Ken A. Lindstedt

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.

Activation of paracrine TGF-β1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase

As a source of transforming growth factor β1 (TGF‐β1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF‐β1 have remained vague. We show here by means of RT‐PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF‐β1 in their chymase 1‐containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF‐β1, which is converted by chymase 1 into an active form recognized by the type II TGF‐β serine/ threonine kinase receptor (TβRII). Thus, mast cells secrete active TGF‐β1 by a unique secretory mechanism in which latent TGF‐β1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF‐β1 specifically by chymase was verified using recombinant human latent TGF‐β1 and recombinant human chymase. In isolated TβRI‐ and TβRII‐expressing peritoneal macrophages, the activated TGF‐β1 induces the expression of the plasminogen activator inhibitor 1 (PAI‐1), whereas in the mast cells, the levels of TβRI, TβRII, and PAI‐1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF‐β1 in peritoneal mast cells and of TβRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF‐β1 and by enhancing its response in target cells.—Lindstedt, K. A., Wang, Y., Shiota, N., Saarinen, J., Hyytiäinen, M., Kokkonen, J. O., Keski‐Oja, J., Kovanen, P. T. Activation of paracrine TGF‐β1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase. FASEB J. 15, 1377–1388 (2001)

Mast Cell Chymase Induces Smooth Muscle Cell Apoptosis by a Mechanism Involving Fibronectin Degradation and Disruption of Focal Adhesions

Objective—Chymase released from activated mast cells has been shown to induce apoptosis of vascular smooth muscle cells (SMCs) in vitro. The proteolytic activity of chymase is essential for the proapoptotic effect, but the mechanism of chymase-induced apoptosis has remained unknown. Methods and Results—Here we show by means of FACS analysis, immunohistochemistry, and Western blotting that mast cell–derived chymase induces SMC apoptosis by a mechanism involving degradation of an extracellular matrix component, fibronectin (FN), with subsequent disruption of focal adhesions. The FN degradation products induced SMC apoptosis of similar magnitude and with similar changes in outside-in signaling, as did chymase. Sodium orthovanadate, an inhibitor of tyrosine phosphatases, inhibited the chymase-induced SMC apoptosis. Focal adhesion kinase (FAK), one of the key mediators of integrin–extracellular matrix interactions and cell survival, was rapidly degraded in the presence of chymase or FN degradation products. Loss of phosphorylated FAK (p-FAK) resulted in a rapid dephosphorylation of the p-FAK–dependent downstream mediator Akt. Conclusions—The results suggest that chymase-secreting mast cells can mediate apoptosis of neighboring SMCs through a mechanism involving degradation of pericellular FN and disruption of the p-FAK–dependent cell-survival signaling cascade.

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.

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.

Regulation of smooth muscle cell growth, function and death in vitro by activated mast cells--a potential mechanism for the weakening and rupture of atherosclerotic plaques.

The fibrous cap of a lipid-containing atherosclerotic plaque consists of collagen produced by arterial smooth muscle cells (SMCs) of synthetic phenotype. A thick cap protects the lipid-rich core, whereas a thin cap predisposes it to rupture, with ensuing acute clinical complications, such as myocardial infarction. Among the pathological mechanisms leading to plaque weakening and rupture, one possibility is loss of the matrix-synthesizing SMCs. Indeed, caps of ruptured coronary plaques contain a reduced number of SMCs. In contrast, in such lesions, the number of activated inflammatory cells, such as mast cells, is increased, suggesting that they may regulate the SMC number. We have shown that heparin proteoglycans secreted by activated mast cells can efficiently inhibit proliferation of SMCs in vitro and reduce their ability to produce collagen. Chymase, a neutral serine protease secreted by activated mast cells, can also inhibit SMC-mediated collagen synthesis by a transforming growth factor-beta-dependent and -independent mechanism, and moreover, cause degradation of the collagen matrix by activating latent interstitial collagenase (MMP-1). Furthermore, chymase can induce SMC apoptosis by degrading the extracellular matrix component fibronectin necessary for SMC adhesion, with subsequent disruption of focal adhesions and loss of outside-in survival signaling. Thus, activated mast cells may participate in the weakening and rupture of atherosclerotic plaques by secreting mediators, such as heparin proteoglycans and chymase, which affect the growth, function and death of arterial SMCs.

Mast cells in vulnerable coronary plaques: potential mechanisms linking mast cell activation to plaque erosion and rupture.

Purpose of review A novel link between inflammation and acute coronary syndromes is emerging, in that infiltrating inflammatory cells may convert a clinically silent coronary plaque into a dangerous and potentially lethal plaque. The majority of acute atherothrombotic events now relate to erosion or rupture of such unstable plaques. Here we summarize the molecular mechanisms by which activated mast cells may contribute to plaque erosion or rupture. Recent findings In-vitro experiments have revealed a multitude of paracrine effects exerted by activated mast cells. By secreting heparin proteoglycans and chymase, activated mast cells efficiently inhibit the proliferation of smooth muscle cells in vitro, and reduce their ability to produce collagen by a transforming growth factor beta-dependent and -independent mechanism. Mast cell chymase and tryptase are capable of activating matrix metalloproteinases types 1 and 3, causing degradation of the extracellular matrix component, collagen, necessary for the stability of the plaque. Activated mast cells also secrete matrix metalloproteinases types 1 and 9 themselves. Furthermore, chymase induces SMC apoptosis by degrading fibronectin, a pericellular matrix component necessary for SMC adhesion and survival, with the subsequent disruption of focal adhesions and loss of outside-in survival signaling. By secreting chymase and tumour necrosis factor alpha, activated mast cells also induce endothelial cell apoptosis. Summary Locally activated mast cells may participate in the weakening of atherosclerotic plaques by secreting heparin proteoglycans, chymase, and cytokines, which affect the growth, function and death of arterial endothelial cells and smooth muscle cells, thereby predisposing to plaque erosion or rupture.

Kallidin- and Bradykinin-Degrading Pathways in Human Heart Degradation of Kallidin by Aminopeptidase M–Like Activity and Bradykinin by Neutral Endopeptidase

BACKGROUND Since kinins kallidin (KD) and bradykinin (BK) appear to have cardioprotective effects ranging from improved hemodynamics to antiproliferative effects, inhibition of kinin-degrading enzymes should potentiate such effects. Indeed, it is believed that this mechanism is partly responsible for the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors. In the heart, enzymes other than ACE may contribute to local degradation of kinins. The purpose of this study was to investigate which enzymes are responsible for the degradation of KD and BK in human heart tissue. METHODS AND RESULTS Cardiac membranes were prepared from the left ventricles of normal (n=5) and failing (n=10) hearts. The patients had end-stage congestive heart failure as the result of coronary heart disease or idiopathic dilated cardiomyopathy. Heart tissue was incubated with KD or BK in the presence or absence of enzyme inhibitors. We found no difference in the enzymes responsible for kinin metabolism or their activities between normal and failing hearts. Thus KD was mostly converted into BK by the aminopeptidase M-like activity. When BK was used as substrate, it was converted into an inactive metabolite BK-(1-7) mostly (80% to 90%) by the neutral endopeptidase (NEP) activity, with ACE unexpectedly playing only a minor role. The low enzymatic activity of ACE in the cardiac membranes, compared with that of NEP, was not due to chronic ACE inhibitor therapy, because the cardiac ACE activities of patients, whether receiving ACE inhibitors or not, and of normal subjects were all equal. CONCLUSIONS The present in vitro study shows that in human cardiac membranes, the most critical step in kinin metabolism, that is, inactivation of BK, appears to be mediated mostly by NEP. This observation suggests a role for NEP in the local control of BK concentration in heart tissue. Thus inhibition of cardiac NEP activity could be cardioprotective by elevating the local concentration of BK in the heart.

Inhibiting TRPA1 ion channel reduces loss of cutaneous nerve fiber function in diabetic animals: Sustained activation of the TRPA1 channel contributes to the pathogenesis of peripheral diabetic neuropathy

Peripheral diabetic neuropathy (PDN) is a devastating complication of diabetes mellitus (DM). Here we test the hypothesis that the transient receptor potential ankyrin 1 (TRPA1) ion channel on primary afferent nerve fibers is involved in the pathogenesis of PDN, due to sustained activation by reactive compounds generated in DM. DM was induced by streptozotocin in rats that were treated daily for 28 days with a TRPA1 channel antagonist (Chembridge-5861528) or vehicle. Laser Doppler flow method was used for assessing axon reflex induced by intraplantar injection of a TRPA1 channel agonist (cinnamaldehyde) and immunohistochemistry to assess substance P-like innervation of the skin. In vitro calcium imaging and patch clamp were used to assess whether endogenous TRPA1 agonists (4-hydroxynonenal and methylglyoxal) generated in DM induce sustained activation of the TRPA1 channel. Axon reflex induced by a TRPA1 channel agonist in the plantar skin was suppressed and the number of substance P-like immunoreactive nerve fibers was decreased 4 weeks after induction of DM. Prolonged treatment with Chembridge-5861528 reduced the DM-induced attenuation of the cutaneous axon reflex and loss of substance P-like immunoreactive nerve fibers. Moreover, in vitro calcium imaging and patch clamp results indicated that reactive compounds generated in DM (4-hydroxynonenal and methylglyoxal) produced sustained activations of the TRPA1 channel, a prerequisite for adverse long-term effects. The results indicate that the TRPA1 channel exerts an important role in the pathogenesis of PDN. Blocking the TRPA1 channel provides a selective disease-modifying treatment of PDN.

Proteolysis of the Pericellular Matrix: A Novel Element Determining Cell Survival and Death in the Pathogenesis of Plaque Erosion and Rupture

The 2 major general concepts about the cell biology of atherogenesis, growth of smooth muscle cells, and lipid accumulation in macrophages, ie, foam cell formation, have not been able to satisfactorily explain the genesis of acute coronary syndromes. Rather, the basic pathology behind the acute atherothrombotic events relates to erosion and rupture of unstable coronary plaques. At the cellular level, we now understand that a switch from cellular growth to cellular death, notably apoptosis, could be involved in turning at least some types of atherosclerotic plaques unstable. Because intimal cells require a proper matrix environment for normal function and survival, the vulnerability of an atherosclerotic plaque may critically depend on the integrity of the pericellular matrix of the plaque cells. In vitro studies have revealed that plaque-infiltrating inflammatory cells, such as macrophages, T-lymphocytes, and mast cells, by secreting a variety of proteases capable of degrading pericellular matrix components, induce death of endothelial cells and smooth muscle cells, and so provide a mechanistic explanation for inflammation-dependent plaque erosion and rupture. Thus, a novel link between inflammation and acute coronary syndromes is emerging. For a more explicit understanding of the role of proteases released by inflammatory cells in the conversion of a clinically silent plaque into a dangerous and potentially killing plaque, animal models of plaque erosion and rupture need to be established.