Acute Intravenous Injection Research Articles

Hinokitiol for hypertensive emergencies: effects on peripheral resistance, cardiac load, baroreflex sensitivity, and electrolytes balance

Hinokitiol, a natural monoterpenoid, has been shown previously to possess a potent vasodilating activity in vitro in both control and hypertensive aortae. Here, the antihypertensive and cardioprotective effects of an intravenous hinokitiol injection were fully investigated in angiotensin II-induced hypertensive emergency in rats. Hinokitiol intravenous injection was prepared in the form of self-nanoemulsifying drug delivery system. Rat’s arterial and ventricular hemodynamics were measured in real-time recordings in addition to surface electrocardiogram while slow injection of cumulative doses of hinokitiol or vehicle as well as time control. Hinokitiol at dose 10 mg/kg showed a considerable reduction in the raised systolic blood pressure (30 mmHg) within only 30 min. The decrease in blood pressure seems to be mediated through a reduction in peripheral resistance, as appears from the decreases in diastolic pressure, dicrotic notch pressure, and pulse pressure. In addition, hinokitiol injection reduced heart load due to the decrease in heart rate, increases in cycle duration (particularly the non-ejection duration) and diastolic duration, and decreases in end-diastolic pressure. An effect most likely mediated via prolongation of ventricular repolarization as appears from the increases in PR, QTc, and JT intervals. However, acute intravenous injection of hinokitiol neither affected the baroreflex sensitivity nor sodium/potassium balance. In conclusion, acute hinokitiol intravenous injection markedly reduced severe hypertension in rats. This effect seems to be mediated through decreasing peripheral resistance and decreasing cardiac load, suggesting that it is an effective treatment in hypertensive emergencies after clinical evaluation.

Natriuretic Peptides-New Targets for Neurocontrol of Blood Pressure via Baroreflex Afferent Pathway.

Natriuretic peptides (NPs) induce vasodilation, natriuresis, and diuresis, counteract the renin-angiotensin-aldosterone system and autonomic nervous system, and are key regulators of cardiovascular volume and pressure homeostasis. Baroreflex afferent pathway is an important reflex loop in the neuroregulation of blood pressure (BP), including nodose ganglion (NG) and nucleus tractus solitarius (NTS). Dysfunction of baroreflex would lead to various hypertensions. Here, we carried out functional experiments to explore the effects of NPs on baroreflex afferent function. Under physiological and hypertensive condition (high-fructose drinking-induced hypertension, HFD), BP was reduced by NPs through NG microinjection and baroreflex sensitivity (BRS) was enhanced via acute intravenous NPs injection. These anti-hypertensive effects were more obvious in female rats with the higher expression of NPs and its receptor A/B (NPRA/NPRB) and lower expression of its receptor C (NPRC). However, these effects were not as obvious as those in HFD rats compared with the same gender control group, which is likely to be explained by the abnormal expression of NPs and NPRs in the hypertensive condition. Our data provide additional evidence showing that NPs play a crucial role in neurocontrol of BP regulation via baroreflex afferent function and may be potential targets for clinical management of metabolic-related hypertension.

Phytochemical Screening and In vivo Antileukemic Activity of Methanolic Extract of Seeds of Nigella Sativa L. on Benzene-Induced Leukemia in Wistar Rats

Nigella sativa is exquisite bounty of nature to humankind. It is an annual flowering plant of family Ranunculaceae majorly found in Southwest Asia, seeds are used as flavor food. The present work was directed to evaluate antileukemic effect of N. sativa seeds extract in benzene-induced leukemia in female Wistar rats. Seed powder was extracted using petroleum ether, chloroform, ethyl acetate, and methanol. OECD guidelines 423 were used to study acute oral toxicity study and intravenous injection of benzene was given on every 2nd day up to 21 days to induce leukemia. To assess burden of leukemia, various parameters analyzed are body weight (B.W), organ weight, complete blood picture, serum potassium, phosphate, uric acid, and glutathione. The presence of alkaloids, carbohydrates, cardiac glycosides, tannins, phytosterols, and flavonoids is confirmed by phytochemical screening. Acute oral toxicity study found 2000 mg/kg as a safe dose of N. sativa seed extract. Significant decrease in potassium, phosphate, uric acid, WBC, and increase in B.W, glutathione demonstrated therapeutic efficacy of extract in leukemia. Our results confirmed significant antileukemic effect of N. sativa seed extract, suggesting role of this herb for the treatment of malignancies such as leukemia.

Eukaryotic elongation factor 2 kinase inhibitor, A484954 induces diuretic effect via renal vasorelaxation in spontaneously hypertensive rats

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K), alternatively known as calmodulin-dependent protein kinase III, inhibits protein translation via phosphorylating its sole substrate, eEF2. We previously demonstrated that expression and activity of eEF2K change in mesenteric artery from spontaneously hypertensive rats (SHR) with aging and that eEF2K is involved in pathogenesis of essential hypertension. In addition, we have recently revealed that acute intravenous injection with A484954, a selective eEF2K inhibitor, lowers blood pressure specifically in SHR partly via inducing vasorelaxation. In this study, we examined whether A484954 induces diuretic effect. After male SHR and normotensive Wistar Kyoto rats (WKY) were given a single intraperitoneal injection of A484954 (2.5 mg/kg, 0.5–9 h), urine was collected using metabolic cage. Contraction of isolated renal arteries form SHR was isometrically measured. While A484954 did not induce diuretic effect in WKY, it increased urine output, water intake, and urinary sodium excretion in SHR. A484954 (10 μM) induced vasorelaxation in isolated renal arteries, which was inhibited by a β-adrenergic receptor antagonist, propranolol. It was confirmed that A484954 increased renal blood flow in SHR as measured by renal ultrasonography. In summary, it was for the first time revealed that A484954 induces diuretic effect in SHR at least partly via renal vasorelaxation through β-adrenergic receptor.

Acute titanium dioxide nanoparticles exposure impaired spatial cognitive performance through neurotoxic and oxidative mechanisms in Wistar rats

Context Titanium dioxide nanoparticles (TiO2-NPs) are used in many commercial products. However, their effects on human and animal organism remained to be clarified. Objective The present study aimed to investigate the effects of TiO2-NPs on the behavioural performance, monoamine neurotransmitters and oxidative stress in the rat brain. Material and methods Rats were injected intravenously with a single dose of TiO2-NPs (20 mg/kg body weight) and were subjected to cognitive and emotional tests using Morris water maze and elevated plus maze. Results Cognitive capacity as well as the emotional reactivity were significantly disrupted, in TiO2-NPs-administered rats compared to control group. These behavioural effects were correlated with changes in brain neurotransmitter contents reflected by a significant increase in dopamine and a decrease in serotonin levels. TiO2-NPs also induced oxidative stress in the brain manifested by increased levels of H2O2 and malondialdehyde, associated with antioxidant enzymes activities disturbance, in particular, superoxide dismutase and catalase activities. Moreover, TiO2-NPs administration caused histological damages in the brain tissue with abundant lymphocytic clusters, capillary dilations, vascular congestion and oedema. Conclusions Acute intravenous injection of TiO2-NPs impaired behaviour performances through brain biochemical and structural changes and precautions should be taken to their usage in food additive and medical applications.

Platelet-like particles reduce coagulopathy-related and neuroinflammatory pathologies post-experimental traumatic brain injury.

Coagulopathy may occur following traumatic brain injury (TBI), thereby negatively affecting patient outcomes. Here, we investigate the use of platelet-like particles (PLPs), poly(N-isopropylacrylamide-co-acrylic-acid) microgels conjugated with a fibrin-specific antibody, to improve hemostasis post-TBI. The objective of this study was to diminish coagulopathy in a mouse TBI model (controlled cortical impact) via PLP treatment, and subsequently decrease blood-brain barrier (BBB) permeability and neuroinflammation. Following an acute intravenous injection of PLPs post-TBI, we analyzed BBB permeability, ex vivo coagulation parameters, and neuroinflammation at 24 hr and 7 days post-TBI. Both PLP-treatment and control particle-treatment had significantly decreased BBB permeability and improved clot structure 24 hr post-injury. Additionally, no significant change in tissue sparing was observed between 24 hr and 7 days for PLP-treated cohorts compared to that observed in untreated cohorts. Only PLP-treatment resulted in significant reduction of astrocyte expression at 7 days and percent difference from 24 hr to 7 days. Finally, PLP-treatment significantly reduced the percent difference from 24 hr to 7 days in microglia/macrophage density compared to the untreated control. These results suggest that PLP-treatment addressed acute hypocoagulation and decreased BBB permeability followed by decreased neuroinflammation and fold-change tissue loss by 7 days post-injury. These promising results indicate that PLPs could be a potential therapeutic modality for TBI.

BRAIN PERIVASCULAR MACROPHAGES CONTRIBUTE TO THE DEVELOPMENT OF HYPERTENSION THROUGH SYMPATHETIC ACTIVATION IN STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS

Objective: Hypertension is associated with systemic inflammation. The activation of the sympathetic nervous system is critically involved in the pathogenesis of hypertension. Brain perivascular macrophages (PVMs) can be affected by circulating inflammatory cytokines and contribute to sympathoexcitaion in a heart failure model. We thus investigated whether brain PVMs contribute to the development of hypertension through sympathoexcitation. Design and method: We measured blood pressure and evaluated the number of brain PVMs and the plasma levels of proinflammatory cytokines in stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar–Kyoto rats (WKY) at the ages of 4, 8, and 12 weeks. To determine the contribution of brain PVMs to blood pressure elevation, we intracerebroventricularly injected liposome-encapsulated clodronate, which eliminates macrophages by inducing apoptosis, or vehicle into 8-week-old SHRSP and WKY; we then assessed its effects in 10-week-old rats. We also investigated the effects of clodronate treatment on responses to the acute intravenous injection of proinflammatory cytokines in WKY. Results: SHRSP developed hypertension over 8 weeks from 4 to 12 weeks of age. The number of brain PVMs and plasma interleukin-1β levels significantly increased at the ages of 8 and 12 weeks in SHRSP compared with WKY. Clodronate treatment attenuated the increase in blood pressure in SHRSP (δmean blood pressure, 11.6 ± 3.8 mmHg vs. 23.8 ± 1.9 mmHg; clodronate vs. vehicle, n = 7, p < 0.05) but not in WKY. Clodronate treatment reduced the depressor effect of hexamethonium, an index of sympathetic activity; it also reduced the neuronal activity marker c-Fos in sympathetic regulatory nuclei such as the hypothalamic paraventricular nucleus and rostral ventrolateral medulla and reduced the expression of cyclooxygenase-2 and prostaglandin E2, a downstream pathway in activated macrophages, in SHRSP but not in WKY. Furthermore, clodronate treatment attenuated the increase in blood pressure and renal sympathetic nerve activity (RSNA) in response to an acute intravenous injection of interleukin-1β in WKY (δmean blood pressure, 8.8 ± 2.5 mmHg vs. 21.2 ± 4.6 mmHg; integrated RSNA, 125.6 ± 10.4 % vs. 213.1 ± 22.2 %; clodronate vs vehicle, n = 5, p < 0.05). Conclusions: Brain PVMs contribute to the development of hypertension via sympathetic activation. PVMs may be activated by increased levels of circulating interleukin-1β.

Extracellular Vesicles Mediate Neuroprotection and Functional Recovery after Traumatic Brain Injury

The lack of effective therapies for moderate-to-severe traumatic brain injuries (TBIs) leaves patients with lifelong disabilities. Neural stem cells (NSCs) have demonstrated great promise for neural repair and regeneration. However, direct evidence to support their use as a cell replacement therapy for neural injuries is currently lacking. We hypothesized that NSC-derived extracellular vesicles (NSC EVs) mediate repair indirectly after TBI by enhancing neuroprotection and therapeutic efficacy of endogenous NSCs. We evaluated the short-term effects of acute intravenous injections of NSC EVs immediately following a rat TBI. Male NSC EV-treated rats demonstrated significantly reduced lesion sizes, enhanced presence of endogenous NSCs, and attenuated motor function impairments 4 weeks post-TBI, when compared with vehicle- and TBI-only male controls. Although statistically not significant, we observed a therapeutic effect of NSC EVs on brain lesion volume, nestin expression, and behavioral recovery in female subjects. Our study demonstrates the neuroprotective and functional benefits of NSC EVs for treating TBI and points to gender-dependent effects on treatment outcomes, which requires further investigation.

Brain perivascular macrophages contribute to the development of hypertension in stroke-prone spontaneously hypertensive rats via sympathetic activation.

Hypertension is associated with systemic inflammation. The activation of the sympathetic nervous system is critically involved in the pathogenesis of hypertension. Brain perivascular macrophages (PVMs) can be affected by circulating inflammatory cytokines, and the contribution of brain PVMs to sympathoexcitation has been demonstrated in a heart failure model. We thus investigated whether brain PVMs contribute to the development of hypertension through sympathoexcitation. Stroke-prone spontaneously hypertensive rats (SHRSP) developed hypertension over an 8-week period from 4 to 12 weeks of age. The number of brain PVMs and plasma interleukin-1β levels significantly increased at the ages of 8 and 12 weeks in SHRSP compared with normotensive Wistar-Kyoto rats (WKY). To determine the contribution of brain PVMs to blood pressure elevation, we intracerebroventricularly injected liposome-encapsulated clodronate, which eliminates macrophages by inducing apoptosis, into 8-week-old rats; we then assessed its effects in 10-week-old rats. Clodronate treatment attenuated the increase in mean blood pressure in SHRSP but not in WKY. Clodronate treatment reduced the depressor effect of hexamethonium, an index of sympathetic activity; it also reduced neuronal activity in sympathetic regulatory nuclei such as the hypothalamic paraventricular nucleus and rostral ventrolateral medulla and reduced the expression of cyclooxygenase-2 and prostaglandin E2, a downstream pathway in activated macrophages, in SHRSP but not in WKY. Furthermore, clodronate treatment attenuated the increase in blood pressure and renal sympathetic nerve activity in response to an acute intravenous injection of interleukin-1β in WKY. In conclusion, brain PVMs contribute to the development of hypertension via sympathetic activation. PVMs may be activated by increased levels of circulating interleukin-1β.

Abstract P3060: Cardiovascular Regulatory Actions of Apelin in Normotensive Rats: Central versus Peripheral Effects

Apelin and its receptor APJ are expressed in both cardiovascular system (CVS) and central nervous system (CNS). Although the apelin/APJ system have already been shown to be involved in cardiovascular homeostasis, the mechanisms in the regulation of CVS are still not fully clear. Thus, this study was undertaken to compare central versus peripheral effect of apelin in the regulation of hemodynamic in SD rats. The chronic recording of blood pressure (BP) and heart rate (HR) were performed using radiotelemetry. Chronic infusion of apelin-13 (100 μg/kg/day) into the lateral cerebral ventricle using osmotic minipumps significantly elevated BP by 15.2±0.9 mmHg, The water intake was also increased from 38±2 to 51±3 ml/day before and after brain infusion of apelin-13. However, the HR was not significantly altered. Furthermore, this chronic pressor effect of apelin-13 was associated with a dramatic increase in the number of Fra-LI positive neurons within the paraventricular nucleus (PVN) and nucleus tractus solitarii (NTS), which were measured with immunohistochemistry using c-Fos antibodies, suggesting an elevated neuronal activity in the brain cardiovascular regulatory areas. Thus, apelin-13 was microinjected into the PVN to confirm the neuronal effect on the BP. The BP of rats was recorded using femoral arterial cannulation. The results demonstrated that acute microinjection of apelin-13 into the PVN significantly increased BP and HR. In peripheral CVS, acute intravenous injections of apelin-13 induced a transient reduction in BP by 25±3 mmHg as expected. Surprisingly, in the same group of rats, the cardiac contractility (dP/dt Max from 8131±512 to 7822±484 mmHg/s) was not significantly altered by apelin-13. In addition, chronic subcutaneous infusion of apelin-13 did not significantly alter either BP or cardiac contractility, which were detected with ratiotelemetry and echocardiography in rats. In conclusion, peripheral administration of apelin-13 only induced a transient decrease in BP without chronic BP regulatory and cardiotonic effects. In contrast, central application of apelin-13 leaded to a chronic elevation in the BP, suggesting that apelin-13 acting as an important neuropeptide in the CNS may contribute to the long-term control of BP.

Combination of acute intravenous methamphetamine injection and LPS challenge facilitate leukocyte infiltration into the central nervous system of C57BL/6 mice

Methamphetamine (METH) is a stimulant of the central nervous system (CNS) that causes behavioral changes in users. METH is slowly cleared from brain tissue and its chronic use is neurotoxic. METH also alters the cellular and chemical components of inflammation. However, little is known about the effect of a single intravenous dose of METH followed by bacterial lipopolysaccharide (LPS) injection on cellular infiltration and cytokine release in brain tissue. Using a murine model of acute METH administration and flow cytometry, we found that combination of METH and LPS stimulate the infiltration of macrophages (F4/80+cells) and neutrophils (Ly-6G+cells) into the CNS. Histological sections of the brainstem of METH-treated and LPS-challenged C57BL/6 mice demonstrated considerable leukocyte infiltration relative to untreated, LPS, and METH groups. Moreover, rodents treated with LPS alone or combined with METH showed elevated levels of pro-inflammatory cytokines mRNA in brain tissue. Our observations are important because recognizing neuroinflammatory changes after acute METH administration might help us to understand METH-induced neurotoxicity in users.

A11957 Irisin Lowers Blood Pressure by Improvement of Endothelial Dysfunction via AMPK-Akt-eNOS-NO Pathway in the Spontaneously Hypertensive Rat

Objectives: Exercise is a major nonpharmacological treatment for hypertension, but its underlying mechanisms are still not completely elucidated. Irisin, a polypeptide containing 112 amino acids, which is secreted mainly by skeletal muscle cells during exercise, exerts a protective role in metabolic diseases, such as diabetes and obesity. Due to the close relationship between irisin and metabolic diseases, we hypothesized that irisin may play a role in the regulation of blood pressure. Methods: Blood pressures of male WKY rats and SHRs were monitored via the carotid artery. The vascular response was checked by tension measurement of the artery rings. Protein expression was tested by immunoblot. Results: Our study found that the acute intravenous injection of irisin reduced blood pressure in SHRs but not WKY rats. Irisin, by itself, had no direct vasorelaxing effect in phenylephrine-pre-constricted mesenteric arteries from SHRs. However, irisin augmented the acetylcholine-induced vasorelaxation in mesenteric arteries from SHRs that could be reversed by Nω-nitro-L-arginine-methyl ester (L-NAME, 100 μM), indicating a role of NO in this action. Indeed, irisin increased NO production and phosphorylation of eNOS in endothelial cells. 5¢-AMP-activated protein kinase (AMPK) was involved in the vasorelaxing effect of irisin because compound C (20 μM), an AMPK inhibitor, blocked the irisin-mediated increase in phosphorylation of eNOS and Akt in endothelial cells and vasodilation in mesenteric arteries. Conclusion: We conclude that acute administration of irisin lowers blood pressure of SHRs by amelioration of endothelial dysfunction of the mesenteric artery via AMPK-Akt-eNOS-NO signaling pathway.

Abstract 002: Macula Densa SGLT1-NOS1-TGF Pathway -- A New Mechanism for Glomerular Hyperfiltration in Diabetes

Glomerular hyperfiltration is a common observation in early diabetes and considered as risk factor for diabetic nephropathy. The mechanisms underlying glomerular hyperfiltration have not been fully clarified. In the present study, we tested a novel SGLT1-NOS1 -TGF (sodium-glucose cotransporter 1 - neuronal nitric oxide synthase – tubuloglomerular feedback) pathway with a hypothesis that tubular glucose is sensed by SGLT1 of the macula densa, which enhances macula densa NOS1 activity and blunts TGF response, therefore increasing glomerular filtration rate (GFR). NO generation by the macula densa, measured by DAF-2 DA in isolated perfused juxtaglomerular apparatus (JGA), increased by 78.4±9.1% when tubular glucose (glucose) was increased from 100 mg/dl to 300 mg/dl (n=4, p<0.01 vs 100 mg/dl). A selective SGLT1 inhibitor KGA-2727 (10 -6 M) blocked this glucose-induced NO generation. Human kidney biopsy tissues were cultured in normal glucose (100 mg/dl) and high glucose (300 mg/dl) media for 30 minutes with hanging drop technique. Protein levels of P-NOS1 ser1417 increased by 63.1±9.2% and P-NOS1 ser847 decreased by 47.4±5.9% (n=4, p<0.01 vs normal glucose) with high glucose. TGF response was significantly reduced from 3.8±0.2 μm to 2.4±0.2 μm (n=4, p<0.05 vs 100 mg/dl), measured in isolated and double perfused JGAs when tubular glucose was increased from 100 mg/dl to 300 mg/dl. The glucose-induced TGF inhibition was blocked in the presence of SGLT1 inhibition (n=4). GFR, measured in conscious mice with a single bolus injection of FITC-inulin, increased by 19.1±3.5% in response to an acute intravenous injection of 50 μl 2M glucose in C57BL/6 mice (n=4, p<0.01 vs baseline). Then, we repeated the experiments in macula densa specific NOS1 KO (KO) mice. Both glucose-induced TGF (ΔP sf was from 7.8±1.3 to 7.3±2.1 mmHg) and GFR (from 223±15.7 to 240±6.9 μl/min) (n=5) were blocked in the KO mice. We conclude that increase of tubular glucose concentration activates NOS1 by phosphorylation of NOS1 ser1417 via SGLT1 in the macula densa, which increases GFR by inhibiting TGF response.

The Favorable Effect of Empagliflozin on Erectile Function in an Experimental Model of Type 2 Diabetes

IntroductionFollowing the results of the EMPA-REG Outcome trial, we hypothesized that empagliflozin, a highly potent and specific sodium/glucose cotransporteur 2 inhibitor, could improve type 2 diabetes mellitus (T2DM)-associated erectile dysfunction (ED), a highly prevalent complication of T2DM, very often coexisting with cardiovascular complications and considered as a prognostic factor of cardiovascular disease in men with diabetes. AimTo investigate the effects of chronic treatment with empagliflozin on ED in a T2DM rat model in the presence or absence of sildenafil. MethodsMale Goto-Kakizaki (GK), a model of T2DM, and age-matched Wistar rats received placebo or empagliflozin treatment at 25.3 ± 0.9 mg/kg/d for 4 weeks. Then, the in vivo effect of empagliflozin on erectile function was assessed by electrical stimulation of the cavernous nerve at different frequencies under anesthesia in the presence or absence of acute intravenous injection of sildenafil. Endothelium-dependent, -independent, and nitrergic relaxations of cavernosal strips from the rats were studied. Main Outcome MeasuresBody weight, food consumption, metabolic parameters, plasma inflammation biomarkers, and in vivo erectile responses elicited by electrical stimulation of the cavernous nerve in empagliflozin-treated and untreated GK rats and control Wistar rats were assessed and followed by concentration or frequency response curves to endothelium-dependent, -independent, and nitrergic relaxations of cavernosal strips from these rats. ResultsChronic empagliflozin followed by acute sildenafil significantly improved erectile responses in adult GK rats (n = 12–15/group). Ratios of intracavernous pressure and area under the curve/mean arterial pressure during the electrical stimulation were significantly increased in empagliflozin-treated vs untreated GK rats. Nitrergic relaxations of cavernosal strips from GK rats were significantly increased with empagliflozin compared with placebo. Moreover, the effect of sildenafil on erectile function was not altered by empagliflozin treatment. Clinical ImplicationsEmpagliflozin may benefit T2DM patient with ED. Strengths & LimitationsThe mechanism(s) by which empagliflozin shows favorable effect on erectile function in GK rats needs to be further elucidated. ConclusionEmpagliflozin shows favorable effect on erectile function in diabetic GK rats mediated by an improvement of nitrergic relaxation of erectile tissue. Whether this favorable effect on ED in the experimental context of T2DM is due to better glycemic control or to another effect of empagliflozin deserves further investigation.Assaly R, Gorny D, Compagnie S, et al. The Favorable Effect of Empagliflozin on Erectile Function in an Experimental Model of Type 2 Diabetes. J Sex Med 2018;15:1224–1234.

From pregnancy to cardiovascular disease: Lessons from relaxin-deficient animals to understand relaxin actions in the vascular system.

Early maternal vascular adaptations to pregnancy are predominantly driven by changes in vascular tone, reactivity, and remodeling. Failure of the maternal systemic vasculature to adapt sufficiently can lead to serious complications of pregnancy. The hormone relaxin is widely recognized for its contribution to the essential renal and systemic hemodynamic adaptations in early pregnancy through direct actions on the maternal vasculature. Studies in relaxin gene knockout mice revealed that endogenous relaxin is not only a "pregnancy hormone" but has pleiotropic actions in various tissues in males and non-pregnant females. There is strong interest in relaxin's actions in the vasculature and its utility in the treatment of vascular diseases. Relaxin treatment in rodents for 2-5days or acute intravenous injection enhances endothelium-dependent relaxation and decreases myogenic tone in resistance arteries. These vascular actions are prolonged, even in the absence of circulating relaxin, and are underpinned by the production of endothelium-derived relaxing factors including nitric oxide, endothelium-derived hyperpolarization, and prostacyclin. Relaxin is also capable of remodeling the vascular wall in a variety of blood vessels in disease conditions. Lessons learned in pregnancy research have aided studies investigating the potential therapeutic potential of relaxin in cardiovascular disease.

Acute or Delayed Systemic Administration of Human Amnion Epithelial Cells Improves Outcomes in Experimental Stroke.

Human amnion epithelial cells (hAECs) are nonimmunogenic, nontumorigenic, anti-inflammatory cells normally discarded with placental tissue. We reasoned that their profile of biological features, wide availability, and the lack of ethical barriers to their use could make these cells useful as a therapy in ischemic stroke. We tested the efficacy of acute (1.5 hours) or delayed (1-3 days) poststroke intravenous injection of hAECs in 4 established animal models of cerebral ischemia. Animals included young (7-14 weeks) and aged mice (20-22 months) of both sexes, as well as adult marmosets of either sex. We found that hAECs administered 1.5 hours after stroke in mice migrated to the ischemic brain via a CXC chemokine receptor type 4-dependent mechanism and reduced brain inflammation, infarct development, and functional deficits. Furthermore, if hAECs administration was delayed until 1 or 3 days poststroke, long-term functional recovery was still augmented in young and aged mice of both sexes. We also showed proof-of-principle evidence in marmosets that acute intravenous injection of hAECs prevented infarct development from day 1 to day 10 after stroke. Systemic poststroke administration of hAECs elicits marked neuroprotection and facilitates mechanisms of repair and recovery.

Skeletal muscle insulin resistance is induced by 4-hydroxy-2-hexenal, a by-product of n-3 fatty acid peroxidation

Aims/hypothesisOxidative stress is involved in the pathophysiology of insulin resistance and its progression towards type 2 diabetes. The peroxidation of n-3 polyunsaturated fatty acids produces 4-hydroxy-2-hexenal (4-HHE), a lipid aldehyde with potent electrophilic properties able to interfere with many pathophysiological processes. The aim of the present study was to investigate the role of 4-HHE in the development of insulin resistance.Methods4-HHE concentration was measured in plasma from humans and rats by GC–MS. Insulin resistance was estimated in healthy rats after administration of 4-HHE using hyperinsulinaemic–euglycaemic clamps. In muscle cells, glucose uptake was measured using 2-deoxy-d-glucose and signalling pathways were investigated by western blotting. Intracellular glutathione was measured using a fluorimetric assay kit and boosted using 1,2-dithiole-3-thione (D3T).ResultsCirculating levels of 4-HHE in type 2 diabetic humans and a rat model of diabetes (obese Zucker diabetic fatty rats), were twice those in their non-diabetic counterparts (33 vs 14 nmol/l, p < 0.001), and positively correlated with blood glucose levels. During hyperinsulinaemic–euglycaemic clamps in rats, acute intravenous injection of 4-HHE significantly altered whole-body insulin sensitivity and decreased glucose infusion rate (24.2 vs 9.9 mg kg−1 min−1, p < 0.001). In vitro, 4-HHE impaired insulin-stimulated glucose uptake and signalling (protein kinase B/Akt and IRS1) in L6 muscle cells. Insulin-induced glucose uptake was reduced from 186 to 141.9 pmol mg−1 min−1 (p < 0.05). 4-HHE induced carbonylation of cell proteins and reduced glutathione concentration from 6.3 to 4.5 nmol/mg protein. Increasing intracellular glutathione pools using D3T prevented 4-HHE-induced carbonyl stress and insulin resistance.Conclusions/interpretation4-HHE is produced in type 2 diabetic humans and Zucker diabetic fatty rats and blunts insulin action in skeletal muscle. 4-HHE therefore plays a causal role in the pathophysiology of type 2 diabetes and might constitute a potential therapeutic target to taper oxidative stress-induced insulin resistance.

Abstract 11804: GsMTx4-D is a Cardioprotectant Against Myocardial Infarction During Ischemia and Reperfusion

Introduction: GsMTx4 is a selective inhibitor of cationic mechanosensitive ion channels (MSCs) and has helped establish the role of MSCs in cardiac physiology. Inhomogeneous local mechanical stresses due to hypercontracture and swelling, likely induce elevated MSC activity that can contribute to the cation imbalance observed during ischemic reperfusion injury. This study was to determine if the D enantiomer of GsMTx4 can act as a cardioprotectant during ischemia and reperfusion and to evaluate its effects on cation influx and inflammatory kinases that are regulated by this influx. Hypothesis: GsMTx4 protects heart from ischemic insults through inhibiting the cationic mechanosensitive ion channels. Methods: Ischemia and reperfusion in the mouse heart involved ligating a coronary artery followed by release of the ligature. Results: GsMTx4-D was administered by acute intravenous injection during the ischemic event. Based on pharmacokinetic studies, GsMTx4-D dosage was set to achieve expected plasma concentrations between 50-5000 nM and heart tissue concentrations between 1-200 nM. Relative to vehicle injected animals, GsMTx4-D reduced infarct area by ~40% for acute and pretreated animals, and for both 20 and 45 min ischemic challenges. Many indicators of cardiac output were indistinguishable from sham-treated control hearts after GsMTx4-D treatment and most showed improvement at both 4 and 48 hrs post ischemia. Premature ventricular beats immediately following reperfusion were also significantly reduced by intravenous injections of GsMTx4-D. Myocytes cultured under hypoxic conditions treated with 10 μM GsMTx4-D showed improved contractility and near normal contraction-related Ca 2+ influx. GsMTx4-D inhibited indicators of ischemic damage such as the apoptotic signaling system JNK/c-Jun, but also inhibited the energy response signaling system Akt kinase. Conclusions: GsMTx4-D is a potent cardioprotectant that decreases infarct area, increases cardiac output and decreases arrhythmias that are caused by ischemia and reperfusion. GsMTx4-D improved cardiomyocyte function and suppresses apoptosis, making it potentially useful in multidrug strategies to treat ischemic heart disease.

Irisin Lowers Blood Pressure by Improvement of Endothelial Dysfunction via AMPK-Akt-eNOS-NO Pathway in the Spontaneously Hypertensive Rat.

BackgroundExercise is a major nonpharmacological treatment for hypertension, but its underlying mechanisms are still not completely elucidated. Irisin, a polypeptide containing 112 amino acids, which is secreted mainly by skeletal muscle cells during exercise, exerts a protective role in metabolic diseases, such as diabetes mellitus and obesity. Because of the close relationship between irisin and metabolic diseases, we hypothesized that irisin may play a role in the regulation of blood pressure.Methods and ResultsBlood pressures of male Wistar‐Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were monitored through the carotid artery. Our study found that acute intravenous injection of irisin reduced blood pressure in SHRs, but not WKY rats. Irisin, by itself, had no direct vasorelaxing effect in phenylephrine‐preconstricted mesenteric arteries from SHRs. However, irisin augmented the acetylcholine‐induced vasorelaxation in mesenteric arteries from SHRs that could be reversed by Nω‐nitro‐l‐arginine‐methyl ester (L‐NAME; 100 μmol/L), indicating a role of nitric oxide (NO) in this action. Indeed, irisin increased NO production and phosphorylation of endothelial nirtic oxide synthase (eNOS) in endothelial cells. 5′‐AMP‐activated protein kinase (AMPK) was involved in the vasorelaxing effect of irisin because compound C (20 μmol/L), an AMPK inhibitor, blocked the irisin‐mediated increase in phosphorylation of eNOS and protein kinase B (Akt) in endothelial cells and vasodilation in mesenteric arteries.ConclusionsWe conclude that acute administration of irisin lowers blood pressure of SHRs by amelioration of endothelial dysfunction of the mesenteric artery through the AMPK‐Akt‐eNOS‐NO signaling pathway.

GsMTx4-D is a cardioprotectant against myocardial infarction during ischemia and reperfusion

GsMTx4 is a selective inhibitor of cationic mechanosensitive ion channels (MSCs) and has helped establish the role of MSCs in cardiac physiology. Inhomogeneous local mechanical stresses due to hypercontracture and swelling during ischemic reperfusion injury (IRI) likely induce elevated MSC activity that can contribute to cation imbalance. The aim of this study was to determine if the D enantiomer of GsMTx4 can act as a cardioprotectant in a mouse IRI model. Ischemia and reperfusion involved ligating a coronary artery followed by release of the ligature. GsMTx4-D was tested by either acute intravenous injection during the ischemic event or by two day pretreatment by intraperitoneal injection, both methods achieving similar results. Based on pharmacokinetic studies, GsMTx4-D dosage was set to achieve expected plasma concentrations between 50 and 5000nM and heart tissue concentrations between 1 and 200nM by intravenous injection. Relative to vehicle injected animals, GsMTx4-D reduced infarct area by ~40% for acute and pretreated animals for both 20 and 45min ischemic challenges. Many indicators of cardiac output were indistinguishable from sham-treated control hearts after GsMTx4-D treatment showing improvement at both 4 and 48h post ischemia, and premature ventricular beats immediately following reperfusion were also significantly reduced. To determine if GsMTx4-D cardioprotection could act directly at the level of cardiomyocytes, we tested its effects in vitro on indicators of IRI damage like cation influx and activation of inflammatory kinases in isolated myocytes cultured under hypoxic conditions. Hypoxia challenged cardiomyocytes treated with 10μM GsMTx4-D showed improved contractility and near normal contraction-related Ca2+ influx. GsMTx4-D inhibited indicators of ischemic damage such as the apoptotic signaling system JNK/c-Jun, but also inhibited the energy response signaling system Akt kinase. We conclude that GsMTx4-D is a potent cardioprotectant in vivo that may act directly on cardiomyocytes and potentially be useful in multidrug strategies to treat IRI.