Jorma O. Kokkonen

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)

Regulation of local angiotensin II formation in the human heart in the presence of interstitial fluid : Inhibition of chymase by protease inhibitors of interstitial fluid and of angiotensin-converting enzyme by Ang-(1-9) formed by heart carboxypeptidase A-like activity

BACKGROUND Data from in vitro studies suggest that both chymase and ACE contribute to the local generation of angiotensin (Ang) II in the heart. The enzyme kinetics under in vivo conditions are unclear. We thus studied the generation of Ang II by cardiac tissue in the presence of interstitial fluid (IF) that contains a variety of naturally occurring protease inhibitors. METHODS AND RESULTS Ang I was incubated with heart homogenate in the presence of IF. IF obtained from human skin contained substantial amounts of protease inhibitors and ACE activity, the concentration of alpha 1-antitrypsin being 35% and the activity of ACE 24% of the corresponding serum values. When heart homogenate was incubated with Ang I, three enzymes were responsible for its metabolism: heart chymase and heart ACE converted Ang I to Ang II, and heart carboxypeptidase A (CPA)-like activity degraded Ang I to Ang-(1-9). Incubation of heart homogenate in the presence of IF led to practically full inhibition of heart chymase-mediated Ang II formation by the natural protease inhibitors present in IF. In contrast, heart CPA-like activity was not blocked, as reflected by the continued generation of Ang-(1-9). In addition, both heart ACE- and IF ACE-mediated Ang II formation were strongly inhibited. This inhibition was shown to be due to the Ang-(1-9) formed. CONCLUSIONS The present experimental study defines two novel inhibitory mechanisms of Ang II formation in the human heart interstitium. Heart chymase-mediated Ang II formation is strongly inhibited by the natural protease inhibitors present in the IF. Similarly, both heart ACE- and IF ACE-mediated Ang II formation appear to be inhibited by the endogenous inhibitor Ang-(1-9) formed by heart CPA-like activity. These inhibitory mechanisms provide additional information about how the Ang II concentration in the heart interstitium may be controlled.

Cardiac Sarcoidosis: Epidemiology, Characteristics and Outcome over 25 Years in a Nationwide Study

Background— This study was designed to assess the epidemiology, characteristics, and outcome of cardiac sarcoidosis (CS) in Finland. Methods and Results— We identified in retrospect all adult (>18 years of age) patients diagnosed with histologically confirmed CS in Finland between 1988 and 2012. A total of 110 patients (71 women) 51±9 years of age (mean±SD) were found and followed up for outcome events to the end of 2013. The annual detection rate of CS increased >20-fold during the 25-year period, reaching 0.31 in 1×105 adults between 2008 and 2012. The 2012 prevalence of CS was 2.2 in 1×105. Nearly two thirds of patients had clinically isolated CS. Altogether, 102 of the 110 patients received immunosuppressive therapy, and 56 received an intracardiac defibrillator. Left ventricular function was impaired (ejection fraction <50%) in 65 patients (59%) at diagnosis and showed no overall change over 12 months of steroid therapy. During follow-up (median, 6.6 years), 10 patients died of a cardiac cause, 11 patients underwent transplantation, and another 11 patients suffered an aborted sudden cardiac death. The Kaplan–Meier estimates for 1-, 5-, and 10-year transplantation-free cardiac survival were 97%, 90%, and 83%, respectively. Heart failure at presentation predicted poor outcome (log-rank P=0.0001) with a 10-year transplantation-free cardiac survival of only 53%. Conclusions— The detection rate of CS has increased markedly in Finland over the last 25 years. With current therapy, the prognosis of CS appears better than generally considered, but patients presenting with heart failure still have poor long-term outcome.Background— This study was designed to assess the epidemiology, characteristics, and outcome of cardiac sarcoidosis (CS) in Finland. Methods and Results— We identified in retrospect all adult (>18 years of age) patients diagnosed with histologically confirmed CS in Finland between 1988 and 2012. A total of 110 patients (71 women) 51±9 years of age (mean±SD) were found and followed up for outcome events to the end of 2013. The annual detection rate of CS increased >20-fold during the 25-year period, reaching 0.31 in 1×105 adults between 2008 and 2012. The 2012 prevalence of CS was 2.2 in 1×105. Nearly two thirds of patients had clinically isolated CS. Altogether, 102 of the 110 patients received immunosuppressive therapy, and 56 received an intracardiac defibrillator. Left ventricular function was impaired (ejection fraction <50%) in 65 patients (59%) at diagnosis and showed no overall change over 12 months of steroid therapy. During follow-up (median, 6.6 years), 10 patients died of a cardiac cause, 11 patients underwent transplantation, and another 11 patients suffered an aborted sudden cardiac death. The Kaplan–Meier estimates for 1-, 5-, and 10-year transplantation-free cardiac survival were 97%, 90%, and 83%, respectively. Heart failure at presentation predicted poor outcome (log-rank P =0.0001) with a 10-year transplantation-free cardiac survival of only 53%. Conclusions— The detection rate of CS has increased markedly in Finland over the last 25 years. With current therapy, the prognosis of CS appears better than generally considered, but patients presenting with heart failure still have poor long-term outcome. # CLINICAL PERSPECTIVE {#article-title-42}

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.

Role For Chymase in Heart Failure Angiotensin II-Dependent or -Independent Mechanisms?

One of the major questions regarding the role of angiotensin II (Ang II) in the pathophysiology of heart failure has been whether other enzymes, in addition to angiotensin-converting enzyme (ACE), could contribute to the local production of Ang II in the heart. Specifically, there is controversy as to whether the major Ang II-forming enzyme within the heart is ACE or chymase, a chymotrypsin-like serine protease that is synthesized and stored in the cardiac mast cells and is not affected by ACE inhibitors. See p 2555 Formation of Ang II in the heart has been studied both in vitro and in vivo, but the results of these experiments are inconsistent. In vitro experiments with human1,2 or animal heart extracts,3,4 derived from either normal or failing hearts, have demonstrated unequivocally that the major Ang II-forming enzyme, responsible for 80% to 90% of the Ang II-forming capacity in the heart extracts, is chymase. In striking contrast, experiments in vivo with normal5,6 or failing dog hearts7,8 have demonstrated that most (>70%) of the Ang II formation can be inhibited by an ACE inhibitor, indicating that, under these conditions, the major Ang II-forming enzyme is ACE. Moreover, in normal human hearts, Zisman et al9 have demonstrated that, in vivo, the major Ang II-forming enzyme is ACE, as 90% of the Ang II formation could be inhibited by an ACE inhibitor. However, no experiments with intact failing human hearts have been performed. The obvious explanation for the discrepancy between the in vitro and in vivo studies is that the chymase-mediated Ang II formation is subjected to local regulation, a fact that has been overlooked in the studies performed in vitro. Thus, chymase activity is known to be regulated by at least 2 factors: Those that lead to stimulation and degranulation of …

Regulation of the activity of secreted human lung mast cell tryptase by mast cell proteoglycans

When mast cells from human lungs were stimulated in vitro to degranulate, all of the tryptase secreted was found to be complexed with proteoglycans, three quarters with heparin proteoglycans and one quarter with chondroitin sulphate proteoglycans. Isolation of the tryptase-proteoglycan complexes by fibronectin affinity chromatography and gel filtration on a Sephacryl S-200 column gave the complexes an apparent Mr of 200000, suggesting the presence of heparin and chondroitin sulphate proteoglycans (Mrs=60000) and tryptase (Mr=134000) in a molar ratio of 1:1, equivalent to a mass ratio of about 0.45:1. However, analysis of the total mast cell releasate showed that it contained more proteoglycans (mass ratio of about 2:1) than was needed to complex tryptase. We could demonstrate that the releasate contained two proteoglycan fractions, one complexed (20%) with tryptase and the other not (80%). Incubation of the isolated tryptase-proteoglycan complexes led to rapid monomerisation and inactivation of tryptase, whereas the releasate, containing both complexed and free proteoglycans, retained its tryptase activity for up to at least 18 h. The results indicate that the majority of the proteoglycans secreted by stimulated lung mast cells, although not complexed with the secreted tryptase, are critical for the preservation of its activity.

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.

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% O2) induced a fourfold temporal increase in BK‐2R mRNA expression in HCAECs, which was also observed at the protein level, whereas severe hypoxia (1% O2) 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. J. Cell. Physiol. 221: 359–366, 2009.

Kinin-Degrading Pathways in the Human Heart

In experimental animals, kinins protect the myocardium from ischemia-reperfusion injuries and reduce left ventricular hypertrophy and progression of heart failure. This suggests that in humans, also, the presence of an intact kinin system is critical for the prevention of heart failure. In addition to the kinin-generating system, the concentration of kinins, and consequently the extent of their actions, is regulated by their degradation. In the vascular bed of the human heart, bradykinin (BK) is degraded by angiotensin-converting enzyme (ACE). In contrast, in the interstitium of the human heart, BK is degraded by neutral endopeptidase (NEP). For potentiating the beneficial effects of BK, one strategy is elevation of the BK concentration by inhibition of BK-degrading enzymes. An even more effective form of pharmacological control of BK elevation than inhibition of ACE alone might be the combined inhibition of ACE and NEP.

Inhibition of copper-mediated oxidation of LDL by rat serosal mast cells. A novel cellular protective mechanism involving proteolysis of the substrate under oxidative stress.

Rat serosal mast cells, when stimulated to exocytose their cytoplasmic granules, effectively blocked the copper-mediated oxidation of low density lipoproteins (LDLs) in vitro. This effect depended on the proteolytic activity of the formed extracellular granule remnants, since specific inhibition of chymase, the neutral protease that they contain, blocked the protective effect of the mast cells. The mechanism of this chymase-mediated inhibition of LDL oxidation was found to be binding of the copper ions present in the incubation medium by peptides released from LDL on proteolytic degradation of their apolipoprotein B (apoB) component. This was verified by demonstrating that addition of such peptides to LDL--copper ion mixtures completely prevented oxidation of LDL and that this protective effect could be overcome by adding copper ions in excess. Furthermore, proteolytic degradation of the apoB of LDL, with concomitant release of copper-containing peptides, left the partially degraded apoB without the copper ions necessary for propagation of LDL oxidation. These observations provide the first evidence for cell-mediated inhibition of LDL oxidation.