Miriam Lee

Chymase bound to heparin is resistant to its natural inhibitors and capable of proteolyzing high density lipoproteins in aortic intimal fluid

Degranulated mast cells are present in the human arterial intima. After degranulation of rat serosal mast cells, the secreted neutral serine protease chymase remains bound to the heparin proteoglycan matrix of the exocytosed granules, forming granule remnants. Addition of granule remnants to human aortic intimal fluid results in proteolysis of the apoAI present in the intimal fluid, which contains physiological inhibitors of chymase. To study the physiological mechanism of this protection of granule remnant-bound chymase against its inhibitors, we performed experiments using HDL3 as substrate. Chymase, when bound to the heparin proteoglycans of granule remnants, but not when released from them, resisted inhibition by the mammalian protease inhibitors alpha1-antitrypsin, alpha2-antichymotrypsin, alpha2-macroglobulin, and eglin C. Importantly, the heparin proteoglycan-bound chymase, but not unbound chymase, degraded its inhibitor (alpha1-antitrypsin) in the presence of its substrate (HDL3). Finally, binding to heparin proteoglycans of a physiological inhibitor of chymase (mucus protease inhibitor (MPI)) or of another substrate of chymase (LDL) did not inhibit the degradation of HDL3 by granule remnant-bound chymase. This study demonstrates that binding of chymase to the heparin proteoglycan chains of the exocytosed mast cell granules allows the protease to remain active and degrade HDL3 in the presence of its physiological inhibitors and in the presence of high concentrations of LDL, such as are found in the interstitial fluid of the arterial intima.

Mast Cell Tryptase Degrades HDL and Blocks Its Function as an Acceptor of Cellular Cholesterol

Objective—In human atherosclerotic lesions, degranulated mast cells are found in the vicinity of macrophage foam cells. Mast cell granules contain tryptase, a tetrameric serine protease requiring glycosaminoglycans for stabilization. No endogenous inhibitors have been described for tryptase, and the physiological functions of the enzyme are poorly understood. Here, we investigated the effects of human tryptase on the integrity of high density lipoprotein (HDL)3 and on its ability to release cholesterol from cultured mouse macrophage foam cells. Methods and Results—Incubation of HDL3 with tryptase led to degradation of its apolipoproteins. Tryptase predominantly degraded a quantitatively minor subfraction of HDL3 that is lipid poor, exhibits electrophoretic pre-&bgr; mobility, and contains either apolipoprotein A-I or apolipoprotein A-IV as its sole apolipoprotein. Moreover, tryptase caused functional changes in HDL3 by destroying its ability to promote high-affinity efflux of cholesterol from macrophage foam cells, ie, the pre-&bgr;-HDL-dependent component of the process. Human aortic proteoglycans increased the ability of tryptase to proteolyze HDL3, suggesting that the proteoglycan-rich extracellular matrix of the arterial intima provides an appropriate environment for the extracellular actions of tryptase. Conclusions—By depleting pre-&bgr;-HDL, mast cell tryptase may impair the initial step of reverse cholesterol transport and will then favor cellular accumulation of cholesterol during atherogenesis.

Mast Cell Chymase Degrades ApoE and ApoA-II in ApoA-I–Knockout Mouse Plasma and Reduces Its Ability to Promote Cellular Cholesterol Efflux

Objective—Mast cell chymase is a chymotryptic heparin proteoglycan–bound neutral protease that exerts its activity in extracellular fluids. We studied the effect of chymase on the apolipoprotein compositions and the abilities of plasmas from apolipoprotein (apo)A-I–knockout (A-I-KO) and wild-type (C57BL/6J) mice to stimulate efflux of cellular cholesterol from mouse macrophage foam cells. Methods and Results—The A-I-KO apolipoproteins compared with the wild-type (apoA-I, apoA-II, apoA-IV, and apoE) showed total lack of apoA-I, unaltered apoA-II, an absence of apoA-IV, and an increase of apoE. Despite these major differences, the 2 plasmas induced similar high-affinity efflux of cholesterol from the foam cells. Quantitative analysis of chymase-treated plasmas revealed (1) in A-I-KO plasma, complete loss of apoE and apoA-II, and (2) in wild-type plasma, slight reduction of apoA-I associated with complete depletion of the minor pre-&bgr;-high density lipoprotein fraction, strong reduction of apoA-II, and complete depletion of apoA-IV and apoE. Both proteolyzed plasmas had lost the ability to induce cellular cholesterol efflux with high affinity. Addition of discoidal pre-&bgr;-migrating reconstituted high density lipoprotein particles containing human apoA-I or apoA-II to the chymase-treated A-I-KO plasma fully restored its cholesterol efflux–inducing ability, indicating functional replacement of the proteolyzed apoE and apoA-II. Thus, chymase degraded all the nondeleted apolipoproteins of the A-I-KO plasma involved in the high-affinity efflux of cellular cholesterol. Conclusions—This is the first indication that genetically engineered mice could be used as models for examining the hypothesis that extracellular proteases are involved in the development of atherosclerosis by inhibiting the apolipoprotein-mediated removal of macrophage cholesterol.

Apolipoprotein composition and particle size affect HDL degradation by chymase effect on cellular cholesterol efflux

Mast cell chymase, a chymotrypsin-like neutral protease, can proteolyze HDL3. Here we studied the ability of rat and human chymase to proteolyze discoidal preβ-migrating reconstituted HDL particles (rHDLs) containing either apolipoprotein A-I (apoA-I) or apoA-II. Both chymases cleaved apoA-I in rHDL at identical sites, either at the N-terminus (Tyr18 or Phe33) or at the C-terminus (Phe225), so generating three major truncated polypeptides that remained bound to the rHDL. The cleavage sites were independent of the size of the rHDL particles, but small particles were more susceptible to degradation than bigger ones. Chymase-induced truncation of apoA-I yielded functionally compromised rHDL with reduced ability to promote cellular cholesterol efflux. In sharp contrast to apoA-I, apoA-II was resistant to degradation. However, when apoA-II was present in rHDL that also contained apoA-I, it was degraded by chymase. We conclude that chymase reduces the ability of apoA-I in discoidal rHDL particles to induce cholesterol efflux by cleaving off either its amino- or carboxy-terminal portion. This observation supports the concept that limited extracellular proteolysis of apoA-I is one pathophysiologic mechanism leading to the generation and maintenance of foam cells in atherosclerotic lesions.

Mast cell-mediated inhibition of reverse cholesterol transport.

Net cholesterol efflux from cholesterol-loaded macrophages, i.e., foam cells, was induced by incubating the foam cells with high density lipoprotein3 (HDL3). However, when the incubation system included rat serosal mast cells stimulated to trigger exocytosis of their cytoplasmic secretory granules, the ability of HDL3 to induce cholesterol efflux was largely lost. This loss was found to be due to the proteolytic action of chymase, the neutral protease of the granules, which degraded the apolipoproteins of HDL3, so rendering them unable to mediate cholesterol efflux from the foam cells. The observation defines a novel cell-dependent mechanism that blocks the initial steps of reverse cholesterol transport and suggests a role for mast cell chymase in cellular accumulation of cholesterol, an early stage in atherogenesis.

Depletion of Preβ1LpA1 and LpA4 Particles by Mast Cell Chymase Reduces Cholesterol Efflux From Macrophage Foam Cells Induced by Plasma

Exposure of the LpA1-containing particles present in HDL3 and plasma to a minimal degree of proteolysis by the neutral protease chymase from exocytosed rat mast cell granules (granule remnants) leads to a reduction in the high-affinity component of cholesterol efflux from macrophage foam cells. In this study, we demonstrate for the first time, a role for mast cell chymase in the depletion of the lipid-poor minor components of HDL that are specifically involved in reverse cholesterol transport as initial acceptors of cellular cholesterol. Thus, addition of proteolytically active granule remnants or human skin chymase to cholesterol-loaded macrophages of mouse or human origin incubated with human apoA1, ie, a system in which prebeta1LpA1 is generated, resulted in a sharp reduction in the high-affinity cholesterol efflux promoted by apoA1. As determined by nondenaturing 2-dimensional polyacrylamide gradient gel electrophoresis, the granule remnants effectively depleted the prebeta1LpA1, but not the alphaLpA1, in HDL3 and in plasma during incubation at 37 degrees C for <1 hour. Incubation of plasma with granule remnants for 1 hour also led to near disappearance of the LpA4-1 and LpA4-2 particles, but did not affect the distribution of the apoA2-containing lipoproteins present in the plasma. We conclude that the reduced ability of granule remnant-treated HDL3 and granule remnant-treated plasma to induce cholesterol efflux from macrophage foam cells is caused by selective depletion by mast cell chymase of quantitatively minor A1- and A4-containing subpopulations of HDL. Because these particles, ie, prebeta1LpA1 and LpA4, are efficient acceptors of cholesterol from cell surfaces, their depletion by mast cells may block the initiation of reverse cholesterol transport in vivo and thereby favor foam cell formation in the arterial intima, the site of atherogenesis.

Mast Cells in Atherosclerotic Human Coronary Arteries: Implications for Coronary Fatty Streak Formation and Plaque Erosion or Rupture

Atherosclerosis is a slowly progressing disease of the intima, the innermost arterial layer. Fatty streaks appear first, then atheromas or atherosclerotic plaques develop, and, finally, the fibrous cap of a plaque may erode or rupture, triggering thrombus formation. Data emerging from clinical, pathological, and experimental studies on atherogenesis have revived a paradigm, according to which atherosclerosis is an inflammatory disease.! The inflammatory cells, notably macrophages, T lymphocytes, and mast cells are key players in the production of local inflammation in an atherosclerotic lesion. Importantly, immunohistochemical observations of atherosclerotic lesions in human coronary arteries have revealed that these lesions contain not only more macrophages and more T lymphocytes, but also more mast cells than does the normal coronary intima. Most importantly, the fraction of degranulated mast cells in the lesions is increased. Biochemical and cell culture experiments with degranulated rat serosal mast cells have suggested several mechanisms which, if operative in vivo, would provide mechanisms explaining how mast cells could influence the development of coronary atherosclerosis in man. First, the heparin proteoglycans and the neutral protease chymase in exocytosed mast cell granules can induce the formation of foam cells, the hallmarks of fatty streak lesions. Second, granule heparin proteoglycans can inhibit the proliferation of cultured smooth muscle cells. Third, granule chymase can induce their apoptosis. Since smooth muscle cells produce collagen, thus providing tensile strength in the fibrous cap, their loss would weaken the cap and render it susceptible to rupture. Together, these experimental observations have identified diverse functions by which mast cells may affect the development of atherosclerosis.

Mast Cells in Atherogenesis: A Model for Studying Proteolytic Modification of Low and High Density Lipoproteins

Immunohistochemical observations on human atherosclerotic lesions have revealed that the lesions contain, not only foam cells, but also mast cells. Studies taking advantage of in vitro and in vivo models that use rodent mast cells and human lipoproteins have disclosed a series of metabolic events by which mast cells can induce the formation of foam cells. The mast cells are filled with cytoplasmic secretory granules containing histamine, heparin proteoglycans, and neutral proteases. When activated, mast cells degranulate, i.e. they expel their granules and in this way influence the metabolism of lipoproteins in their vicinity. In the extracellular fluid, histamine is released from the exocytosed granules, but the heparin proteoglycans and neutral proteases (notably the chymotryptic enzyme chymase) remain tightly bound together in the form of granule remnants. Of the three granule remnant components, (1) histamine increases endothelial permeability, thereby enhancing transendothelial transport of low-density lipoproteins (LDL) into the tissues; (2) the heparin proteoglycans bind LDL; and (3) the chymase degrades the apolipoprotein B-100 component of the granule remnant-bound LDL. Such proteolytically modified LDL particles are unstable and fuse into larger lipid droplets (up to 100 LDL per droplet), and so allow the granule remnants to bind new LDL particles and to increase their maximal LDL binding capacity. Ultimately, macrophages or smooth muscle cells phagocytose the granule remnants loaded with fused LDL particles, degrade them within phagosomes, and become converted into typical foam cells. The chymase also attacks the HDL3 particles, effectively proteolyzing their apolipoprotein components, and thereby reducing the ability of HDL to induce efflux of cholesterol from the foam cells. Notably, the high-affinity component of cholesterol efflux is blocked, this being due to specific proteolytic depletion of the small apolipoprotein A-I- and A-IV-containing particles present in plasma. Taken together, experimental work carried out with rodent mast cells and immunohistochemical studies on human arterial samples suggest that stimulated mast cells can accelerate foam cell formation both by promoting the uptake of LDL cholesterol and by inhibiting release of cellular cholesterol, i.e. the initial step of reverse cholesterol transport.