The role of mast cells in cutaneous wound healing in streptozotocin-induced diabetic mice.

Mast cells (MCs) reside in cutaneous tissue, and an increment of MCs is suggested to induce vascular regression in the process of wound healing. To clarify participation of MCs in diabetic cutaneous wound healing, we created an excisional wound on diabetic mice 4 weeks after streptozotocin injections and subsequently investigated the healing processes for 49 days, comparing them with control mice. The rate of wound closure was not markedly different between the diabetic and control mice. In the proliferative phase at days 7 and 14, neovascularization in the wound was weaker in diabetic mice than in control mice. In the remodeling phase at day 21 and afterward, rapid vascular regression occurred in control mice; however, neovascularization was still observed in diabetic mice where the number of vessels in granulation tissues was relatively higher than in control mice. In the remodeling phase of the control mice, MCs within the wound began to increase rapidly and resulted in considerable accumulation, whereas the increment of MCs was delayed in diabetic mice. In addition, the number of fibroblast growth factor (FGF)- or vascular endothelial growth factor (VEGF)-immunopositive hypertrophic fibroblast-like spindle cells and c-Kit-positive/VEGFR2-positive/FcεRIα-negative endothelial progenitor cells (EPCs) were higher in diabetic wounds. In conclusion, neovascularization in the proliferative phase and vascular regression in the remodeling phase were impaired in diabetic mice. The delayed increment of MCs and sustained angiogenic stimuli by fibroblast-like spindle cells and EPCs may inhibit vascular regression in the remodeling phase and impair the wound-healing process in diabetic mice.

Pranlukast, a cysteinyl leukotriene type 1 receptor antagonist, attenuates the progression but not the onset of silica-induced pulmonary fibrosis in mice.

BACKGROUND: Although cysteinyl leukotrienes (CysLTs) have been implicated in the etiology of acute inflammatory diseases, recent studies have suggested that they also directly stimulate fibroblasts. However, their precise role in the pathogenesis of pulmonary fibrosis is unclear. METHODS: In this study, we evaluated the effect of both short- and long-term treatment with pranlukast, a CysLT type 1 (CysLT(1)) receptor antagonist, on silica-induced pulmonary fibrosis in mice, which is characterized by persistent progression of fibrosis in the chronic phase. Pranlukast (30 mg/kg/day) was administered orally to mice for 2 or 10 weeks after intratracheal silica instillation. RESULTS: Pranlukast treatment for 10 weeks significantly attenuated the progression of pulmonary fibrosis, and decreased the content of CysLTs and LTB(4), which were markedly increased in the bronchoalveolar lavage fluid (BALF) and lung tissues of silica-instilled mice in the chronic phase. However, pranlukast treatment for 2 weeks neither affected the acute inflammatory response induced by silica instillation nor inhibited the onset of fibrosis. The expression of TGF-ß1 and TNF-α was not affected by pranlukast treatment for either 2 or 10 weeks. CONCLUSIONS: Pranlukast attenuates the progression of pulmonary fibrosis in the chronic phase but has no effect on the acute inflammatory response or on the onset of pulmonary fibrosis. The antifibrotic effect of pranlukast may be exhibited by antagonizing the direct profibrotic effect of CysLTs, without affecting the expression of other profibrotic cytokines such as TGF-ß1 and TNF-α, and also by decreasing the production of CysLTs and LTB(4).

Bradykinin type-2 receptor expression correlates with age and is subjected to transcriptional regulation.

Accumulating work in experimental animals suggests that bradykinin (BK) exerts cardioprotective effects via bradykinin type-2 receptors (BK-2Rs). In human end-stage heart failure, BK-2Rs are significantly downregulated by mechanisms that have remained elusive. Heart tissues from idiopathic dilated cardiomyopathy (IDC; n = 7), coronary heart disease (CHD; n = 6), and normal patients (n = 6) were analyzed by RT-PCR, SSCP, and Western blotting. In normal and IDC hearts, BK-2R expression increased with age, with a lower relative increase in IDC hearts. BK-2R mRNA and protein levels showed a positive linear correlation, suggesting transcriptional regulation. Two known BK-2R promoter polymorphisms, -58T/C and -9/+9, were found to be present in the study population. The allelic frequencies for the C-allele in -58T/C were 0.58 in normal and CHD hearts and 0.81 in IDC hearts. Furthermore, the allelic frequencies for the -9 and +9 alleles were 0.42 and 0.58 in normal hearts and 0.64 and 0.36 in IDC hearts, respectively. All analyzed CHD hearts were homozygous for the -9 allele. Thus, the expression of cardioprotective BK-2Rs in human hearts is increased with age in normal and IDC hearts and may be regulated on the transcriptional level. Moreover, comparison of normal subjects and patients with failing hearts revealed different allelic frequencies in each of two known BK-2R gene polymorphisms.

Effects of montelukast, a cysteinyl-leukotriene type 1 receptor antagonist, on the pathogenesis of bleomycin-induced pulmonary fibrosis in mice.

Cysteinyl-leukotrienes are potent mediators involved in various inflammatory diseases and lung disorders such as asthma. However, their precise role in the pathogenesis of pulmonary fibrosis is unknown. In the present study, we investigated the effect of montelukast, a cysteinyl-leukotriene type 1 receptor antagonist, on bleomycin-induced pulmonary fibrosis in mice. Montelukast (10mg/kg/day) was orally administered to the bleomycin-induced pulmonary fibrosis mice for 3days before and 14days after intratracheal instillation of bleomycin. We evaluated the effects of montelukast on the development of pulmonary fibrosis in these mice and investigated the expression of various cytokines and two cysteinyl-leukotriene receptors. Treatment with montelukast significantly attenuated the increased fibrotic area and hydroxyproline content in the fibrotic lungs of bleomycin-instilled mice. Montelukast treatment also decreased mRNA levels of IL-6, IL-10, IL-13, and TGF-ß1, all of which were elevated in fibrotic lungs. In fibrotic lungs, TNF-α and IL-1ß mRNA levels were increased and IFN-γ mRNA levels were decreased, but montelukast did not affect these mRNA levels. Furthermore, cysteinyl-leukotriene type 1 receptor mRNA levels were increased, whereas cysteinyl-leukotriene type 2 receptor mRNA levels were decreased in fibrotic lungs. Montelukast treatment induced the recovery of cysteinyl-leukotriene type 2 receptor mRNA levels to normal control levels but did not change cysteinyl-leukotriene type 1 receptor mRNA levels. These results suggest that montelukast exhibits its beneficial effects by inhibiting the overexpression of IL-6, IL-10, IL-13, and TGF-ß1 and by modulating the homeostatic balance between the cysteinyl-leukotriene type 1 and type 2 receptors.

Involvement of leukotrienes in the pathogenesis of silica-induced pulmonary fibrosis in mice.

The authors investigated the role of leukotrienes (LTs) in the pathogenesis of silica-induced pulmonary fibrosis in mice during the progression from acute to chronic phases. Intratracheal instillation of silica particles induced progressive pulmonary fibrosis. The tissue content of cysteinyl (Cys) LTs and LTB(4) was markedly increased in the acute phase after silica instillation, concurrently with the up-regulation of LTB(4) receptor, transforming growth factor (TGF)-beta1, and tumor necrosis factor (TNF)-alpha, along with down-regulation of the CysLT type 2 receptor. Importantly, the tissue content of CysLTs and mRNA levels of TGF-beta1 and TNF-alpha were increased in the fibrotic lung in the chronic phase. Furthermore, strong immunohistochemical staining for the CysLT type 1 receptor, TNF-alpha, and TGF-beta1, but not for the CysLT type 2 receptor, was codetected in the pathological lesions during both acute and chronic phases. These findings suggest that an increase in LT production in the lung and modulation of homeostatic balance among LT receptors may contribute to the progression of pulmonary fibrosis.

Pathophysiological role of skin mast cells in wound healing after scald injury: study with mast cell-deficient W/W(V) mice.

BACKGROUND: The major role of mast cells in wound healing process has not been identified. In this study, we used mast cell-deficient W/W(V) mice and their congenic control (+/+) mice to examine the role of mast cells in scald wound healing. METHODS: The size of the scald wound, thickness of the dermis, collagen deposition, vascularization, number of mast cells and chymase activity were measured before and at 3, 7, 14 and 21 days after inducing scald injury. RESULTS: Although the process of wound closure and re-epithelialization was not markedly different between W/W(V) mice and +/+ mice, the degree of fibrous proliferation at the wound edge and wound vascularization in the proliferative phase was significantly lower in W/W(V) mice than in +/+ mice, and no vascular regression in the late remodeling phase was observed in W/W(V) mice. Mast cells producing chymase, FGF2, TGF-beta1 and VEGF were highly accumulated at the edge of scald wound in +/+ mice during the proliferative and remodeling phases at days 14 and 21. Chymase activity in the injured tissues of +/+ mice decreased in the acute phase, but recovered to no-injury level at days 14 and 21. The number of mast cells and chymase activity were very low in the injured tissues of W/W(V) mice throughout the experiment. CONCLUSIONS: Wound healing after skin scald injury was partially impaired in mast cell-deficient mice. Mast cells may contribute to the wound healing process, especially in the proliferative and remodeling phases after scald injury.

Pathophysiological role of mast cells in collagen-induced arthritis: study with a cysteinyl leukotriene receptor antagonist, montelukast.

Our previous study showed that the number of mast cells was increased in the inflamed paws of collagen-induced arthritis in mice, and treatment with a mast cell-stabilizing compound effectively suppressed the development of collagen-induced arthritis. A recent in vitro study showed that mast cells express cysteinyl leukotriene type 1 receptor, and that a cysteinyl leukotriene type 1 receptor antagonist inhibits the production of TNF-alpha by mast cells. To further investigate the role of mast cells in vivo, we evaluated the therapeutic effects of a cysteinyl leukotriene type 1 receptor antagonist, montelukast, on the development of collagen-induced arthritis in mice. Montelukast (10 mg/kg/day) or vehicle was orally administered to mice for 12 weeks, starting 6 weeks after immunization with bovine type II collagen. Treatment with montelukast significantly reduced clinical scores and X-ray scores of collagen-induced arthritis, and decreased the number of mast cells in the inflamed paws of collagen-induced arthritic mice. Immunohistochemical analysis revealed that mast cells in the inflamed synovium were one of the major cells producing TNF-alpha and that the number of TNF-alpha positive mast cells was significantly reduced by treatment with montelukast. Furthermore, TNF-alpha and SCF mRNA levels in the paws of collagen-induced arthritic mice were markedly decreased by montelukast treatment. Montelukast may lead to a beneficial therapeutic effect by inhibiting TNF-alpha production by mast cells.

Effect of mast cell chymase inhibitor on the development of scleroderma in tight-skin mice.

1 Although the pathogenesis of scleroderma is not fully understood, activation of connective-tissue-type mast cells (CTMCs) has been implicated in various fibrotic diseases. 2 Our previous study showed that the number of CTMCs was markedly increased during fibrous proliferation in the skin of a scleroderma model, namely tight-skin (Tsk) mice. Because mast cells express numerous bioactive factors, such as cytokines, growth factors, proteases, and others, it is crucial to identify the primary factors that may be involved in the pathogenesis of scleroderma. Our previous study also showed that a CTMC-specific protease, chymase-4, was selectively upregulated in accordance with the development of skin fibrosis in Tsk mice. 3 To further elucidate the role of chymase secreted from CTMCs, we evaluated the therapeutic effects of a synthetic chymase-specific inhibitor, SUN-C8257, on the development of skin fibrosis in Tsk mice. SUN-C8257 (50 mg kg-1 day-1) was administered via intraperitoneal injection in 13-week-old Tsk mice for a period of 2 weeks. 4 Treatment with SUN-C8257 significantly reduced chymase activity by 43% and the chymase-4 mRNA level by 47%, and also decreased the thickness of the subcutaneous fibrous layer of Tsk mice by 42% compared with that of Tsk mice injected with vehicle. 5 Furthermore, immunohistochemical analysis revealed that transforming growth factor (TGF)-beta1 staining in the fibrous layer of Tsk skin was markedly reduced by the treatment with SUN-C8257. This chymase inhibitor may prevent the chymase-dependent pathway that activates the latent TGF-beta1 in fibrous tissue, and may exhibit beneficial effects that inhibit the development of fibrosis. 6 In conclusion, our results strongly support the assumption that CTMC-derived chymase may play a key role in the pathogenesis of scleroderma.

Increased expression of bradykinin type-1 receptors in endothelium of intramyocardial coronary vessels in human failing hearts.

In experimental animals, bradykinin type-1 receptors (BK-1Rs) are induced during inflammation and ischemia, and, by exerting either cardioprotective or cardiotoxic effects, they may contribute to the pathogenesis of heart failure. Nothing is known about the expression of BK-1Rs in human heart failure. Human heart tissue was obtained from excised hearts of patients undergoing cardiac transplantation (n = 13), due to idiopathic dilated cardiomyopathy (IDC; n = 7) or to coronary heart disease (CHD; n = 6), and from normal hearts (n = 6). The expression of BK-1Rs was analyzed by means of competitive RT-PCR, Western blot analysis, and immunohistochemistry. Expression of BK-1R mRNA was increased in both IDC (2.8-fold) and CHD (2.1-fold) hearts compared with normal hearts. The observed changes were verified at the protein level. Expression of BK-1Rs in failing hearts localized to the endothelium of intramyocardial coronary vessels and correlated with an increased expression of TNF-alpha in the vessel wall. Treatment of human coronary artery endothelial cells with TNF-alpha increases their BK-1R expression. These novel results show that BK-1Rs are induced in the endothelium of intramyocardial coronary vessels in failing human hearts and so may participate in the pathogenesis of heart failure.

Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo.

APJ is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin, was identified recently. They are highly expressed in the cardiovascular system, suggesting that APJ is important in the regulation of blood pressure. To investigate the physiological functions of APJ, we have generated mice lacking the gene encoding APJ. The base-line blood pressure of APJ-deficient mice is equivalent to that of wild-type mice in the steady state. The administration of apelin transiently decreased the blood pressure of wild-type mice and a hypertensive model animal, a spontaneously hypertensive rat. On the other hand, this hypotensive response to apelin was abolished in APJ-deficient mice. This apelin-induced response was inhibited by pretreatment with a nitric-oxide synthase inhibitor, and apelin-induced phosphorylation of endothelial nitric-oxide synthase in lung endothelial cells from APJ-deficient mice disappeared. In addition, APJ-deficient mice showed an increased vasopressor response to the most potent vasoconstrictor angiotensin II, and the base-line blood pressure of double mutant mice homozygous for both APJ and angiotensin-type 1a receptor was significantly elevated compared with that of angiotensin-type 1a receptor-deficient mice. These results demonstrate that APJ exerts the hypotensive effect in vivo and plays a counterregulatory role against the pressor action of angiotensin II.

A role for cardiac mast cells in the pathogenesis of hypertensive heart disease.

OBJECTIVE: Cardiac mast cells participate in myocardial dysfunction, but the mechanisms are presently unknown. DESIGN: By examining spontaneously hypertensive rats (SHRs) during their entire lifespan, we attempted to define the role of mast cells in the induction of cardiac hypertrophy and transition to heart failure. METHODS AND RESULTS: By contrast to normotensive littermates, hearts of newborn SHRs already contained mast cells. In the prehypertensive (2-week-old) SHRs, the increased expression of c-kit and soluble stem cell factor correlated with an increased number of cardiac mast cells. The mast cells contained tumour necrosis factor-alpha which, together with nuclear factor kappa-B (NF-kappaB) and interleukin (IL)-6, was significantly induced in the prehypertensive SHRs. Stimulation of cardiac mast cells with compound 48/80 in an ex-vivo Langendorff heart perfusion system resulted in increased expression of nuclear factor Kappa-B (NF-kappaB) (four-fold) and IL-6 (nine-fold) mRNA in the left ventricles of adult rat hearts. In the presence of an inhibitor of mast cell degranulation, disodium cromoglycate, the induced expression of NF-kappaB and IL-6 was inhibited. In the late hypertensive stage, the hearts of SHRs with advanced cardiac hypertrophy (12-month-old) and heart failure (20-month-old) had significantly increased levels of transforming growth factor (TGF)-beta1 and basic fibroblast growth factor (bFGF), and displayed increased myocardial fibrosis. Activated mast cells were a major source of TGF-beta1 and bFGF, and localized to areas of myocardial fibrosis. CONCLUSIONS: By synthesizing and secreting prohypertrophic cytokines and profibrotic growth factors, cardiac mast cells participate in the induction of cardiac hypertrophy and cardiac fibrosis, which are the key steps in the transition to heart failure.

Expression of bradykinin receptors in the left ventricles of rats with pressure overload hypertrophy and heart failure.

OBJECTIVES: Bradykinin exerts cardioprotective effects through bradykinin type-2 receptors (BK-2Rs). After acute myocardial infarction in rat, the heart adapts by increasing its number of BK-2Rs. However, in human chronic end-stage heart failure, the number of BK-2Rs is significantly decreased. Thus, the presence of a cardioprotective BK-2R signaling system may be critical in the prevention of pressure overload-induced heart failure. DESIGN: To explain differences in myocardial BK-2R expression during cardiac overload, we studied: (1). spontaneously hypertensive rats (SHRs) of different ages, and (2). normotensive Sprague-Dawley rats subjected to aortic banding or angiotensin II infusion. METHODS AND RESULTS: The mRNA levels of BK-2Rs were found to be significantly (P < 0.05) increased in the aging (12 and 20-month-old) SHRs (2.9- and 3-fold, respectively). Similarly, in the Sprague-Dawley rats, the expression of BK-2Rs was increased at 12 h (1.8-fold, P < 0.05) and at 3 days (3.1-fold, P < 0.05) after aortic banding, and at 2 weeks (2.2-fold) after angiotensin II infusion. In the 12-month-old SHRs, with compensated left ventricular hypertrophy (no fibrosis or left ventricular dysfunction), the amount of BK-2Rs was also significantly increased (1.8-fold, P < 0.05). However, in the 20-month-old SHRs, with a dramatic increase in fibrosis and development of diastolic dysfunction and heart failure, the amount of BK-2Rs were significantly decreased (63%, P < 0.05) specifically in the cardiac endothelial cells. CONCLUSIONS: The present results show that, during pressure overload and compensated left ventricular hypertrophy, the expression of BK-2Rs is increased. However, ongoing pressure overload leads to a loss of BK-2Rs with a dramatic increase in left ventricular fibrosis followed by diastolic dysfunction and heart failure.

Mast cell-mediated apoptosis of endothelial cells in vitro: a paracrine mechanism involving TNF-alpha-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-alpha. 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-alpha 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-alpha, may contribute to the erosion of atherosclerotic plaques by inducing apoptosis in adjacent endothelial cells. Published 2003 Wiley-Liss, Inc.

Down-regulation of cardioprotective bradykinin type-2 receptors in the left ventricle of patients with end-stage heart failure.

OBJECTIVES: We sought to study the expression of bradykinin type-2 receptors (BK-2Rs) in patients with heart failure (HF). BACKGROUND: Recent work in experimental animals has suggested that bradykinin (BK) exerts cardioprotective effects through specific BK-2Rs. However, nothing is known about the regulation of BK-2R expression in the pathogenesis of human HF. METHODS: Human heart tissue was obtained from excised hearts of patients undergoing cardiac transplantation (n = 13) and from normal hearts (n = 6) unsuitable for donation. The patients had HF due to idiopathic dilated cardiomyopathy (IDC) (n = 7) or coronary heart disease (CHD) (n = 6). Tissue samples from the left ventricles were analyzed by competitive reverse-transcriptase-polymerase chain reaction and Western blotting for the expression of BK-2R messenger ribonucleic acid (mRNA) and protein. RESULTS: In both the IDC and CHD hearts, the level of BK-2R mRNA expression was found to be significantly lower (30% and 38% of control values, respectively) than that in normal hearts. Correspondingly, the BK-2R protein level was significantly reduced in both the IDC and CHD hearts (45% and 62% of control values, respectively) and apparently involved all myocardial cell types. The down-regulation of BK-2R expression in failing hearts did not correlate with decreased cellularity or with the expression pattern of other members of the G-protein-coupled receptor superfamily. However, BK-2R down-regulation in the failing hearts was associated with a decrease in endothelial nitric oxide synthase in both IDC (53% of control value) and CHD (43% of control value) hearts. CONCLUSIONS: These results are the first to suggest that a loss of BK-2Rs is involved in the pathogenesis of human HF.