Isoform-selective upregulation of mast cell chymase in the development of skin fibrosis in scleroderma model mice.

The involvement of connective-tissue-type mast cells and chymase, a protease unique to their secretory granules, has been implicated in fibrotic diseases. To elucidate the role of chymase in fibroproliferative inflammation, in this study we examined the enzymatic activity and mRNA expression of chymase in the sclerotic skin of tight-skin mice; syngeneic Pallid mice served as the control. Dorsal skin specimens from mice aged 5, 10, and 20 wk were evaluated by morphometric and biochemical analyses. At ages 10 and 20 wk, the hydroxyproline concentration in tight-skin dermis was higher than that in Pallid. At any age, the subcutaneous fibrous layer was thicker in tight-skin than in Pallid. In accordance with these fibrous changes, both connective-tissue-type mast cell counts and chymase activity were higher in tight-skin skin than in Pallid skin up to 20 wk of age. Age-matched (10-wk-old) tight-skin and Pallid were quantified for their mRNA of connective-tissue-type mast-cell-specific chymase, mouse mast cell protease-4, by the competitive reverse transcriptase polymerase chain reaction technique, which revealed its higher level in tight-skin than Pallid. In contrast, the mRNA level of mouse mast cell protease-5, the chymase isoform of undifferentiated mast cells, in tight-skin skin was only a tenth that of mouse mast cell protease-4 and no different from the mouse mast cell protease-5 mRNA level of Pallid mice. An in situ hybridization study confirmed the higher expression of mouse mast cell protease-4 by connective-tissue-type mast cells in tight-skin skin than Pallid skin. These results strongly support the contention that the connective-tissue-type mast cell chymase plays a crucial role in fibroproliferative remodeling of the skin.

Differential suppression of pressure-overload cardiac and aortic hypertrophy in rats by angiotensin-converting enzyme inhibitors.

Role of tissue angiotensin-converting enzyme (ACE) in the development of pressure-overload cardiovascular hypertrophy was examined in rats by comparing the inhibitory effect of trandolapril (high efficiency on tissue ACE) with that of enalapril (low efficiency) at equally antihypertensive doses. Rats with abdominal aorta banded or sham-operated were orally treated with trandolapril (0.5 mg/kg per day), enalapril (20 mg/kg per day) or vehicle for 8 weeks after the surgical maneuvers. In vehicle-treated rats, the banding raised the intra-aortic systolic pressure by 58%, diastolic pressure by 31%, maximum velocity of pressure rise by 65%, left ventricular (LV) weight by 41%, LV hydroxyproline concentration by 56%, aortic mass by 46%, LV ACE activity by 45%, and aortic ACE activity by 265%. Although both drugs equally reduced the aortic systolic pressure to approx. 70% and diastolic pressure to approx. 80% that of banded rats receiving vehicle, trandolapril partially prevented the LV hypertrophy, whereas enalapril yielded nonsignificant suppression. Trandolapril completely prevented the LV increments in hydroxyproline and ACE activity, whereas enalapril partially inhibited the LV hydroxyproline increase with little inhibition of LV ACE activity. In contrast, both inhibitors almost completely prevented the aortic hypertrophy, with the ACE activity of the aorta being potently inhibited. These results suggest that tissue ACE is the principal factor for pressure-induced aortic hypertrophy and an important yet non-essential factor for LV hypertrophy.

Increases in mast cells and chymase in fibroproliferative paws of collagen-induced arthritic mice.

OBJECTIVE AND DESIGN: To investigate whether mast cells (MCs) and chymase, the major protease of murine MCs, were involved in a chronic fibroproliferative disorder of the paws associated with type II collagen (CII)-induced arthritis. MATERIALS: Eighteen DBA/1J mice were divided into 3 groups and were used to study fibroproliferative changes in paws elicited by immunization. TREATMENT: Arthritis was induced by immunization with CII, which was intradermally injected as an emulsion made with adjuvant. A booster shot was done 3 weeks after the initial shot. A group with no treatment and that received adjuvant alone served as control. METHODS: Twelve weeks after the booster shot, inflammation of the paws was evaluated for pathological and biochemical indices. Chymase activity was determined with a chromogenic peptide substrate. RESULTS: In CII-immunized group, collagen bundles accumulated around the destructed joints. In accordance with the pathological findings, MC density in the affected paws was increased (154.8+/-13.3/mm2; p<0.05 vs. control) and chymase activity was also increased (29.5+/-2.8 mU/mg protein; p<0.01 vs. control). CONCLUSIONS: The present results demonstrate increases in MCs and chymase in fibroproliferative paws of collagen-induced arthritic mice.

[Effects of orally available prodrug of cromoglycic acid on collagen-induced arthritis mice].

Cromoglicate lisetil (CL) is an orally deliverable prodrug of cromoglycic acid, having diethyl promoieties and a lysyl promoiety for its optimum drug-delivery. We examined the effects of CL on bovine type II collagen (CII)-induced arthritis (CIA) of male DBA/1J mice, an experimental model for human rheumatoid arthritis, and its action mechanism. CL (100 mg/kg/day) was given by gavage to CII-immunized mice once daily for 6 weeks, starting when arthritic symptoms became evident. Symptomatic scores of arthritis obviously elevated in non-treated CIA mice at week 6.5 after initial immunization and continued elevated thereafter throughout the experiment, the elevation which was reduced by CL. CL also improved radiographic score of phalangeal destruction and pathohistological indexes at the end of treatment period. Serum anti-CII antibody titer was increased in non-treated CIA mice and the elevation was reduced by CL treatment. Mast cells (MCs) number in arthritic region was increased in non-treated CIA mice but not by CL treatment. In conclusion, oral CL treatment proved beneficial in CIA mice. Observed correlation between the CL effect on CIA and that on MCs number suggests the potential contribution of MCs to accelerate chronic arthritic processes and may further implicate potential action mechanism of CL, which may act by regulating MC functions for chronic inflammation.

Skin mast cell promotion of matrix remodeling in burn wound healing in mice: relevance of chymase.

Inflammation, granulation, and collagen accumulation, which are observed in the wound healing process, occasionally lead to hypertrophic scarring. Several in vitro reports have suggested that skin mast cells (MCs) and their major protease, chymase, participate in the healing process as well as in fibrotic skin diseases. The present study examined the potential involvement of MCs and MC chymase in the healing of burns in mouse dorsal skin. The size of the burn wounds, density of the capillaries, collagen accumulation, MC number, and chymase activity were measured before and 1, 3, 7, and 14 days after burning. The healing process corresponded strongly with MC density and chymase activity in both acute and subacute phases. The maximum decrease in MC number and chymase activity occurred on day 3 when tissue loss due to necrosis was maximal. From day 7 to 14, the burn wounds retracted rapidly accompanied by increases in capillaries and collagen fibers, in correspondence with fast increments in MC numbers and chymase activity at the wound edges. The present results combined with previous in vitro results strongly support the contention that skin MC chymase plays a role in the normal wound healing process, and presumably in dermal fibrotic disorders.

Acetylcholine-induced systemic vasodilation resistant to NG-nitro-L-arginine in an anaesthetized rats.

1. The effects of NG-nitro-L-arginine (L-NNA; 20 mg/kg bodyweight (BW), i.v.) and metyrapone (300 mg/kg BW, s.c.) on acetylcholine (ACh)-induced depressor responses were investigated in anaesthetized rats. 2. Acetylcholine (0.05, 0.5, 5 micrograms/kg BW, i.v.) dose-dependently evoked a sharp fall in mean blood pressure (BP) followed by a slow recovery under control conditions. 3. Basal BP level was elevated when rats were treated with L-NNA, indicating endogenous nitric oxide (NO) participated in BP regulation. However, pretreatment with L-NNA did not attenuate but rather augmented the ACh-induced maximum vasodilation. In contrast, the time for recovery of mean BP to the pre-ACh administration level was shortened by L-NNA. These observations suggested that ACh-induced vasodilation consisted of two phases: a sharp and transient fall (phase 1) that was resistant to L-NNA followed by a longer depressor response (phase 2) that was suppressed by L-NNA. 4. To examine whether augmentation of phase 1 by L-NNA resulted from the elevation of basal BP, an appropriate dose of phenylephrine was infused to obtain similar BP elevation. Phenylephrine infusion augmented the phase 1 in a similar manner to L-NNA pretreatment but showed little effect on phase 2, supporting the selective inhibition of phase 2 by L-NNA. 5. The s.c. pretreatment with metyrapone for 3 days failed to attenuate phase 1. Thus, the involvement of endothelium-derived hyperpolarizing factor that could be formed by a metyrapone-sensitive oxidase in phase 1 was unlikely. 6. These results suggest that some factor(s), which is not inhibitable by L-NNA or metyrapone, may induce the phase 1 depressor response to ACh while NO is responsible for the phase 2 response. The mechanism inducing the phase 1 response remains to be identified.

Synergistic interactions between neuropeptide and histamine on the capillary permeability in rat skin: evaluation by reflectance spectrophotometry.

Effects of neuropeptides on capillary permeability and their interactions with histamine (HIS) in rat skin were investigated. The capillary permeability was measured continuously by reflectance spectrophotometry after intravenous (iv) injection of Evans blue dye. The capillary permeability was increased dose-dependently by the intradermal injection of HIS (0.3-100 microg/site) and substance P (SP; 25-250 ng/site). Calcitonin gene-related peptide (80-800 ng/site) elicited a significant but less increase than did SP. Capsaicin (30 microg/site) also increased capillary permeability slightly but significantly, suggesting the release of endogenous neuropeptides. Both diphenhydramine (DPH; 3 mg/kg, iv) and cimetidine (CIM; 30 mg/kg, iv) reduced HIS-induced responses. DPH also reduced the SP-induced response significantly, but CIM did not. An SP antagonist, [D-Pro2, D-Trp7,9]-SP (1.3 mg/kg, iv), reduced not only SP- but also HIS-induced responses. Furthermore, the HIS-induced response was attenuated by pretreatment with epicutaneous capsaicin for 4 days, depleting endogenous SP. These results delineate the synergistic interactions between SP and HIS in rat skin and suggest the participation of neuropeptides in increasing capillary permeability.

Effect of NG-monomethyl-L-arginine on the microcirculation of rat skin.

1. The effects of NG-monomethyl-L-arginine (L-NMMA), a nitric oxide (NO) synthase inhibitor, on the microcirculation of rat dorsal skin were studied to examine the role of NO in the regulation of regional haemodynamics. 2. The blood volume in the skin microcirculation, which was continuously measured by reflectance spectrophotometry, was significantly decreased in response to L-NMMA (10 mg/kg bodyweight, i.v.), suggesting vasoconstriction, and the response continued for more than 20 min. In contrast, the increase in systemic blood pressure (BP) disappeared soon after administration of L-NMMA. 3. These results show that L-NMMA elicits long-lasting responses in the skin microcirculation, whereas the systemic BP rapidly recovers, suggesting a large contribution of the NO system to the regulation of rat skin microcirculation.

[Suppressive effects of a plant-origin polyol, dulcitol on collagen-induced arthritis in mice].

Dulcitol was isolated chemically from Celastrus obiculatus Thumb and determined by HPLC. Effects of dulcitol were examined on collagen-induced arthritis (CIA) in DBA/1J mice. From 6 weeks after the first immunization with bovine type II collagen, dulcitol (100 mg/kg body weight/day) was administered orally to immunized mice for 9 weeks. Clinical score of CIA was improved significantly by dulcitol intervention compared with the non-treated CIA mice. Radiographic score of phalangeal destruction was also improved by dulcitol treatment. These findings suggest that dulcitol may play a role in regulation of some inflammatory responses in the present arthritis model. Significant reduction of percentage of CD4+ and CD8+ T cell subsets in the spleen leucocytes of CIA mice was observed by flow cytometry. Almost normal level of CD4+ and CD8+ T cells was observed in dulcitol-treated groups, suggesting T cell-modifying effect of dulcitol in CIA. Weight of spleen was larger in CIA mice and it was not affected by dulcitol. Anti-collagen antibody titer was increased in CIA mice, and it was not affected by dulcitol, either. Improvement of the changes of CD4+ and CD8+ T cells in the spleen by dulcitol may suggest its modulatory effect on cellular immunity.

Spectrophotometric study of alpha-adrenoceptors affecting microcirculation of rat skin.

1. To determine the alpha-adrenergic receptor subtypes that affect the microcirculation of skin, the relative absorption (RA) spectra of the skin on the backs of rats were measured using reflectance spectrophotometric methods. We injected alpha-adrenergic agonists, noradrenaline (NA), phenylephrine (PE) and clonidine (CL), intravenously and determined changes in the RA value at 569 nm, one of the isosbestic points of the oxyhaemoglobin and deoxyhaemoglobin absorption. 2. NA reduced the RA value and the reduction was inhibited significantly by pretreatment with phenoxybenzamine (PBZ; P < 0.01). These findings suggested that the haemoglobin content in the skin tissue decreased as a result of vasoconstriction through alpha-adrenoceptors. 3. NA, PE and CL produced dose-dependent reductions in RA. CL and NA produced virtually equipotent reductions except at the highest dose used. PE produced smaller effects. The potency of these drugs in terms of changes in RA did not correlate with their potency in terms of rises in systemic blood pressure (NA > PE > or = CL). 4. Yohimbine (YO) inhibited the NA-induced reduction in RA to a greater degree than bunazosin (BU). Midaglizole, a specific alpha 2-adrenergic antagonist, significantly and dose dependently inhibited the NA-induced reduction in RA. 5. Although BU inhibited NA-induced reduction in RA only slightly, the effect was significant (P < 0.05). BU significantly inhibited PE-induced reduction (P < 0.01), but did not inhibit CL-induced reduction. 6. These observations suggest that the microcirculation of the skin of the rat is affected mainly by alpha 2-adrenoceptor mediated vasoconstriction. However, alpha 1-adrenoceptor mediated vasoconstriction also has some effect.

Real-time measurement of microcirculation of skin by reflectance spectrophotometry.

A reflectance spectrophotometric measurement was developed to analyze the microcirculation of the skin in real time. The relative absorption (RA) spectra were obtained from skin tissue 10 times per second using a spectro-multi-channel-photodetector system. In this system, white light was projected onto the skin of the back of the anesthetized rat and the spectrum of the reflected light between 450 and 643 nm was analyzed. Two absorption peaks at wavelengths of about 540 and 577 nm were observed, corresponding to the absorption peaks in the oxyhemoglobin (HbO2) spectrum. Either phlebotomy or the i.v. injection of norepinephrine (NE) reduced the RA spectrum. Peaks of the reduction in spectrum [(RA spectrum before the treatment) - (RA spectrum after the treatment)] were observed at about 540 and 577 nm. Injection of NE (0.75-48 ng/100 g B.W., i.v.) reduced the RA value at 577 nm dose-dependently, suggesting a decrease in the skin blood flow due to vasoconstriction. In addition, the content of HbO2 and capillary permeation were measured at the same time after the i.v. injection of Evans blue (EB) dye. As indices of HbO2 content and capillary permeation, RA changes at 540 and 610 nm (the latter is an absorption peak in the EB spectrum) were measured in real time, respectively. Both RA values increased dose-dependently after the intradermal injection of histamine (0.3-100 micrograms/site), suggesting the presence of vasodilation and an increase in permeability. The time at which the RA value for HbO2 reached a maximum was shorter than that for EB. These observations suggest that the method described here can detect changes in the HbO2 content and permeation of skin microcirculation at the same time, and in real time.

Comparison of vasopressor effects of nitro arginine in stroke-prone spontaneously hypertensive rats and Wistar-Kyoto rats.

1. NG-nitro-L-arginine (NO2Arg) is a guanidine nitro arginine derivative and an inhibitor of endothelium-dependent vascular relaxation. Significant rise of the systolic blood pressure was observed after 1 week administration of NO2Arg in food (0.023% in weight, about 2.8 mg of NO2Arg/rat per day) in female rats of stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). The rises were not different between SHRSP (21 mmHg) and WKY (23 mmHg). 2. In ring preparations of the thoracic aorta of NO2Arg-administered rats of both strains, relaxation by acetylcholine decreased markedly compared with those of the control rats (to 43-44%). On the contrary, glyceryltrinitrate-induced relaxation was slightly but significantly increased in the aorta of WKY after NO2Arg administration and the same tendency was observed in SHRSP. 3. The rise of blood pressure and the decrease of acetylcholine-induced relaxation suggested that NO2Arg inhibited the endothelium-dependent relaxation not only in WKY but also in SHRSP. The relaxation of the thoracic aorta preparation of SHRSP by acetylcholine was much less (ca 38%) than that of WKY; however, that of SHRSP by glyceryltrinitrate was slightly less (ca 74%), indicating that endothelium-dependent relaxation declined in vascular preparation of SHRSP. 4. The present results suggest that endothelium-dependent relaxation has some contribution on blood pressure regulation in the hypertensive state, although a decline of endothelium-dependent relaxation is evident in vascular preparation of SHRSP compared with WKY.