Spontaneous remodeling of HDL particles at acidic pH enhances their capacity to induce cholesterol efflux from human macrophage foam cells

HDL particles may enter atherosclerotic lesions having an acidic intimal fluid. Therefore, we investigated whether acidic pH would affect their structural and functional properties. For this purpose, HDL(2) and HDL(3) subfractions were incubated for various periods of time at different pH values ranging from 5.5 to 7.5, after which their protein and lipid compositions, size, structure, and cholesterol efflux capacity were analyzed. Incubation of either subfraction at acidic pH induced unfolding of apolipoproteins, which was followed by release of lipid-poor apoA-I and ensuing fusion of the HDL particles. The acidic pH-modified HDL particles exhibited an enhanced ability to promote cholesterol efflux from cholesterol-laden primary human macrophages. Importantly, treatment of the acidic pH-modified HDL with the mast cell-derived protease chymase completely depleted the newly generated lipid-poor apoA-I, and prevented the acidic pH-dependent increase in cholesterol efflux. The above-found pH-dependent structural and functional changes were stronger in HDL(3) than in HDL(2). Spontaneous acidic pH-induced remodeling of mature spherical HDL particles increases HDL-induced cholesterol efflux from macrophage foam cells, and therefore may have atheroprotective effects.     

Biological functions of serine proteases in mast cells in allergic inflammation

Serine proteases in mast cell granules, such as chymase, atypical chymase, and tryptase, which are major proteins in the granules, may play important roles in the process of immunoglobulin E (IgE)-mediated degranulation and in pathobiological alterations in tissues. Indeed, inhibitors of chymase, substrate analogs, and antichymase F(ab')2, but not inhibitors of tryptase, markedly inhibited histamine release induced by IgE-receptor bridging but not that induced by Ca ionophore. In contrast, inhibitors of metalloprotease inhibited histamine release induced not only by IgE-receptor bridging but also by Ca ionophore. These results suggest that chymase and metalloprotease are involved at different steps in the process of degranulation. The extents of inhibition of histamine release were closely correlated with the amounts of the inhibitors of chymase accumulated in the granules. After degranulation, the released proteases may in part contribute to pathobiological alterations in allergic disorders through generations of C3a anaphylatoxin and thrombin by human and rat tryptase, respectively, and those of angiotensin II and a chemotactic factor of neutrophils by human and rat chymase, respectively. Moreover, chymase and atypical chymase from rat were shown to destroy type IV collagen, and human tryptase was found to hydrolyze various plasma proteins, such as fibrinogen and high-molecular-weight kininogen. The biological activities of tryptase and chymase from rat may be regulated by their dissociation from and association with trypstatin, an endogenous inhibitor of these proteases.     

A simple method for purification of chymase from rat tongue and rat peritoneal cells

Chymase was purified from rat tongue and rat peritoneal cells by a simple new method involving hydrophobic chromatography on octyl-Sepharose 4B and hydroxylapatite column chromatography. This procedure can be completed in 1 or 2 days and the recovery is 45-60% from rat tongue and 32-47% from rat peritoneal cells. The specific activity of the purified enzyme is higher than that of crystallized enzyme previously reported (Y. Sanada, N. Yasogawa, and N. Katunuma (1978) Biochem. Biophys. Res. Commun. 82, 108-113). This procedure should be particularly useful for purifying chymase on a large scale from tissues in which it is present in relatively low concentrations.     

Design and Synthesis of Indole and Tetrahydroisoquinoline Hydantoin Derivatives as Human Chymase Inhibitors

The synthesis of new potential inhibitors of human chymase is described. Treatment of dihydroimidazo[1,5-a]indole and [1,5-b]isoquinoline-dione with thioaryl followed by oxidation gave the N-arylsulfonylmethyl of polycyclic hydantoin derivatives 3, 5 and 6.     

Finding off‐targets,biological pathways,and target diseases for chymase inhibitors via structure‐based systems biology approach

Off‐target binding connotes the binding of a small molecule of therapeutic significance to a protein target in addition to the primary target for which it was proposed. Progressively such off‐targeting is emerging to be regular practice to reveal side effects. Chymase is an enzyme of hydrolase class that catalyzes hydrolysis of peptide bonds. A link between heart failure and chymase is ascribed, and a chymase inhibitor is in clinical phase II for treatment of heart failure. However, the underlying mechanisms of the off‐target effects of human chymase inhibitors are still unclear. Here, we develop a robust computational strategy that is applicable to any enzyme system and that allows the prediction of drug effects on biological processes. Putative off‐targets for chymase inhibitors were identified through various structural and functional similarity analyses along with molecular docking studies. Finally, literature survey was performed to incorporate these off‐targets into biological pathways and to establish links between pathways and particular adverse effects. Off‐targets of chymase inhibitors are linked to various biological pathways such as classical and lectin pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system. Tissue kallikreins, granzyme M, neutrophil elastase, and mesotrypsin are also identified as off‐targets. These off‐targets and their associated pathways are elucidated for the effects of inflammation, cancer, hemorrhage, thrombosis, and central nervous system diseases (Alzheimer's disease). Prospectively, our approach is helpful not only to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses for these inhibitors. Proteins 2015; 83:1209–1224. © 2014 Wiley Periodicals, Inc.     

Mast cell chymase regulates dermal mast cell number in mice

Chymase inhibitor reduced the increase in the number of dermal mast cells in 2,4-dinitrofluorobenzene-induced dermatitis in a dose-dependent manner. Intradermal injection of human chymase to mouse ear significantly increased histamine content, the marker for mast cell number in the skin. These results suggest that chymase released by mast cells may participate in local mast cell accumulation in a positive feedback fashion. Immunohistochemical analysis revealed that the intradermal injection of chymase reduces expression of stem cell factor (SCF) on surface of the skin keratinocytes. In addition, incubation of human keratinocytes with chymase in vitro resulted in release of SCF into the culture medium. Since soluble SCF is thought to regulate mast cell number, the chymase-induced mast cell accumulation may occur via the ability of chymase to process membrane-bound SCF on the epidermal keratinocytes.     

Chymase increases glomerular albumin permeability via protease-activated receptor-2

Increased infiltration of the kidney by mast cells is associated with proteinuria, and interstitial fibrosis in various renal diseases. Mast cells produce serine proteases including tryptase and chymase (MCC) that act via protease-activated receptors (PARs) to induce synthesis of fibrogenic cytokines by renal cells. In the present study, we investigated direct effect of MCC and role of PARs on glomerular albumin permeability (P_alb). Isolated rat glomeruli were incubated with MCC (0.1, 1, 10, and 100 ng/ml) for 5–30 min in presence or absence of PAR-1 and PAR-2 blocking antibodies. P_alb was determined from the change in glomerular volume in response to an albumin oncotic gradient. The effect of direct activation of PARs on P_alb was verified by incubating glomeruli with synthetic hexapeptide known to activate PAR-1 and PAR-2. MCC increased P_alb of isolated rat glomeruli in a dose- and time-dependent manner. Blocking PAR-2 prevented MCC-mediated increase in P_alb. RT-PCR analysis of glomerular RNA demonstrated the presence of constitutively expressed PAR-1, -2, and -3 and low levels of PAR-4. In addition, direct activation of PAR-2 by hexapeptide SLIGKV increased P_alb comparable to MCC, whereas PAR-1 activation by TFLLRN had no effect on P_alb. Our results document that MCC induces increase in P_alb and that this effect is mediated through PAR-2. MCC may also play a role in renal scarring. We propose that inhibiting MCC activity or blocking the activation of PAR-2 may provide new targets for therapy in renal diseases.     

IgE-Independent Activation of Human Mast Cells Indicates their Role in the Late Phase Reaction of Allergic Inflammation

Mast cells are tissue dwelling cells that have a clear-cut pathologic role in allergy. Besides that, they participate in several chronic inflammatory conditions, helminitic parasitosis, and in some solid tumor reactions, but also in physiological situations, such as wound healing and innate immunity. Mast cells produce and release various mediators after activation induced by either IgE-dependent or IgE-independent mechanisms. Although much information has been gathered on the immunological (IgE-dependent) mast cell activation both in vivo and in vitro, not much is known about the non-immunological (IgE-independent) activation particularly in human mast cells. Mast cell IgE-independent activation is mediated through Gi3alpha which has been identified in rat mast cells as the pertussis toxin (Ptx)-sensitive heterotrimeric G protein that interacts with cationic secretagogues inducing PLC-independent mast cell exocytosis. Mast cell IgE-independent activation in allergy most likely occurs when mast cells encounter eosinophils, the main inflammatory cells that persist throughout the late and chronic phases of the allergic reaction. This review summarizes the influence of eosinophils on mast cell activation demonstrating that IgE-independent activation has a significant role in pathophysiological processes.     

Generation and characterization of alpha-chymase-Cre transgenic mice

The Cre-loxP technology allows the introduction of somatic gene alterations in a tissue and/or cell type specific manner. The development of transgenes that target Cre expression to specific cell types is a critical component in this system. Here, we describe the generation and characterization of transgenic mouse lines expressing Cre recombinase under the control of the baboon alpha-chymase promoter, designated Chm:Cre, in order to direct Cre expression specifically to mouse mast cells. Chm:Cre expression was detected in mast cells in lung and colon tissue. Cre-mediated recombination in these mice identified a population of mature tissue resident mast cells using ROSA26R reporter mice. No Cre-expression and Cre-mediated recombination was induced in in vitro generated bone marrow derived mast cells or mast cells isolated from the peritoneal cavity indicating that Cre-expression under the control of the alpha-chymase promoter is solely activated in tissue resident mast cells. These Chm:Cre transgenic mice represent a useful tool to specifically inactivate genes of interest in mast cells of these tissues.