Effects of probiotic Lactobacillus rhamnosus GG and Propionibacterium freudenreichii ssp. shermanii JS supplementation on intestinal and systemic markers of inflammation in ApoE*3Leiden mice consuming a high-fat diet.

A high-fat diet disturbs the composition and function of the gut microbiota and generates local gut-associated and also systemic responses. Intestinal mast cells, for their part, secrete mediators which play a role in the orchestration of physiological and immunological functions of the intestine. Probiotic bacteria, again, help to maintain the homeostasis of the gut microbiota by protecting the gut epithelium and regulating the local immune system. In the present study, we explored the effects of two probiotic bacteria, Lactobacillus rhamnosus GG (GG) and Propionibacterium freudenreichii spp. shermanii JS (PJS), on high fat-fed ApoE*3Leiden mice by estimating the mast cell numbers and the immunoreactivity of TNF-α and IL-10 in the intestine, as well as plasma levels of several markers of inflammation and parameters of lipid metabolism. We found that mice that received GG and PJS exhibited significantly lower numbers of intestinal mast cells compared with control mice. PJS lowered intestinal immunoreactivity of TNF-α, while GG increased intestinal IL-10. PJS was also observed to lower the plasma levels of markers of inflammation including vascular cell adhesion molecule 1, and also the amount of gonadal adipose tissue. GG lowered alanine aminotransferase, a marker of hepatocellular activation. Collectively, these data demonstrate that probiotic GG and PJS tend to down-regulate both intestinal and systemic pro-inflammatory changes induced by a high-fat diet in this humanised mouse model.

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.

Downregulation of Bradykinin type 2 receptor expression in cardiac endothelial cells during senescence.

OBJECTIVES: Bradykinin type 2 receptor (BK-2R) knockout mice develop microvascular dysfunction and cardiac hypertrophy. In aged human cardiac microvascular endothelium, dysfunction develops before heart failure symptoms. Since endothelial aging is an independent risk factor for cardiovascular disease, we aimed to clarify the role of kinin receptors in age-related endothelial senescence. METHODS AND RESULTS: Using qRT-PCR, a downregulation of BK-2Rs during senescence of cultured human coronary artery endothelial cells (HCAECs) and rat cardiac microvascular endothelial cells (RCMECs) was observed. BK-2R downregulation was associated with a decreased cell proliferation rate, with a growth arrest phenotype and reduced angiogenic potential. By staining senescence-associated ß-galactosidase, RCMECs from old spontaneously hypertensive rats (SHRs) were found to be significantly more senescent than those derived from age-matched WKY rats, albeit their telomere lengths were similar. Despite downregulation of BK-2Rs and BK-1Rs, a novel family member GPR-100 was highly expressed in HCAECs throughout the culture period. CONCLUSIONS: Aging cardiac endothelial cells gradually lose their capacity to express BK-2Rs, and this loss appears to be parallel with a loss of the angiogenic potential of the aging cells. Since RCMECs from hypertensive rats showed premature senescence, hypertension may predispose to cardiac dysfunction by accelerating endothelial aging.

Probiotic Lactobacillus rhamnosus downregulates FCER1 and HRH4 expression in human mast cells.

AIM: To investigate the effects of four probiotic bacteria and their combination on human mast cell gene expression using microarray analysis. METHODS: Human peripheral-blood-derived mast cells were stimulated with Lactobacillus rhamnosus (L. rhamnosus) GG (LGG(®)), L. rhamnosus Lc705 (Lc705), Propionibacterium freudenreichii ssp. shermanii JS (PJS) and Bifidobacterium animalis ssp. lactis Bb12 (Bb12) and their combination for 3 or 24 h, and were subjected to global microarray analysis using an Affymetrix GeneChip(®) Human Genome U133 Plus 2.0 Array. The gene expression differences between unstimulated and bacteria-stimulated samples were further analyzed with GOrilla Gene Enrichment Analysis and Visualization Tool and MeV Multiexperiment Viewer-tool. RESULTS: LGG and Lc705 were observed to suppress genes that encoded allergy-related high-affinity IgE receptor subunits α and γ (FCER1A and FCER1G, respectively) and histamine H4 receptor. LGG, Lc705 and the combination of four probiotics had the strongest effect on the expression of genes involved in mast cell immune system regulation, and on several genes that encoded proteins with a pro-inflammatory impact, such as interleukin (IL)-8 and tumour necrosis factor alpha. Also genes that encoded proteins with anti-inflammatory functions, such as IL-10, were upregulated. CONCLUSION: Certain probiotic bacteria might diminish mast cell allergy-related activation by downregulation of the expression of high-affinity IgE and histamine receptor genes, and by inducing a pro-inflammatory response.

OxLDL-IgG immune complexes induce expression and secretion of proatherogenic cytokines by cultured human mast cells.

OBJECTIVE: Human atherosclerotic lesions contain mast cells and immunoglobulin G immune complexes containing oxidized low-density lipoproteins (oxLDL-IgG ICs). Here we studied whether such oxLDL-IgG ICs can activate human mast cells and induce them to express and secrete pro-inflammatory cytokines that are potentially capable of inducing and amplifying atherogenic processes. METHODS AND RESULTS: Incubation of cultured human mast cells in the presence of oxLDL-IgG ICs led to a significant dose-dependent upregulation of the expression and secretion of tumor necrosis factor-alpha (TNF-a) and interleukin-8 (IL-8), and the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). The secretory responses were dose-dependent and associated with moderate release of histamine and tryptase, which are preformed mast cell mediators contained in the cytoplasmic secretory granules of the cells. Also native LDL-IgG ICs induced similar pro-inflammatory cytokine response, suggesting that ICs per se are important for the IgG IC-induced mast cell activation. CONCLUSION: Mast cells in atherosclerotic lesions which also contain oxLDL-IgG ICs may become activated by the ICs and secrete many pro-inflammatory cytokines. Our results suggest that intimal mast cells act as a cellular link between oxLDL-IgG ICs and the inflammatory response in atherosclerosis.

Vascular endothelial growth factor-secreting mast cells and myofibroblasts: a novel self-perpetuating angiogenic pathway in aortic valve stenosis.

OBJECTIVE: To examine the proangiogenic potential of myofibroblasts and mast cells, 2 types of cells present in human aortic valves. METHODS AND RESULTS: Aortic valve stenosis is an active atheroinflammatory disease, characterized by the accumulation of inflammatory cells and the neovascularization of the valves. A total of 85 stenotic valves and 20 control valves were obtained during valve replacement surgery. The results of immunohistochemistry analysis revealed stenotic aortic valves that contained 3 types of neovessels: small microvessels, medium microvessels, and organized arterioles. The distribution density of the neovessels was significantly higher in stenotic valves than in control valves (P<0.001) and correlated positively with valvular calcification gradus (r=0.26, P=0.02) and mast cell density (r=0.38, P<0.001). In the neovascularized areas of stenotic aortic valves, mast cells contained vascular endothelial growth factor and were degranulated, indicating their activation. The stimulation of cultured myofibroblasts derived from aortic valves with a mast cell-preconditioned medium, hypoxic culture conditions, or tobacco smoke all induced vascular endothelial growth factor secretion in the myofibroblasts. Finally, mast cell tryptase was able to degrade the antiangiogenic molecule endostatin in vitro. CONCLUSIONS: Mast cells and myofibroblasts may accelerate the progression of aortic valve stenosis by altering the balance between angiogenic and antiangiogenic factors in the valves, thus promoting valvular neovascularization.

Mast cells promote atherosclerosis by inducing both an atherogenic lipid profile and vascular inflammation.

Accumulating in vitro and in vivo studies have proposed a role for mast cells in the pathogenesis of atherosclerosis. Here, we studied the role of mast cells in lipoprotein metabolism, a key element in the atherosclerotic disease. Male mice deficient in low-density lipoprotein receptors and mast cells on a Western diet for 26 weeks had significantly less atherosclerotic changes both in aortic sinus (55%, P = 0.0009) and in aorta (31%, P = 0.049), as compared to mast cell-competent littermates. Mast cell-deficient female mice had significantly less atherosclerotic changes in aortic sinus (43%, P = 0.011). Furthermore, we found a significant positive correlation between the extent of atherosclerosis and the number of adventitial/perivascular mast cells in aortic sinus of mast cell-competent mice (r = 0.615, P = 0.015). Serum cholesterol and triglyceride levels were significantly lower in both male (63%, P = 0.0005 and 57%, P = 0.004) and female (73%, P = 0.00009 and 54%, P = 0.007) mast cell-deficient mice, with a concomitant decrease in atherogenic apoB-containing particles and serum prebeta-high-density lipoprotein and phospholipid transfer protein activity in both male (69% and 24%) and female (74% and 54%) mast cell-deficient mice. Serum soluble intercellular adhesion molecule was decreased in both male (32%, P = 0.004) and female (28%, P = 0.003) mast cell-deficient mice, whereas serum amyloid A was similar between mast cell-deficient and competent mice. In conclusion, mast cells participate in the pathogenesis of atherosclerosis in ldlr(-/-) mice by inducing both an atherogenic lipid profile and vascular inflammation.

Hypoxia-induced expression of bradykinin type-2 receptors in endothelial cells triggers NO production, cell migration, and angiogenesis.

Bradykinin receptors are differentially expressed in the coronary vascular endothelium of rat and human hearts during the pathogenesis of heart failure, but the mechanisms responsible for this regulation have remained vague. Here we show by quantitative real-time PCR, Western blot analysis, and immunohistochemistry, that hypoxia triggers the expression of bradykinin type-2 receptors (BK-2Rs) in cultured human coronary artery endothelial cells (HCAECs), in isolated rat cardiac microvascular endothelial cells (RCMECs), and in rat hearts subjected to ligation of the left anterior descending coronary artery. Mild hypoxia (5% O(2)) induced a fourfold temporal increase in BK-2R mRNA expression in HCAECs, which was also observed at the protein level, whereas severe hypoxia (1% O(2)) slightly inhibited the mRNA expression of BK-2Rs. In addition, HOE-140, a BK-2R antagonist, inhibited mRNA and protein expression of BK-2Rs. The BK-2Rs induced by mild hypoxia were biologically active, that is, capable of inducing intracellular production of nitric oxide (NO) upon activation of HCAECs with bradykinin (BK), a response attenuated by HOE-140. In rat hearts recovering from myocardial infarction, BK-2Rs were upregulated in the endothelium of vessels forming at the border zone between fibrotic scar tissue and healthy myocardium. Furthermore, in an in vitro wound-healing assay, RCMEC migration was increased under mild hypoxic culture conditions in the presence of BK and was attenuated with HOE-140. Our present results show that mild hypoxia triggers a temporal expression of functional BK-2Rs in human and rat endothelial cells and support a role for BK-2Rs in hypoxia-induced angiogenesis.

Pro-atherogenic lung and oral pathogens induce an inflammatory response in human and mouse mast cells.

A broad variety of microbes are present in atherosclerotic plaques and chronic bacterial infection increases the risk of atherosclerosis by mechanisms that have remained vague. One possible mechanism is that bacteria or bacterial products activate plaque mast cells that are known to participate in the pathogenesis of atherosclerosis. Here, we show by real-time PCR analysis and ELISA that Chlamydia pneumoniae (Cpn) and a periodontal pathogen, Aggregatibacter actinomycetemcomitans (Aa), both induce a time and concentration-dependent expression and secretion of interleukin 8 (IL-8), tumour necrosis factor-alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1) by cultured human peripheral blood-derived mast cells, but not anti-inflammatory molecules, such as IL-10 or transforming growth factor beta 1 (TGF-beta 1). The IL-8 and MCP-1 responses were immediate, whereas the onset of TNF-alpha secretion was delayed. The Cpn-mediated pro-inflammatory effect was attenuated when the bacteria were inactivated by UV-treatment. Human monocyte-derived macrophages that were pre-infected with Cpn also induced a significant pro-inflammatory response in human mast cells, both in cocultures and when preconditioned media from Cpn-infected macrophages were used. Intranasal and intravenous administration of live Cpn and Aa, respectively induced an accumulation of activated mast cells in the aortic sinus of apolipoprotein E-deficient mice, however, with varying responses in the systemic levels of lipopolysaccharide (LPS) and TNF-alpha. Pro-atherogenic Cpn and Aa induce a pro-inflammatory response in cultured human connective tissue-type mast cells and activation of mouse aortic mast cells in vivo.

Activated mast cells induce endothelial cell apoptosis by a combined action of chymase and tumor necrosis factor-alpha.

OBJECTIVE: Activated mast cells (MCs) induce endothelial cell (EC) apoptosis in vitro and are present at sites of plaque erosions in vivo. To further elucidate the role of MCs in endothelial apoptosis and consequently in plaque erosion, we have studied the molecular mechanisms involved in MC-induced EC apoptosis. METHODS AND RESULTS: Primary cultures of rat cardiac microvascular ECs (RCMECs) and human coronary artery ECs (HCAECs) were treated either with rat MC releasate (ie, mediators released on MC activation), rat chymase and tumor necrosis factor-alpha (TNF-alpha), or with human chymase and TNF-alpha, respectively. MC releasate induced RCMEC apoptosis by inactivating the focal adhesion kinase (FAK) and Akt-dependent survival signaling pathway, and apoptosis was partially inhibited by chymase and TNF-alpha inhibitors. Chymase avidly degraded both vitronectin (VN) and fibronectin (FN) produced by the cultured RCMECs. In addition, MC releasate inhibited the activation of NF-kappaB (p65) and activated caspase-8 and -9. Moreover, in HCAECs, human chymase and TNF-alpha induced additive levels of apoptosis. CONCLUSIONS: Activated MCs induce EC apoptosis by multiple mechanisms: chymase inactivates the FAK-mediated cell survival signaling, and TNF-alpha triggers apoptosis. Thus, by inducing EC apoptosis, MCs may contribute to plaque erosion and complications of atherosclerosis.

Complement system is activated in stenotic aortic valves.

OBJECTIVE: To examine the role of the complement system, a source of powerful proinflammatory mediators, in aortic valve stenosis (AS). METHODS AND RESULTS: Stenotic aortic valves (n=24) were obtained at valve replacement surgery, and non-stenotic (n=12) and early sclerotic (n=4) valves at cardiac transplantations. The terminal complement complex C5b-9 was stained by immunohistochemistry. Expression of the anaphylatoxin receptors C3aR and C5aR was studied in the valves by immunohistochemistry and RT-PCR, and in isolated valve myofibroblasts after stimulation with potential AS-accelerating factors (TNF-alpha and cigarette smoke) by RT-PCR. Cultured myofibroblasts were exposed to C3a, and their secretion of proinflammatory cytokines was assessed by ELISA. C5b-9 was found already in early aortic valve lesions, and its deposition was augmented in advanced stenotic valves. In stenotic valves, expression of C3aR mRNA was upregulated (p<0.05) and strong staining of C3aR and C5aR was detected. Myofibroblasts in stenotic, but not in control valves, expressed C3aR, and, in isolated myofibroblasts, TNF-alpha and cigarette smoke induced C3aR mRNA expression (p<0.05 for both). Stimulation of myofibroblasts with C3a resulted in enhanced secretion of MCP-1 (p<0.001), IL-6 (p=0.003), and IL-8 (p=0.03). CONCLUSIONS: In stenotic aortic valves, complement is activated leading to generation of the anaphylatoxins C3a and C5a. Upregulation of C3aR in the valves as a result of inflammation and external risk factors, such as cigarette smoke, leads to an inflammatory response in aortic valve myofibroblasts. Complement activation in stenotic valves emerges as a novel pathogenic component of AS and may serve as a therapeutic target in this disease.

Inhibition of smooth muscle cell proliferation by a chemically modified tetracycline.

INTRODUCTION: Chemically modified tetracyclines (CMTs) are a group of nonantimicrobial derivatives of tetracycline, which exert antiproliferative and anticollagenolytic properties. The molecular mechanisms, however, are poorly understood. MATERIALS AND METHODS: The effect of CMT-3 on cultured, subconfluent rat aortic smooth muscle cells (SMCs) was analyzed by [(3)H]-thymidine incorporation, counting cell numbers, and flow cytometry analysis. RESULTS: CMT-3 inhibited the incorporation of [(3)H]-thymidine and reduced the cell number dose-dependently, with approximately 60% inhibition at the maximal CMT-3 concentration used (20 mumol/l). CMT-3 decreased the SMC proportion in S-phase and gradually increased the proportion at G2/M. Initially, the proportion of cells in G1-phase increased and then gradually decreased back to baseline as the CMT-3 concentration increased. CMT-3 treatment of confluent SMCs for 24 h did not induce apoptosis. CONCLUSIONS: CMT-3 inhibited SMC proliferation by inducing cell cycle arrest at the G2/M restriction point. Nonetheless, CMT-3 did not induce SMC apoptosis.

Aortic valve stenosis: an active atheroinflammatory process.

PURPOSE OF REVIEW: To summarize the current understanding of the pathobiology of aortic valve stenosis and portray the major advances in this field. RECENT FINDINGS: Stenotic aortic valves are characterized by atherosclerosis-like lesions, consisting of activated inflammatory cells, including T lymphocytes, macrophages, and mast cells, and of lipid deposits, calcific nodules, and bone tissue. Active mediators of calcification and cells with osteoblast-like activity are present in diseased valves. Extracellular matrix remodeling, including collagen synthesis and elastin degradation by matrix metalloproteinases and cathepsins, contributes to leaflet stiffening. In experimental animals, hypercholesterolemia induces calcification and bone formation in aortic valves, which can be inhibited by statin treatment. The potential of statins to retard progression of aortic valve stenosis has also been recognized in clinical studies; however, further prospective trials are needed. Angiotensin II-forming enzymes are upregulated in stenotic valves. Angiotensin II may participate in profibrotic progression of aortic valve stenosis and may serve as a possible therapeutic target. SUMMARY: Recent findings regarding the interaction of inflammatory cells, lipids, mediators of calcification, and renin-angiotensin system in stenotic valves support the current opinion of aortic valve stenosis being an actively regulated disease, potentially amenable to targeted molecular therapy. Evidence from prospective clinical studies is eagerly awaited.

Mast cells in vulnerable atherosclerotic plaques--a view to a kill.

The aim of the present review is to discuss the participation of mast cells in the pathogenesis of erosion and rupture of atherosclerotic plaques, the major causes behind acute coronary syndromes and myocardial infarction. We present ex vivo observations describing mast cells and their activation in human atherosclerotic plaques and discuss in vitro and in vivo data showing that mast cells are potential regulators of inflammation, immunity and adverse remodeling, including matrix remodeling and cell death. Furthermore, we focus on studies that have been performed with human tissues and human mast cells, but when appropriate, we also discuss observations made in animal models. Finally, we present potential pharmacological means to modulate mast cell responses in the arterial vessel walls.

Lovastatin induces the expression of bradykinin type 2 receptors in cultured human coronary artery endothelial cells.

Cardioprotective bradykinin type-2 receptors (BK-2Rs) are downregulated in the myocardial endothelium of both human and rat failing hearts. Statins are cardioprotective drugs that reduce the level of plasma cholesterol but also exert cholesterol-independent pleiotropic effects. Here we examined the effect of lovastatin on BK-2R expression in cultured human coronary artery endothelial cells. The effect of lovastatin on the expression of BK receptors in human coronary artery endothelial cells (HCAECs) was examined by real-time PCR, Western blot analysis and immunocytochemistry. Lovastatin induced a time- and concentration-dependent increase in both BK-2R and BK-1R mRNA expression in the cultured HCAECs. Also, the number of functional BK-2Rs capable of inducing BK-mediated NO production and cGMP signaling was increased in the lovastatin-treated HCAECs. Mevalonate, the direct metabolite of HMG-CoA reductase, reversed the effect of lovastatin. Furthermore, lovastatin inhibited Rho activation and a selective inhibitor of Rho-associated kinases, Y-27632, induced a similar increase in BK-2R expression as lovastatin. In contrast, a specific inhibitor of COX-2, NS398, significantly inhibited the lovastatin-induced expression of BK-2Rs. Here we show for the first time that lovastatin induces the expression of BK-2Rs in cultured human coronary artery endothelial cells through a novel cholesterol-independent pleiotropic mechanism that involves RhoA kinase inhibition and COX-2 activation. Thus, reported beneficial effects of statins in cardiovascular diseases may be partly mediated by an increased expression of cardioprotective BK-2Rs in the endothelial cells of the coronary tree. Moreover, the use of COX-2 inhibitors may affect the level of endothelial BK-2Rs in a negative fashion.

Improved identification of endothelial erosion by simultaneous detection of endothelial cells (CD31/CD34) and platelets (CD42b).

Loss of endothelial cells (ECs) with ensuing exposure of thrombogenic subendothelial surface is a common cause of thromboembolic complications in atherosclerotic arteries. Thus, endothelial denudation has emerged as a major contributor to the pathogenesis of atherosclerosis and its complications. Despite ongoing efforts in elucidating the pathogenesis of endothelial erosions in human atherosclerotic arteries, the mechanisms of erosion have remained enigmatic, partly due to lack of well-established methods for its identification. Here the authors point out plausible pitfalls in the current methodology and provide an improved immunohistochemical method for identifying endothelial erosion; i.e., immunofluorescence double staining with antibodies against CD42b and CD31/CD34. This method enables reliable detection of ECs and platelets in the same staining by allowing detection of "pseudoendothelium" caused by CD31 staining of a thin platelet layer covering sites of endothelial erosion. As erosion with a luminal platelet thrombus is likely to represent an in vivo erosion, and erosion without platelets an ex vivo artefact, the method makes it possible to exclude artefactual erosions resulting from sample processing. The novel immunostaining protocol presented here allows more reliable detection of endothelial erosions and so may facilitate studies on the mechanisms involved in the pathogenesis of plaque erosion and acute coronary syndromes.

Increased expression of profibrotic neutral endopeptidase and bradykinin type 1 receptors in stenotic aortic valves.

AIMS: In aortic stenosis (AS), adverse remodelling of the valves may depend on altered local regulation of pro- and antifibrotic systems. We have recently shown that angiotensin-converting enzyme (ACE), which generates profibrotic angiotensin II and inactivates antifibrotic bradykinin (BK), is upregulated in stenotic aortic valves. Here, we analyse the expression of neutral endopeptidase (NEP), another profibrotic and BK-degrading enzyme, and of BK receptors in aortic valves in AS. METHODS AND RESULTS: Stenotic aortic valves (n = 86) were obtained at valve replacement surgery and control valves (n = 13) at cardiac transplantation. Expression levels of NEP and BK type 1 and 2 receptors (BK-1R and BK-2R) in aortic valves and in isolated valvular myofibroblasts were analysed by real-time PCR and immunohistochemistry, and NEP activity was quantified by autoradiography. NEP, BK-1R, and BK-2R mRNA levels were higher in stenotic than in non-stenotic valves (P < 0.05 for each) and the respective proteins localized to valvular endothelial cells and myofibroblasts. In stenotic valves, the proteolytic activity of NEP was significantly increased (4.5-fold, P < 0.001), and tumour necrosis factor-alpha induced the expression of NEP in cultured myofibroblasts. Finally, treatment of cultured myofibroblasts with an NEP inhibitor (phosphoramidon) downregulated the expression of profibrotic transforming growth factor-beta1, whereas addition of BK decreased the expression of collagens I and III which was reversed by a BK-2R antagonist. CONCLUSION: NEP activity is increased in stenotic aortic valves in parallel with increased expression of BK-receptors. The upregulation of NEP and BK-1R have the potential to promote valvular fibrosis and remodelling while the increase in BK-2R may represent a compensatory antifibrotic response. These findings add novel pathogenic insight and raise potential new therapeutic targets in AS.

Perivascular mast cells promote atherogenesis and induce plaque destabilization in apolipoprotein E-deficient mice.

BACKGROUND: Mast cells are major effector cells in allergy and host defense responses. Their increased number and state of activation in perivascular tissue during atherosclerosis may point to a role in cardiovascular disorders. In the present study, we investigated the contribution of perivascular mast cells to atherogenesis and plaque stability in apolipoprotein E-deficient mice. METHODS AND RESULTS: We show here that episodes of systemic mast cell activation during plaque progression in mice leads to robust plaque expansion. Targeted activation of perivascular mast cells in advanced plaques sharply increases the incidence of intraplaque hemorrhage, macrophage apoptosis, vascular leakage, and CXCR2/VLA-4-mediated recruitment of leukocytes to the plaque. Importantly, treatment with the mast cell stabilizer cromolyn does prevent all the adverse phenomena elicited by mast cell activation. CONCLUSIONS: This is the first study to demonstrate that mast cells play a crucial role in plaque progression and destabilization in vivo. We propose that mast cell stabilization could be a new therapeutic approach to the prevention of acute coronary syndromes.

Increased circulating concentrations and augmented myocardial extraction of osteoprotegerin in heart failure due to left ventricular pressure overload.

BACKGROUND: Osteoprotegerin (OPG) and the receptor activator of nuclear factor-kappaB ligand (RANKL), two cytokines regulating bone remodeling, have recently been raised as potential pathogenetic factors in cardiovascular diseases. We have studied circulating and myocardial OPG and RANKL in patients having severe aortic stenosis (AS) with or without heart failure (HF). METHODS: We studied 131 adults with AS. Blood was sampled from the aortic root, coronary sinus, and femoral vein at cardiac catheterization. LV myocardial biopsies were taken at surgery. Plasma OPG and soluble (s)RANKL were analyzed by ELISA, and myocardial OPG and RANKL by RT-PCR and immunohistochemistry. RESULTS: Circulating OPG was elevated in AS patients with HF, the association being independent of age, sex, and presence of coronary artery disease (beta=0.17, p=0.033). Elevated plasma OPG decreased after valve replacement in patients with preoperative HF (p=0.0005). Relative to its concentration in the aortic root, plasma OPG was reduced in the coronary sinus (p<0.05) and in the femoral vein (p<0.001), these arteriovenous gradients being accentuated in HF (p=0.003). CONCLUSIONS: HF due to LV pressure overload in AS increases circulating OPG and augments OPG extraction by the heart and peripheral tissues. OPG may be involved in the pathogenesis of HF and could serve as a useful biomarker in HF due to LV pressure overload.

Angiotensin II increases expression and secretion of cathepsin F in cultured human monocyte-derived macrophages: an angiotensin II type 2 receptor-mediated effect.

Increasing evidence suggests that cathepsins and angiotensin II (AngII) participate in atherosclerosis, particularly in remodeling of the extracellular matrix of the inflamed arterial intima. Here, we show that AngII induces mRNA expression of cathepsin F, a member of the cysteine protease family, in human monocyte-derived macrophages. AngII did not affect the amount of intracellular cathepsin F protein, but significantly enhanced its secretion by the treated cells. The stimulatory effect of AngII was mediated by the AngII type 2 (AT(2)) receptor, as demonstrated by the ability of the AT(2)-receptor antagonist PD123319 to block the AngII-induced increase in cathepsin F secretion. Our present data demonstrate a novel proatherogenic role for AngII, namely its ability to enhance secretion of lysosomal cathepsin F by monocyte-derived macrophages.