Pharmacological Inhibition of Mammalian Target of Rapamycin Attenuates Deoxycorticosterone Acetate Salt-Induced Hypertension and Related Pathophysiology: Regulation of Oxidative Stress, Inflammation, and Cardiovascular Hypertrophy in Male Rats.

ABSTRACT: The present study aimed to explore the contribution of mammalian target of rapamycin (mTOR) in deoxycorticosterone acetate (DOCA) salt-induced hypertension and related pathophysiological changes in cardiovascular and renal tissues. DOCA salt loading resulted in an increase in systolic blood pressure, diastolic blood pressure, and mean blood pressure along with the activity of ribosomal protein S6, the effector protein of mTOR. Treatment with rapamycin, the selective inhibitor of mTOR, initiated at the fourth week of DOCA- salt administration normalized the systolic blood pressure and attenuated ribosomal protein S6 activity in the heart, aorta, and kidney. Cardiac and vascular hypertrophy, oxidative stress, and infiltration of macrophages (CD68+), the marker of inflammation, were also reduced in rapamycin-treated, DOCA-salt, hypertensive rats. In addition, renal hypertrophy and dysfunction were also reduced with rapamycin-treated hypertensive rats. Moreover, these pathophysiological changes in DOCA-salt hypertensive rats were associated with increased NADPH oxidase (NOX) activity, gp91phox (formerly NOX2) expression, ERK1/2, and p38 MAPK activities in the heart, aorta, and kidney were minimized by rapamycin. These data indicate that mTOR plays an important role in regulating blood pressure and the development of cardiovascular and renal pathophysiological changes, most likely due to increased NOX expression/activity, ERK1/2, and p38 MAPK activity with macrophages infiltration in the heart, kidney, and aorta. Pharmacological inhibition of mTOR and related signaling pathways could serve as a novel target for the treatment of hypertension.

Adrenomedullin expression in aortic artery wall of diabetic rats given alpha lipoic acid.

BACKGROUND: Diabetes mellitus (DM) is a major health problem predisposing to cardiovascular diseases. The aim of this study was to investigate the effects of alpha lipoic acid (ALA) on both the arterial wall of diabetic rats and the adrenomedullin (ADM) gene expression. METHODS: Twenty-four Wistar Albino rats were divided into three groups as Control, DM + S, and DM + ALA. For DM model, a single dose of 40 mg/kg streptozotocin, for DM + ALA group, 100 mg/kg/day/4 weeks was administered. Hematoxylin & Eosin (H&E) staining was done and vascular endothelial growth factor (VEGF) was detected by immunohistochemical analysis in the artery wall. Total damage score of vessel wall (endothelial cell damage, media layer smooth muscle cell damage, and internal elastic lamina damage) and H score (immunoreactivity intensity) were calculated. Expression of ADM gene was measured by qRT-PCR. RESULTS: In DM + S group, Total damage score of vessel wall were detected by light microscopy. There were statistically significant differences between the groups Control/DM + S and DM + S/DM + ALA in terms of the vessel total damage score and H score (p < 0.005). ADM expression was increased threefold in both DM + S and DM + ALA groups compared to the control group (p < 0.05). CONCLUSIONS: ALA may have positive effect on the vessel damage in diabetic rats. However, no significant decrease in ADM expression levels was observed in diabetic rats after ALA administration and we considered that the protective effect of ALA is independent of adrenomedullin. Further studies with different doses and durations of ALA administrations are required to investigate the changes in ADM expression.

A light microscopic investigation of the renoprotective effects of α-lipoic acid and α-tocopherol in an experimental diabetic rat model.

We investigated the effects of α-lipoic acid (AL) and α-tocopherol (AT) on renal histopathology in a streptozotocin (STZ) induced diabetic rat model. Adult male rats were divided into six groups: group 1, saline only; group 2, AL only; group 3, AT only; group 4, STZ only; group 5, STZ + AL; group 6 STZ + AT. Experimental diabetes was induced by STZ. AL and AT were administered for 15 days. Kidney sections were examined using a light microscope after hematoxylin and eosin (H & E), periodic acid-Schiff (PAS) and caspase-3 staining. Histological damage to glomeruli, tubule epithelial cells and basement membrane was observed in group 4. Administration of AT and AL reduced renal injury in the diabetic rats. Group 5 exhibited a greater curative effect on diabetic rats than group 6. AT and AL may be useful for preventing diabetic renal damage.

Modulation of oxidative-nitrosative stress and inflammatory response by rapamycin in target and distant organs in rats exposed to hindlimb ischemia-reperfusion: the role of mammalian target of rapamycin.

Mammalian target of rapamycin (mTOR) has been recognized with potential immunomodulatory properties playing an important role in various physiopathological processes including ischemia-reperfusion (I/R) injury. I/R injury stimulate reactive oxygen and nitrogen species by activating nicotinamide adenine dinucleotide phosphate oxidase and inducible nitric oxide synthase, respectively. Controversial results have been obtained in different I/R models following localized I/R; however, the precise role of the mTOR signaling pathway remains undefined. The objective of the current study was to evaluate the role of the mTOR in oxidative-nitrosative stress and inflammation in hindlimb I/R-induced injury in target and remote organ injuries. In rats subjected to I/R, an increased expression of ribosomal protein S6 (rpS6), inhibitor κB (IκB)-α, nuclear factor-κB (NF-κB) p65, inducible nitric oxide synthase, cyclooxygenase 2, gp91phox, and levels of tumor necrosis factor α, nitrite, nitrotyrosine, malondialdehyde and the activities of myeloperoxidase and catalase in the tissues and (or) sera were detected. Treatment with rapamycin, a selective inhibitor of mTOR, reversed all the I/R-induced changes as manifested by its anti-inflammatory and antioxidant effects in kidney and gastrocnemius muscle of rats. Collectively, these findings suggest that rapamycin protects against I/R-induced oxidative-nitrosative stress and inflammation leading to organ injuries via suppression of mTOR/IκB-α/NF-κB signaling pathway.

N-Acetylcysteine-induced vasodilatation is modulated by KATP channels, Na+/K+-ATPase activity and intracellular calcium concentration: An in vitro study.

BACKGROUND: In this study, we aimed to investigate the role of ATP-sensitive potassium (KATP) channel, Na+/K+-ATPase activity, and intracellular calcium levels on the vasodilatory effect of N-acetylcysteine (NAC) in thoracic aorta by using electrophysiological and molecular techniques. METHODS: Rat thoracic aorta ring preparations and cultured thoracic aorta cells were divided into four groups as control, 2mM NAC, 5mM NAC, and 10mM NAC. Thoracic aorta rings were isolated from rats for measurements of relaxation responses and Na+/K+-ATPase activity. In the cultured thoracic aorta cells, we measured the currents of KATP channel, the concentration of intracellular calcium and mRNA expression level of KATP channel subunits (KCNJ8, KCNJ11, ABCC8 and ABCC9). RESULTS: The relaxation rate significantly increased in all NAC groups compared to control. Similarly, Na+/K+- ATPase activity also significantly decreased in NAC groups. Outward KATP channel current significantly increased in all NAC groups compared to the control group. Intracellular calcium concentration decreased significantly in all groups with compared control. mRNA expression level of ABCC8 subunit significantly increased in all NAC groups compared to the control group. Pearson correlation analysis showed that relaxation rate was significantly associated with KATP current, intracellular calcium concentration, Na+/K+-ATPase activity and mRNA expression level of ABCC8 subunit. CONCLUSION: Our findings suggest that NAC relaxes vascular smooth muscle cells through a direct effect on KATP channels, by increasing outward K+ flux, partly by increasing mRNA expression of KATP subunit ABCC8, by decreasing in intracellular calcium and by decreasing in Na+/K+-ATPase activity.

Role of ACE I/D gene polymorphisms on the effect of ramipril in inflammatory response and myocardial injury in patients undergoing coronary artery bypass grafts.

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors block angiotensin II formation and release bradykinin, which is effective in the regulation of oxidoinflammatory injury. Some reports denote alterations in the effectiveness of ACE inhibitors in association with ACE insertion/deletion (I/D) gene polymorphisms. This study investigates the effects of ramipril on the oxidoinflammatory cytokines (IL-6, IL-8, TNF-alpha) and TnT (myocardial injury marker) and their alteration in association with ACE I/D gene polymorphisms. METHODS: The study group (n = 51) patients received ramipril before coronary artery bypass grafting (CABG), while patients not receiving ramipril (n = 51) constituted the controls. TNFα, IL-6, and IL-8 were evaluated using ELISA and TnT by electrochemiluminescence methods before the induction of anesthesia (t1), at the 20th minute following cross-clamping (t2), at the end of the operation (t3), and at the 24th hour from the commencement of anesthesia (t4). Genotyping was performed by PCR. RESULTS: Differences between the groups were significant at t4 for the TNFα and at t3 for IL-6 (p < 0.05). The TnT levels increased from t2 onward in the control group and were highest in t3. Changes in t3 and t4 values in both groups according to their t1 values were significant (p < 0.05). However, differences between the groups were insignificant (p > 0.05). The IL-6, IL-8, TNFα, and TnT serum levels had no correlation with the ACE I/D gene polymorphism. CONCLUSION: Low cytokine and TnT levels in the study group, especially after cross-clamping, may indicate the protective effect of ramipril from oxidoinflammatory injury. This effect did not appear to be associated with the ACE I/D gene polymorphism.

Contribution of RhoA/Rho-kinase/MEK1/ERK1/2/iNOS pathway to ischemia/reperfusion-induced oxidative/nitrosative stress and inflammation leading to distant and target organ injury in rats.

The small G protein RhoA and its downstream effector Rho-kinase play an important role in various physiopathological processes including ischemia/reperfusion (I/R) injury. Reactive oxygen and nitrogen species produced by iNOS and NADPH oxidase are important mediators of inflammation and organ injury following an initial localized I/R event. The aim of this study was to determine whether RhoA/Rho-kinase signaling pathway increases the expression and activity of MEK1, ERK1/2, iNOS, gp91(phox), and p47(phox), and peroxynitrite formation which result in oxidative/nitrosative stress and inflammation leading to hindlimb I/R-induced injury in kidney as a distant organ and gastrocnemius muscle as a target organ. I/R-induced distant and target organ injury was performed by using the rat hindlimb tourniquet model. I/R caused an increase in the expression and/or activity of RhoA, MEK1, ERK1/2, iNOS, gp91(phox), p47(phox), and 3-nitrotyrosine and nitrotyrosine levels in the tissues. Although Rho-kinase activity was increased by I/R in the kidney, its activity was decreased in the muscle. Serum and tissue MDA levels and MPO activity were increased following I/R. I/R also caused an increase in SOD and catalase activities associated with decreased GSH levels in the tissues. Y-27632, a selective Rho-kinase inhibitor, (100µg/kg, i.p.; 1h before reperfusion) prevented the I/R-induced changes except Rho-kinase activity in the muscle. These results suggest that activation of RhoA/Rho-kinase/MEK1/ERK1/2/iNOS pathway associated with oxidative/nitrosative stress and inflammation contributes to hindlimb I/R-induced distant organ injury in rats. It also seems that hindlimb I/R induces target organ injury via upregulation of RhoA/MEK1/ERK1/2/iNOS pathway associated with decreased Rho-kinase activity.

Pressure applied during surgery alters the biomechanical properties of human saphenous vein graft.

Pressure applied during harvesting of the saphenous vein (SV) graft in coronary artery bypass surgery might change its mechanical properties and thereby decrease the patency. This study was performed to assess the mechanical properties of the SV graft distended manually with different levels of pressure and to determine the pressure level that induces changes in its structure and mechanics. Saphenous vein graft segments, collected from 36 patients undergoing coronary artery bypass surgery, were distended with pressures of either 50-60, 75-100, or 130-150 mmHg. Grafts were tested for the stress-strain relationship; the Young's moduli at the low- and high-strain regions were calculated, and their structures were examined by light and electron microscopy. Pressures of 50-60 mmHg did not influence the mechanics of the vein graft, whereas pressures of 75-100 mmHg elevated the elastic modulus of the vein at the low-strain region while pressures above 130 mmHg increased the elastic moduli at both low- and high-strain regions. There was a prominent loss of microfibrils at all distending pressure levels. The mechanical results suggest that distending pressures above 75 mmHg might play a role in graft failure. Furthermore, the absence of microfibrils surrounding elastin suggests that application of distending pressures, even as low as 50 mmHg, can cause degeneration of the elastic fibers following implantation, increasing the stiffness of the graft and thus impairing the graft's function under its new hemodynamic conditions.

Apoptotic and anti-angiogenic effects of benzimidazole compounds: relationship with oxidative stress mediated ischemia/reperfusion injury in rat hind limb.

Ischemia/reperfusion (I/R)-induced injury is a pathophysiological process consisting of a complex cascade characterized by an increase in reactive oxygen species production, pro-inflammatory cytokine release, and activated endothelial cells leading to cell damage and death. The aim of this study was to investigate effects of substituted 2-benzylbenzimidazole derivatives, 2-(3-methoxybenzyl)benzimidazole (BB3) and 2-(4-methoxybenzyl)benzimidazole (BB4), on I/R-induced changes in the markers of oxidative stress, apoptosis, and angiogenesis in rats. BB3 and BB4 were synthesized with microwave irradiation and conventional Phillips methods. I/R was performed by occlusion of femoral artery. Catalase activity and reduced glutathione (GSH) levels as well as caspase-3, -8, and -9 activities were measured in muscle tissues as an index for oxidative stress and apoptosis, respectively. Vascular endothelial growth factor (VEGF) levels as an index for angiogenesis were also measured in the muscle tissues and sera. I/R decreased GSH levels, increased catalase activity and VEGF levels, and did not change caspase-3, -8, and -9 activities compared to control groups. BB3 and BB4 caused a further decrease in GSH levels and increased caspase-3, -8, and -9 activities in I/R group. These compounds caused a further increase in catalase activity and prevented the increase in VEGF levels induced by I/R. These data suggest that BB3 and BB4 exhibit apoptotic and anti-angiogenic activity with pro-oxidative effects resulting in oxidative stress in pathophysiological process of I/R-induced hind limb injury in rats.

Affirmative effects of iloprost on apoptosis during ischemia-reperfusion injury in kidney as a distant organ.

OBJECTIVE: Apoptosis and its regulatory mechanisms take part in renal ischemia-reperfusion (I/R) injury which can result in acute renal failure and the inhibition of the caspase is considered as a new therapeutic strategy. In this context, we investigated the antiapoptotic and cytoprotective effects of iloprost, a prostacyclin analog, in kidney as a distant organ. METHODS: Wistar albino rats were randomized into five groups (n = 12 in each) as sham, ischemia, I/R, iloprost (10 µg kg(-1)), and I/R + iloprost (10 µg kg(-1)). A 4 h reperfusion procedure was carried out after 4 h of ischemia. Caspase-8 was evaluated for death receptor-induced pathways, whereas caspase-9 was evaluated for mitochondria-dependent pathways and caspase-3 was investigated for overall apoptosis. Superoxide dismutase (SOD) enzyme activity and nitrite content as an indicator of nitric oxide (NO) production were also analyzed in kidney tissues. RESULTS: Caspases-3, -8, and -9 were all significantly elevated in both ischemia and I/R groups compared to the sham group; however, treatment with iloprost reduced caspases-3, -8, and -9. SOD enzyme activity was attenuated by iloprost when compared to ischemic rats. The different effects of NO were found which change according to the present situation in ischemia, I/R, and treatment with iloprost. CONCLUSIONS: These findings suggested that iloprost prevents apoptosis in both receptor-induced and mitochondria-dependent pathways in renal I/R injury and it may be considered as a cytoprotective agent for apoptosis. Understanding the efficiency of iloprost on the pathways for cell death may lead to an opportunity in the therapeutic approach for renal I/R injury.

Molecular mechanism of vasorelaxant and antiatherogenic effects of the statins in the human saphenous vein graft.

In this study we aimed to investigate the vasorelaxant and antiatherogenic effects of the statins (fluvastatin and pravastatin) in the human saphenous vein grafts at the molecular level by using histopathologic, pharmacological and immunochemical techniques. The saphenous vein grafts evaluated histopathologically displayed a loss in their endothelium up to a ratio of 30% and set forth indications of functional deterioration. The pharmacological evaluations proved that the relaxation responses induced by fluvastatin and pravastatin were significantly inhibited by nitric oxide synthase inhibitor, N(G)-nitro-l-arginine, and cyclooxygenase inhibitor, indomethacin, while these responses were significantly increased by angiotensin converting enzyme inhibitors, captopril and enalapril, and rho kinase inhibitor, Y27632. The results of immunochemical studies are in accordance with the results of the pharmacological studies that the related statins increased the levels of nitric oxide, phospholipase A(2) and they decreased the levels of angiotensin II and active rho kinase. On the other hand mevalonolactone, a substrate of lipid metabolism, failed to change the effects of fluvastatin and pravastatin in the related tissue. The experimental results indicate that activation of nitric oxide synthase and phospholipase A(2)-cyclooxygenase pathway and inhibition of angiotensin converting enzyme and rho kinase may have a role on the effects of fluvastatin and pravastatin in the human saphenous vein grafts. It seems that the vasorelaxant and antiatherogenic effects of the related statins are independent of their lipid lowering mechanism.

Role of neopterin, C-reactive protein and myeloperoxidase in patients undergoing cardiopulmonary bypass.

OBJECTIVE: The aim of the present study was to investigate the role of neopterin (NP), C-reactive protein (CRP) and myeloperoxidase (MPO) in patients undergoing coronary artery bypass grafting with or without cardiopulmonary bypass (CPB). PATIENTS AND METHODS: Forty patients submitted for elective coronary artery bypass grafting were included in this prospective study. Patients were divided into two groups of 20 individuals, those who did not undergo CPB (group 1), aged 54.1 ± 13.5 years, and those who did (group 2), aged 60.2 ± 11.7 years. In group 1, there were 17 males and 3 females, while in group 2, there were 16 males and 4 females. Serum CRP, serum and urine NP and leukocyte MPO activity were measured preoperatively, at the end of surgery, and 4, 24 and 72 h after surgery using high-performance liquid chromatography, immunoturbidimetry and the reduction in o-dianizidine, respectively. RESULTS: The level of serum NP was higher preoperatively and at the end of surgery (0 h), 4, 24, and 72 h after the operation in those who underwent CPB compared to those who did not. However, there was no significant difference in NP concentrations between the two groups at any time except 24 h after surgery (p = 0.002). Urine NP concentrations showed similar values preoperatively but increased postoperatively in both groups of patients. The only significant difference in urine NP concentration between the two groups occurred at 0 and 24 h after surgery (p = 0.001, p = 0.000). Serum CRP concentrations showed similar values preoperatively, at the end of surgery and 72 h after the operation and increased at 4 and 24 h postoperatively in both groups. The only significant difference in CRP concentration between the two groups occurred 4 and 24 h after surgery (p = 0.024 and p = 0.000, respectively). MPO levels were found to be increased in the CPB patients when compared to those patients who did not undergo CPB. However, the difference between the groups was not statistically significant. CONCLUSION: Our data show that CPB induced a rise in NP and CRP levels.

Admission levels of C-reactive protein and plasminogen activator inhibitor-1 in patients with acute myocardial infarction with and without cardiogenic shock or heart failure on admission.

Scarce data exist on the relationship of C-reactive protein (CRP) or plasminogen activator inhibitor-1 (PAI-1) to the occurrence of heart failure (HF) or cardiogenic shock (CS) after acute myocardial infarction (AMI) and on the relationship between these biomarkers and mortality in CS patients. Thus, we compared high-sensitivity CRP and PAI-1 antigen plasma levels on admission among 3 age- and gender-matched AMI patients groups (consisting of 60 patients with CS, 60 with HF, and 60 without HF on admission), after determining that PAI-1 levels did not vary significantly diurnally in these groups by comparing the data among subgroups which were divided according to admission time within the groups. For CS patients, we also conducted regression analyses to examine the relations of these biomarkers to mortality. CRP levels both in CS (P < 0.001) and HF (P < 0.05) patients were significantly higher compared to those without HF, PAI-1 levels in CS patients were significantly higher compared to both those with (P < 0.05) and without HF (P > 0.01), and CRP and PAI-1 were independent predictors of in-hospital (Odds ratio [OR] = 6.12, 95% confidence intervals [95%CI] = 1.47-25.54 and OR = 5.92, 95%CI = 1.31-26.77, respectively) and 1-year mortality (OR = 5.53, 95%CI = 1.21-25.17 and OR = 5.48, 95%CI = 1.09-27.52, respectively) in CS patients. In conclusion, at admission, CRP is associated with the occurrence of CS and HF and PAI-1 is associated with the occurrence of CS after AMI, and they are of prognostic value in CS complicating AMI.

The role of CYP2C9 gene polymorphisms on anticoagulant therapy after heart valve replacement.

OBJECTIVE: The aim of the present study was to investigate the role of CYP2C9 gene polymorphisms after heart valve replacement in a group of patients on warfarin therapy. MATERIALS AND METHODS: The study population consisted of 74 patients with heart valve replacement. Peripheral blood was collected into evacuated tubes containing EDTA, and DNA was extracted from circulating leukocytes by using a high pure PCR template preparation kit. CYP2C9*2, CYP2C9*3 alleles were detected by using real-time PCR. RESULTS: The patients with CYP2C9*1/*3 and CYP2C9*2/*3 genotypes were taking 28.21 and 24.47 mg, respectively, as mean weekly warfarin dose, whereas patients with CYP2C9*1/*1 genotype were taking 33.90 mg. CONCLUSION: The data show that patients with CYP2C9*1/*3 and CYP2C9*2/*3 genotypes needed a lower maintenance dose of warfarin than patients with CYP2C9*1/*1 wild-type genotype.

Factor VLeiden and prothrombin G20210A gene polymorphisms in patients with coronary artery disease.

PURPOSE: The precise molecular mechanisms culminating in coronary artery disease (CAD) are not well understood, despite a wealth of knowledge on predisposing risk factors and pathomechanisms. CAD and myocardial infarction (MI) are complex genetic diseases; neither the environment alone, nor a single gene, cause disease, rather, a mix of environmental and genetic factors lead to atherosclerosis of the coronary arteries. MATERIALS AND METHODS: In the present study, our aim was to investigate the roles of prothrombin G20210A mutation and Factor VLeiden mutation in atherosclerotic coronary artery disease. 287 subjects (106 control subjects, who were angiographically normal, and 181 angiographically documented coronary atherosclerotic patients who exhibited coronary artery narrowing to a degree of >or=50%) were included in this study. The mutations were assessed with LightCycler Real-Time PCR mutation detection kits (Roche Diagnostics, GmbH, Germany). RESULTS: 6.6% of control subjects, and 6.1% of patients with (50% coronary artery narrowing were determined to have the Factor VLeiden heterozygote mutation. 6.6% of control subjects had the Prothrombin G20210A heterozygote mutation, while 7.7% of patients with (50% coronary artery narrowing had this mutation. The OR for Factor VLeiden was 1.52 (CI: 0.240-9.602) and for Prothrombin G20210A mutation, the OR was 1.415 (CI: 0.287-6.962). CONCLUSION: Although both the heterozygote Factor VLeiden and Prothrombin gene mutations were more frequent in patients with CAD than in control subjects, there was no statistical relationship found to exist between coronary artery disease and the Factor VLeiden and Prothrombin G20210A mutations.

Citric acid as a decalcifying agent for the excised calcified human heart valves.

OBJECTIVE: Cardiac valvular pathologies are frequently encountered as mechanical and functional disorders due to the calcification of the valves whatever the etiologies are. This pathophysiologic table usually ends up with valvular replacement. In this study, we aimed to decrease/eliminate the calcium in the excised calcified human heart valves by using citric acid in vitro hence bringing about the question for possible oral treatment of calcification of the valves by citric acid ingestion. METHODS: Fourteen pieces of mitral and/or aortic valves excised from 12 patients undergoing valve replacement were placed in a freshly prepared phosphate buffered saline solution containing 0.625% glutaraldehyde at +4 0C for 48 h. They were rinsed with 0.9% NaCl and divided into two groups; study and control. Control tissues were further treated in a freshly prepared solution with identical properties for another 5 days. Study tissues were placed into a solution containing 3.8% citric acid (pH 7.4) and kept for 48 h at +37 degrees C, then rinsed with 0.9% NaCl and transferred into a fresh solution containing 0.625% glutaraldehyde with phosphate buffer at 37 0C for 3 more days. Specimens were biochemically and histopathologically evaluated and compared using Mann Whitney U test. RESULTS: Calcium and phosphate levels in the study group were lower than in the control group (852.5+/-913.41 microg g-1 vs 413.05+/-519.53 microg g-1, p=0.001 and 207.6+/-321.86 microg g-1 vs 124.4+/-289.48 microg g-1, p=0.035, respectively). Malondialdehyde and protein level values were changed insignificantly in the control and study groups. Histopathologic evaluation showed that collagen and elastin fibers were similar in both groups. In the study group, irregular and fusiform calcific formations around the collagen fibers were significantly decreased. CONCLUSIONS: Decalcifying human heart valves in vitro conditions with citric acid without an adverse change to the morphology of the valvular tissue specimens is meaningful. We believe that forwarding and looking for the answer to the question "whether systemic application of citric acid could lead to the decalcification and/or reduction of calcification in the native human heart valves" would be expressive.