Urinary excretion of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine is positively related to nitric oxide level in tissues of normotensive and hypertensive rats.

Nitric oxide (NO) is one of the gaseous transmitters which play a very important role in the regulation of the circulatory system. Decreased NO availability is associated with hypertension, cardiovascular and kidney diseases. Endogenous NO is generated enzymatically by nitric oxide synthase (NOS) depending on the availability of the substrate, cofactors, or presence/absence of inhibitors, such as asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). The objective of this study was to evaluate the potential relationship between NO level in rat tissues (heart and kidneys) and the concentrations of endogenous metabolites related to NO in plasma and urine. The experiment was carried out with 16- and 60-week-old male Wistar Kyoto (WKY) and age-matched male Spontaneously Hypertensive Rats (SHR). NO level in tissue homogenates was determined by the colorimetric method. RT-qPCR was used to verify the expression of the eNOS (endothelial NOS) gene. Plasma and urine concentrations of arginine, ornithine, citrulline, and dimethylarginines were examined by the UPLC-MS/MS method. 16-week-old WKY rats had the highest tissue NO and plasma citrulline levels. Furthermore, 16-week-old WKY rats showed higher urinary excretion of ADMA/SDMA compared to other experimental groups, however, plasma concentrations of arginine, ADMA, and SDMA were comparable between the groups. In conclusion, our research shows that hypertension and aging decrease tissue NO levels and are associated with reduced urinary excretion of NOS inhibitors, i.e., ADMA and SDMA.

Electrophoretic Determination of Trimethylamine (TMA) in Biological Samples as a Novel Potential Biomarker of Cardiovascular Diseases Methodological Approach.

In competitive athletes, the differential diagnosis between nonpathological changes in cardiac morphology associated with training (commonly referred to as "athlete's heart") and certain cardiac diseases with the potential for sudden death is an important and not uncommon clinical problem. The use of noninvasive, fast, and cheap analytical techniques can help in making diagnostic differentiation and planning subsequent clinical strategies. Recent studies have demonstrated the role of gut microbiota and their metabolites in the onset and the development of cardiovascular diseases. Trimethylamine (TMA), a gut bacteria metabolite consisting of carnitine and choline, has recently emerged as a potentially toxic molecule to the circulatory system. The present work aims to develop a simple and cost-effective capillary electrophoresis-based method for the determination of TMA in biological samples. Analytical characteristics of the proposed method were evaluated through the study of its linearity (R2 > 0.9950) and the limit of detection and quantification (LOD = 1.2 µg/mL; LOQ = 3.6 µg/mL). The method shows great potential in high-throughput screening applications for TMA analysis in biological samples as a novel potential biomarker of cardiovascular diseases. The proposed electrophoretic method for the determination of TMA in biological samples from patients with cardiac disease is now in progress.

Keratin Biomaterials in Skin Wound Healing, an Old Player in Modern Medicine: A Mini Review.

Impaired wound healing is a major medical problem. To solve it, researchers around the world have turned their attention to the use of tissue-engineered products to aid in skin regeneration in case of acute and chronic wounds. One of the primary goals of tissue engineering and regenerative medicine is to develop a matrix or scaffold system that mimics the structure and function of native tissue. Keratin biomaterials derived from wool, hair, and bristle have been the subjects of active research in the context of tissue regeneration for over a decade. Keratin derivatives, which can be either soluble or insoluble, are utilized as wound dressings since keratins are dynamically up-regulated and needed in skin wound healing. Tissue biocompatibility, biodegradability, mechanical durability, and natural abundance are only a few of the keratin biomaterials' properties, making them excellent wound dressing materials to treat acute and chronic wounds. Several experimental and pre-clinical studies described the beneficial effects of the keratin-based wound dressing in faster wound healing. This review focuses exclusively on the biomedical application of a different type of keratin biomaterials as a wound dressing in pre-clinical and clinical conditions.

Heart Failure Disturbs Gut-Blood Barrier and Increases Plasma Trimethylamine, a Toxic Bacterial Metabolite.

Trimethylamine (TMA) is a gut bacteria product oxidized by the liver to trimethylamine-N-oxide (TMAO). Clinical evidence suggests that cardiovascular disease is associated with increased plasma TMAO. However, little headway has been made in understanding this relationship on a mechanistic and molecular level. We investigated the mechanisms affecting plasma levels of TMAO in Spontaneously Hypertensive Heart Failure (SHHF) rats. Healthy Wistar Kyoto (WKY) and SHHF rats underwent metabolic, hemodynamic, histopathological and biochemical measurements, including tight junction proteins analysis. Stool, plasma and urine samples were evaluated for TMA and TMAO using ultra performance liquid chromatography-mass spectrometry. SHHF presented disturbances of the gut-blood barrier including reduced intestinal blood flow, decreased thickness of the colonic mucosa and alterations in tight junctions, such as claudin 1 and 3, and zonula occludens-1. This was associated with significantly higher plasma levels of TMA and TMAO and increased gut-to-blood penetration of TMA in SHHF compared to WKY. There was no difference in kidney function or liver oxidation of TMA to TMAO between WKY and SHHF. In conclusion, increased plasma TMAO in heart failure rats results from a perturbed gut-blood barrier and increased gut-to-blood passage of TMAO precursor, i.e., TMA. Increased gut-to-blood penetration of bacterial metabolites may be a marker and a mediator of cardiovascular pathology.

His-Leu, an angiotensin I-derived peptide, does not affect haemodynamics in rats.

INTRODUCTION:: The dipeptide histidine-leucine (His-Leu) is formed in the process of converting angiotensin I into angiotensin II. Several studies show that short peptides containing His-Leu may produce significant haemodynamic effects; however, to the best of our knowledge, data on haemodynamic effects of His-Leu are not available in medical databases. MATERIALS AND METHODS:: We evaluated acute haemodynamic effects of intravenous administration of either 0.9% NaCl (vehicle) or His-Leu at a dose of 3-15 mg/kg body weight in anaesthetized 15-16-week-old, male, normotensive Wistar Kyoto and spontaneously hypertensive rats. Chronic effects of treatment with either the vehicle or His-Leu at a dose of 15 mg/kg body weight given subcutaneously daily were determined during continuous telemetry recordings in freely moving rats. RESULTS:: In anaesthetized rats both the vehicle and His-Leu produced a mild and transient increase in blood pressure and no change in plasma renin activity. There was no significant difference in haemodynamics between the rats infused with the vehicle and the rats infused with His-Leu. In chronic studies, seven-day treatment with vehicle and with His-Leu did not affect arterial blood pressure in freely moving rats. CONCLUSION:: His-Leu does not produce either acute or chronic changes in arterial blood pressure in normotensive and hypertensive rats.

Chronic, low-dose TMAO treatment reduces diastolic dysfunction and heart fibrosis in hypertensive rats.

Several studies have suggested negative effects of trimethylamine oxide (TMAO) on the circulatory system. However, a number of studies have shown protective functions of TMAO, a piezolyte and osmolyte, in animals exposed to high hydrostatic and/or osmotic stress. We evaluated the effects of TMAO treatment on the development of hypertension and its complications in male spontaneously hypertensive rats (SHRs) maintained on water (SHR-Water) and SHRs drinking TMAO water solution from weaning (SHR-TMAO). Wistar-Kyoto (WKY) rats were used as normotensive controls to discriminate between age-dependent and hypertension-dependent changes. Telemetry measurements of blood pressure were performed in rats between the 7th and 16th weeks of life. Anesthetized rats underwent echocardiographic, electrocardiographic, and direct left ventricular end-diastolic pressure (LVEDP) measurements. Hematoxylin and eosin as well as van Gieson staining for histopathological evaluation were performed. Plasma TMAO measured by chromatography coupled with mass spectrometry was significantly higher in the SHR-Water group compared with the WKY group (~20%). TMAO treatment increased plasma TMAO by four- to fivefold and did not affect the development of hypertension in SHRs. Sixteen-week-old rats in the SHR-Water and SHR-TMAO groups (12-wk TMAO treatment) showed similar blood pressures, angiopathy, and cardiac hypertrophy. However, the SHR-TMAO group had lower plasma NH2-terminal pro-B-type natriuretic peptide, LVEDP, and cardiac fibrosis. In contrast to age-matched WKY rats, 60-wk-old SHRs showed hypertensive angiopathy and heart failure with preserved ejection fraction. Compared with the SHR-Water group, the SHR-TMAO group (56-wk TMAO treatment) showed significantly lower plasma NH2-terminal pro-B-type natriuretic peptide and vasopressin, significantly lower LVEDP, and cardiac fibrosis. In conclusion, a four- to fivefold increase in plasma TMAO does not exert negative effects on the circulatory system. In contrast, increased dietary TMAO seems to reduce diastolic dysfunction in pressure-overloaded hearts in rats. NEW & NOTEWORTHY Chronic, low-dose trimethylamine oxide (TMAO) treatment that increases plasma TMAO by four- to fivefold reduces plasma NH2-terminal pro-B-type natriuretic peptide and vasopressin, left ventricular end-diastolic pressure, and cardiac fibrosis in pressure-overloaded hearts in hypertensive rats. Our study provides evidence that a moderate increase in plasma TMAO does not have a negative effect on the circulatory system. In contrast, increased dietary TMAO seems to reduce diastolic dysfunction in the pressure-overloaded heart.

Indole and indoxyl sulfate, gut bacteria metabolites of tryptophan, change arterial blood pressure via peripheral and central mechanisms in rats.

Arterial blood pressure (BP) is regulated by a complex network of peripheral and central (brain) mechanisms. Research suggests that gut bacteria-derived compounds may affect the circulatory system. We evaluated hemodynamic effects of indole, a gut bacteria-derived product of tryptophan, and indoxyl sulfate (indoxyl), a liver metabolite of indole. BP and heart rate (HR) were recorded in anesthetized, male, Wistar rats at baseline and after the administration of either a vehicle, indole, or indoxyl into the femoral vein (IV) or into the lateral ventricle of the brain (ICV). Besides, we evaluated the effect of pretreatment with flupentixol, a non-selective D1, D2, α1 and 5 HT2A receptor blocker; pizotifen, a non-selective 5-HT1, 5-HT2A and 5HT2C receptor blocker; and ondansetron, a 5-HT3 blocker, on hemodynamic responses to indole and indoxyl. Vehicle infused IV and ICV did not affect hemodynamics. Indole administered IV produced a dose-dependent increase in BP but not HR. In contrast, the ICV infusion of indole produced a decrease in BP and HR. Indoxyl infused IV produced an increase in BP and HR, whereas indoxyl infused ICV did not affect BP and HR. The hemodynamic effects of indole and indoxyl were inhibited by pretreatment with ondansetron and pizotifen but not flupentixol. In conclusion, indole and indoxyl sulfate affect arterial blood pressure via peripheral and central mechanisms dependent on serotonin signalling. We propose that indole and indoxyl sulfate may be mediators in the interaction between gut bacteria and the circulatory system.

Na2S, a fast-releasing H2S donor, given as suppository lowers blood pressure in rats.

BACKGROUND: Hydrogen sulfide (H2S) is involved in blood pressure control. The available slow-releasing H2S-donors are poorly soluble in water and their ability to release H2S in biologically relevant amounts under physiological conditions is questionable. Therefore, new slow-releasing donors or new experimental approaches to fast-releasing H2S donors are needed. METHODS: Hemodynamics and ECG were recorded in male, anesthetized Wistar Kyoto rats (WKY) and in Spontaneously hypertensive rats (SHR) at baseline and after: 1) intravenous (iv) infusion of vehicle or Na2S; 2) administration of vehicle suppositories or Na2S suppositories. RESULTS: Intravenously administered vehicle and vehicle suppositories did not affect mean arterial blood pressure (MABP) and heart rate (HR). Na2S administered iv caused a significant, but transient (2-5min) decrease in MABP. Na2S suppositories produced a dose-dependent hypotensive response that lasted ∼45min in WKY and ∼75-80min in SHR. It was accompanied by a decrease in HR in WKY, and an increase in HR in SHR. Na2S suppositories did not produce a significant change in corrected QT, an indicator of cardiotoxicity. Na2S suppositories increased blood level of thiosulfates, products of H2S oxidation. CONCLUSIONS: Na2S administered in suppositories exerts a prolonged hypotensive effect in rats, with no apparent cardiotoxic effect. SHR and WKY differ in hemodynamic response to the H2S donor. Suppository formulation of fast-releasing H2S donors may be useful in research, if a reference slow-releasing H2S donor is not available.

Parenteral Na2S, a fast-releasing H2S donor, but not GYY4137, a slow-releasing H2S donor, lowers blood pressure in rats.

Hydrogen sulfide (H2S) is involved in blood pressure regulation. We evaluated hemodynamic effects of Na2S and morpholin-4-ium (4-methoxyphenyl)(morpholino)phosphinodithioate (GYY4137), H2S donors. GYY4137 is the most widely studied slow-releasing H2S donor, however, its ability to release H2S under physiological conditions is unclear. Hemodynamics were recorded in anaesthetized Wistar-Kyoto rats at baseline and after intravenous (IV) or intraperitoneal (IP) administration of either a vehicle (20% dimethyl sulfoxide), GYY4137 or Na2S. The stability of GYY4137 in buffers and in plasma was evaluated with nuclear magnetic resonance. The vehicle, as well as GYY4137, given IV did not affect mean arterial blood pressure (MABP), whereas Na2S produced a significant decrease in MABP. Similarly, IP given Na2S, but not GYY4137, lowered MABP. In the buffers at pH of 7.4 and 5.5 and in rat plasma no reaction of GYY4137 was found during 18 hours of observation. In contrast, rapid decomposition of GYY4137 occurred in buffers at pH 2.0. In conclusion, parenteral GYY4137 does not exert a hemodynamic effect in Wistar-Kyoto rats. This seems to be due to the high stability of GYY4137 at physiological pH. Therefore, it is likely that widely reported biological effects of GYY4137 are not H2S-dependent but may depend on GYY4137 itself. However, the H2S-dependent biological effects of GYY4137 may be expected in tissues characterized by low pH.

Characterization of Disopyramide derivative ADD424042 as a non-cardiotoxic neuronal sodium channel blocker with broad-spectrum anticonvulsant activity in rodent seizure models.

It was reported that antiarrhythmic drugs (AADs) can be useful in controlling refractory seizures in humans or in enhancing the action of antiepileptic drugs (AEDs) in animal models. Disopyramide phosphate (DISO) is an AAD that blocks sodium channels in cardiac myocytes. We evaluated a DISO derivative, 2-(2-chlorophenyl)-2-(pyridin-2-yl)acetamide (ADD424042) for its anticonvulsant activity in a battery of rodent models of epileptic seizures. The compound displayed a broad spectrum of activity in the 'classical' models as well as in the models of pharmacoresistant seizures. Furthermore, ADD424042 showed good therapeutic indices between the anticonvulsant activity and the motor impairment. On the contrary, no anticonvulsant effects but severe lethality were observed in the primary anticonvulsant testing of the parent DISO. By performing the whole-cell voltage-clamp experiments in dispersed cortical neurons we demonstrated that ADD424042 decreased the maximal amplitude of voltage-gated sodium channels with an IC50 value in nM range. Moreover, the compound enhanced use-dependent block and decreased excitability in pyramidal neurons in the current-clamp experiments in cortical slices. Importantly, we found that ADD424042 possessed either no, or very small cardiotoxic effect. In contrast to DISO, ADD424042 did not produce any apparent changes in electrocardiogram (ECG) and arterial blood pressure recordings. ADD424042 had no effect on QT and corrected QT intervals, at a dose which was 15 times higher than ED50 for the anticonvulsant effect in the MES model. Taken together, these data suggest that ADD424042 has the potential to become a lead structure for novel broadly acting AEDs with wide margin of cardiac safety.

The effect of simvastatin and pravastatin on arterial blood pressure, baroreflex, vasoconstrictor, and hypertensive effects of angiotensin II in Sprague-Dawley rats.

Research suggests that statins affect the regulation of arterial blood pressure (BP), however, the mechanisms remain obscure. We maintained male, 12-week-old, Sprague-Dawley rats on tap water (controls) or water containing simvastatin or pravastatin for 4 weeks. Subsequently, we measured mean arterial blood pressure and heart rate at baseline and after intravenous infusion of either saline or angiotensin II (Ang II). Additionally, we tested baroreflex function and the effect of statins on vasoconstrictor response to Ang II on isolated femoral artery branches. Controls and simvastatin and pravastatin groups showed a significant increase in mean arterial BP and heart rate in response to Ang II. The increase was significantly smaller in the simvastatin group than in controls and in the pravastatin group. In contrast, when pretreated with hexamethonium, a ganglionic blocker, simvastatin and pravastatin groups showed a similar hypertensive response to Ang II, which was smaller than in controls. Likewise, the Ang II-induced vasoconstrictor response of femoral artery branches was comparable between simvastatin and pravastatin groups and smaller than in controls. We found no effect of statins on the baroreflex. This study shows that simvastatin and pravastatin differ in their effects on the Ang II-dependent mechanisms controlling BP.

Trimethylamine-N-oxide: a carnitine-derived metabolite that prolongs the hypertensive effect of angiotensin II in rats.

BACKGROUND: Recent evidence suggests that an elevated plasma trimethylamine N-oxide (TMAO) level is associated with an increased risk of adverse cardiovascular events in humans; however, the mechanism is not clear. The aims of this study were to establish the plasma TMAO level in rats and to evaluate the effect of TMAO on arterial blood pressure (BP) and the hemodynamic effects of angiotensin II (Ang II). METHODS: Twelve-week-old, Sprague-Dawley rats were implanted with telemetric transmitters, and continuous recordings of heart rate, systolic BP (SBP), and diastolic BP (DBP) were made for 7 days before and 14 days during osmotic minipump-driven subcutaneous infusion of saline (controls), TMAO, low-dose Ang II, or Ang II + TMAO. Plasma TMAO concentration was evaluated using liquid chromatography coupled with triple-quadrupole mass spectrometry. RESULTS: The plasma TMAO concentration in controls was 0.57 µmol/L, whereas in TMAO-infused rats it was 58 µmol/L. Neither saline nor TMAO infusion affected SBP and DBP. Infusion of Ang II significantly increased SBP and DBP for the first 5 days of infusion only. In contrast, infusion of Ang II + TMAO produced a hypertensive response that lasted until the end of the experiment. TMAO infusions did not affect body weight and motor activity. CONCLUSIONS: We showed that physiological plasma TMAO concentration in rats was approximately 10 times lower than that reported in humans. Furthermore, the new finding of the study is that TMAO does not affect BP in normotensive animals. However, it prolongs the hypertensive effect of Ang II.

Exogenous hydrogen sulfide causes different hemodynamic effects in normotensive and hypertensive rats via neurogenic mechanisms.

BACKGROUND: Increasing evidence suggests that disturbances in H2S homeostasis may participate in the development of hypertension. In this study we compared hemodynamic responses to intracerebroventricular (ICV) infusions of sodium hydrosulfide (NaHS), a H2S donor, between normotensive rats (WKY), spontaneously hypertensive rats (SHR) and angiotensin II - induced hypertensive rats (WKY-Ang II). METHODS: We tested the effects of NaHS on mean arterial blood pressure (MABP) and heart rate (HR) in 12-14-week-old, male rats. MABP and HR were continuously recorded at baseline and during ICV infusion of either vehicle (Krebs-Henseleit buffer) or NaHS. RESULTS: ICV infusions of the vehicle did not affect MABP and HR. WKY rats infused with 30 nmol/h of NaHS showed a mild decrease in MABP and HR. ICV infusion of 100 nmol/h produced a biphasic response i.e. mild hypotension and bradycardia followed by an increase in MABP and HR, whereas, the infusion of 300 nmol/h of the H2S donor caused a monophasic increases in MABP and HR. In contrast, SHR rats as well as WKY-Ang II rats showed a decrease in MABP and HR during ICV infusions of NaHS. CONCLUSIONS: The results provide further evidence for the involvement of H2S in the neurogenic regulation of the circulatory system and suggest that alterations in H2S signaling in the brain could be associated with hypertension.

Statins, the renin-angiotensin-aldosterone system and hypertension - a tale of another beneficial effect of statins.

BACKGROUND: Statins, a class of lipid lowering drugs, decrease mortality associated with cardiovascular events. As hypercholesterolemia is often accompanied by hypertension, a large number of patients receive therapy with statins and antihypertensive drugs which act via the renin-angiotensin-aldosterone system (RAAS). New guidelines published by the American Heart Association and American College of Cardiology on the treatment of dyslipidaemia and the reduction of atherosclerotic cardiovascular risk, which use a risk prediction algorithm based on risk factors such as hypertension but not low-density lipoprotein (LDL) level, may even further increase the number of patients receiving such concomitant therapy. METHOD: In this paper we review studies on an interaction between statins, the RAAS and antihypertensive drugs acting via the RAAS. RESULT: Accumulating evidence suggests that the combination of statins and drugs affecting the RAAS exerts a synergistic effect on the circulatory system. For example, statins may lower arterial blood pressure and augment the effect of antihypertensive drugs acting via the RAAS. Statins may interact with the RAAS in a number of ways i.e. to decrease the expression of receptors for angiotensin II (Ang II), inhibit the Ang II-dependent intracellular signalling, reduce the RAAS-dependent oxidative stress and inflammation as well as inhibit the synthesis of Ang II and aldosterone. CONCLUSION: Although statins given either alone or together with antihypertensive drugs acting via the RAAS may lower arterial blood pressure, further research is needed to evaluate the mechanisms and their therapeutic significance.

Oral simvastatin reduces the hypertensive response to air-jet stress.

Brain angiotensin (Ang) II and vasopressin play important roles in the neurogenic regulation of the circulatory system, such as in cardiovascular responses to stress. Recently, it has become evident that the positive effects of statins are not limited to their lipid-lowering actions; for example, it has been found that statins interact with angiotensin peptides. In the present study we tested the hypothesis that simvastatin affects haemodynamic responses to air-jet stress and intracerebroventricular infusions of vasopressin and AngII. We maintained 12-week-old male Sprague-Dawley rats either on tap water (control) or on water containing simvastatin (20, 40 or 60 mg/L) for 4 weeks. Subsequently, we measured arterial blood pressure and heart rate (HR) at baseline and after air-jet stress or intracerebroventricular infusions over 30 s of 10 ng AngII, 20 ng vasopressin or their antagonists (10 µg losartan and 400 ng d(CH(2) )(5) [Tyr(Me)(2) ,Ala-NH(2) (9) ] vasopressin, respectively). There were no significant differences between the control and simvastatin groups in terms of baseline mean arterial blood pressure (MAP) and HR. In rats given 60 mg/L simvastatin, the hypertensive response to air-jet stress was significantly smaller than in controls, as was the increase in MAP in response to AngII. In contrast, there was no significant difference between the groups in terms of the hypertensive response to vasopressin. These findings show that simvastatin affects the hypertensive response to air-jet stress and provide evidence that statins may affect the brain's regulation of the circulatory system.

Early high-sodium solid diet does not affect sodium intake, sodium preference, blood volume and blood pressure in adult Wistar-Kyoto rats.

A high-Na diet may lead to the development of hypertension in both humans and rats; however, the causes of Na intake in amounts greater than physiologically needed as well as the mechanisms whereby high-Na food elevates blood pressure are not clear. Therefore, we decided to test the hypothesis that a high-Na diet introduced after suckling affects Na intake, food preference, resting blood pressure and blood volume in adult rats. Male Wistar-Kyoto (WKY) rats, 4 weeks old, were divided into three groups and placed on either a high-Na (3.28%), a medium-Na (0.82%) or a regular diet (0.22%) with the same energy content for 8 weeks. Subsequently, food preference, resting arterial blood pressure, blood volume, plasma osmolality and Na blood level were evaluated. When offered a choice of diets, all the groups preferred the regular chow, and there was no significant difference in total Na intake between the groups. When the rats experienced the change from their initial chow to a new one with different Na content, they continued to eat the same amount of food. Body weight, resting arterial blood pressure, blood volume, plasma osmolality and Na blood level were comparable between the groups. In conclusion, the results show that a high-Na diet introduced immediately after suckling does not affect Na preference and Na intake in adult WKY rats. Furthermore, the findings provide evidence that both blood volume and arterial blood pressure are highly protected in normotensive rats on a high-Na diet.