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Angiotensin-converting enzyme (ACE) metabolizes a number of important peptides participating in blood pressure regulation and vascular remodeling. Elevated ACE expression in tissues (which is generally reflected by blood ACE levels) is associated with an increased risk of cardiovascular diseases. Elevated blood ACE is also a marker for granulomatous diseases. Decreased blood ACE activity is becoming a new risk factor for Alzheimer's disease. We applied our novel approach-ACE phenotyping-to characterize pairs of tissues (lung, heart, lymph nodes) and serum ACE in 50 patients. ACE phenotyping includes (1) measurement of ACE activity with two substrates (ZPHL and HHL); (2) calculation of the ratio of hydrolysis of these substrates (ZPHL/HHL ratio); (3) determination of ACE immunoreactive protein levels using mAbs to ACE; and (4) ACE conformation with a set of mAbs to ACE. The ACE phenotyping approach in screening format with special attention to outliers, combined with analysis of sequencing data, allowed us to identify patient with a unique ACE phenotype related to decreased ability of inhibition of ACE activity by albumin, likely due to competition with high CCL18 in this patient for binding to ACE. We also confirmed recently discovered gender differences in sialylation of some glycosylation sites of ACE. ACE phenotyping is a promising new approach for the identification of ACE phenotype outliers with potential clinical significance, making it useful for screening in a personalized medicine approach., Competing Interests: The authors declare no conflicts of interest.

Autoantibodies targeting the lung tissue were identified in severe COVID-19 patients in this retrospective study. Fifty-three percent of 104 patients developed anti-pulmonary antibodies, the majority of which were IgM class, suggesting that they developed upon infection with SARS-CoV-2. Anti-pulmonary antibodies correlated with worse pulmonary function and a higher risk of multiorgan failure that was further aggravated if 3 or more autoantibody clones were simultaneously present (multi-producers). Multi-producer patients were older than the patients with less or no autoantibodies. One of the identified autoantibodies (targeting a pulmonary protein of ~ 50 kDa) associated with worse clinical outcomes, including mortality. In summary, severe COVID-19 is associated with the development of lung-specific autoantibodies, which may worsen the clinical outcome. Tissue proteome-wide tests, such as the ones applied here, can be used to detect autoimmunity in the post-COVID state to identify the cause of symptoms and to reveal a new target for treatment., (© 2023. The Author(s).)

Severe SARS-CoV-2 elicits a hyper-inflammatory response that results in intravascular inflammation with endothelial injury, which contributes to increased mortality in COVID-19. To predict the outcome of severe SARS-CoV-2 infection, we analyzed the baseline level of different biomarkers of vascular disorders in COVID-19 subjects upon intensive care unit (ICU) admission and prior to any vaccination. A total of 70 severe COVID-19 patients (37 survivors and 33 non-survivors) were included with 16 age- and sex-matched controls. Vascular dysfunction was monitored via soluble VCAM-1, E-selectin, ACE2 and Lp-PLA2, while abnormal platelet activation was evaluated by soluble P-selectin and CD40L in parallel. These results were correlated with routine laboratory parameters and disease outcomes. Among these parameters, VCAM-1 and ACE2 showed significantly higher serum levels in COVID-19 patients with early death vs. convalescent subjects. VCAM-1 was significantly correlated with the Horowitz index (r = 0.3115) and IL-6 (r = 0.4599), while ACE2 was related to E-selectin (r = 0.4143) and CD40L (r = 0.2948). Lp-PLA2 was altered in none of these COVID-19 subcohorts and showed no relationship with the other parameters. Finally, the pre-treatment level of VCAM-1 (≥1420 ng/mL) and ACE2 activity (≥45.2 μU/mL) predicted a larger risk for mortality (Log-Rank p = 0.0031 and p = 0.0117, respectively). Vascular dysfunction with endothelial cell activation is linked to lethal COVID-19, and highly elevated soluble VCAM-1 and ACE2 at admission to ICU may predict unfavorable outcomes.

Objectives: Serum angiotensin-converting enzyme (ACE) is the only biomarker routinely used in the laboratory diagnostics of sarcoidosis, and ACE inhibitor (ACEi) drugs are among the most prescribed drugs worldwide. Taking ACEi can mislead medical teams by lowering ACE activity, delaying diagnosis and giving a false impression of disease activity of sarcoidosis. We aimed to develop a simple method to detect the presence of ACEi drugs in samples, to investigate the ACEi medication-caused interference and consequences in a retrospective study., Methods: ACE activity and the level of ACE inhibition were determined for 1823 patients with suspected sarcoidosis. These values were compared with the therapeutic information at the first and follow-up visits., Results: A total of 302 patients had biochemical evidence of an ACEi drug effect during diagnostic ACE activity testing. In their case, ACE activity was significantly lower (median(IQR): 4.41 U/L(2.93-6.72)) than in patients not taking ACEi (11.32 U/L(8.79-13.92), p<0.01). In 62 sarcoidosis patients, the ACEi reduced ACE activity to the reference range or below. Only in 40 % of the cases was the medication list recorded in the outpatient chart and only in 3 cases was low ACE activity associated with ACEi use. 67 % of the repeated ACE activity measurements were also performed during ACEi therapy., Conclusions: Our study revealed that the use of ACEi is common in patients with suspected sarcoidosis. The ACE activity lowering effect of ACEi drugs may escape the attention of medical teams which can lead to diagnostic errors and unnecessary tests. Nevertheless, these pitfalls can be avoided by using a method suggested by our team., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Background: We retrospectively analyzed serum level of human epididymis protein 4 (HE4) as a pulmonary inflammatory biomarker in patients with COVID-19 pneumonia in association with disease severity and outcome., Methods: Ninety-nine (40 critically ill, 40 severe and 19 mild) COVID-19 patients and as controls 25 age- and sex-matched non-COVID-19 bacterial sepsis subjects were included. Serum HE4 was measured by an immunoassay (Architect ® i1000SR, Abbott) in the baseline samples of all study participants obtained at intensive care unit (ICU) admission or during outpatient clinic visit and follow-up sera were available in case of 30 COVID-19 subjects with life-threating conditions. Associations were studied between serum HE4, routinely available laboratory parameters, clinical characteristics, and disease progression., Results: Baseline HE4 level was significantly higher (P < 0.0001) in critically ill (524.7 [300.1-1153.0] pmol/L) than severe COVID-19 subjects (157.4 [85.2-336.9] pmol/L) and in mild SARS-CoV-2 infection (46.7 [39.1-57.2] pmol/L). Similarly increased HE4 concentrations were found in bacterial sepsis (1118.0 [418.3-1953.0] pmol/L, P = 0.056) compared to critically ill COVID-19 individuals. Serum HE4 levels significantly correlated with age, SOFA-score, inflammation-dependent biomarkers, and the degree of lung manifestation evaluated by chest CT examination in ICU COVID-19 individuals. Based on ROC-AUC curve analysis, baseline HE4 independently indicated the severity of COVID-19 with an AUC value of 0.816 (95% CI [0.723-0.908]; P < 0.0001), while binary logistic regression test found HE4 as an independent prognostic parameter for death (OR: 10.618 [2.331-48.354]; P = 0.002). Furthermore, COVID-19 non-survivors showed much higher baseline HE4 levels without a substantial change under treatment vs. survivors (P < 0.0001). Finally, pre-treatment HE4 level of ≥ 331.7 pmol/L effectively predicted a larger risk for mortality (Log-Rank P < 0.0001) due to severe COVID-19 pneumonia., Conclusion: Elevated serum HE4 level at ICU admission highly correlates with COVID-19 severity and predicts disease outcome., (© 2023. The Author(s).)

Angiotensin-converting enzyme (ACE) inhibitors are the primarily chosen drugs to treat various cardiovascular diseases, such as hypertension. Although the most recent guidelines do not differentiate among the various ACE inhibitory drugs, there are substantial pharmacological differences., Goal: Here, we tested if lipophilicity affects the efficacy of ACE inhibitory drugs when used as the first therapy in newly identified hypertensives in a prospective study., Methods: We tested the differences in the cardiovascular efficacy of the hydrophilic lisinopril (8.3 ± 3.0 mg/day) and the lipophilic enalapril (5.5 ± 2.3 mg/day) ( n = 59 patients). The cardiovascular parameters were determined using sonography (flow-mediated dilation (FMD) in the brachial artery, intima-media thickness of the carotid artery), 24 h ambulatory blood pressure monitoring (peripheral arterial blood pressure), and arteriography (aortic blood pressure, augmentation index, and pulse wave velocity) before and after the initiation of ACE inhibitor therapy., Results: Both enalapril and lisinopril decreased blood pressure. However, lisinopril failed to improve arterial endothelial function (lack of effects on FMD) when compared to enalapril. Enalapril-mediated improved arterial endothelial function (FMD) positively correlated with its blood-pressure-lowering effect. In contrast, there was no correlation between the decrease in systolic blood pressure and FMD in the case of lisinopril treatment., Conclusion: The blood-pressure-lowering effects of ACE inhibitor drugs are independent of their lipophilicity. In contrast, the effects of ACE inhibition on arterial endothelial function are associated with lipophilicity: the hydrophilic lisinopril was unable to improve, while the lipophilic enalapril significantly improved endothelial function. Moreover, the effects on blood pressure and endothelial function did not correlate in lisinopril-treated patients, suggesting divergent mechanisms in the regulation of blood pressure and endothelial function upon ACE inhibitory treatment.

Background: Inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis (UC), are associated with higher thrombotic risk and enhanced thrombin generation (TG) in adults. Despite encouraging data reporting vaccine safety and low IBD flare rates in adults with IBD, vaccine hesitancy was demonstrated to be high in families of children with IBD. We aimed to find out whether TG is increased in children with IBD as compared to healthy controls and whether TG parameters show significant changes following SARS-CoV-2 mRNA vaccination., Patients and Methods: In this observational case-control study, 38 children with IBD (CD:18, UC: 20) aged 12-18 years and 62 healthy age-and sex-matched children were enrolled. Blood was collected before the first dose and 2-6 weeks after the second dose of BNT162b2 (Pfizer-BioNTech) mRNA vaccine dose. Blood cell counts, fibrinogen, inflammatory markers (hsCRP, ferritin), anti-SARS-CoV-2 antibody levels were investigated, TG assay was carried-out using platelet-poor plasma. Detailed clinical parameters including disease activity scores (PUCAI, PCDAI) were registered pre-and post- vaccination. A guided questionnaire was used to collect data on adverse reactions (AEs) post- vaccination., Results: Baseline TG parameters did not differ between patients and controls. Endogenous thrombin potential showed a significant positive correlation with markers of inflammation and with PCDAI. Inflammatory parameters and TG did not increase in patients and controls post-vaccination. Vaccination significantly increased antibody levels in all three investigated groups, but post-vaccination anti-SARS-CoV-2 S IgG/IgM levels were below the 5 th percentile value of healthy children in more than one third of patients. Those receiving TNFα inhibitor therapy presented significantly lower SARS-CoV-2 S IgG/IgM levels as compared to patients on other immunosuppressive regimens. Systemic AEs did not differ between patients and controls while lower rate of local symptoms was found post-vaccination in children with IBD. Only 2 IBD flares were detected 2-6 weeks after the second dose of vaccination., Conclusion: Our study is the first to support the safety and efficacy of anti-SARS-CoV-2 BNT162b2 vaccination in children with IBD with detailed pre-and post-vaccination laboratory data including TG. Results of this study may further increase confidence and reduce vaccine hesitancy in caretakers of pediatric IBD patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stercel, Lóczi, Kadenczki, Nemes, Nagy, Hodossy-Takács, Szabó, Fagyas, Kappelmayer, Szabó and Bagoly.)

Introduction: The Renin-Angiotensin-Aldosterone system (RAAS) has been implicated in the regulation of the cardiovascular system and linked to rheumatoid arthritis (RA). Little information has become available on the effects of Janus kinase (JAK) inhibition on RAAS. Here we studied the effects of 12-month tofacitinib treatment on angiotensin converting enzyme (ACE), ACE2 production and ACE/ACE2 ratios in RA along with numerous other biomarkers., Patients and Methods: Thirty RA patients were treated with tofacitinib in this prospective study. Serum ACE concentrations were assessed by ELISA. ACE2 activity was determined by a specific quenched fluorescent substrate. ACE/ACE2 ratios were calculated. We also determined common carotid intima-media thickness (ccIMT), brachial artery flow-mediated vasodilation (FMD) and carotid-femoral pulse-wave velocity (cfPWV) by ultrasound. C-reactive protein (CRP), rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) were also determined. All measurements were performed at baseline, as well as after 6 and 12 months of tofacitinib treatment., Results: After the dropout of 4 patients, 26 completed the study. Tofacitinib treatment increased ACE levels after 6 and 12 months, while ACE2 activity only transiently increased at 6 months. The ACE/ACE2 ratio increased after 1 year of therapy ( p < 0.05). Logistic regression analyses identified correlations between ACE, ACE2 or ACE/ACE2 ratios and RF at various time points. Baseline disease duration also correlated with erythrocyte sedimentation rate (ESR) ( p < 0.05). One-year changes of ACE or ACE2 were determined by tofacitinib treatment plus ACPA or RF, respectively ( p < 0.05)., Conclusion: JAK inhibition increases serum ACE and ACE/ACE2 ratio in RA. Baseline inflammation (ESR), disease duration and ACPA, as well as RF levels at various time points can be coupled to the regulation of ACE/ACE2 ratio. The effect of tofacitinib on RAAS provides a plausible explanation for the cardiovascular effects of JAK inhibition in RA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kacsándi, Fagyas, Horváth, Végh, Pusztai, Czókolyová, Soós, Szabó, Hamar, Pethő, Bodnár, Kerekes, Hodosi, Szamosi, Szűcs, Papp and Szekanecz.)

Background: Atrial fibrillation (AF) is accompanied by inflammation and fibrosis to variable extent. The biomarkers of fibrosis were measured in patients with different forms of AF and cardiac status. Herein, we assessed the associations of the baseline concentrations of different biomarkers with the long-term success of pulmonary vein isolation (PVI) in patients with a structurally normal heart. Furthermore, we compared biomarker levels before and 3 years after ablation to gain further insights into the AF mechanism., Methods: Patients, undergoing PVI for paroxysmal/persistent AF were enrolled prospectively. Blood samples were obtained 24 hours before and 3 years after ablation. Serum cancer antigen 125 (CA-125), plasma Caspase-3, Galectin-3 and Cathepsin L concentrations were measured. Follow-up visits every 6 months included 12-lead electrocardiogram, 24-hour Holter, trans-telephonic monitoring as well as transthoracic echocardiography after ablation. Biomarker levels, left ventricular ejection fraction and left atrial (LA) diameters at baseline and at the 3-year follow-up were compared in patients with versus without AF recurrence., Results: A total of 63 patients were enrolled (23 women; age 61.4 ( ± 8.8) years). The acute isolation of all pulmonary veins was achieved in all patients. During a mean follow-up of 36.3 ± 6.3 months, AF recurrence was demonstrated in 26 (41.3%) patients. No significant differences were demonstrated in the levels of CA-125, Galectin-3, Caspase-3 and Cathepsin L pre- and post-ablation in patients with versus without AF recurrence. A significant decrease was detected in the concentrations of Caspase-3, Galectin-3 and Cathepsin L during follow-up with no difference in patients with versus without AF recurrence. A positive correlation was found between Caspase-3 levels and LA diameters in the AF recurrence group both before (r = 0.477; p = 0.018) and after the procedure (r = 0.533; p = 0.019)., Conclusions: Our results demonstrated that the levels of CA-125, Caspase-3, Cathepsin L and Galectin-3 are not associated with AF recurrence after PVI in patients with a structurally normal heart and mainly paroxysmal AF. Except for CA-125, all the other biomarkers demonstrated a significant decrease during a 3-year follow-up post-ablation. Furthermore, Caspase-3 levels demonstrated a positive correlation with LA dimensions in patients with AF recurrence., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2023 The Author(s). Published by IMR Press.)

Background: Angiotensin-converting enzyme (ACE) is highly expressed in renal proximal tubules, but ACE activity/levels in the urine are at least 100-fold lower than in the blood. Decreased proximal tubular ACE has been associated with renal tubular damage in both animal models and clinical studies. Because ACE is shed into urine primarily from proximal tubule epithelial cells, its urinary ACE measurement may be useful as an index of tubular damage., Objective and Methodology: We applied our novel approach-ACE phenotyping-to characterize urinary ACE in volunteer subjects. ACE phenotyping includes (1) determination of ACE activity using two substrates (ZPHL and HHL); (2) calculation of the ratio of hydrolysis of the two substrates (ZPHL/HHL ratio); (3) quantification of ACE immunoreactive protein levels; and (4) fine mapping of local ACE conformation with mAbs to ACE., Principal Findings: In normal volunteers, urinary ACE activity was 140-fold less than in corresponding plasma/serum samples and did not differ between males and females. However, urinary ACE immunoreactivity (normalized binding of 25 mAbs to different epitopes) was strongly sex-dependent for the several mAbs tested, an observation likely explained by differences in tissue ACE glycosylation/sialylation between males and females. Urinary ACE phenotyping also allowed the identification of ACE outliers. In addition, daily variability of urinary ACE has potential utility as a feedback marker for dieting individuals pursuing weight loss., Conclusions/significance: Urinary ACE phenotyping is a promising new approach with potential clinical significance to advance precision medicine screening techniques.

Severe cases of COVID-19 are characterized by an inflammatory burst, which is accompanied by multiorgan failure. The elderly population has higher risk for severe or fatal outcome for COVID-19. Inflammatory mediators facilitate the immune system to combat viral infection by producing antibodies against viral antigens. Several studies reported that the pro-inflammatory state and tissue damage in COVID-19 also promotes autoimmunity by autoantibody generation. We hypothesized that a subset of these autoantibodies targets cardiac antigens. Here we aimed to detect anti-cardiac autoantibodies in severe COVID-19 patients during hospitalization. For this purpose, 104 COVID-19 patients were recruited, while 40 heart failure patients with dilated cardiomyopathy and 20 patients with severe aortic stenosis served as controls. Patients were tested for anti-cardiac autoantibodies, using human heart homogenate as a bait. Follow-up samples were available in 29 COVID-19 patients. Anti-cardiac autoantibodies were detected in 68% (71 out of 104) of severe COVID-19 patients. Overall, 39% of COVID-19 patients had anti-cardiac IgG autoantibodies, while 51% had anti-cardiac autoantibodies of IgM isotype. Both IgG and IgM anti-cardiac autoantibodies were observed in 22% of cases, and multiple cardiac antigens were targeted in 38% of COVID-19 patients. These anti-cardiac autoantibodies targeted a diverse set of myocardial proteins, without apparent selectivity. As controls, heart failure patients (with dilated cardiomyopathy) had similar occurrence of IgG (45%, p = 0.57) autoantibodies, while significantly lower occurrence of IgM autoantibodies (30%, p = 0.03). Patients with advanced aortic stenosis had significantly lower number of both IgG (11%, p = 0.03) and IgM (10%, p < 0.01) type anti-cardiac autoantibodies than that in COVID-19 patients. Furthermore, we detected changes in the anti-cardiac autoantibody profile in 7 COVID-19 patients during hospital treatment. Surprisingly, the presence of these anti-cardiac autoantibodies did not affect the clinical outcome and the prevalence of the autoantibodies did not differ between the elderly (over 65 years) and the patients younger than 65 years of age. Our results demonstrate that the majority of hospitalized COVID-19 patients produce novel anti-cardiac IgM autoantibodies. COVID-19 also reactivates resident IgG autoantibodies. These autoantibodies may promote autoimmune reactions, which can complicate post-COVID recuperation, contributing to post-acute sequelae of COVID-19 (long COVID)., (© 2022. The Author(s).)

Introduction: The respiratory illness triggered by severe acute respiratory syndrome virus-2 (SARS-CoV-2) is often particularly serious or fatal amongst patients with pre-existing heart conditions. Although the mechanisms underlying SARS-CoV-2-related cardiac damage remain elusive, inflammation (i.e. 'cytokine storm') and oxidative stress are likely involved., Methods and Results: Here we sought to determine: 1) if cardiomyocytes are targeted by SARS-CoV-2 and 2) how inflammation and oxidative stress promote the viral entry into cardiac cells. We analysed pro-inflammatory and oxidative stress and its impact on virus entry and virus-associated cardiac damage from SARS-CoV-2 infected patients and compared it to left ventricular myocardial tissues obtained from non-infected transplanted hearts either from end stage heart failure or non-failing hearts (donor group). We found that neuropilin-1 potentiates SARS-CoV-2 entry into human cardiomyocytes, a phenomenon driven by inflammatory and oxidant signals. These changes accounted for increased proteases activity and apoptotic markers thus leading to cell damage and apoptosis., Conclusion: This study provides new insights into the mechanisms of SARS-CoV-2 entry into the heart and defines promising targets for antiviral interventions for COVID-19 patients with pre-existing heart conditions or patients with co-morbidities., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objectives: Angiotensin-converting enzyme 2 (ACE2) represents the primary receptor for SARS-CoV-2 to enter endothelial cells. Here we investigated circulating ACE2 activity to predict the severity and mortality of COVID-19., Methods: Serum ACE2 activity was measured in COVID-19 (110 critically ill and 66 severely ill subjects at hospital admission and 106 follow-up samples) and in 32 non-COVID-19 severe sepsis patients. Associations between ACE2, inflammation-dependent biomarkers, pre-existing comorbidities, and clinical outcomes were studied., Results: Initial ACE2 activity was significantly higher in critically ill COVID-19 patients (54.4 [36.7-90.8] mU/L) than in severe COVID-19 (34.5 [25.2-48.7] mU/L; P<0.0001) and non-COVID-19 sepsis patients (40.9 [21.4-65.7] mU/L; P=0.0260) regardless of comorbidities. Circulating ACE2 activity correlated with inflammatory biomarkers and was further elevated during the hospital stay in critically ill patients. Based on ROC-curve analysis and logistic regression test, baseline ACE2 independently indicated the severity of COVID-19 with an AUC value of 0.701 (95% CI [0.621-0.781], P<0.0001). Furthermore, non-survivors showed higher serum ACE2 activity vs. survivors at hospital admission (P<0.0001). Finally, high ACE2 activity (≥45.4 mU/L) predicted a higher risk (65 vs. 37%) for 30-day mortality (Log-Rank P<0.0001)., Conclusions: Serum ACE2 activity correlates with COVID-19 severity and predicts mortality., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Angiotensin-converting enzyme (ACE) and ACE2 have been implicated in the regulation of vascular physiology. Elevated synovial and decreased or normal ACE or ACE2 levels have been found in rheumatoid arthritis (RA). Very little is known about the effects of tumor necrosis factor α (TNF-α) inhibition on ACE or ACE2 homeostasis. In this study, we assessed the effects of one-year anti-TNF therapy on ACE and ACE2 production in RA and ankylosing spondylitis (AS) in association with other biomarkers., Patients and Methods: Forty patients including 24 RA patients treated with either etanercept (ETN) or certolizumab pegol (CZP) and 16 AS patients treated with ETN were included in a 12-month follow-up study. Serum ACE levels were determined by commercial ELISA, while serum ACE2 activity was assessed using a specific quenched fluorescent substrate. Ultrasonography was performed to determine flow-mediated vasodilation (FMD), common carotid intima-media thickness (ccIMT) and arterial pulse-wave velocity (PWV) in all patients. In addition, CRP, rheumatoid factor (RF) and ACPA were also measured. All assessments were performed at baseline and 6 and 12 months after treatment initiation., Results: Anti-TNF therapy increased ACE levels in the full cohort, as well as in the RA and AS subsets. ACE2 activity increased in the full cohort, while the ACE/ACE2 ratio increased in the full cohort and in the RA subset ( p < 0.05). Uni- and multivariable regression analyses determined associations between ACE or ACE/ACE2 ratios at different time points and disease duration, CRP, RF, FMD and IMT ( p < 0.05). ACE2 activity correlated with CRP. The changes of ACE or ACE2 over 12 months were determined by treatment together with either RF or FMD (p < 0.05)., Conclusions: Anti-TNF treatment may increase ACE and ACE2 in the sera of RA and AS patients. ACE and ACE2 may be associated with disease duration, markers of inflammation and vascular pathophysiology. The effects of TNF inhibition on ACE and ACE2 may reflect, in part, the effects of these biologics on the cardiovascular system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Soós, Fagyas, Horváth, Végh, Pusztai, Czókolyová, Csongrádi, Hamar, Pethő, Bodnár, Kerekes, Hodosi, Szekanecz, Szamosi, Szántó, Szűcs, Papp and Szekanecz.)

Introduction: Serum total lactate dehydrogenase (LDH) activity was elevated and showed a positive correlation with disease severity and outcome in severe COVID-19 disease. However, it is still unknown whether the relative abundance or calculated activity of any LDH isoenzyme is predominately increased in COVID-19 subjects., Methods: Twenty-two consecutive patients suffered from moderate or severe COVID-19 pneumonia were recruited into this study who showed enhanced total LDH activity. The ratio of LDH isoenzyme activities was further investigated using gel electrophoresis (Hydragel ® , Sebia) with densitometric evaluation. Calculated activity values of these isoenzymes were correlated with routine laboratory parameters, the degree of lung parenchymal affection based on chest CT and clinical outcome., Results: Total LDH activity was raised in the range of 272-2141 U/L and significantly correlated with calculated LDH-3 and LDH-4 activities (r=0.765, P=0.0001; and r=0.783, P=0.0001, respectively). In contrast, the relative abundance of neither LDH isoenzyme was exclusively abnormal in COVID-19 patients. Calculated activity of LDH-3 and LDH-4 demonstrated a modest but statistically significant association with serum ferritin (r=0.437, P=0.042; r=0.505, P=0.016, respectively). When the relationship between the severity of pulmonary affection by SARS-CoV-2 infection and relative abundance of LDH isoenzymes was studied, a larger ratio of mid-zone fractions was observed in the presence of ≥ 50% lung parenchymal involvement. Finally, regardless of LDH isoenzyme pattern, abnormal relative ratio of LDH-4 and higher calculated LDH-3 and LDH-4 activity values were detected in subjects with unfavorable outcome., Conclusion: No characteristic profile of LDH isoenzymes can be detected in COVID-19 pneumonia, however, elevated activities of LDH-3 and LDH-4 are associated with worse clinical outcomes., Competing Interests: This work was funded by grants from the National Research, Development and Innovation Office (FK 135327 to BN and FK 128809 to MF). M.P. and M.F. were supported by the ÚNKP-21-3-I-DE-255 and ÚNKP-21-5-DE-458 New National Excellence Program of The Ministry for Innovation and Technology, respectively. This paper was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00069/21/5). No funding source had any role in the writing of the manuscript or the decision to submit. Conflict of interest There are no competing interests to declare among the authors of this work., (Copyright © 2021 International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). All rights reserved.)

Post-ischemic left ventricular (LV) remodeling and its hypothetical prevention by repeated remote ischemic conditioning (rRIC) in male Sprague-Dawley rats were studied. Myocardial infarction (MI) was evoked by permanent ligation of the left anterior descending coronary artery (LAD), and myocardial characteristics were tested in the infarcted anterior and non-infarcted inferior LV regions four and/or six weeks later. rRIC was induced by three cycles of five-minute-long unilateral hind limb ischemia and five minutes of reperfusion on a daily basis for a period of two weeks starting four weeks after LAD occlusion. Sham operated animals served as controls. Echocardiographic examinations and invasive hemodynamic measurements revealed distinct changes in LV systolic function between four and six weeks after MI induction in the absence of rRIC (i.e., LV ejection fraction (LVEF) decreased from 52.8 ± 2.1% to 50 ± 1.6%, mean ± SEM, p < 0.05) and in the presence of rRIC (i.e., LVEF increased from 48.2 ± 4.8% to 55.2 ± 4.1%, p < 0.05). Angiotensin-converting enzyme (ACE) activity was about five times higher in the anterior LV wall at six weeks than that in sham animals. Angiotensin-converting enzyme 2 (ACE2) activity roughly doubled in post-ischemic LVs. These increases in ACE and ACE2 activities were effectively mitigated by rRIC. Ca 2+ -sensitivities of force production (pCa 50 ) of LV permeabilized cardiomyocytes were increased at six weeks after MI induction together with hypophosphorylation of 1) cardiac troponin I (cTnI) in both LV regions, and 2) cardiac myosin-binding protein C (cMyBP-C) in the anterior wall. rRIC normalized pCa 50 , cTnI and cMyBP-C phosphorylations. Taken together, post-ischemic LV remodeling involves region-specific alterations in ACE and ACE2 activities together with changes in cardiomyocyte myofilament protein phosphorylation and function. rRIC has the potential to prevent these alterations and to improve LV performance following MI.

Angiotensin-converting enzyme 2 (ACE2) is essential for SARS-CoV-2 cellular entry. Here we studied the effects of common comorbidities in severe COVID-19 on ACE2 expression. ACE2 levels (by enzyme activity and ELISA measurements) were determined in human serum, heart and lung samples from patients with hypertension (n = 540), heart transplantation (289) and thoracic surgery (n = 49). Healthy individuals (n = 46) represented the controls. Serum ACE2 activity was increased in hypertensive subjects (132%) and substantially elevated in end-stage heart failure patients (689%) and showed a strong negative correlation with the left ventricular ejection fraction. Serum ACE2 activity was higher in male (147%), overweight (122%), obese (126%) and elderly (115%) hypertensive patients. Primary lung cancer resulted in higher circulating ACE2 activity, without affecting ACE2 levels in the surrounding lung tissue. Male sex resulted in elevated serum ACE2 activities in patients with heart transplantation or thoracic surgery (146% and 150%, respectively). Left ventricular (tissular) ACE2 activity was unaffected by sex and was lower in overweight (67%), obese (62%) and older (73%) patients with end-stage heart failure. There was no correlation between serum and tissular (left ventricular or lung) ACE2 activities. Neither serum nor tissue (left ventricle or lung) ACE2 levels were affected by RAS inhibitory medications. Abandoning of ACEi treatment (non-compliance) resulted in elevated blood pressure without effects on circulating ACE2 activities. ACE2 levels associate with the severity of cardiovascular diseases, suggestive for a role of ACE2 in the pathomechanisms of cardiovascular diseases and providing a potential explanation for the higher mortality of COVID-19 among cardiovascular patients. Abandoning RAS inhibitory medication worsens the cardiovascular status without affecting circulating or tissue ACE2 levels., (© 2021. The Author(s).)

Objective: Inhibitors of the angiotensin converting enzyme (ACE) are the primarily chosen drugs to treat heart failure and hypertension. Moreover, an imbalance in tissue ACE/ACE2 activity is implicated in COVID-19. In the present study, we tested the relationships between circulating and tissue (lung and heart) ACE levels in men. Methods: Serum, lung ( n = 91) and heart ( n = 72) tissue samples were collected from Caucasian patients undergoing lung surgery or heart transplantation. ACE I/D genotype, ACE concentration and ACE activity were determined from serum and tissue samples. Clinical parameters were also recorded. Results: A protocol for ACE extraction was developed for tissue ACE measurements. Extraction of tissue-localized ACE was optimal in a 0.3% Triton-X-100 containing buffer, resulting in 260 ± 12% higher ACE activity over detergent-free conditions. SDS or higher Triton-X-100 concentrations inhibited the ACE activity. Serum ACE concentration correlated with ACE I/D genotype (II: 166 ± 143 ng/mL, n = 19, ID: 198 ± 113 ng/mL, n = 44 and DD: 258 ± 109 ng/mL, n = 28, p < 0.05) as expected. In contrast, ACE expression levels in the lung tissue were approximately the same irrespective of the ACE I/D genotype (II: 1423 ± 1276 ng/mg, ID: 1040 ± 712 ng/mg and DD: 930 ± 1273 ng/mg, p > 0.05) in the same patients (values are in median ± IQR). Moreover, no correlations were found between circulating and lung tissue ACE concentrations and activities (Spearman's p > 0.05). In contrast, a significant correlation was identified between ACE activities in serum and heart tissues (Spearman's Rho = 0.32, p < 0.01). Finally, ACE activities in lung and the serum were endogenously inhibited to similar degrees (i.e., to 69 ± 1% and 53 ± 2%, respectively). Conclusion: Our data suggest that circulating ACE activity correlates with left ventricular ACE, but not with lung ACE in human. More specifically, ACE activity is tightly coordinated by genotype-dependent expression, endogenous inhibition and secretion mechanisms.

Omecamtiv mecarbil (OM) is a promising novel drug for improving cardiac contractility. We tested the therapeutic range of OM and identified previously unrecognized side effects. The Ca 2+ sensitivity of isometric force production (pCa 50 ) and force at low Ca 2+ levels increased with OM concentration in human permeabilized cardiomyocytes. OM (1 µM) slowed the kinetics of contractions and relaxations and evoked an oscillation between normal and reduced intracellular Ca 2+ transients, action potential lengths and contractions in isolated canine cardiomyocytes. Echocardiographic studies and left ventricular pressure-volume analyses demonstrated concentration-dependent improvements in cardiac systolic function at OM concentrations of 600-1200 µg/kg in rats. Administration of OM at a concentration of 1200 µg/kg was associated with hypotension, while doses of 600-1200 µg/kg were associated with the following aspects of diastolic dysfunction: decreases in E/A ratio and the maximal rate of diastolic pressure decrement (dP/dt min ) and increases in isovolumic relaxation time, left atrial diameter, the isovolumic relaxation constant Tau, left ventricular end-diastolic pressure and the slope of the end-diastolic pressure-volume relationship. Moreover, OM 1200 µg/kg frequently evoked transient electromechanical alternans in the rat in vivo in which normal systoles were followed by smaller contractions (and T-wave amplitudes) without major differences on the QRS complexes. Besides improving systolic function, OM evoked diastolic dysfunction and pulsus alternans. The narrow therapeutic window for OM may necessitate the monitoring of additional clinical safety parameters in clinical application.

Serum chitotriosidase (CTO) activity was proposed as a biomarker in sarcoidosis being potentially useful in diagnostics. Nevertheless, a common duplication polymorphism (c.1049&#95;1072dup24, Dup24) of the CTO gene influences CTO activity and thereby compromises its use in sarcoidosis. Here we aimed to substitute CTO activity with CTO concentration to prevent the confounding effect of Dup24. CTO activity, concentration and genetic backgrounds were determined in 80 histopathology proven sarcoidosis patients and 133 healthy individuals. CTO activities were lower in healthy individuals and sarcoidosis patients heterozygous for Dup24 mutation (472 ± 367 mU/L, n = 49; 2300 ± 2105 mU/L, n = 29) than in homozygous wild types (838 ± 856 mU/L, n = 81; 5125 ± 4802 mU/L, n = 48; p < 0.001, respectively). Sera of Dup24 homozygous individuals had no CTO activity. CTO concentrations were also lower in healthy individuals and sarcoidosis patients heterozygous for Dup24 mutation (7.2 ± 1.9 µg/L, n = 11; 63.16 ± 56.5 µg/L, n = 29) than in homozygous wild types (18.9 ± 13.0 µg/L, n = 36; 157.1 ± 132.4 µg/L, n = 47, p < 0.001, respectively) suggestive for an interaction between Dup24 mutation and CTO concentration determinations. We also identified a healthy Hungarian male subject without CTO activity carrying a rare mutation (c.(965&#95;993)del), which mutation has been considered unique for Cypriot population to date. Taken together, CTO concentration determination does not add to the CTO activity measurement when CTO is used as a biomarker in sarcoidosis. Therefore, genotyping of CTO gene should be involved in the interpretation of laboratory findings., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Coronavirus disease 2019 (COVID-19) has a high mortality in elderly patients with pre-existing cardiovascular diseases. The cellular receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the angiotensin-converting enzyme 2 (ACE2), thereby implicating a link between cardiovascular diseases and SARS-CoV-2 susceptibility. Aortic stenosis (AS) represents a chronic inflammatory state with severe cardiovascular complications in the elderly, a prime condition for COVID-19 mortality. The circulating ACE2 levels were measured in 111 patients with severe AS and compared to patients with hypertension and healthy individuals. About 4 times higher circulating ACE2 activity was found in patients with severe AS than in hypertensives or healthy individuals (88.3 ± 61.6., n = 111, 20.6 ± 13.4, n = 540, and 16.1 ± 7.4 mU/L, n = 46, respectively). Patients with severe AS were older than patients with hypertension (80 ± 6 years vs. 60 ± 15 years, P < 0.05). Serum ACE2 activity correlated negatively with the left ventricular ejection fraction, aortic root area, TAPSE, and positively with the right ventricular systolic pressure, cardiac diameters in patients with AS. In contrast, circulating ACE2 activity was independent of the blood pressure, peak flow velocity at the aortic root, kidney function (GFR), and inflammatory state (CRP). We found no effect of RAAS inhibitory drugs on the serum ACE2 activity in this group of patients. Our results illustrate circulating ACE2 as a potential interface between chronic inflammation, cardiovascular disease, and COVID-19 susceptibility. Elderly patients with AS have markedly elevated ACE2 levels together with altered left and right ventricular functions, which may pose higher risks during COVID-19. Our clinical data do not support a role for RAAS inhibitors in regulating circulating ACE2 levels.

Background: Cardiomyopathy is a common side effect of doxorubicin (DOX) chemotherapy. Despite intensive research efforts in the field, there is still no evidence available for routine cardioprotective prophylaxis to prevent cardiotoxicity in the majority of oncological patients at low risk of cardiovascular disease. We have recently demonstrated the advantages of a prophylactic, combined heart failure therapy in an experimental model of DOX-induced cardiomyopathy. In the current work, we focus on individually applied prophylactic medications studied in the same translational environment to clarify their distinct roles in the prevention of DOX cardiotoxicity., Methods: Twelve-week-old male Wistar rats were divided into 5 subgroups. Prophylactic β-blocker (BB, bisoprolol), angiotensin-converting enzyme inhibitor (ACEI, perindopril) or aldosterone antagonist (AA, eplerenone) treatments were applied 1 week before DOX administration, then 6 cycles of intravenous DOX chemotherapy were administered. Rats receiving only intravenous DOX or saline served as positive and negative controls. Blood pressure, heart rate, body weight, and echocardiographic parameters were monitored in vivo. Two months after the last DOX administration, the animals were sacrificed, and their heart and serum samples were frozen in liquid nitrogen for histological, mechanical, and biochemical measurements., Results: All prophylactic treatments increased the survival of DOX-receiving animals. The lowest mortality rates were seen in the BB and ACEI groups. The left ventricular ejection fraction was only preserved in the BB group. The DOX-induced increase in the isovolumetric relaxation time could not be prevented by any prophylactic treatment. A decreased number of apoptotic nuclei and a preserved myocardial ultrastructure were found in all groups receiving prophylactic cardioprotection, while the DOX-induced fibrotic remodelling and the increase in caspase-3 levels could only be substantially prevented by the BB and ACEI treatments., Conclusion: Primary prophylaxis with cardioprotective agents like BB or ACEI has a key role in the prevention of DOX-induced cardiotoxicity in healthy rats. Future human studies are necessary to implement this finding in the clinical management of oncological patients free of cardiovascular risk factors.

One of the most abundant coagulation proteins is β 2 -glycoprotein I (β 2 GPI) that is present in humans at a concentration of around 200 mg/L. Its physiological role is only partially understood, but it adopts several different structural forms the majority of which are the open and closed forms. We isolated native (circular) β 2 GPI and converted it into an open conformation. The effectiveness of these procedures was assessed by Western blot and negative-staining electron microscopy. We found that in coagulation assays the open form of β 2 GPI had a significant prolonging effect on fibrin formation in a dilute prothrombin time test (p < 0.001). In the dilute activated partial thromboplastin time test, both conformations had a significant prolonging effect (p < 0.001) but the open conformation was more effective. In a fluorescent thrombin generation assay both conformations slightly delayed thrombin generation with no significant effect on the quantity of formed thrombin. By using surface plasmon resonance assays, the equilibrium dissociation constants of both the open and closed conformations of β 2 GPI showed a similar and strong affinity to isolated anti-β 2 GPI autoantibodies (K d closed β 2 GPI = 5.17 × 10 -8  M, K d open β 2 GPI = 5.56 × 10 -8  M) and the open form had one order of magnitude stronger affinity to heparin (K d  = 0.30 × 10 -6  M) compared to the closed conformation (K d  = 3.50 × 10 -6  M). The two different forms of β 2 GPI have distinct effects in functional tests and in ligand binding, which may considerably affect the intravascular events related to this abundant plasma protein in health and disease., (Copyright © 2020. Published by Elsevier B.V.)

Aims: Tenascin-C (TN-C) is suggested to be detrimental in cardiac remodelling after myocardial infarction (MI). The aim of this study is to reveal the effects of TN-C on extracellular matrix organization and its haemodynamic influence in an experimental mouse model of MI and in myocardial cell culture during hypoxic conditions., Methods and Results: Myocardial infarction was induced in TN-C knockout (TN-C KO) and wild-type mice. Six weeks later, cardiac function was studied by magnetic resonance imaging and under isolated working heart conditions. Myocardial mRNA levels and immunoreactivity of TN-C, TIMP-1, TIMP-3, and matrix metalloproteinase (MMP)-9, as well as serum and tissue activities of angiotensin-converting enzyme (ACE), were determined at 1 and 6 weeks after infarction. Cardiac output and external heart work were higher, while left ventricular wall stress and collagen expression were decreased (P < 0.05) in TN-C KO mice as compared with age-matched controls at 6 weeks after infarction. TIMP-1 expression was down-regulated at 1 and 6 weeks, and TIMP-3 expression was up-regulated at 1 week (P < 0.01) after infarction in knockout mice. MMP-9 level was lower in TN-C KO at 6 weeks after infarction (P < 0.05). TIMP-3/MMP-9 ratio was higher in knockout mice at 1 and 6 weeks after infarction (P < 0.01). ACE activity in the myocardial border zone (i.e. between scar and free wall) was significantly lower in knockout than in wild-type mice 1 week after MI (P < 0.05)., Conclusions: Tenascin-C expression is induced by hypoxia in association with ACE activity and MMP-2 and MMP-9 elevations, thereby promoting left ventricular dilatation after MI., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Establishing the diagnosis of sarcoidosis most often requires biopsy and histopathologic evaluation, since there is no single marker with sufficient specificity and sensitivity for the disease. Our aims were to determine and compare the diagnostic accuracies of several potential biomarkers and to develop a combined biomarker analysis tool for the diagnosis of sarcoidosis. 133 healthy individuals and 104 patients with suspected sarcoidosis and diagnostic thoracic surgery were enrolled into this study. Histopathologic results were contrasted to biomarker levels of chitotriosidase (CTO), serum amyloid-A (SAA), soluble interleukin-2 receptor (sIL-2R), lysozyme (LZM) or angiotensin converting enzyme (ACE). Sarcoidosis was confirmed by histopathology in 69 patients. CTO activity, sIL-2R concentration and ACE activity could discriminate between sarcoidosis and control patients, while SAA and LZM concentrations could not. A new combined parameter, which was derived from the multiplication of ACE by CTO activities (double product) showed the best diagnostic accuracy in this clinical study: (AUC = 0.898, sensitivity: 90.5%, specificity: 79.3%, positive and negative predictive values: 90.5% and 79.3%, respectively). Sarcoidosis can be diagnosed with the combined analysis of ACE and CTO activities more accurately than with single serum biomarkers in the absence of invasive biopsy in the majority of cases with pulmonary manifestation of sarcoidosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Serum angiotensin-converting enzyme (ACE) activity determination can aid the early diagnosis of sarcoidosis. We aimed to optimize a fluorescent kinetic assay for ACE activity by screening the confounding effects of endogenous ACE inhibitors and interfering factors. Genotype-dependent and genotype-independent reference values of ACE activity were established, and their diagnostic accuracies were validated in a clinical study., Methods: Internally quenched fluorescent substrate, Abz-FRK(Dnp)P-OH was used for ACE-activity measurements. A total of 201 healthy individuals and 59 presumably sarcoidotic patients were enrolled into this study. ACE activity and insertion/deletion (I/D) genotype of the ACE gene were determined., Results: Here we report that serum samples should be diluted at least 35-fold to eliminate the endogenous inhibitor effect of albumin. No significant interferences were detected: up to a triglyceride concentration of 16 mM, a hemoglobin concentration of 0.71 g/L and a bilirubin concentration of 150 μM. Genotype-dependent reference intervals were considered as 3.76-11.25 U/L, 5.22-11.59 U/L, 7.19-14.84 U/L for II, ID and DD genotypes, respectively. I/D genotype-independent reference interval was established as 4.85-13.79 U/L. An ACE activity value was considered positive for sarcoidosis when it exceeded the upper limit of the reference interval. The optimized assay with genotype-dependent reference ranges resulted in 42.5% sensitivity, 100% specificity, 100% positive predictive value and 32.4% negative predictive value in the clinical study, whereas the genotype-independent reference range proved to have inferior diagnostic efficiency., Conclusions: An optimized fluorescent kinetic assay of serum ACE activity combined with ACE I/D genotype determination is an alternative to invasive biopsy for confirming the diagnosis of sarcoidosis in a significant percentage of patients.

It was shown recently that angiotensin-converting enzyme activity is limited by endogenous inhibition in vivo, highlighting the importance of angiotensin II (ACE2) elimination. The potential contribution of the ACE2 to cardiovascular disease progression was addressed. Serum ACE2 activities were measured in different clinical states (healthy, n=45; hypertensive, n=239; heart failure (HF) with reduced ejection fraction (HFrEF) n=141 and HF with preserved ejection fraction (HFpEF) n=47). ACE2 activity was significantly higher in hypertensive patients (24.8±0.8 U/ml) than that in healthy volunteers (16.2±0.8 U/ml, p=0.01). ACE2 activity further increased in HFrEF patients (43.9±2.1 U/ml, p=0.001) but not in HFpEF patients (24.6±1.9 U/ml) when compared with hypertensive patients. Serum ACE2 activity negatively correlated with left ventricular systolic function in HFrEF, but not in hypertensive, HFpEF or healthy populations. Serum ACE2 activity had a fair diagnostic value to differentiate HFpEF from HFrEF patients in this study. Serum ACE2 activity correlates with cardiovascular disease development: it increases when hypertension develops and further increases when the cardiovascular disease further progresses to systolic dysfunction, suggesting that ACE2 metabolism plays a role in these processes. In contrast, serum ACE2 activity does not change when hypertension progresses to HFpEF, suggesting a different pathomechanism for HFpEF, and proposing a biomarker-based identification of these HF forms., (© The Author(s) 2016.)

Introduction: Renal transplantation provides longer life expectancy in patients with renal failure. Nonetheless, this improved life expectancy is still shorter than that for the general population. The main couse of death in renal transplant patients is cardiovascular disease, and chronic allograft nephropathy is the most significant cause of graft loss. Genetic polymorphisms of the renin angiotensin system have been implicated in both chronic allograft nephropathy and fatal cardiovascular diseases., Aim: The long term goal of the authors was to improve the survival of renal transplanted patients. The authors aimed to identify novel biomarkers which correlate with the survival of the transplant organ and the recipient with a special attention to elements of the renin-angiotensin system., Method: A retrospective clinical trial was performed involving 72 renal transplanted patients. Angiotensin-converting enzyme I/D genotypes and activity, kidney function and morphological properties of the heart were determined., Results: A significant positive correlation was found between the DD genotype of the angiotensin-converting enzíme gene, and the DD genotype predicted severe left ventricular hypertrophy., Conclusions: These findings suggest that the I/D genotypes of the angiotensin-converting enzyme gene predict not only the expected survival of the transplanted organ, but also that of the patient. Patients with the DD genotype are more susceptible for transplant failure. These patients should be identified and a special attention should be made on their pharmacological treatment (renin-angiotensin system inhibition), and their complience should also be maintained.

Hypertension (HTN) is a major risk factor for heart failure. We investigated the influence of HTN on cardiac contraction and relaxation in transgenic renin overexpressing rats (carrying mouse Ren-2 renin gene, mRen2, n = 6). Blood pressure (BP) was measured. Cardiac contractility was characterized by echocardiography, cellular force measurements, and biochemical assays were applied to reveal molecular mechanisms. Sprague-Dawley (SD) rats (n = 6) were used as controls. Transgenic rats had higher circulating renin activity and lower cardiac angiotensin-converting enzyme two levels. Systolic BP was elevated in mRen2 rats (235.11 ± 5.32 vs. 127.03 ± 7.56 mmHg in SD, P < 0.05), resulting in increased left ventricular (LV) weight/body weight ratio (4.05 ± 0.09 vs. 2.77 ± 0.08 mg/g in SD, P < 0.05). Transgenic renin expression had no effect on the systolic parameters, such as LV ejection fraction, cardiomyocyte Ca(2+)-activated force, and Ca(2+) sensitivity of force production. In contrast, diastolic dysfunction was observed in mRen2 compared with SD rats: early and late LV diastolic filling ratio (E/A) was lower (1.14 ± 0.04 vs. 1.87 ± 0.08, P < 0.05), LV isovolumetric relaxation time was longer (43.85 ± 0.89 vs. 28.55 ± 1.33 ms, P < 0.05), cardiomyocyte passive tension was higher (1.74 ± 0.06 vs. 1.28 ± 0.18 kN/m(2), P < 0.05), and lung weight/body weight ratio was increased (6.47 ± 0.24 vs. 5.78 ± 0.19 mg/g, P < 0.05), as was left atrial weight/body weight ratio (0.21 ± 0.03 vs. 0.14 ± 0.03 mg/g, P < 0.05). Hyperphosphorylation of titin at Ser-12742 within the PEVK domain and a twofold overexpression of protein kinase C-α in mRen2 rats were detected. Our data suggest a link between the activation of renin-angiotensin-aldosterone system and increased titin-based stiffness through phosphorylation of titin's PEVK element, contributing to diastolic dysfunction., (Copyright © 2016 the American Physiological Society.)

We set out to characterize the mechanical effects of myeloperoxidase (MPO) in isolated left-ventricular human cardiomyocytes. Oxidative myofilament protein modifications (sulfhydryl (SH)-group oxidation and carbonylation) induced by the peroxidase and chlorinating activities of MPO were additionally identified. The specificity of the MPO-evoked functional alterations was tested with an MPO inhibitor (MPO-I) and the antioxidant amino acid Met. The combined application of MPO and its substrate, hydrogen peroxide (H2O2), largely reduced the active force (Factive), increased the passive force (Fpassive), and decreased the Ca(2+) sensitivity of force production (pCa50) in permeabilized cardiomyocytes. H2O2 alone had significantly smaller effects on Factive and Fpassive and did not alter pCa50. The MPO-I blocked both the peroxidase and the chlorinating activities, whereas Met selectively inhibited the chlorinating activity of MPO. All of the MPO-induced functional effects could be prevented by the MPO-I and Met. Both H2O2 alone and MPO + H2O2 reduced the SH content of actin and increased the carbonylation of actin and myosin-binding protein C to the same extent. Neither the SH oxidation nor the carbonylation of the giant sarcomeric protein titin was affected by these treatments. MPO activation induces a cardiomyocyte dysfunction by affecting Ca(2+)-regulated active and Ca(2+)-independent passive force production and myofilament Ca(2+) sensitivity, independent of protein SH oxidation and carbonylation. The MPO-induced deleterious functional alterations can be prevented by the MPO-I and Met. Inhibition of MPO may be a promising therapeutic target to limit myocardial contractile dysfunction during inflammation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: To compare cardiometabolic risk-related biochemical markers and sexual hormone and leptin receptors in the adrenal gland of rat males, non-ovariectomized females (NON-OVX), and ovariectomized females (OVX) under chronic stress., Methods: Forty six 16-week-old Sprague-Dawley rats were divided into male, NON-OVX, and OVX group and exposed to chronic stress or kept as controls. Weight, glucose tolerance test (GTT), serum concentration of glucose, and cholesterol were measured. Adrenal glands were collected at the age of 28 weeks and immunohistochemical staining against estrogen beta (ERβ), progesterone (PR), testosterone (AR), and leptin (Ob-R) receptors was performed., Results: Body weight, GTT, serum cholesterol, and glucose changed in response to stress as expected and validated the applied stress protocol. Stressed males had significantly higher number of ERβ receptors in comparison to control group (P = 0.028). Stressed NON-OVX group had significantly decreased AR in comparison to control group (P = 0.007). The levels of PR did not change in any consistent pattern. The levels of Ob-R increased upon stress in all groups, but the significant difference was reached only in the case of stressed OVX group compared to control (P = 0.033)., Conclusion: Chronic stress response was sex specific. OVX females had similar biochemical parameters as males. Changes upon chronic stress in adrenal gland were related to an increase in testosterone receptor in females and decrease in estrogen receptor in males.

Aim: To assess how ovarian-derived sex hormones (in particular progesterone) modify the effects of single acute stress on the mechanical and biochemical properties of left ventricular cardiomyocytes in the rat., Methods: Non-ovariectomized (control, n=8) and ovariectomized (OVX, n=8) female rats were kept under normal conditions or were exposed to stress (control-S, n=8 and OVX-S, n=8). Serum progesterone levels were measured using a chemiluminescent immunoassay. Left ventricular myocardial samples were used for isometric force measurements and protein analysis. Ca(2+)-dependent active force (Factive), Ca(2+)-independent passive force (Fpassive), and Ca(2+)-sensitivity of force production were determined in single, mechanically isolated, permeabilized cardiomyocytes. Stress- and ovariectomy-induced alterations in myofilament proteins (myosin-binding protein C [MyBP-C], troponin I [TnI], and titin) were analyzed by sodium dodecyl sulfate gel electrophoresis using protein and phosphoprotein stainings., Results: Serum progesterone levels were significantly increased in stressed rats (control-S, 35.6±4.8 ng/mL and OVX-S, 21.9±4.0 ng/mL) compared to control (10±2.9 ng/mL) and OVX (2.8±0.5 ng/mL) groups. Factive was higher in the OVX groups (OVX, 25.9±3.4 kN/m(2) and OVX-S, 26.3±3.0 kN/m(2)) than in control groups (control, 16.4±1.2 kN/m(2) and control-S, 14.4±0.9 kN/m(2)). Regarding the potential molecular mechanisms, Factive correlated with MyBP-C phosphorylation, while myofilament Ca(2+)-sensitivity inversely correlated with serum progesterone levels when the mean values were plotted for all animal groups. Fpassive was unaffected by any treatment., Conclusion: Stress increases ovary-independent synthesis and release of progesterone, which may regulate Ca(2+)-sensitivity of force production in left ventricular cardiomyocytes. Stress and female hormones differently alter Ca(2+)-dependent cardiomyocyte contractile force production, which may have pathophysiological importance during stress conditions affecting postmenopausal women.