Mikko I. Mäyränpää

Circulating and cardiac levels of apelin, the novel ligand of the orphan receptor APJ, in patients with heart failure.

The orphan receptor APJ and its recently identified endogenous ligand, apelin, are expressed in the heart. However, their importance in the human cardiovascular system is not known. This study shows that apelin-like immunoreactivity is abundantly present in healthy human heart and plasma. Gel filtration HPLC analysis revealed that atrial and plasma levels of high molecular weight apelin, possibly proapelin, were markedly higher than those of mature apelin-36 itself. As assessed by quantitative RT-PCR analysis, left ventricular apelin mRNA levels were increased 4.7-fold in chronic heart failure (CHF) due to coronary heart disease (p<0.01) and 3.3-fold due to idiopathic dilated cardiomyopathy (p<0.05), whereas atrial apelin mRNA levels were unchanged. Atrial and plasma apelin-like immunoreactivity as well as atrial and ventricular APJ receptor mRNA levels were significantly decreased in CHF. Our results suggest that a new cardiac regulatory peptide, apelin, and APJ receptor may contribute to the pathophysiology of human CHF.

OSBP-related Protein 8 (ORP8) Suppresses ABCA1 Expression and Cholesterol Efflux from Macrophages

ORP8 is a previously unexplored member of the family of oxysterol-binding protein-related proteins (ORP). We now report the expression pattern, the subcellular distribution, and data on the ligand binding properties and the physiological function of ORP8. ORP8 is localized in the endoplasmic reticulum (ER) via its C-terminal transmembrane span and binds 25-hydroxycholesterol, identifying it as a new ER oxysterol-binding protein. ORP8 is expressed at highest levels in macrophages, liver, spleen, kidney, and brain. Immunohistochemical analysis revealed ORP8 in the shoulder regions of human coronary atherosclerotic lesions, where it is present in CD68(+) macrophages. In advanced lesions the ORP8 mRNA was up-regulated 2.7-fold as compared with healthy coronary artery wall. Silencing of ORP8 by RNA interference in THP-1 macrophages increased the expression of ATP binding cassette transporter A1 (ABCA1) and concomitantly cholesterol efflux to lipid-free apolipoprotein A-I but had no significant effect on ABCG1 expression or cholesterol efflux to spherical high density lipoprotein HDL2. Experiments employing an ABCA1 promoter-luciferase reporter confirmed that ORP8 silencing enhances ABCA1 transcription. The silencing effect was partially attenuated by mutation of the DR4 element in the ABCA1 promoter and synergized with that of the liver X receptor agonist T0901317. Furthermore, inactivation of the E-box in the promoter synergized with ORP8 silencing, suggesting that the suppressive effect of ORP8 involves both the liver X receptor and the E-box functions. Our data identify ORP8 as a negative regulator of ABCA1 expression and macrophage cholesterol efflux. ORP8 may, thus, modulate the development of atherosclerosis.

Increased Expression of Elastolytic Cathepsins S, K, and V and Their Inhibitor Cystatin C in Stenotic Aortic Valves

Objective—To investigate the possible role of elastolytic cathepsins S, K, and V and their endogenous inhibitor cystatin C in adverse extracellular matrix remodeling of stenotic aortic valves. Methods and Results—Stenotic aortic valves were collected at valve replacement surgery and control valves at cardiac transplantations. The expression of cathepsins S, K, and V and cystatin C was studied by conventional and real-time polymerase chain reaction and by immunohistochemistry. Total cathepsin activity in the aortic valves was quantified by a fluorometric microassay. When compared with control valves, stenotic valves showed increased mRNA expression of cathepsins S, K, and V (P<0.05 for each) and a higher total cathepsin activity (P<0.001). In stenotic valves, cystatin C mRNA was increased (P<0.05), and cystatin C protein was found particularly in areas with infiltrates of inflammatory cells. Both cathepsin S and cystatin C were present in bony areas of the valves, whereas cathepsin V localized to endothelial cells in areas rich of neovascularization. Incubation of thin sections of aortic valves with cathepsins S, K, and V resulted in severe disruption of elastin fibers, and this cathepsin effect could be blocked by adding cystatin C to the incubation system. Conclusions—Stenotic aortic valves show increased expression and activity of elastolytic cathepsins S, K, and V. These cathepsins may accelerate the destruction of aortic valvular extracellular matrix, so promoting the progression of aortic stenosis.

Mast cells associate with neovessels in the media and adventitia of abdominal aortic aneurysms

OBJECTIVE Mast cells (MCs) are inflammatory cells present in atherosclerotic lesions and neovascularized tissues. Recently, MCs were shown to modulate abdominal aortic aneurysm (AAA) formation in a mouse model. Progression of aneurysmatic disease process may also depend on intraluminal thrombus and neovascularization of the aneurysm wall. Here we investigated the relationship between MCs and inflammation, neovascularization, and the presence of intraluminal thrombus in human AAA. METHODS AND RESULTS Specimens from AAAs and normal control aortas were analyzed with basic histology, immunohistochemical staining, and quantitative real-time polymerase chain reaction (PCR). Double immunostainings with endothelial cell markers CD31/CD34 and MC tryptase showed that, in contrast to histologically normal aorta, MCs in AAA were abundant in the media, but absent from the intima. Medial MCs and (CD31/CD34)(+) neovessels increased significantly in AAA compared with normal aorta (P < .0001 for both), and the highest densities of neovessels and MCs were observed in the media of thrombus-covered AAA samples. Also, the proportional thickness of aortic wall penetrated by the neovessels was significantly higher in the AAA samples (P < .0001), and the neovascularized area correlated with the density of medial MCs (P < .0001). In histologic analysis, the medial MCs were mainly located adjacent to the stem cell factor (SCF)(+) medial neovessels. Real-time PCR analysis also showed that mRNA levels of genes associated with neovascularization (vascular endothelial growth factor [VEGF], FLT1, VE-cadherin, CD31), and MCs (tryptase, chymase, cathepsin G) were higher in AAA samples than in controls. Demonstration of adhered platelets by CD42b staining and lack of endothelial cell (CD31/CD34) staining in the luminal surface of AAA specimens suggest endothelial erosion of the aneurysm walls. CONCLUSIONS The results support participation of MCs in the pathogenesis of AAA, particularly regarding neovascularization of aortic wall.

VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart.

Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain‐ and loss‐of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor‐B (VEGF‐B) in the heart. A cardiomyocyte‐specific VEGF‐B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia‐reperfusion. VEGF‐B increased VEGF signals via VEGF receptor‐2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF‐B transgenic, gene‐targeted or wildtype rats. Importantly, we also show that VEGF‐B expression is reduced in human heart disease. Our data indicate that VEGF‐B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.

The R-Ras interaction partner ORP3 regulates cell adhesion

Oxysterol-binding protein (OSBP)-related protein 3 (ORP3) is highly expressed in epithelial, neuronal and hematopoietic cells, as well as in certain forms of cancer. We assessed the function of ORP3 in HEK293 cells and in human macrophages. We show that ORP3 interacts with R-Ras, a small GTPase regulating cell adhesion, spreading and migration. Gene silencing of ORP3 in HEK293 cells results in altered organization of the actin cytoskeleton, impaired cell-cell adhesion, enhanced cell spreading and an increase of β1 integrin activity–effects similar to those of constitutively active R-Ras(38V). Overexpression of ORP3 leads to formation of polarized cell-surface protrusions, impaired cell spreading and decreased β1 integrin activity. In primary macrophages, overexpression of ORP3 leads to the disappearance of podosomal structures and decreased phagocytotic uptake of latex beads, consistent with a role in actin regulation. ORP3 is phosphorylated when cells lose adhesive contacts, suggesting that it is subject to regulation by outside-in signals mediated by adhesion receptors. The present findings demonstrate a new function of ORP3 as part of the machinery that controls the actin cytoskeleton, cell polarity and cell adhesion.

Tissue inhibitor of metalloproteinases 4 (TIMP4) is involved in inflammatory processes of human cardiovascular pathology

Tissue inhibitors of matrix metalloproteinases (TIMPs) comprise a family of four members, of which TIMP4 is characterized by being primarily restricted to cardiovascular structures. We demonstrate with immunohistochemical analysis of healthy human tissue that TIMP4 is present in medial smooth muscle cells and adventitial capillaries of arteries as well as in cardiomyocytes. Animal studies have suggested a role for TIMP4 in several inflammatory diseases and cardiovascular pathologies. We therefore examined whether TIMP4 is involved in human inflammatory cardiovascular disorders, specifically atherosclerosis, giant cell arteritis and chronic rejection of heart allografts. TIMP4 was most clearly visible in cardiovascular tissue areas populated by abundant inflammatory cells, mainly macrophages and CD3+ T cells. Using western blotting and immunocytochemistry, human blood derived lymphocytes, monocytes/macrophages and mast cells were shown to produce TIMP4. In advanced atherosclerotic lesions, TIMP4 was detected around necrotic lipid cores, whereas TIMP3 and caspase 3 resided within and around the core regions, indicating different roles for TIMP3 and TIMP4 in inflammation-induced apoptosis and in matrix turnover. In conclusion, the data demonstrate upregulation of TIMP4 in human cardiovascular disorders exhibiting inflammation, suggesting its future use as a novel systemic marker for vascular inflammation.

Mast Cells Important Players in the Orchestrated Pathogenesis of Abdominal Aortic Aneurysms

Mast cells (MCs) regulate inflammation and immunity. Their granular content includes heparin, histamine, and several enzymes (tryptase, chymase, carboxypeptidase, and cathepsin G). In addition, activated MCs synthesize and release eicosanoids and a large number of cytokines and chemokines. Recent findings suggest a role of MCs in abdominal aortic aneurysms (AAAs) in humans, where they are found in the media and adventitia. Experimentally induced AAA in MC-deficient animals and animals treated with MC inhibitors demonstrate that MCs are involved in the pathogenesis of AAA via several different mechanisms. MC-dependent activation of metalloproteinases and the renin-angiotensin system, contribution to smooth muscle cell apoptosis, and release of proteolytic enzymes are some key examples. Human studies indicate that MCs are the main source of cathepsin G in AAAs and contribute to activation of the renin-angiotensin system via chymase and cathepsin G. Activated MCs also contribute to neovascularization, inflammation, and atherosclerosis, all hallmarks of AAA. Thus, we may envision that MC stabilizing agents, as well as leukotriene receptor antagonists and histamine receptor blockers already in clinical use for treatment of other diseases, could also be tested for their efficacy in preventing development and growth of AAA.

Mast cells in vulnerable atherosclerotic plaques – a view to a kill

•  Mast cells – an introduction ‐  The origin of mast cells ‐  Mast cell subtypes ‐  Mast cell localization and physiological function •  Mast cell – a potent effector cell ‐  Histamine ‐  Mast cell‐derived proteoglycans ‐  Mast cell‐derived proteases ‐  Growth factors and preformed cytokines ‐  Newly formed cytokines and chemokines ‐  Newly formed lipid mediators ‐  Mast cells in the vessel wall •  Mast cells and atherosclerosis ‐  A short history ‐  Activated mast cells in human atherosclerotic plaques ‐  Mast cell activation without degranulation ‐  Mast cell – an inflammatory cell •  Coronary mast cells in acute human myocardial infarction •  Animal models of plaque erosion and rupture •  Clinical approaches to stabilize mast cells in the atherosclerotic plaque •  Conclusions

Increased expression of leukotriene C4 synthase and predominant formation of cysteinyl-leukotrienes in human abdominal aortic aneurysm

Leukotrienes (LTs) are arachidonic acid-derived lipid mediators involved in the pathogenesis and progression of diverse inflammatory disorders. The cysteinyl-leukotrienes LTC4, LTD4, and LTE4 are important mediators of asthma, and LTB4 has recently been implicated in atherosclerosis. Here we report that mRNA levels for the three key enzymes/proteins in the biosynthesis of cysteinyl-leukotrienes, 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), and LTC4 synthase (LTC4S), are significantly increased in the wall of human abdominal aortic aneurysms (AAAs). In contrast, mRNA levels of LTA4 hydrolase, the enzyme responsible for the biosynthesis of LTB4, are not increased. Immunohistochemical staining of AAA wall revealed focal expression of 5-LO, FLAP, and LTC4S proteins in the media and adventitia, localized in areas rich in inflammatory cells, including macrophages, neutrophils, and mast cells. Human AAA wall tissue converts arachidonic acid and the unstable epoxide LTA4 into significant amounts of cysteinyl-leukotrienes and to a lesser extent LTB4. Furthermore, challenge of AAA wall tissue with exogenous LTD4 increases the release of matrix metalloproteinase (MMP) 2 and 9, and selective inhibition of the CysLT1 receptor by montelukast blocks this effect. The increased expression of LTC4S, together with the predominant formation of cysteinyl-leukotrienes and effects on MMPs production, suggests a mechanism by which LTs may promote matrix degradation in the AAA wall and identify the components of the cysteinyl-leukotriene pathway as potential targets for prevention and treatment of AAA.