Mitochondrial Homeostasis and Mast Cells in Experimental Hepatic Ischemia-Reperfusion Injury of Rats.

BACKGROUND: Ischemia-reperfusion injury is a histopathological event and is an important cause of morbidity and mortality after hepatobiliary surgery. We aimed to investigate the protective effect of uridine on hepatic ischemia-reperfusion injury in rats. METHODS: The animals were divided into 4 groups (n = 8): group I (control), group II: ischemia-reperfusion (30 minutes ischemia and 120 minutes reperfusion), group III: ischemia-reperfusion+uridine (at the beginning of reperfusion), and group IV: ischemia-reperfusion+uridine (5 minutes before ischemia-reperfusion). Uridine was administered a single dose of 30 mg/kg IV. The 3 elements of the hepatoduodenal ligament (hepatic artery, portal vein, and biliary tract) were obliterated for 30 minutes. Then hepatic reperfusion was achieved for 120 minutes. RESULTS: In the ischemia-reperfusion group, both liver tissues and serum chymase activity and high-temperature requirement A2 levels were higher. Severe central vein dilatation and congestion, widening sinusoidal range, diffuse necrotic hepatocytes and dense erythrocyte accumulation in sinusoids, and strongly inducible nitric oxide synthase expression were seen in the ischemia-reperfusion group. A clear improvement was seen in both uridine co-administration and pretreatment groups. CONCLUSION: Our results revealed that uridine limits the development of liver damage under conditions of ischemia-reperfusion, thus contributing to an increase in hepatocyte viability.

Chronic allergic lung inflammation negatively influences neurobehavioral outcomes in mice.

BACKGROUND: Asthma is a major public health problem worldwide. Emerging data from epidemiological studies show that allergies and allergic diseases may be linked to anxiety, depression and cognitive decline. However, little is known about the effect of asthma, an allergic lung inflammation, on cognitive decline/behavioral changes. Therefore, we investigated the hypothesis that allergic lung inflammation causes inflammation in the brain and leads to neurobehavioral changes in mice. METHODS: Wild-type C57BL/6J female mice were sensitized with nasal house dust mite (HDM) antigen or control PBS for 6 weeks to induce chronic allergic lung inflammation. A series of neurocognitive tests for anxiety and/or depression were performed before and after the intranasal HDM administration. After the behavior tests, tissues were harvested to measure inflammation in the lungs and the brains. RESULTS: HDM-treated mice exhibited significantly increased immobility times during tail suspension tests and significantly decreased sucrose preference compared with PBS controls, suggesting a more depressed and anhedonia phenotype. Spatial memory impairment was also observed in HDM-treated mice when assessed by the Y-maze novel arm tests. Development of lung inflammation after 6 weeks of HDM administration was confirmed by histology, bronchoalveolar lavage (BAL) cell count and lung cytokine measurements. Serum pro-inflammatory cytokines and Th2-related cytokines levels were elevated in HDM-sensitized mice. In the brain, the chemokine fractalkine was increased in the HDM group. The c-Fos protein, a marker for neuronal activity, Glial Fibrillary Acidic Protein (GFAP) and chymase, a serine protease from mast cells, were increased in the brains from mice in HDM group. Chymase expression in the brain was negatively correlated with the results of sucrose preference rate in individual mice. CONCLUSIONS: 6 weeks of intranasal HDM administration in mice to mimic the chronic status of lung inflammation in asthma, caused significant inflammatory histological changes in the lungs, and several behavioral changes consistent with depression and altered spatial memory. Chymase and c-Fos proteins were increased in the brain from HDM-treated mice, suggesting links between lung inflammation and brain mast cell activation, which could be responsible for depression-like behavior.

Glia Maturation Factor and Mast Cell-Dependent Expression of Inflammatory Mediators and Proteinase Activated Receptor-2 in Neuroinflammation.

Parkinson's disease (PD) is characterized by the presence of inflammation-mediated dopaminergic neurodegeneration in the substantia nigra. Inflammatory mediators from activated microglia, astrocytes, neurons, T-cells and mast cells mediate neuroinflammation and neurodegeneration. Administration of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induces PD like motor deficits in rodents. 1-methyl-4-phenylpyridinium (MPP+), a toxic metabolite of MPTP activates glial cells, neurons and mast cells to release neuroinflammatory mediators. Glia maturation factor (GMF), mast cells and proteinase activated receptor-2 (PAR-2) are implicated in neuroinflammation. Alpha-synuclein which induces neurodegeneration increases PAR-2 expression in the brain. However, the exact mechanisms are not yet understood. In this study, we quantified inflammatory mediators in the brains of MPTP-administered wild type (Wt), GMF-knockout (GMF-KO), and mast cell knockout (MC-KO) mice. Additionally, we analyzed the effect of MPP+, GMF, and mast cell proteases on PAR-2 expression in astrocytes and neurons in vitro. Results show that the levels of interleukin-1beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and the chemokine (C-C motif) ligand 2 (CCL2) were lesser in the brains of GMF-KO mice and MC-KO mice when compared to Wt mice brain after MPTP administration. Incubation of astrocytes and neurons with MPP+, GMF, and mouse mast cell protease-6 (MMCP-6) and MMCP-7 increased the expression of PAR-2. Our studies show that the absence of mast cells and GMF reduce the expression of neuroinflammatory mediators in the brain. We conclude that GMF along with mast cell interactions with glial cells and neurons during neuroinflammation can be explored as a new therapeutic target for PD and other neuroinflammatory disorders.

Effect of Mast Cell Chymase on Activation, Proliferation and Transdifferentiation of Hepatic Stellate Cells.

BACKGROUND/AIMS: This work aims to observe the effects of mast cell (MC) chymase on activation, proliferation and transdifferentiation of hepatic stellate cells (HSCs) and to discuss the role of chymase in development of liver fibrosis. METHODOLOGY: Rat HSCs were isolated by in situ perfusion using collagenase IV and proteinase E and density gradient centrifugation (Percoll method), followed by culture. After intervention by chymase with series of concentration (1, 10 and 100 ng/mL), MTT assay was performed to determine HSC proliferation. Expressions of α-SMA and TGF-ß1 protein in HSCs were determined by Western blotting method, and TGF-ß1 and Col-I mRNA expression were determined using semi-quantitive RT-PCR method. RESULTS: MC Chymase could significantly promote the proliferation of HSCs (P < 0.05), with chymase concentration dependence from 1 ng/mL to 100 ng/mL. The OD value of HSCs in 100 ng/ml chymase group was 1.45 times to control group. Compared with control group, expressions of α-SMA and TGF-ß1 protein and expressions of TGF-ß1 and Col-I mRNA in chymase groups were significantly increased, respectively (P < 0.05), with chymase concentration dependence. CONCLUSIONS: MC chymase can promote the activation, proliferation and transdifferentiation of HSCs, and plays an important role in occurrence and development of liver fibrosis.

Involvement of chymase in allergic conjunctivitis of guinea pigs.

It has been reported that chymase activity was increased in allergic conjunctivitis patients and this activity was correlated with the severity of the disease. However, the precise roles of chymase in allergic conjunctivitis are unclear, and whether chymase inhibitors are effective for allergic conjunctivitis has not been reported even in experimental animal models. In this study, the roles of chymase in the pathogenesis were evaluated using a selective chymase inhibitor, ONO-WH-236, in a guinea pig model of allergic conjunctivitis induced by cedar pollen. Sensitized guinea pigs were challenged by the pollen, followed by assessing redness and edema in the conjuntiva, and counting the frequency of eye scratching as an itch-associated response. Treatment with the ONO-WH-236 (40 and 80 mg/kg, p.o.) dose-dependently inhibited the induction of redness, edema and scratching behavior. An anti-histaminic drug, ketotifen (3 mg/kg, p.o.), also significantly inhibited conjunctivitis symptoms. Chymase activity was increased in ophthalmic lavage fluid immediately after the pollen challenge. The increase in chymase activity was inhibited by in vivo treatment with ONO-WH-236. Interestingly, increased histamine in the ophthalmic lavage fluid immediately after the challenge was also inhibited by the chymase inhibitor. Administration of human recombinant chymase by eye dropping (0.09 and 0.9 µg/eye) dose-dependently induced scratching behavior, which was inhibited by not only ONO-WH-236 but also ketotifen; however, chymase administration induced only weak redness in the conjunctiva, which was resistant to treatment with anti-histaminic drugs. In conclusion, it was suggested that chymase was released from mast cells after antigen challenge, followed by the induction of conjunctivitis symptoms through histamine release from mast cells. Thus, chymase could be a potential target for pharmacotherapy for allergic conjunctivitis.

Stem cell factor is responsible for the rapid response in mature mast cell density in the acutely stressed heart.

In the abdominal aortocaval (AV) fistula model of heart failure, we have shown that the acute doubling of cardiac mature mast cell (MC) density involved the maturation, but not proliferation, of a resident population of immature cardiac MCs. An increase in stem cell factor (SCF) may be responsible for this MC maturation process. Thus, the purpose of this study was to determine if: 1) myocardial SCF levels are increased following the initiation of cardiac volume overload; 2) the incubation of left ventricular (LV) tissue slices with SCF results in an increase in mature MC density; and 3) chemical degranulation of mature cardiac MCs in LV tissue slices results in an increase in SCF and mature MC density via MC chymase. Male rats with either sham or AV fistula surgery were studied at 6h and 1 and 3 days post-surgery. LV slices from normal male rat hearts were incubated for 16h with media alone or media containing one of the following: 1) recombinant rat SCF (20 ng/ml) to determine the effects of SCF on MC maturation; 2) the MC secretagogue compound 48/80 (20 µg/ml) to determine the effects of MC degranulation on SCF levels and mature MC density; 3) media containing compound 48/80 and anti-SCF (5 µg/ml) to block the effects of SCF; 4) chymase (100 nM) to determine the effects of chymase on SCF; and 5) compound 48/80 and chymostatin (chymase inhibitor, 10 µM) to block the effects of MC chymase. In AV fistula animals, myocardial SCF was significantly elevated above that in the sham group at 6h and 1 day post fistula by 2 and 1.8 fold, respectively, and then returned to normal by 3 days; this increase slightly preceded significant increases in MC density. Incubation of LV slices with SCF resulted in a doubling of mature MC density and this was concomitant with a significant decrease in the number of immature mast cells. Incubation of LV slices with compound 48/80 increased media SCF levels and mature MC density and with anti-SCF and chymostatin prevented these compound 48/80-induced increases. Incubation with chymase increased media SCF levels and mature MC density. These findings indicate that activated mature cardiac mast cells are responsible, in a paracrine fashion, for the increase in mature MC density post AV fistula by rapidly increasing SCF levels via the release of chymase.

Mast cell chymase promotes cell proliferation and expression of certain cytokines in a dose-dependent manner.

Wound healing is a complex process, with various intracellular molecules and cytokines involved. Chymase is a chymotrypsin-like serine protease originally found to be a mast cell protease. In this study, to further investigate the role of chymase in the process of wound healing, the effects of chymase on cell proliferation and cytokine activation in human skin fibroblasts were determined. To determine cell proliferation, MTT assay was employed. The cells were harvested and total proteins were extracted and detected by western blotting. It was found that in vitro cell treatments with chymase led to dose-dependent increases of skin fibroblast proliferation. Moreover, the treatment of cells with chymase for 6 h induced dose-dependent increases in the expression levels of transforming growth factor (TGF)-ß1 and interleukin (IL)-1ß, although higher doses (120 ng/ml for TGF-ß1; 60 and 120 ng/ml for IL-1ß) did not facilitate the detected cytokine expression. In addition, treatment for longer periods of time (12 or 24 h) did not increase TGF-ß1 or IL-1ß expression. The results from our study will strongly facilitate the understanding of the roles of chymase in the process of wound healing.

Tryptase and chymase are angiogenic in vivo in the chorioallantoic membrane assay.

Human mast cells (MCs) are divided in two types depending on the expression of tryptase and chymase in their granules. Literature data indicate that both tryptase and chymase are angiogenic, but there is currently no evidence of their direct angiogenic activity in vivo. In this study, we have investigated the capacity of tryptase and chymase to promote vasoproliferation in chick embryo chorioallantoic membrane (CAM), a well established in vivo assay to study angiogenesis and anti-angiogenesis. The results showed that both tryptase and chymase stimulate angiogenesis and that the response is similar to that obtained with vascular endothelial growth factor (VEGF), a well-known angiogenic cytokine, and confirm the angiogenic activity of these two proteases stored in MC granules.

The inflammatory response after an epidermal burn depends on the activities of mouse mast cell proteases 4 and 5.

A second-degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to nonmuscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-Kit(W)/Kit(Wv) mice, which lack MCs in a context of other defects due to a mutation of the Kit receptor. To address further an MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury after a second-degree scald burn, whereas mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficient mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first hour postburn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing nonredundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.

Mast cells regulate homeostatic intestinal epithelial migration and barrier function by a chymase/Mcpt4-dependent mechanism.

Altered intestinal barrier function is postulated to be a central predisposing factor to intestinal diseases, including inflammatory bowel diseases and food allergies. However, the mechanisms involved in maintaining homeostatic intestinal barrier integrity remain undefined. In this study, we demonstrate that mice deficient in mast cells (Kit(W-sh/W-sh) [Wsh]) or mast cell chymase (Mcpt4(-/-)) have significantly decreased basal small intestinal permeability compared with wild-type (WT) mice. Altered intestinal barrier function was linked to decreased intestinal epithelial cell migration along the villus/crypt axis, altered intestinal morphology, and dysregulated claudin-3 crypt expression. Remarkably, engraftment of Wsh mice with WT but not Mcpt4(-/-) mast cells restored intestinal epithelial cell migration, morphology, and intestinal epithelial barrier function. Collectively, these findings identify a mechanism by which mast cells regulate homeostatic intestinal epithelial migration and barrier function.

Contribution of chymase-dependent angiotensin II formation to the progression of tubulointerstitial fibrosis in obstructed kidneys in hamsters.

Recent studies indicate a role of chymase in the regulation of angiotensin II (AngII) formation in cardiovascular and renal tissues. We investigated a possible contribution of chymase to AngII formation and to renal fibrosis in unilateral ureteral obstruction (UUO). Eight-week-old Syrian hamsters were subjected to UUO and treated with vehicle, the specific chymase inhibitor (CI) 4-[1-(4-methyl-benzo[b]thiophen-3-ylmethyl)-1H-benzimidazol-2-ylsulfanyl]-butyric acid (50 mg/kg, twice a day, p.o.), or the selective AT(1)-receptor blocker olmesartan (10 mg/kg per day, p.o.) for 14 days. UUO-induced renal interstitial fibrosis was associated with increases in renal mRNA levels of alpha-smooth muscle actin (SMA), type I collagen, and transforming growth factor (TGF)-beta. The UUO hamsters showed markedly higher AngII contents and increased AT(1)-receptor mRNA level in the obstructed kidney than sham-operated ones. In contrast, angiotensin-converting enzyme (ACE) protein expression was significantly lower in UUO hamsters. In UUO hamsters, treatment with CI or olmesartan significantly decreased AngII levels in renal tissue and mRNA levels of alpha-SMA, type I collagen, and TGF-beta and ameliorated tubulointerstitial injury. On the other hand, neither CI nor olmesartan changed systolic blood pressure, renal ACE, and AT(1)-receptor protein levels. These data suggest that chymase-dependent intrarenal AngII formation contributes to the pathogenesis of interstitial fibrosis in obstructed kidneys of hamsters.

Chymase induces profibrotic response via transforming growth factor-beta 1/Smad activation in rat cardiac fibroblasts.

Mast cell-derived chymase is implicated in myocardial fibrosis (MF), but the underlying mechanism of intracellular signaling remains unclear. Transforming growth factor-beta 1 (TGF-beta1) is identified as the most important profibrotic cytokine, and Smad proteins are essential, but not exclusive downstream components of TGF-beta 1 signaling. Moreover, novel evidence indicates that there is a cross talk between Smad and mitogen-activated protein kinase (MAPK) signaling cascade. We investigated whether chymase activated TGF-beta 1/Smad pathway and its potential role in MF by evaluating cardiac fibroblasts (CFs) proliferation and collagen synthesis in neonatal rats. MTT assay and 3H-Proline incorporation revealed that chymase induced CFs proliferation and collagen synthesis in a dose-dependent manner. RT-PCR and Western blot assay demonstrated that chymase not only increased TGF-beta1 expression but also upregulated phosphorylated-Smad2/3 protein. Furthermore, pretreatment with TGF-beta 1 neutralizing antibody suppressed chymase-induced cell growth, collagen production, and Smad activation. In contrast, the blockade of angiotensin II receptor had no effects on chymase-induced production of TGF-beta 1 and profibrotic action. Additionally, the inhibition of MAPK signaling had no effect on Smad activation elicited by chymase. These results suggest that chymase can promote CFs proliferation and collagen synthesis via TGF-beta 1/Smad pathway rather than angiotensin II, which is implicated in the process of MF.

The effects of chymase on matrix metalloproteinase-2 activation in neointimal hyperplasia after balloon injury in dogs.

Chymase is known to generate angiotensin II in the vascular wall. In this study we investigated a novel role for chymase other than angiotensin II production in vascular proliferation after balloon injury. Chymase promoted the migration of vascular smooth muscle cells in the matrix-coated invasion chambers and activated promatrix metalloproteinase-2 obtained from the culture medium of vascular smooth muscle cells. Two weeks after balloon injury, significant neointimal formation was found in dog carotid arteries. After injury, active matrix metalloproteinase-2 was increased in parallel with the augmentation of chymase activity that was seen in the proliferating region of the vascular wall. The oral administration of NK3201 (1 mg/kg per day), a chymase inhibitor, prevented neointimal formation and significantly suppressed both active matrix metalloproteinase-2 and chymase activities 2 weeks after injury. These results suggest that chymase inhibitors can prevent the development of intimal hyperplasia via the inhibition of matrix metalloproteinase-2 activation in balloon-injured arteries.

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.