenesis.

Mast Cell Granule Remnants Carry LDL Into Smooth Muscle Cells of the Synthetic Phenotype and Induce Their Conversion Into Foam Cells

We report the effect of mast cells on the uptake of LDL by smooth muscle cells (SMCs) and their conversion into foam cells in vitro. The mast cells were stimulated to exocytose their cytoplasmic secretory granules, and the granule remnants formed were recovered from the extracellular fluid and added to cultures of SMCs of either the synthetic or contractile phenotype in LDL-containing medium. In the presence but not in the absence of granule remnants, SMCs of the synthetic phenotype took up LDL with ensuing stimulation of intracellular cholesteryl ester synthesis and cytoplasmic accumulation of neutral lipid droplets. Using methylated LDL (mLDL), a modified species of LDL that binds to granule remnants but not to LDL receptors, we demonstrated that this uptake (leading to foam cell formation) occurred only when LDL was bound to granule remnants. After the addition of colloidal gold-LDL and granule remnants to the incubation system, electron microscopy revealed that within phagosomes of the SMCs there were granule remnants (diameter, 0.5 to 1 micron) coated with LDL, confirming that LDL had been carried into the cells with the remnants. SMCs of the contractile phenotype were less efficient than their synthetic counterparts at phagocytosing LDL-coated granule remnants and were not converted into foam cells. This difference in the rate of phagocytosis of granule remnants was present even in the absence of LDL, revealing that the more active phagocytosis by SMCs of the synthetic phenotype was not specifically related to uptake of lipids but rather reflected a general phenotype characteristic of these cells. These observations indicate a phagocytic mechanism by which SMCs of the synthetic phenotype are converted into cholesteryl ester-filled foam cells, and they also suggest that degranulation of mast cells plays a role in the development of fatty streak lesions.

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.