Bartosz Malinowski

Calcium blockers inhibit cyclosporine A-induced hyperreactivity of vascular smooth muscle cells

Cyclosporine belongs to the group of the most commonly used immunosuppressants. Hypertension occurs in approximately 30% of patients treated with this drug. However, the pathogenesis of this occurrence has not been explained to date. The purpose of our study was to clarify the mechanisms leading to the evolution of hypertension induced by cyclosporine A (CsA). We examined the changes in transmission within receptors and around the receptors. We also aimed to elucidate the mechanisms responsible for averting arterial hyperresponsiveness induced by the drug. Experiments were performed on isolated and perfused tail arteries of Wistar rats. Tissues surrounding the artery were removed and the proximal segment (length of 2-3 cm) was used for cannulation. Cannulated arteries were placed in a 20-ml glass chamber (vertical position). The contraction force in our model was measured by an increased degree of perfusion pressure with a constant flow rate (approximately 1 ml/min). The results showed that in the presence of CsA, the concentration-response curves/phenylephrine (PHE) curve shifted to the left. Cyclosporine increased the reactivity of the arteries to PHE. This effect was directly linked to the increase in the receptor reserve. The analysis of the reactivity of vascular smooth muscle showed that CsA increased the influx of calcium ions from the extracellular to the intracellular area. No difference was found between the contraction triggered by Bay-K8644 in the presence of CsA and the control probe. The increase in perfusion pressure induced by CsA was blocked by L-type calcium channel blockers (nifidipine and diltiazem). The results from our experiments show that CsA increases the reactivity of vessels to the effect of catecholamines. CsA also enhances signal transmission between G-protein coupled receptors (GPCRs) and calcium channels. The activation of protein kinase C also plays a significant role in this process. Our results suggest that the best choice for the pharmacotherapy of hypertension induced by CsA would be calcium channel antagonists.

Cyclosporine-A, but not tacrolimus significantly increases reactivity of vascular smooth muscle cells

BACKGROUND Application of cyclosporine-A (CsA) or tacrolimus is associated with numerous side effects. One of the main reasons for restricting usage of CsA is hypertension. In tacrolimus treated subjects the frequency of these phenomena is significantly lower. The known molecular mechanism of action of tacrolimus and cyclosporine-A seems to be the same, thus we decided to compare modulatory effect of drugs on vascular smooth muscle contractility. METHODS Experiments were performed on isolated and perfused tail artery of Wistar rats. Contraction force was measured by increased degree of perfusion pressure with a constant flow rate. RESULTS Concentration-response curves for agonist in the presence CsA were significantly shifted to the left with increase in maximal responses. This effect was due to increased calcium influx from extracellular calcium stores whereas there were no significant changes in calcium influx in the presence of tacrolimus; concentration-response curve was comparable to controls. CONCLUSION Our results strongly support the idea that main difference between effects on smooth muscle contractility of calcineurin-dependent immunosuppressants: CsA and tacrolimus is related to the different level of extracellular calcium influx to the cytoplasm. The elucidation of these mechanisms may permit the identification of new therapeutic strategies against CsA-induced hypertension.

PCSK9 signaling pathways and their potential importance in clinical practice

In the following review, authors described the structure and biochemical pathways of PCSK9, its involvement in LDL metabolism, as well as significances of proprotein convertase subtilisin/kexin type 9 targeted treatment. PCSK9 is a proprotein convertase, which plays a crucial role in LDL receptor metabolism. Transcription and translation of PCSK9 is controlled by different nuclear factors, such as, SREBP and HNF1α. This review focuses on interactions between PCSK9 and LDL receptor, VLDLR, ApoER2, CD36, CD81, and others. The role of PCSK9 in the inflammatory process is presented and its influence on cytokine profile (IL-1, IL-6, IL-10, TNF) in atherosclerotic plaque. Cholesterol metabolism converges also with diabetes by mTORC1 pathways. PCSK9 can be altered by oncologic pathways with utilization of kinases, such as Akt, JNK, and JAK/STAT. Finally, the article shows that blocking PCSK9 has proapoptotic capabilities. Administration of monoclonal antibodies against PCSK9 reduced mortality rate and cardiovascular events in randomized trials. On the other hand, immunogenicity of new drugs may play a crucial role in their efficiency. Bococizumab ended its career following SPIRE-1,2 outcome. PCSK9 inhibitors have enormous potential, which had been reflected by introducing them (as a new class of drugs reducing LDL concentration cholesterol) into New Lipid Guidelines from Rome 2016. Discoveries in drugs development are focused on blocking PCSK9 on different levels. For example, silencing messenger RNA (mRNA of PCSK9) is a new alternative against hypercholesterolemia. Peptides mimicking EGF-A domain of the LDL receptor are gaining significance and hopefully they will soon join others. The significance of PCSK9 has just been uncovered and further data is still required to understand their activity.

Resveratrol Increases Serum BDNF Concentrations and Reduces Vascular Smooth Muscle Cells Contractility via a NOS-3-Independent Mechanism

Resveratrol is a polyphenol that presents both antineuroinflammatory properties and the ability to interact with NOS-3, what contributes to vasorelaxation. Brain-derived neurotrophic factor (BNDF), a molecule associated with neuroprotection in many neurodegenerative disorders, is considered as an important element of maintaining stable cerebral blood flow. Vascular smooth muscle cells (VSMCs) are considered to be an important element in the pathogenesis of neurodegeneration and a potential preventative target by agents which reduce the contractility of the vessels. Our main objectives were to define the relationship between serum and long-term oral resveratrol administration in the rat model, as well as to assess the effect of resveratrol on phenylephrine- (PHE-) induced contraction of vascular smooth muscle cells (VSMCs). Moreover, we attempt to define the dependence of contraction mechanisms on endothelial NO synthase. Experiments were performed on Wistar rats (n = 17) pretreated with resveratrol (4 weeks; 10 mg/kg p.o.) or placebo. Serum BDNF levels were quantified after 2 and 4 weeks of treatment with ELISA. Contraction force was measured on isolated and perfused tail arteries as the increase of perfusion pressure with a constant flow. Values of serum BNDF in week 0 were 1.18 ± 0.12 ng/mL (treated) and 1.17 ± 0.13 ng/mL (control) (p = ns). After 2 weeks of treatment, BDNF in the treatment group was higher than in controls, 1.52 ± 0.23 ng/mL and 1.24 ± 0.13 ng/mL, respectively. (p = 0.02) Following 4 weeks of treatment, BDNF values were higher in the resveratrol group compared to control 1.64 ± 0.31 ng/mL and 1.32 ± 0.26 ng/mL, respectively (p = 0.031). EC50 values obtained for PHE in resveratrol pretreated arteries were significantly higher than controls (5.33 ± 1.7 × 10−7 M/L versus 4.53 ± 1.2 × 10−8 M/L, p < 0.05). These results show a significant increase in BDNF concentration in the resveratrol pretreated group. The reactivity of resistant arteries was significantly reduced for resveratrol pretreated vessels and this effect was partially NOS-3 independent.

Influence of celecoxib on the vasodilating properties of human mesenteric arteries constricted with endothelin-1.

The mitogenic and vasoconstrictive properties of the vascular system are attributed to endothelin-1 (ET-1). ET-1 serum concentration increases in a number of pathological conditions, particularly in those associated with blood vessel constriction. ET-1 is also associated with the underlying pathomechanisms of primary pulmonary hypertension, arterial hypertension and eclampsia. The aim of this study was to compare the vasodilating properties of selected phosphodiesterase (PDE) inhibitors and celecoxib in human mesenteric arteries constricted with ET-1, and investigate the role of the endothelium in relaxation. Perfused human mesenteric arteries were collected and stored under the same conditions as organs for transplantation. The mesenteric arteries (with and without the endothelium) were constricted by the addition of ET-1 and treated with one of the following: sildenafil (PDE5 inhibitor), zaprinast (PDE5 and 6 inhibitor), rolipram (PDE4 inhibitor) and celecoxib [cyclooxygenase-2 (COX-2) inhibitor]. Based on the observed changes of the perfusion pressure, concentration response curves (CRCs) were prepared for the respective inhibitors and the EC50 (concentration causing an effect equal to half of the maximum effect), pD2 (negative common logarithm of EC50) and relative potency (RP) were calculated. The results suggested that all the inhibitors triggered a concentration-dependent decrease in the perfusion pressure in isolated human superior mesenteric arteries with endothelium constricted by the addition of ET-1. In the arteries without endothelium, CRCs for celecoxib and rolipram were shifted to the right without a significant decrease in the maximum dilating effect. Moreover, CRCs for sildenafil and zaprinast were shifted to the right with a simultaneous significant decrease in the maximum dilating effect and with an increased inclination angle in reference to the concentration axis. In the presence of the endothelium, all of the evaluated PDE inhibitors, as well as celecoxib, reduced the reactivity of the mesenteric arteries caused by ET-1. Sildenafil indicated the lowest efficacy in the presence of the endothelium, but showed a higher potency compared to that of the other compounds. Removing the endothelium significantly reduced the vasodilating efficacy of PDE5 and 6 inhibitors and a statistically significant influence on the vasodilating efficacy of PDE4 inhibitor and celecoxib was observed. The high vasorelaxing efficacy of celecoxib at the background of the PDE inhibitors was observed, not only in the presence, but also in the absence of the endothelium and may be evidence for the relaxation induced by this COX-2 inhibitor in the cAMP- and cGMP-dependent pathways.

Role of endothelium, acetylocholine and calcium ions in Bay K8644- and KCl-induced contraction

The aim of this study was to establish the involvement of acetylcholine (Ach) and calcium ions in modulating contractions induced by Bay K8644 (an agonist of calcium channels located in the cell membrane) and KCl (at depolarizing concentrations), and also to examine the importance of the vascular endothelium in the activity of Bay K8644. The study was performed on perfused Wistar rat tail arteries. Contraction induced by Bay K8644 with the participation of intracellular (in calcium‑free physiological salt solution, FPSS) and extracellular (in physiological salt solution, PSS, following the emptying of the cellular Ca2+ stores) pools of Ca2+ and the addition of nitro-L-arginine (L‑NNA; nitric oxide synthase inhibitor) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; an inhibitor of soluble guanylyl cyclase) was studied. In addition, the effect of Ach on the contraction response was analyzed and the results were compared with the depolarizing action of KCl. The effects of 8Br‑cGMP on the artery contraction induced by Bay K8644 prior to and following removal of the endothelium were compared. Bay K8644 and KCl in PSS induced vascular contraction, which was reduced with the addition of Ach. The spasmolytic Ach action did not occur in the presence of L‑NNA and ODQ. 8Br‑cGMP reduced the contraction of arterial walls (with and without endothelium) induced by Bay K8644. The increase in vascular tone induced by Bay K8644 and KCl was independent of the intracellular calcium ion pool. The relaxant effect of Ach on the responses stimulated by Bay K8644 and KCl indicated the participation of nitric oxide in modulating the reactivity of the arteries to the factors examined, resulting in an influx of Ca2+ into the cell.

Selected atherosclerosis risk factors in youth aged 13-15 years .

INTRODUCTION The high frequency of cases of circulatory system conditions in Europe and other countries around the world requires scientific research to define risk factors of early atherosclerotic changes. The aim of the present study was to define which students are at danger of developing atherosclerosis by means of measuring cholesterol and triglyceride levels in blood as well as defining the correlation between atherosclerosis risk factors and arterial blood pressure, physical fitness and efficiency of the subjects. MATERIAL/METHODS The research covered 167 students of Public Junior High School 1 in Biala Podlaska aged 13-15 years. Accutrend GCT was employed to define the levels of total cholesterol and triglycerides in the screen test. Those students who were found to have increased values of biochemical parameters of capillary blood were subjected to additional blood tests aiming to define complete lipid profile of venous blood. The blood pressure in subjects was tested three times. The Moderate-to-Vigorous Physical Activity (MVPA) test, suggested by American authors, was employed to define physical activity in subjects. EUROFIT was employed to define physical efficiency. RESULTS Among the 167 subjects there were found 42 students (25.1%) whose lipid level in capillary blood proved to be increased. Full lipid profile tests proved that 16 students (9.6%) had increased blood lipid levels; those subjects constituted the risk group. Subjects in the risk group were characterized by lower levels of physical activity and physical efficiency compared to subjects with normal blood lipid level. Moreover, the frequency of hypertension was greater in risk group subjects compared to subjects with normal blood lipid levels. INFERENCES Students diagnosed with atherosclerosis risk factors require observation and early prophylactics by adopting habits of healthy physical activity.

Effect of reperfusion on vascular smooth muscle reactivity in three contraction models

BACKGROUND Ischemia and reperfusion remain inseparable elements of numerous medical procedures such as by-pass surgery, organ transplantation or other cardiology and intervention radiology. The contraction of the smooth muscle of the vessel is considered to be one of the basic components leading to impaired perfusion, an increase in the oxygen deficit of the endothelium of the vessel, and subsequently also to tissues vascularized by the vessel. Main aim of this study was to evaluate the effect of ischemia and reperfusion on vascular smooth muscle cells stimulated pharmacologically with mastoparan-7 (direct G-protein activator) in comparison to stimulation of G-protein coupled receptor agonist - phenylephrine, and direct calcium channel activator - Bay K8644. MATERIAL AND METHODS Experiments were performed on isolated and perfused tail artery of Wistar rats. Contraction force in our model was measured by increased level of perfusion pressure with a constant flow. RESULTS Concentration-response curves obtained for phenylephrine, mastoparan-7 and Bay K8644 presented a sigmoidal relation. Ischemia induced hyporreactivity of vessels, whereas during reperfusion the significant time related hyperreactivity for phenylephrine and mastoparan-7 only but not for Bay K8644. These reactions were secondary to the modulation of calcium influx from intra- and extracellular calcium stores. CONCLUSIONS Results of our experiments suggest that mastoparan-7 significantly induces contraction of vascular smooth muscle cells not only for controls but in the presence of ischemia and reperfusion too. Potential therapeutic applications of the observed reactions are important. They may include regenerative processes within the nervous system, studies on the improvement of blood flow within the microcirculation, or antimicrobial activity. Modulation of the G protein-phospholipase C response may also be an interesting point of action of future drugs modifying the response to stimulation during ischemia in particular, such activities could take place during the transport of organs for transplantation.

The role of Rho-kinase and calcium ions in constriction triggered by ET-1

Endothelin-1 (ET-1) is one of the key factors regulating tension of smooth muscles in blood vessels. It is believed that ET-1 plays an important role in pathogenesis of hypertension, and cardiovascular diseases; therefore, research in order to limit ET-1-mediated action is still in progress. The main objective of this paper was to evaluate the role of Rho-kinase in the ET-1-induced constriction of arteries. The analysis also included significance of intra- and extracellular pool of calcium ions in constriction triggered by ET-1. The studies were performed on perfused Wistar rat tail arteries. Concentration response curve (CRC) was determined for ET-1 in the presence of increased concentrations of Rho-kinase inhibitor (Y-27632) and IP3-receptor antagonist (2APB), both in reference to constriction triggered by solely ET-1. Afterwards, the influence of calcium ions present in the perfusion fluid was evaluated in terms of the effect triggered by 2APB and occurring in arteries constricted by ET-1. ET-1, in concentration dependent manner, leads to increase in perfusion pressure. Y-27632 and 2APB lead to shift of the concentration response curve for ET-1 to the right with simultaneously lowered maximum effect. There was no difference in reaction of the artery constricted by ET-1 and treated with 2APB in solution containing calcium and in calcium-free solution. Vasoconstrictive action of endothelin is not significantly dependent on the inflow of extracellular calcium, but it is proportional to inflow of Ca2+ related to activation of IP3 receptors and to Rho-kinase activity.

Neuroprotective Activity of Sitagliptin via Reduction of Neuroinflammation beyond the Incretin Effect: Focus on Alzheimer’s Disease

Sitagliptin is a member of a class of drugs that inhibit dipeptidyl peptidase (DPP-4). It increases the levels of the active form of incretins such as GLP-1 (glucagon-like peptide-1) or GIP (gastric inhibitory polypeptide) and by their means positively affects glucose metabolism. It is successfully applied in the treatment of diabetes mellitus type 2. The most recent scientific reports suggest beneficial effect of sitagliptin on diseases in which neuron damage occurs. Result of experimental studies may indicate a reducing influence of sitagliptin on inflammatory response within encephalon area. Sitagliptin decreased the levels of proinflammatory factors: TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), IL-17 (interleukin-17), and CD-163 (cluster of differentiation 163), and contributed to an increase in levels of anti-inflammatory factors: IL-10 (interleukin-10) and TGF-β (transforming growth factor β). Moreover, sitagliptin demonstrated antioxidative and antiapoptotic properties by modifying glutamate and glutathione levels within the region of hippocampus in mice. It has been observed that sitagliptin decreases accumulation of β-amyloid within encephalon structures in experimental models of Alzheimers dementia. This effect may be connected with SDF-1α (stromal cell-derived factor 1α) concentration. Administration of sitagliptin caused a significant improvement in MMSE (Mini–Mental State Examination) tests used for assessment of dementias. The paper presents potential mechanisms of sitagliptin activity in conditions connected with neuroinflammation with special emphasis on Alzheimers disease.