Familial hypercholesterolemia and elevated lipoprotein(a): double heritable risk and new therapeutic opportunities.

There is compelling evidence that the elevated plasma lipoprotein(a) [Lp(a)] levels increase the risk of atherosclerotic cardiovascular disease (ASCVD) in the general population. Like low-density lipoprotein (LDL) particles, Lp(a) particles contain cholesterol and promote atherosclerosis. In addition, Lp(a) particles contain strongly proinflammatory oxidized phospholipids and a unique apoprotein, apo(a), which promotes the growth of an arterial thrombus. At least one in 250 individuals worldwide suffer from the heterozygous form of familial hypercholesterolemia (HeFH), a condition in which LDL-cholesterol (LDL-C) is significantly elevated since birth. FH-causing mutations in the LDL receptor gene demonstrate a clear gene-dosage effect on Lp(a) plasma concentrations and elevated Lp(a) levels are present in 30-50% of patients with HeFH. The cumulative burden of two genetically determined pro-atherogenic lipoproteins, LDL and Lp(a), is a potent driver of ASCVD in HeFH patients. Statins are the cornerstone of treatment of HeFH, but they do not lower the plasma concentrations of Lp(a). Emerging therapies effectively lower Lp(a) by as much as 90% using RNA-based approaches that target the transcriptional product of the LPA gene. We are now approaching the dawn of an era, in which permanent and significant lowering of the high cholesterol burden of HeFH patients can be achieved. If outcome trials of novel Lp(a)-lowering therapies prove to be safe and cost-effective, they will provide additional risk reduction needed to effectively treat HeFH and potentially lower the CVD risk in these high-risk patients even more than currently achieved with LDL-C lowering alone.

Prompt impact of first prospective statin mega-trials on postoperative lipid management of CABG patients: a 20-year follow-up in a single hospital.

BACKGROUND: The long-term success of coronary artery bypass grafting (CABG) depends on secondary prevention. Vast evidence provided by the results of cholesterol mega-trials over two decades has shown that effective reduction of LDL cholesterol improves the prognosis of patients with coronary heart disease. However, the implementation of these results into the clinical practice has turned out to be challenging. We analysed how the information derived from clinical statin trials and international recommendations affected the local treatment practices of dyslipidaemia of CABG patients during a 20-year time period. METHODS: The cohort includes all CABG patients (n = 953) treated in Kanta-Häme Central Hospital during the time period 1990-2009. At the postoperative visits in the cardiology outpatient clinic, each patient's statin prescription was recorded, and blood lipids were determined. RESULTS: During 1990-1994, 12.0 % of patients were on statins and during the following 5-year time periods the proportion was 57.2, 82.2 and 96.8 %, respectively. During the 20-year observation period (1990-2009), the effective statin dose increased progressively during these 5-year periods up to 36-fold, while the mean concentration of LDL cholesterol decreased from 3.7 to 2.1 mmol/l and that of apolipoprotein B from 1.3 to 0.8 g/l. In the very last year of follow-up, the mean concentrations of LDL-C and apoB were 1.83 mmol/l and 0.78 g/l, respectively. The most prominent increase in statin use and dosage took place during 1994-1996 and 2003-2005, respectively. CONCLUSIONS: Among CABG patients the lipid-lowering efficacy of statin therapy improved dramatically since 1994. This progress was accompanied by significant and favourable changes of lipid and apolipoprotein-B values. This study shows that it is possible to effectively improve lipid treatment policy once the results of relevant trials are available, and that this may happen even before international or national guidelines have been updated.

Electronegative LDL induces priming and inflammasome activation leading to IL-1ß release in human monocytes and macrophages.

BACKGROUND: Electronegative LDL (LDL(−)), a modified LDL fraction found in blood, induces the release of inflammatory mediators in endothelial cells and leukocytes. However, the inflammatory pathways activated by LDL(−) have not been fully defined. We aim to study whether LDL(−) induced release of the first-wave proinflammatory IL-1ß in monocytes and monocyte-derived macrophages (MDM) and the mechanisms involved. METHODS: LDL(−) was isolated from total LDL by anion exchange chromatography. Monocytes and MDM were isolated from healthy donors and stimulated with LDL(+) and LDL(−) (100 mg apoB/L). RESULTS: In monocytes, LDL(−) promoted IL-1ß release in a time-dependent manner, obtaining at 20 h-incubation the double of IL-1ß release induced by LDL(−) than by native LDL. LDL(−)-induced IL-1ß release involved activation of the CD14-TLR4 receptor complex. LDL(−) induced priming, the first step of IL-1ß release, since it increased the transcription of pro-IL-1ß (8-fold) and NLRP3 (3-fold) compared to native LDL. Several findings show that LDL(−) induced inflammasome activation, the second step necessary for IL-1ß release. Preincubation of monocytes with K+ channel inhibitors decreased LDL(−)-induced IL-1ß release. LDL(−) induced formation of the NLRP3-ASC complex. LDL(−) triggered 2-fold caspase-1 activation compared to native LDL and IL-1ß release was strongly diminished in the presence of the caspase-1 inhibitor Z-YVAD. In MDM, LDL(−) promoted IL-1ß release, which was also associated with caspase-1 activation. CONCLUSIONS: LDL(−) promotes release of biologically active IL-1ß in monocytes and MDM by induction of the two steps involved: priming and NLRP3 inflammasome activation. SIGNIFICANCE: By IL-1ß release, LDL(−) could regulate inflammation in atherosclerosis.

Intracellular RNA recognition pathway activates strong anti-viral response in human mast cells.

Mast cells have been implicated in the first line of defence against parasites and bacteria, but less is known about their role in anti-viral responses. Allergic diseases often exacerbate during viral infection, suggesting an increased activation of mast cells in the process. In this study we investigated human mast cell response to double-stranded RNA and viral infection. Cultured human mast cells were incubated with poly(I:C), a synthetic RNA analogue and live Sendai virus as a model of RNA parainfluenza virus infection, and analysed for their anti-viral response. Mast cells responded to intracellular poly(I:C) by inducing type 1 and type 3 interferons and TNF-α. In contrast, extracellular Toll-like receptor 3 (TLR)-3-activating poly(I:C) failed to induce such response. Infection of mast cells with live Sendai virus induced an anti-viral response similar to that of intracellular poly(I:C). Type 1, but not type 3 interferons, up-regulated the expression of melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-inducible gene-1 (RIG-1), and TLR-3, demonstrating that human mast cells do not express functional receptors for type 3 interferons. Furthermore, virus infection induced the anti-viral proteins MxA and IFIT3 in human mast cells. In conclusion, our results support the notion that mast cells can recognize an invading virus through intracellular virus sensors and produce high amounts of type 1 and type 3 interferons and the anti-viral proteins human myxovirus resistance gene A (MxA) and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in response to the virus infection.

Immune functions of serum amyloid A.

Serum amyloid A (SAA) is a highly conserved, acute-phase protein synthesized predominantly by the liver. After secretion into the circulation, it associates with high-density lipoprotein (HDL) particles. During acute inflammation, serum SAA levels may rise up to 1000-fold, and under these conditions, SAA displaces apolipoprotein A-I from HDL, thus becoming the major apolipoprotein of circulating HDL3. SAA exhibits significant immunological activity by, for example, inducing the synthesis of several cytokines and by being chemotactic for neutrophils and mast cells. It exerts many of its immunological activities by binding and activating cell-surface receptors, including Toll-like receptor (TLR) 2 and TLR4, formyl peptide receptor-like 1 (FPRL1), class B scavenger receptor CD36, and the ATP receptor P2X7. SAA also recently has been shown to activate the inflammasome cascade, which has a key role in immune activation, thus further stressing the unique role of SAA in immunomodulation. Traditionally, SAA has been considered to have a key role in the pathogenesis of amyloid A-type amyloidosis, but we now understand that it may also participate in the pathogenesis of chronic inflammatory diseases, such as rheumatoid arthritis and atherosclerosis. Thus, SAA is one potential target in the treatment of diseases associated with chronic inflammation. The purpose of this review is to shed light on SAA as an immunologically active protein. We also focus on the recent findings implicating SAA in the regulation of the inflammasome cascade.

Imatinib mesylate alleviates diarrhea in a mouse model of intestinal allergy.

BACKGROUND: When sensitized epicutaneously and challenged orally with ovalbumin, Balb/c mice develop allergen-induced diarrhea. As mast cells play important roles in diarrhea, we studied whether allergic diarrhea could be alleviated with imatinib mesylate. METHODS: Balb/c mice were sensitized and challenged with ovalbumin and treated orally with imatinib. Cytokine mRNA expressions were determined with quantitative RT-PCR and numbers of small intestinal mast cells determined by staining for chloroacetate esterase and mucosal mast cell protease-1. Immunofluorescence staining was used to assess the intestinal CCL1 expression. KEY RESULTS: Ovalbumin-sensitized and challenged Balb/c mice developed diarrhea, which was associated with increased number of mast cells and expression of interleukin (IL)-4 and -13, and chemokines CCL1 and CCL17 in the small intestine. Treatment with imatinib reduced the incidence of diarrhea, inhibited the development of mastocytosis and jejunal mRNA expression of IL-13, CCL1, CCL17 and CCL22. Mast cell-deficient W/W(-V) mice, and surprisingly, also their mast cell-competent control (+/+) littermates failed to develop diarrhea as a response to ovalbumin. This strain-dependent difference was associated with the inability of +/+ and W/W(-V) mice to increase the number of intestinal mast cells and expression of IL-4, IL-13, CCL1 and CCL17 after ovalbumin challenge. CONCLUSIONS & INFERENCES: Development of allergic diarrhea is associated with the ability of mice to develop intestinal mastocytosis. Imatinib inhibited the development of intestinal mastocytosis, reduced the incidence of diarrhea, and reduced the expression of IL-13, CCL1, and CCL17. Targeting intestinal mast cells could be a feasible approach to treat allergic diarrhea.

Mast cells generated from patients with atopic eczema have enhanced levels of granule mediators and an impaired Dectin-1 expression.

BACKGROUND: The disrupted skin barrier of patients with atopic eczema (AE) might facilitate contact between mast cells (MCs) in the skin and environmental triggers of the disease. One such trigger is the skin-colonizing yeast Malassezia sympodialis (M. sympodialis). In this study, we investigated the interaction of MC with M. sympodialis. METHODS: Mast cells were generated from peripheral blood CD34(+) progenitor cells of healthy controls (HC) and M. sympodialis-sensitized AE patients. Biopsy specimens were taken from HC and lesional AE skin for immunohistological stainings. RESULTS: The progenitor-derived MCs expressed the macrophage-inducible C-type lectin receptor Mincle, and exposure of these cells to M. sympodialis induced up-regulation of the mRNA expression of Mincle. Furthermore, we demonstrate that, when compared to HC, the progenitor-derived MCs from AE patients (i) contain more intrinsic granule mediators such as histamine, (ii) exhibit enhanced IL-6 release in response to M. sympodialis exposure, and (iii) have an impaired up-regulation of the fungal recognition receptor Dectin-1. In addition, analysis of skin sections from HC and AE patients revealed MCs as the predominant Dectin-1-expressing cell type in the skin. CONCLUSION: Our data indicate that progenitor-derived MCs from AE patients differ from those from HC. Further investigations with skin-derived MCs are necessary to confirm the observed differences which could provide new insights into the pathogenic mechanisms underlying AE.

The anti-allergic compound tranilast attenuates inflammation and inhibits bone destruction in collagen-induced arthritis in mice.

BACKGROUND AND PURPOSE: Recent findings suggest the importance of mast cells in the pathogenesis of rheumatoid arthritis and their potential as a therapeutic target. Tranilast is an anti-allergic compound with a potent membrane-stabilizing effect on mast cells and a wide range of anti-inflammatory effects, thus may be advantageous in the treatment of arthritis. Here, we have evaluated the effects of tranilast on the progression of collagen-induced arthritis in mice. EXPERIMENTAL APPROACH: Tranilast (400 mg.kg(-1).day(-1)) was orally administered for 8 weeks to mice with established collagen-induced arthritis. Arthritis was assessed by clinical signs and X-ray scores. In paw tissue, the numbers of mast cells and osteoclasts were measured by histological analysis, and several inflammatory factors were assessed by RT-PCR and Western blot analysis.* KEY RESULTS: TNF-alpha-positive mast cells were present extensively throughout the inflamed synovium of vehicle-treated arthritic mice, with some mast cells in close proximity to osteoclasts in areas of marked bone and cartilage destruction. Tranilast significantly reduced clinical and X-ray scores of arthritis and decreased numbers of TNF-alpha-positive mast cells and mRNA levels of TNF-alpha, chymase (mouse mast cell protease 4), tryptase (mouse mast cell protease 6), stem cell factor, interleukin-6, cathepsin-K, receptor activator of nuclear factor-kappaB, and of receptor activator of nuclear factor-kappaB-ligand, but increased interleukin-10 mRNA level in paws of arthritic mice. Osteoclast numbers were decreased by treatment with tranilast. CONCLUSIONS AND IMPLICATIONS: Tranilast possesses significant anti-rheumatic efficacy and, probably, this therapeutic effect is partly mediated by inhibition of mast cell activation and osteoclastogenesis.

Ethanol induces apoptosis in human mast cells.

AIMS: Alcohol abuse is associated with increased frequency of infections attributed to ethanol-induced immune suppression. The precise mechanism of immune suppression is however not known. Mast cells (MC) belong to the innate immune system and they have been implicated in the first line of immune defence against bacteria and parasites. Therefore we studied the effects of ethanol and its first metabolite acetaldehyde on mast cell viability, proliferation and apoptosis. MAIN METHODS: Human mast cell line (HMC)-1 cells, mouse bone marrow derived mast cells (mBMMC) and human peripheral blood derived mast cells (HuMC) were used. Effects of ethanol and acetaldehyde on mast cell proliferation were determined by assessing incorporation of [(3)H]thymidine into cellular DNA and by trypan blue exclusion. Apoptosis was assessed by measuring apoptotic nucleosomes and caspase-3, -8 and -9 activities using ELISA and by using Tunel assay. The expression of anti- and proapoptotic proteins Bcl-2 and Bax was analyzed by RT-PCR and western blot, respectively. KEY FINDINGS: Ethanol, but not acetaldehyde inhibited dose-dependently the proliferation and viability HMC-1 and mBMMC cells. The decreased viability was caused by apoptotic cell death of the MC. Significant apoptosis of HMC-1 cells was observed in the presence of 43mM (2.5 per thousand) ethanol. Induction of apoptosis was associated with clearly increased caspase-3 activity and moderately increased caspase-8 and 9 activities. Ethanol also shifted the Bcl-2/Bax balance towards apoptosis. SIGNIFICANCE: The ethanol-induced reduction of MC viability could contribute to immunosuppression associated with ethanol abuse.

Leukocyte cathepsin S is a potent regulator of both cell and matrix turnover in advanced atherosclerosis.

OBJECTIVE: A dysbalance of proteases and their inhibitors is instrumental in remodeling of atherosclerotic plaques. One of the proteases implicated in matrix degradation is cathepsin-S (CatS). To address its role in advanced lesion composition, we generated chimeric LDLr(-/-) mice deficient in leukocyte CatS by transplantation with CatS(-/-)xLDLr(-/-) or with LDLr(-/-) bone marrow and administered a high-fat diet. METHODS AND RESULTS: No difference in aortic root lesion size could be detected between CatS(+/+) and CatS(-/-) chimeras. However, leukocyte CatS deficiency markedly changed plaque morphology and led to a dramatic reduction in necrotic core area by 77% and an abundance of large foam cells. Plaques of CatS(-/-) chimeras contained 17% more macrophages, 62% less SMCs, and 33% less intimal collagen. The latter two could be explained by a reduced number of elastic lamina fractures. Moreover, macrophage apoptosis was reduced by 60% with CatS deficiency. In vitro, CatS was found to be involved in cholesterol metabolism and in macrophage apoptosis in a collagen and fibronectin matrix. CONCLUSIONS: Leukocyte CatS deficiency results in considerably altered plaque morphology, with smaller necrotic cores, reduced apoptosis, and decreased SMC content and collagen deposition and may thus be critical in plaque stability.

Mast cells associate with T-cells and neointimal microvessels in giant cell arteritis.

OBJECTIVE: Mast cells (MCs) are known to be involved in the neovascularization and regulation of T cell responses. However, the presence of MCs in giant cell arteritis (GCA) is unknown. This prompted us to study the presence and phenotype of MCs in GCA. METHODS: Human GCA specimens collected for diagnostic purposes were examined with immunohistochemistry. Double immunostainings of MC tryptase with cathepsin G, vascular endothelial cell growth factor (VEGF), CD3, and CD31/D34 were performed. RESULTS: Double immunostainings showed that activated tryptase-, cathepsin G- and VEGF-expressing MCs associate with CD3+ T cells and CD31/CD34+ neointimal neovessels in the GCA lesions. CONCLUSIONS: The results suggest that MCs may contribute to the pathogenesis of GCA putatively by regulating the functions of other inflammatory cells and resident vessel wall cells. Importantly, MCs promote neovascularization, which is considered as a prerequisite for the neointimal thickening in GCA.

Mast cells and degradation of pericellular and extracellular matrices: potential contributions to erosion, rupture and intraplaque haemorrhage of atherosclerotic plaques.

Mast cells are present in advanced human atherosclerotic plaques, where they are thought to exert multiple effects on their neighbouring cells and on the extracellular matrix of the plaque. Extensive efforts at delineating their role(s) in atherosclerotic plaques have unravelled mechanisms by which plaque mast cells may render advanced atherosclerotic plaques susceptible to erosion, rupture or intraplaque haemorrhage and so modulate their stability. In these mechanisms, the key effector molecules are mast-cell-derived neutral proteases and pro-inflammatory cytokines. These effector molecules are synthesized and stored in the cytoplasmic secretory granules of mast cells and, once the mast cells are activated to degranulate, are released into the microenvironment surrounding the activated mast cells. In the plaques, the key target cells are endothelial cells and smooth muscle cells and their pericellular matrices. In addition, the various components of the extracellular matrix of the plaques, notably collagen, are degraded when the released mast cell proteases activate matrix metalloproteinases in the plaques. By rendering the plaque susceptible to erosion, to rupture or to intraplaque haemorrhage, the mast cells may contribute to the onset of acute atherothrombotic complications of coronary atherosclerosis, such as myocardial infarction.

A protocol for generating high numbers of mature and functional human mast cells from peripheral blood.

BACKGROUND: Mast cells (MCs) are multi-functional effector cells with an essential role in innate immunity and host defence, and under several pathological conditions, such as allergy. Here, we aimed at defining the culture conditions that would allow efficient generation of mature and functional human MCs from their progenitor cells. METHODS: Human peripheral blood-derived CD34(+) progenitor cells were cultured in vitro under serum-free conditions with human stem cell factor for 9 weeks. Growth and differentiation of the cells into MCs were optimized by selected cytokines and a combination of hypoxic and normoxic conditions. MCs were phenotypically characterized by immunocytochemistry, their preformed mediators were quantified, and their functional ability to degranulate and release histamine was tested. RESULTS: On average, 20 x 10(6) mature MCs were generated from 0.5 x 10(6) progenitor cells during 9 weeks of culture, i.e. at least a 40-fold increase in cell number was achieved. The mature MCs had oval-shaped non-lobular nuclei, contained histamine, heparin, tryptase, chymase, and cathepsin G in their secretory granules, and strongly expressed c-kit (CD117) and Fc epsilon receptor I on their surface. Histamine release from the cells could be brought about by IgE-anti-IgE cross-linkage, compound 48/80, substance P, and anaphylatoxin C3a. The MCs remained functional for several weeks after their maturation. CONCLUSION: This study describes an efficient protocol for generating mature MCs from human peripheral blood with a functional phenotype of connective tissue-type MCs. Use of these cultured human MCs will increase our knowledge and understanding about human MC development and biology in human disease.

Selective activation of mast cells in rheumatoid synovial tissue results in production of TNF-alpha, IL-1beta and IL-1Ra.

OBJECTIVES AND DESIGN: To study the consequences of mast cell activation in human synovial tissue. METHODS: Synovial tissue was obtained from 18 RA patients and mast cells was selectively activated in synovial tissue explant cultures. Expression of TNF-alpha, IL-1beta and IL-1Ra were determined and tissue distribution of IL-1beta was studied. RESULTS: Compared to untreated synovia, selective activation of synovial mast cells increased significantly the production of TNF-alpha (0.49 +/- 0.88 vs. 4.56 +/- 3.18 pg/mg wet tissue, p < 0.001) and IL-1beta (0.058 +/- 0.032 vs. 2.55 +/- 1.98 pg/mg wet tissue, p = 0.013). The expression of TNF-alpha and IL-1beta mRNA increased significantly (19-fold (p = 0.009) and 13-fold (p = 0.031), respectively). Mast cell activation induced IL-1beta expression in particular in nearby CD68 positive synovial macrophages. Secretion of IL-1Ra was also increased but to a lesser degree than that of IL-1beta. CONCLUSIONS: Synovial mast cells produce proinflammmatory cytokines and may thus contribute to the inflammation in RA.

Mast cell proteases: physiological tools to study functional significance of high density lipoproteins in the initiation of reverse cholesterol transport.

The extracellular fluid of the intima is rich in lipid-poor species of high density lipoproteins (HDL) that promote efficient efflux of cholesterol from macrophages. Yet, during atherogenesis, cholesterol accumulates in macrophages, and foam cells are formed. We have studied proteolytic modification of HDL by mast cell proteases as a potential mechanism of reduced cholesterol efflux from foam cells. Mast cells are present in human atherosclerotic lesions and, when activated, they expel cytoplasmic granules that are filled with heparin proteoglycans and two neutral proteases, chymase and tryptase. Both proteases were found to specifically deplete in vitro the apoA-I-containing prebeta-migrating HDL (prebeta-HDL) and other lipid-poor HDL particles that contain only apoA-IV or apoE. These losses led to inhibition of the high-affinity component of cholesterol efflux from macrophage foam cells facilitated by the ATP-binding cassette transporter A1 (ABCA1). In contrast, the diffusional component of efflux promoted by alpha-HDL particles was not changed after proteolysis. Mast cell proteases are providing new insights into the role of extracellular proteolysis of HDL as an inhibiting principle of the initial steps of reverse cholesterol transport in the atherosclerotic intima, where many types of protease-secreting cells are present.

Chemically modified tetracycline (CMT)-3 inhibits histamine release and cytokine production in mast cells: possible involvement of protein kinase C.

OBJECTIVE: To find novel inhibitors of mast cell function we have studied the effect of a potent, non-antimicrobial, chemically modified tetracycline, CMT-3 or COL-3, on key functions of mast cells. METHODS AND RESULTS: In the presence of 25 microM CMT-3, the 48/80-induced histamine release from rat serosal mast cells was inhibited significantly, to 43.0 +/- 7.3% of control. Similarly, the activation-induced secretion of TNF-alpha and IL-8 by HMC-1 cells were decreased in the presence of 25 microM CMT-3 to 13.5 +/- 4.1% and 9.7 +/- 1.1% of control, respectively. CMT-3 did not cause intracellular accumulation of TNF-alpha but instead it reduced the expression of TNF-alpha mRNA in HMC-1 cells. Moreover, CMT-3 was found to significantly inhibit the protein kinase C (PKC) activity with IC(50) value of 31 microM. CMT-3 inhibited effectively both human recombinant PKCalpha and PKCdelta isoforms. In comparison to doxycycline, CMT-3 was more effective as an inhibitor of both cytokine production and PKC activity. CONCLUSIONS: Considering the central role of PKC in mast cell activation, PKC inhibition could, at least partially, explain the observed inhibitory effects of CMT-3. The inhibition of the key proinflammatory functions of mast cells by CMT-3 suggests its potential clinical usefulness in the treatment of allergic and inflammatory disorders.