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Abstract: Studies have examined the therapeutic effect of levosimendan on cardiovascular diseases such as heart failure, perioperative cardiac surgery, and septic shock, but the specific mechanism in mice remains largely unknown. This study aimed to investigate the relaxation mechanism of levosimendan in the thoracic aorta smooth muscle of mice. Levosimendan-induced relaxation of isolated thoracic aortic rings that were precontracted with norepinephrine or KCl was recorded in an endothelium-independent manner. Vasodilatation by levosimendan was not associated with the production of the endothelial relaxation factors nitric oxide and prostaglandins. The voltage-dependent K + channel (K V ) blocker (4-aminopyridine) and selective K Ca blocker (tetraethylammonium) had no effect on thoracic aortas treated with levosimendan, indicating that K V and K Ca channels may not be involved in the levosimendan-induced relaxation mechanism. Although the inwardly rectifying K + channel (K ir ) blocker (barium chloride) and the K ATP channel blocker (glibenclamide) significantly inhibited levosimendan-induced vasodilation in the isolated thoracic aorta, barium chloride had a much stronger inhibitory effect on levosimendan-induced vasodilation than glibenclamide, suggesting that levosimendan-induced vasodilation may be mediated by K ir channels. The vasodilation effect and expression of K ir 2.1 induced by levosimendan were further enhanced by the PKC inhibitor staurosporine. Extracellular calcium influx was inhibited by levosimendan without affecting intracellular Ca 2+ levels in the isolated thoracic aorta. These results suggest that K ir channels play a more important role than K ATP channels in regulating vascular tone in larger arteries and that the activity of the K ir channel is enhanced by the PKC pathway., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Abstract: Paracetamol (PAR) is the most common over-the-counter drug recommended by physicians for treatment of pain and fever during gestation. This drug is not teratogenic, being considered safe for fetus; however, PAR crosses the blood-placental barrier. Considering that, the present study aimed to evaluate the vascular and metabolic safety of PAR exposure during intrauterine and neonatal development in adult male and female-exposed offspring. Wistar female rats were gavaged, with PAR (350 mg/kg/d), from gestational day 6-21 or from gestational day 6 until postnatal day 21. Control dams received water by gavage at the same periods. The male and female offspring were evaluated at adulthood (80 days of life). The thoracic aorta reactivity to acetylcholine, sodium nitroprusside, and phenylephrine was evaluated in male and female adult offspring. It was observed that aortic relaxation was similar between the PAR and control offspring. In addition, the contraction to phenylephrine was similar between the groups. Further, the insulin sensitivity, adipose tissue deposition and blood pressure were not different between PAR and control adult offspring. These results suggest that the protocol of PAR exposure used in the present study did not program vascular and metabolic alterations that would contribute to the development of cardiometabolic diseases in adult life, being safe for the exposed offspring., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The Qingda granule (QDG) formulation was simplified from the Qingxuan Jiangya Decoction, which has been used in China to treat hypertension for decades. However, the molecular mechanisms of QDG in antihypertension remain largely unknown. Therefore, we evaluated the therapeutic efficacy of QDG against elevated blood pressure and explored its underlying mechanism. QDG treatment decreased elevated blood pressure and increased the vascular elasticity of thoracic aortic rings to KCl stimulation in spontaneously hypertensive rats. QDG treatment increased the relaxation of isolated thoracic aortic rings precontracted with norepinephrine (NE) or KCl in an endothelium-independent manner, which was attenuated by treatment with verapamil, but not by treatment with TEA, 4-AP, Gli, or BaCl2. Moreover, QDG pretreatment attenuated the CaCl2-induced constriction of isolated thoracic aortic rings in K- or NE-containing Ca-free solutions. In addition, QDG pretreatment significantly inhibited the influx of Ca in A7r5 cells induced by a K- or NE-containing Ca solution and decreased the levels of p-AKT but had no effect on levels of total AKT protein in isolated thoracic aortic rings. Considering these results, QDG treatment attenuated elevated blood pressure and promoted the vasorelaxation of thoracic aortic rings by inhibiting the influx of Ca and activating the AKT pathway.

Chronic exposure to cold causes arterial hypertension [cold-induce hypertension (CIH)]. Emerging data have indicated that gut barrier dysfunction is involved in the pathogenesis of hypertension. In this study, we explored the effect of gut barrier dysfunction on vascular inflammation induced by cold exposure and the therapeutic effect of atorvastatin in a CIH rat model. The CIH was established by cold exposure for 2 weeks. Two groups of Sprague Dawley rats were exposed to moderate cold (4 ± 1°C), whereas the control group was maintained at room temperature (23 ± 1°C) (10 rats/group). The 2 groups received atorvastatin or vehicle at the beginning of cold exposure, respectively, for 2 weeks. Cold exposure increased mean arterial pressure compared with room temperature group, indicating that animals developed arterial hypertension. Cold exposure induced vascular dysfunction due to decreasing phosphorylated endothelial nitric oxide synthase protein expression in aorta, and these were blunted by atorvastatin. Cold exposure increased the levels of gut-derived inflammatory cytokines, tumor necrosis factor-α, and interleukin-6 production in aorta and resulted in vascular inflammation, whereas atorvastatin prevented these effects. Cold exposure also increased gut permeability, inhibited tight junction protein expression in proximal colon, and resulted in gut barrier dysfunction. Interestingly, atorvastatin eliminated increasing gut permeability, decreasing tight junction protein expression, and gut pathology and reversed gut barrier dysfunction. Atorvastatin attenuated CIH and improved gut barrier function; the beneficial effects might be via inhibiting gut-derived inflammatory cytokines and reversing cold-induced vascular inflammation, suggesting that gut barrier dysfunction may be involved in the pathogenesis of CIH.

This study was designed to characterize the pharmacological profile of DS37001789, which is a structurally novel piperazine derivative that acts as urotensin II (U-II) receptor antagonist. DS37001789 inhibited [I]-U-II binding to human GPR14, U-II receptor, with an IC50 value 0.9 nM. Its potency was superior to that of ACT-058362, a nonpeptide U-II receptor antagonist whose IC50 was 120 nM. Human U-II-induced vascular contraction was blocked by DS37001789. The dose-response curve of DS37001789 in rats and monkeys did not show species differences, and it shifted to the right without any effects on the maximum vascular response. Moreover, orally administered DS37001789 dose-dependently prevented human U-II-induced blood pressure elevation in mice, and this effect was significant at dose and higher dose (30 and 100 mg/kg), and its potency was superior to that of ACT-058362 (100 mg/kg). These results suggest that DS37001789 is a highly potent U-II receptor antagonist both in vitro and in vivo, with no marked species difference. DS37001789 would be a useful tool to clarify the physiological roles of U-II/GPR14 system. In addition, it can serve as a novel therapeutic agent for diseases in which the U-II/GPR14 system is upregulated, such as hypertension, heart failure, renal dysfunction, and diabetes.

Antihypertensive pharmacological treatments focus on the use of angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists, and beta-blockers as single and combined treatments. The effect of single treatments on the mRNA expression of some components of the renin-angiotensin system has been studied, but not the effect of combined treatments. This study determined the expression of the AT1, AT2, B1, and B2 receptors and of the enzymes ACE and ACE2 in hypertensive rats treated with captopril-propranolol or losartan-propranolol. Methods: The mRNA expression of the receptors and enzymes was determined by reverse transcription-quantitative polymerase chain reaction in the aorta of spontaneously hypertensive rats under different treatments. Results: Rats under combined treatments showed a decrease in the expression of AT1 and ACE, and an increase in the expression of the B1 receptor (captopril + propranolol group: 0.43 ± 0.046, 2.243 ± 0.269, 3.356 ± 0.418; Group: losartan + propranolol: 0.727 ± 0.071, 0.852 ± 0.102, 1.277 ± 0.131 compared to the spontaneously hypertensive group: 1 ± 0.212, 1 ± 0.192, 1 ± 0.214). This decrease in the expression of ACE and AT1 suggests a reduction in the expression of Ang II that could be related to a lower response to this vasoconstrictor. An increase in the expression of B1 would improve vasodilation, which would be a beneficial effect of combined therapies for hypertension.

We investigated whether resveratrol (RSV) can attenuate obesity and diabetes progression and improve diabetes-induced vascular dysfunction, and we attempted to delineate its underlying mechanisms. Male C57Bl/6 mice were administered a high-fat diet (HFD) for 17 weeks. Mice developed type 2 diabetes with increased body weight, hyperglycemia, hyperinsulinemia, and hyperlipidemia. Oral gavage with RSV significantly reversed the symptoms induced by the HFD. Insulin sensitivity likewise improved after the RSV intervention in these mice. Phenylephrine-induced cremaster arteriolar constriction was impaired, whereas RSV treatment significantly mitigated the vessel responsiveness to phenylephrine. The obese diabetic mice exhibited increased leukocyte rolling, adhesion, and transmigration in the postcapillary venules of the cremaster muscle. By contrast, RSV treatment significantly attenuated HFD-induced extravasation. RSV significantly recovered phosphorylated Akt and eNOS expression in the thoracic aorta. In addition, activated adenosine monophosphate-activated protein kinase in the thoracic aorta was involved in the improvement of epithelial function after RSV intervention. RSV considerably upregulated the plasma NO level in HFD mice. Moreover, RSV-enhanced human umbilical vein endothelial cells healing through Sirt1/ER pathway may be involved in the prevention of leukocyte extravasation. Collectively, RSV attenuates diabetes-induced vascular dysfunction by activating Akt/eNOS/NO and Sirt1/ER pathway. Our mechanistic study provides a potential RSV-based therapeutic strategy against cardiovascular disease.

Heat shock proteins (HSPs) play an important role in the cellular adaptation to stress, a requisite for cell survival. The aortic wall appears to be a target for increased expression of HSPs during surgical stress. We aimed to define the expression and function of aortic HSP70 in 31 patients with normal ascending thoracic aortic diameter who underwent aortic valve replacement due to aortic valve stenosis and in 35 patients with dilated ascending thoracic aorta who underwent replacement of an ascending thoracic aortic aneurysm. To elucidate responsible signaling mechanisms we used an in vitro model of rat hypoxic aortic vascular smooth muscle cell (AVSMC) cultures. We demonstrated an increase in AVSMC HSP70 and an attenuation of the apoptotic markers (TUNEL-positive nuclei, caspase-3 activity, Bax/Bcl2 ratio) in aortic wall tissue specimens from both aortic valve stenosis and ascending thoracic aortic aneurysm patients on β1 blockade with metoprolol. In vitro, metoprolol treatment of hypoxic rat AVSMCs increased nitric oxide (NO) production, induced heat shock factor 1 transport to the nucleus, upregulated HSP70, decreased p53 phosphorylation and attenuated apoptosis. Blockade of NO production, resulted in decreased HSP70 and prevented the metoprolol-induced anti-apoptotic response of hypoxic AVSMCs. We demonstrate an anti-apoptotic effect of metoprolol dependent on NO-induced HSP70 expression, and thus augmentation of HSP70 expression should be considered as a therapeutic approach to limit apoptosis in the human ascending thoracic aorta of patients undergoing cardiac surgery.

Abnormal expression of microRNAs (miRNAs) has been associated with aortic dissection (AD). Next-generation sequencing was performed to identify the differentially expressed miRNAs in aortic tissue samples between AD and nondiseased individuals. Selected miRNAs, which showed significant variation between the 2 groups, were then transfected into human aortic vascular smooth muscle cells, and assessed for effects on cell migration and induced apoptosis. The changes in gene expression pattern in human aortic vascular smooth muscle cells transfected with the miRNAs were also investigated. Among the 314 miRNAs detected in the aortic tissues from both AD and normal subjects, 46 showed significantly different expression patterns. Only 7 of these differentially expressed miRNAs were found to be enriched in AD, whereas the majority had diminished. hsa-miR-320d and hsa-miR-582 were 2 representative miRNAs that exhibited a decrease of greater than 10-fold. Transfection of hsa-miR-320d and hsa-miR-582 did not affect the migration capability of the vascular smooth muscle cells, but remarkably enhanced the staurosporine and tumor necrosis factor-α-induced apoptosis by 15% and 29%, respectively. Furthermore, the transfection of both miRNAs affected the expression of a vast multitude of genes, most of which were related to apoptotic pathways. The fluorescence reporter assays demonstrated that hsa-miR-320d and hsa-miR-582 bind the 3' UTR region of TRIAP1 and NET1 genes, respectively. These results suggest that hsa-miR-320d and hsa-miR-582 may serve as putative biomarkers for AD research.

Metformin has been used for the treatment of some metabolic diseases during gestation and the beneficial effects of metformin to the vascular system have been described in diabetic and obese animal models. Nevertheless, the long-term consequences to the vascular system of offsprings maternally exposed to metformin have not yet been characterized. Therefore, we want to test the hypothesis that gestational and lactational exposure to metformin would be safe for the vascular reactivity of male adult offsprings. Wistar female rats were treated with metformin 293 mg·kg·d, by gavage, from gestational day (GD) 0 to GD 21 (METG) or GD 0 until postnatal day 21 (METGL). Control dams received water by gavage in the same periods (CTRG and CTRGL). In male offsprings (75 days), the aortic reactivity to phenylephrine, acetylcholine, and sodium nitroprusside in the presence or absence of endothelium were evaluated. The results demonstrated that aortic contraction and relaxation were similar between groups. These data showed that metformin exposure during pregnancy and lactation did not interfere with aortic reactivity, suggesting that metformin exposure during gestational and lactation are safe for the offsprings' vascular system.

Purpose: Roscovitine (Rosc) is a selective inhibitor of cyclin-dependent kinases (CDKs) and a promising therapy for various cancers. However, limited information is available on the biological significance of Rosc in vascular smooth muscle cells (VSMCs), the cell type critical for the development of proliferative vascular diseases. In this study, we address the effects of Rosc in regulating VSMC proliferation, both in vitro and in vivo, exploring the underlying molecular mechanisms., Methods: The proliferations and cell-cycle distributions of in vitro cultured VSMCs, as well as several other cancer cell lines, were examined by cell-counting assay and flow cytometry, respectively. Molecular changes in various CDKs, cyclins, and other regulatory molecules were examined by reverse transcription polymerase chain reaction, Western blot, or immunocytochemistry. The in vivo effects of Rosc were examined on a carotid arterial balloon-injury model., Results: Rosc significantly inhibited VSMC proliferation in response to serum or angiotensin II and arrested these cells at the G0/G1 phase. These changes were associated with a specific and robust decrease in CDK4, cyclin E, c-Jun, and a dramatic increase in p27kip1 in VSMCs, which was also translated in vivo and correlated with the protection of Rosc on injury-induced neointimal hyperplasia., Conclusions: Acting on distinct molecular targets in VSMCs versus cancer cells, Rosc inhibits VSMC proliferation and protects from proliferative vascular diseases.

Numerous studies have found that the age-associated structural and functional alterations in arteries were characterized by increased endothelial dysfunction. In this study, young (3 months), adult (9 months), and aging (20 months) male Sprague-Dawley rats were randomly divided into 6 groups, including control groups and FeTMPyP (peroxynitrite scavenger) groups receiving saline and FeTMPyP, respectively, for 5 administrations once every 3 days through intraperitoneal injection. The aged-related proteins beta-galactosidase, p53, and p16 as well as the nitrotyrosine and endothelial marker endothelial nitric oxide synthase and von Willebrand factor (vWF) in vascular tissues were measured by immunohistochemistry. Endothelium-dependent vasorelaxation and endothelium-independent vasorelaxation of rat thoracic aortas and mesenteric arteries were measured by acetylcholine and sodium nitroprusside, respectively. The amount of circulating endothelial progenitor cells (EPCs) was determined by flow cytometry. The endothelium-dependent/independent relaxation in mesenteric arteries and the amount of circulating EPCs (CD31/CD34) in peripheral blood of aging rats were reduced significantly compared with young and adult rats. Immunohistochemistry results showed that the nitrotyrosine levels and morphological damage in mesenteric arteries were increased significantly in aging rats. Adoption of peroxynitrite scavenger FeTMPyP intervention may not only improve the endothelium-dependent relaxation and the amount of circulating EPCs in aging rats but also reverse endothelial injury. In conclusion, this study demonstrates that enhanced nitrative stress may aggravate the endothelial injury and vascular dysfunction of resistance arteries in aging rats. Antiperoxynitrite treatment can ameliorate the vasorelaxation and may be involved with the protection of circulating EPCs.

The sprouting of new vessels is greatly influenced by the procedure chosen. We sought to optimize the experimental conditions of the angiogenic growth of fresh and cryopreserved vessels cultured in Matrigel with the aim to use this system to analyze the pharmacological modulation of the process. Segments of second-order branches of rat mesenteric resistance arteries, thoracic aorta of rat or mouse, and cryopreserved rat aorta and human femoral arteries were cultured in Matrigel for 7-21 days in different mediums, as well as in the absence of endothelial or adventitia layer. Quantification of the angiogenic growth was performed by either direct measurement of the mean length of the neovessels or by calcein AM staining and determination of fluorescence intensity and area. Fresh and cryopreserved arterial rings incubated in Matrigel exhibited a spontaneous angiogenic response that was strongly accelerated by fetal calf serum. Addition of vascular endothelial growth factor, fibroblast growth factor, endothelial growth factor, or recombinant insulin-like growth factor failed to increase aortic sprouting, unless all were added together. Removal of adventitia, but not the endothelial layer, abrogated the angiogenic response of aortic rings. Determination of the mean neovessel length is an easy and accurate method to quantify the angiogenic growth devoid of confounding factors, such as inclusion of other cellular types surrounding the neovessels. Activity of a α1-adrenoceptor agonist (phenylephrine) and its inhibition by a selective antagonist (prazosin) were analyzed to prove the usefulness of the Matrigel system to evaluate the pharmacological modulation of the angiogenic growth.

Treatment with low, subtherapeutic doses of statins and sartans expresses beneficial pleiotropic effects on the arterial wall. The present study explored whether these effects depend on treatment duration. Wistar rats were randomly divided into 4 groups and received low-dose atorvastatin, low-dose losartan, their combination, or saline (control) daily. After 4, 6, 8, and 10 weeks of treatment, the animals were anesthetized, blood samples taken, and hearts and thoracic aortas isolated. Thoracic aorta endothelium-dependent relaxation and parameters of the isolated heart exposed to ischemic-reperfusion injury were assessed along with blood serum parameters and vasoactive genes expression. Low-dose atorvastatin, losartan, and especially their combination showed the characteristic time dependency of all studied parameters (thoracic aorta relaxation, isolated heart parameters, C-reactive protein values, genes encoding endothelial nitric oxide synthase, and CD40). The peak in efficacy was observed after 6 weeks of treatment and subsequently steadily declined. The peak versus control values were significant for all measured parameters. Only a combination of atorvastatin and losartan increased nitric oxide and decreased asymmetric dimethylarginine. A characteristic time-dependent "rise-peak-fall" pattern of the cardiovascular pleiotropic effects of statins and sartans in subtherapeutic low doses was revealed. Evidently, resistance to the explored treatment occurs after a certain period.

We evaluated the antihypertensive properties of 4-tert-buthyl-2,6-bis(thiomorpholine-4-ilmethyl)phenol (TBTIF). Spontaneously hypertensive rats were treated with TBTIF or captopril (both at 1 mg·kg⁻¹·d⁻¹ intramuscularly for 4 days), and their blood pressure (BP) was assessed. In some experiments, concentration response curves to angiotensin I or angiotensin II were generated in rat aortic rings and in the absence or presence of Ang-(1-7), N(G)-monomethyl L-arginine, or both; additionally, the angiotensin-converting enzyme (ACE) and ACE2 mRNA levels were quantified in the aortic rings using reverse transcription-polymerase chain reaction. TBTIF diminished BP and reduced angiotensin I- or angiotensin II-induced vasoconstriction. The presence of Ang-(1-7) induced a greater reduction in vasoconstriction, and this effect was reversed by L-N(G)-monomethyl arginine. Moreover, TBTIF decreased the mRNA of ACE and increased the mRNA of ACE2. In conclusion, TBTIF diminished rat BP through nitric oxide-dependent and nitric oxide-independent mechanisms. In contrast to captopril, TBTIF exhibits better antihypertensive properties through mechanisms that involve ACE2.

The present investigation examined the effect of pregnancy on the anticontractile effect of perivascular adipose tissue (PVAT) on the rat. Ring segments of the aorta, with and without PVAT, were set up in organ baths for isometric tension recording. In both groups, concentration-response curves to 5-hydroxytryptamine (5-HT) were displaced to the right with a reduction of the maximum response in aorta segments with PVAT. The anticontractile effect of PVAT was attenuated on segments from pregnant rats. 4-Aminopyridine (4-AP), an inhibitor of voltage-gated potassium (Kv) channels, enhanced 5-HT-induced contractions of aorta segments from pregnant and nonpregnant rats only when PVAT was attached. There was no difference in the effect of 4-aminopyridine on 5-HT-induced contractions of aorta segments with PVAT from pregnant and nonpregnant rats. There was also no significant difference in the expression of Kv7.4 channels in aorta segments (with PVAT) between pregnant and nonpregnant rats. Tumor necrosis factor-α (TNF-α) was detected in PVAT from pregnant and nonpregnant rats. The level of TNF-α was significantly greater in PVAT from pregnant rats. Treatment of pregnant rats with pentoxyphyline significantly reduced the level of TNF-α in the PVAT and restored the anticontractile effect of PVAT on aorta segments from pregnant rats. Finally, TNF-α (10 ng/mL) potentiated 5-HT-induced contractions of PVAT-containing pregnant rat aorta. These results would suggest that the loss of anticontractile effect of PVAT in pregnant rat aorta could be due to enhanced production of TNF-α in the PVAT in these rats.

Introduction: The goal of this study was to characterize the role of Endothelin (ET) type B receptors (ETB) on vascular function in healthy and diseased conditions and demonstrate how it affects the pharmacological activity of ET receptor antagonists (ERAs)., Methods: The contribution of the ETB receptor to vascular relaxation or constriction was characterized in isolated arteries from healthy and diseased rats with systemic (Dahl-S) or pulmonary hypertension (monocrotaline). Because the role of ETB receptors is different in pathological vis-à-vis normal conditions, we compared the efficacy of ETA-selective and dual ETA/ETB ERAs on blood pressure in hypertensive rats equipped with telemetry., Results: In healthy vessels, ETB receptors stimulation with sarafotoxin S6c induced vasorelaxation and no vasoconstriction. In contrast, in arteries of rats with systemic or pulmonary hypertension, endothelial ETB-mediated relaxation was lost while vasoconstriction on stimulation by sarafotoxin S6c was observed. In hypertensive rats, administration of the dual ETA/ETB ERA macitentan on top of a maximal effective dose of the ETA-selective ERA ambrisentan further reduced blood pressure, indicating that ETB receptors blockade provides additional benefit., Conclusions: Taken together, these data suggest that in pathology, dual ETA/ETB receptor antagonism can provide superior vascular effects compared with ETA-selective receptor blockade.

Although drug-eluting stents (DES) effectively improve the clinical efficacy of percutaneous coronary intervention, a high risk of late stent thrombosis and in-stent restenosis also exists after DES implantation. Anti-smooth muscle proliferation drugs, such as rapamycin, coating stents, not only inhibit the growth of vascular smooth muscle cells but also inhibit vascular endothelial cells and delay the reendothelialization. Therefore, the development of an ideal agent that protects vascular endothelial cells from rapamycin-eluting stents is of great importance for the next generation of DES. In this study, we demonstrated that rapamycin significantly inhibited the growth of rat aortic endothelial cells in both dose- and time-dependent manner in vitro. Cell apoptosis was increased and migration was decreased by rapamycin treatments in rat aortic endothelial cells in vitro. Surprisingly, treatment with curcumin, an active ingredient of turmeric, significantly reversed these detrimental effects of rapamycin. Moreover, curcumin increased the expression of vascular nitric oxide synthases (eNOS), which was decreased by rapamycin. Furthermore, caveolin-1, the inhibitor of eNOS, was decreased by curcumin. Knockdown of eNOS by small interfering RNA significantly abrogated the protective effects of curcumin. Taken together, our results suggest that curcumin antagonizes the detrimental effect of rapamycin on aortic endothelial cells in vitro through upregulating eNOS. Therefore, curcumin is a promising combined agent for the rescue of DES-induced reendothelialization delay.

Protein kinase C (PKC) isoforms improve endothelial nitric oxide synthase activity and contractile Ca sensitivity in blood vessels. These actions may have opposite effects on propofol-induced vasodilation. This study examines the hypothesis that propofol induces relaxation by enhancing the PKC-mediated nitric oxide synthesis in endothelium and/or inhibiting the PKC-regulated Ca sensitivity in vascular smooth muscle (VSM). Propofol (1-100 μM) induced greater relaxation in endothelium-intact rings compared with denuded rings, and this effect was antagonized by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In contrast, treatment with the general PKC inhibitor GF-109203X augmented both the endothelium-dependent and endothelium-independent relaxation induced by propofol, and this enhancement was more profound in the intact rings at lower propofol concentrations. The enhancement was unaffected by L-NAME. Interestingly, calphostin C (an inhibitor of conventional and novel PKCs) and Gö-6976 (an inhibitor of conventional PKCs) had similar effects in augmenting propofol-induced relaxation in endothelium-denuded rings. Downregulation of novel isoforms not only reduced the norepinephrine-elicited contraction but also decreased the magnitude of propofol-induced relaxation. In vascular smooth muscle cells, propofol prevented norepinephrine-elicited phosphorylation of myosin light chain. Propofol can increase the PKC-mediated availability of nitric oxide but inhibit the novel PKC-regulated Ca-sensitization, which provides a novel explanation for the mechanism of propofol-induced vasodilation.

Ca is a crucial factor in the regulation of smooth muscle contraction. Store-operated Ca entry (SOCE) is one pathway that mediates Ca influx and smooth muscle contraction. Vessel contraction function usually alters with aging to cause severe vascular-related diseases. However, the underlying mechanism is still not fully understood. Here, we assessed intracellular Ca and vessel tension and found that SOCE and SOCE-mediated contraction of vascular smooth muscle cells (VSMCs) was reduced in aorta but increased in mesenteric arteries from aged rats. The results of Western blot and immunofluorescence staining show that the expression levels of Orai1, a store-operated Ca channel, were increased in VSMCs of mesenteric arteries but were reduced in VSMCs of aorta with aging. In conclusion, we demonstrated that the changing pattern of SOCE and SOCE-mediated contraction of VSMCs is completely reversed in mesenteric arteries and aorta with aging, providing a potential therapeutic target for clinical treatment in age-related vascular diseases.

Background: S-equol is known as an estrogenic substance, but its ability to restore vascular endothelial function is unknown. The aim of this study was to investigate the impact of S-equol on endothelial function and intimal thickening under isoflavone- and estrogen-deficient circumstances., Methods: Twelve-week-old female Sprague-Dawley rats were bilaterally ovariectomized and assigned to one of the 3 groups: control, isoflavone-deficient (ID), or ID plus equol (n = 12, respectively). The control group received a normal diet containing isoflavones, while ID and ID plus equol groups received isoflavones-free diet. At 16th week, subcutaneous administration of S-equol (200 μg/d) started in the ID plus equol group. At 18th week, endothelial denudation of the left common carotid artery was performed in all groups, and thoracic and carotid arteries were collected at 20th week., Results: In thoracic artery, endothelium-dependent relaxation, cyclic guanosine monophosphate levels in the tissue, and endothelial nitric oxide (NO) synthase expression and phosphorylation were significantly higher in the groups of ID plus equol and control than in the ID. The ratio of intima to media of the injured carotid artery in the control group was the lowest., Conclusions: Removal of dietary soy isoflavones decreased endothelium-derived NO level in ovariectomized rats. S-equol supplementation partially improved NO-related endothelial function.

Abnormal vascular smooth muscle cell proliferation and migration are key factors in many cardiovascular diseases. Here, we investigated the effects of phloretin on platelet-derived growth factor homodimer (PDGF-BB)-induced rat aortic smooth muscle cell (RASMC) proliferation, migration, and neointimal formation after carotid injury. Phloretin significantly inhibited the PDGF-BB-stimulated RASMC proliferation in a concentration-dependent manner (10-100 μM). Also, PDGF-BB-stimulated RASMC migration was inhibited by phloretin at 50 μM. Pretreating RASMC with phloretin dose-dependently inhibited PDGF-BB-induced Akt and p38 mitogen-activated protein kinases activation. Furthermore, phloretin increased p27 and decreased cyclin-dependent kinase 2, CDK4 expression, and p-Rb activation in PDGF-BB-stimulated RASMC in a concentration-dependent manner (10-50 μM). PDGF-BB-induced cell adhesion molecules and matrix metalloproteinase-9 expression were blocked by phloretin at 50 μM. Preincubation with phloretin dose-dependently reduced the intracellular reactive oxygen species production. In vivo study showed that phloretin (20 mg/kg) significantly reduced neointimal formation 14 days after carotid injury in rats. Thus, phloretin may have potential as a treatment against atherosclerosis and restenosis after vascular injury.

This study investigated whether 3,4-Dihydroxyacetophenone (DHAP) could improve endothelial function in streptozotocin-induced type 2 diabetic rats. Sprague-Dawley rats were randomly divided into control, diabetic, and diabetic DHAP-treated animals. After treatment with DHAP for 8 weeks, endothelial function was determined by measuring endothelium-dependent vasodilatation (EDV) of the thoracic aorta. Endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production in endothelial cells and nuclear transcription factor kappa B (NF-κB) expression and superoxide anion production in the aorta were determined. DHAP treatment reduced serum levels of triglycerides, cholesterol, malondialdehyde, and tumor necrosis factor α, and enhanced serum adiponectin levels. Endothelium-dependent vasodilatation was significantly attenuated in rats with diabetes and increased significantly after DHAP treatment. NO levels and eNOS activity in endothelial cells were significantly reduced, and NF-κB activation and superoxide production increased in rats with diabetes compared with the control group. DHAP treatment enhanced NO levels and eNOS activity and decreased NF-κB activation and superoxide production. These findings suggest that DHAP could improve endothelial function in streptozotocin-induced type 2 diabetic rats. The mechanism may be related to the enhancement of eNOS activity and NO production by reducing plasma lipid levels, oxidative stress, and inflammatory activity.

Recent results indicate that the reduction of β-adrenergic signaling impairs angiogenesis under ischemic conditions. Because angiogenesis may occur in the absence of ischemia, it remains to be determined whether and how β-adrenergic signaling regulates angiogenesis, which develops under normoxic conditions. The effect of β-adrenergic ligands on angiogenesis was investigated using 3-dimensional cultures of mouse aortic rings embedded in collagen type I, in which luminized microvessels develop in response to vascular endothelial growth factor (VEGF). Under normoxic conditions, both isoproterenol, a β-adrenergic receptor (β-AR) agonist, and forskolin, an adenylate cyclase activator, were unable to influence aortic microvessel sprouting. On the contrary, treatment with propranolol, a β-AR antagonist, caused an approximately 70% increase in VEGF-mediated microvessel sprouting. This effect was abolished in rings from both double β-AR and β1-AR knockout mice, but not in rings from β2-AR knockout mice. Significant increases in microvessel sprouting were also observed when mouse aortic rings from C57BL/6 mice were treated with the β1-AR-selective antagonists metoprolol and bisoprolol or with the β2-AR-selective antagonist ICI 118,551. Conversely, carvedilol, a nonselective β-AR antagonist, was unable to affect aortic sprouting. These findings suggest that some β-blockers display proangiogenic activity through a mechanism that is independent of their ability to antagonize catecholamine action. The present results also identify a new function for β-AR signaling as a facilitator for VEGF-mediated angiogenesis and have implications for understanding the mechanisms that regulate angiogenic responses under normoxic conditions.

Background: Phenotypic switching of vascular smooth muscle cells (VSMCs) plays an initial role in neointimal hyperplasia, the main cause of many occlusive vascular diseases. The aim of this study was to measure the effects of resveratrol (RSV) on the phenotypic transformation of VSMCs and to investigate its mechanism of action., Methods: Cultured VSMCs isolated from rat thoracic aorta were prepared with serum starvation for 72 hours followed by RSV treatment (50-200 μmol/L) and 10% serum stimulation. Male Sprague-Dawley rats, subjected to carotid arteries injury from a balloon catheter, were exposed to intraperitoneal injection of RSV (1 mg/kg) or saline and were killed after 7 or 28 days., Results: Compared with cells in the serum-induced group, VSMCs in the RSV or N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment group exhibited significant decreases of proliferation and migration. The total and cytoplasmic Notch-1 levels were declined by RSV, accompanied by a significant increase in smooth muscle α-actin and smooth muscle myosin heavy chain protein. The expression of Notch-1, Jagged-1, Hey-1, and Hey-2 mRNA in balloon-injured arteries at 7 days was decreased by RSV treatment. Arteries from RSV-treated rats showed less neointimal hyperplasia, lower collagen content, and a lower rate of cells positive for proliferating cell nuclear antigen 28 days after injury, compared with saline controls., Conclusions: The results indicate that RSV can attenuate phenotypic switching of VSMCs after arterial injury through inhibition of the Notch pathway.

Xanthine oxidase is an important source of reactive oxygen species; so, it may play a role in the pathogenesis of endothelium dysfunction and its consequences. Allopurinol, a purine analog, is a famous xanthine oxidase inhibitor. This study aimed to investigate possible effects of allopurinol on nitroglycerin tolerance, vasoconstriction, and vasorelaxation in rat aortic ring. Using thoracic aortic rings obtained from male Wistar rats, the effect of allopurinol was examined on nitroglycerin-induced tolerance. In addition, changes of vasoconstriction (by using KCl and phenylephrine) and vasorelaxation (by using carbachol, sodium nitroprusside, and nitroglycerin) were also measured and compared between tissues treated with and without allopurinol. All 3 concentrations of allopurinol (50, 100, and 150 μM) significantly acted against the development of nitroglycerin-induced tolerance in comparison with controls. In terms of vasoconstriction and vasorelaxation, the effect of allopurinol was significant only on carbachol-induced (endothelium related) vasorelaxation in a dose-dependent manner. In conclusion, although allopurinol had no significant effect on the contractile response of the aorta, in accord with the previous data, it significantly intensified endothelium-dependent vasodilation. The inhibitory effect of allopurinol against the development of nitrate-induced tolerance may suggest its clinical benefit and is worth to be studied more extensively.

We previously reported that lysophosphatidic acid (LPA) regulates Ca²⁺ influx of fluid flow in stimulated endothelial cells and that LPA and shear stress showed increment and suppressive effects on phenylephrine-induced vasoconstriction and acetylcholine-induced vasodilatation, respectively. However, a vasoconstrictive effect of LPA alone in the presence of shear stress was not found. The present study examined the effect of LPA alone in the presence of shear stress on Ca²⁺ responses in endothelial and smooth muscle cells and contraction in mouse aortic strip using real-time 2-photon laser scanning microscopy and a custom-made parallel-plate flow chamber. Application of micromolar LPA and high shear stress elicited movement of endothelial cells after Ca²⁺ responses. The endothelial cells moved along the major axis of smooth muscle cells, a direction that was identical to that found during vasoconstriction evoked by the application of phenylephrine. The frequency of Ca²⁺ oscillations in smooth muscle cells was highest according to endothelial movement. Vasoconstriction evoked by LPA and shear stress was significantly reduced by the application of a thromboxane A₂ receptor antagonist, a cyclooxygenase inhibitor, and a thromboxane synthase inhibitor. These results suggest that micromolar LPA and high shear stress elicit vasoconstriction that is caused by Ca²⁺-dependent contraction in medial smooth muscle cells. Thromboxane A₂ may be involved in that response.

Danhong injection (DHI) is the most prescribed injection form of Chinese medicine for the treatment of cardiovascular diseases such as atherosclerosis, angina pectoris, and stroke in China. However, its active components and action mechanisms remain poorly defined. We hypothesized that DHI contains active components that could prevent and restore endothelial dysfunction by improving vascular relaxation activity. DHI increased vasorelaxation in vivo and ex vivo of rat aortas. Vascular reactivity screen identified that danshensu was the major relaxation factor in DHI. DHI-mediated endothelial-dependent vasorelaxation was independent on nitric oxide/endothelial nitric oxide synthase but was via prostacyclin pathway by increasing cyclooxygenase (COX)-2 gene expression and prostacyclin production. Our results revealed a previously unknown endothelium-dependent vasorelaxation mechanism by danshensu and together with previously reported activity on ion channels of vascular smooth muscle cells, demonstrated that its dual actions contribute to a multicomponent Chinese herbal medicine that synergistically targets different pathways to achieve its well-documented cardiovascular protective effects.

Doxorubicin (DOX) is a highly effective anthracycline antibiotic used to treat a wide array of cancers. Its use is limited because of dose-dependent cardiovascular toxicity. Although exercise training has been shown to protect against DOX-induced cardiotoxicity, it is unclear as to whether exercise can attenuate DOX-induced vascular dysfunction. The purpose of this study was to determine if exercise training provides protection against the deleterious vascular effects of DOX treatment and if any changes in vascular function are related to the accumulation of DOX in vascular tissue. Male Sprague-Dawley rats remained sedentary (SED) or engaged in 14 weeks of voluntary wheel running (WR). After the 14-week period, animals received 15 mg DOX per kilogram of body mass or an equivalent volume of saline. Twenty-four hours after DOX/saline exposure, the aorta was isolated and was used to examine vascular function and aortic DOX accumulation. Aortic rings from WR + DOX animals contracted with significantly greater force and showed improved endothelium-independent relaxation when compared with rings from SED + DOX animals. In contrast, no significant differences in endothelium-dependent aortic function were noted between WR + DOX and SED + DOX. Furthermore, no significant differences in aortic DOX accumulation were observed between the DOX groups. These results suggest that chronic exercise attenuates vascular smooth muscle dysfunction associated with DOX treatment and seems to be independent of DOX accumulation in vascular tissue.

This study investigated the role of perivascular adipose tissue (PVAT) in the beneficial effects of andrographolide on vascular reactivity in endotoxaemic rats. After being challenged by lipopolysaccharide (4 mg/kg intraperitoneally), the rats were treated with andrographolide (1 mg/kg intraperitoneally). The response to phenylephrine of aortic rings with or without PVAT was recorded. Vascular relaxing effect of PVAT was determined by bioassay experiments. Inducible nitric oxide synthase (iNOS) in aortic PVAT was tested by Western blot, immunofluorescence, and quantitative polymerase chain reaction. Lipopolysaccharide injection lowered the contraction force induced by phenylephrine in aortic rings with or without PVAT and andrographolide treatment reversed these effects. In bioassay experiments, transferring bathing solution incubated with a PVAT+ ring to a PVAT- ring induced relaxation in the recipient. This relaxing effect of PVAT from endotoxaemic rats was more potent than the rats treated with vehicles. Andrographolide treatment decreased the relaxing effect of PVAT in endotoxaemic rats. The levels of iNOS protein and messenger RNA in PVAT were significantly higher in endotoxaemic rats than in the rats treated with vehicles. Andrographolide treatment decreased PVAT iNOS protein and messenger RNA levels in endotoxaemic rats. Our results suggest that andrographolide restores vascular reactivity in endotoxaemic rats by downregulation of iNOS in PVAT.

Previous studies showed that Gynura procumbens reduced blood pressure by blocking calcium channels and inhibiting the angiotensin-converting enzyme activity. The present experiments were to further explore the effects and mechanisms of a purer aqueous fraction (FA-I) of G. procumbens on angiotensin I (Ang I)-induced and angiotensin II (Ang II)-induced contraction of aortic rings and also on the bradykinin (BK) effect on cardiovascular system. Rat aortic rings suspended in organ chambers were used to investigate the vascular reactivity of FA-I. Effect of FA-I on BK was studied by in vitro and in vivo methods. Results show that FA-I significantly (P < 0.05) decreased the contraction evoked by Ang I and Ang II. In the presence of indomethacin (10 µM) or N-nitro-L-arginine methyl ester (0.1 µM), the inhibitory effect of FA-I on Ang II-induced contraction of aortic rings was reduced. Besides, FA-I potentiated the vasorelaxant effect and enhanced the blood pressure-lowering effect of BK. In conclusion, FA-I reduced the contraction evoked by Ang II probably via the endothelium-dependent pathways, which involve activation of the release of nitric oxide and prostaglandins. The inhibition of angiotensin-converting enzyme activity by FA-I may contribute to the potentiation of the effects of BK on cardiovascular system.

In the mouse aorta, contractions evoked by the α(1)-adrenoceptor agonist phenylephrine are strongly suppressed by the continuous production of nitric oxide (NO). We investigated whether phenylephrine itself stimulated NO production by activating endothelial α(2)-adrenoceptors. On a prostaglandin F(2α) contraction, the α(2)-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) induced 29.3 ± 7.4% relaxation, which was inhibited by 0.1 μM 2-[(4,5-Dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole (BRL44408) with a pKB' corresponding to α(2)-antagonism. In the presence of NO synthase blockers, UK14304 elicited small contractions above 1 μM that were inhibited by 0.1 μM prazosin, but not influenced by 0.1 μM rauwolscine. At 3 μM or higher concentrations, phenylephrine caused only modest relaxation (up to 7.4 ± 2.3%) of segments constricted with prostaglandin F(2α) in the presence of prazosin, which was abolished with 0.1 μM BRL44408. Furthermore, BRL44408 did not increase contractions induced with 1 μM phenylephrine. These results confirm that α(1)- but not α(2)-adrenoceptors are expressed on aortic smooth muscle cells, whereas endothelial cells only express α(2)-adrenoceptors. Moreover, phenylephrine exerted a very modest relaxing effect through nonspecific stimulation of α(2)-adrenoceptors, but only at concentrations higher than 1 μM. It is concluded that the high basal output of NO in the isolated mouse aorta is not due to stimulation of α-adrenoceptors.

The beneficial effects of eplerenone, a specific mineralocorticoid receptor blocker, were previously demonstrated in early atherosclerosis (ATS). The aim of the present study was to evaluate the effect of eplerenone in advanced versus early ATS. Apolipoprotein E knockout mice aged 16 or 32 weeks were randomly divided into eplerenone (100 mg·kg·d) or vehicle treatment for 14 weeks. Eplerenone reduced atherosclerotic lesion size by 51% only in early ATS. In peritoneal macrophages obtained from these mice, eplerenone reduced messenger RNA expression of pro-inflammatory markers, interleukin 6, tumor necrosis factor α, monocyte chemotactic protein 1, and increased anti-inflammatory marker arginase 1 to a greater extent in early compared with advanced ATS. These changes correspond to macrophage polarization toward alternative inflammatory phenotype. Messenger RNA expression of the mineralocorticoid receptor and aldosterone synthase were also reduced by eplerenone to a greater extent in early ATS, and these might increase the sensitivity of macrophages to mineralocorticoid blockade in early ATS. The results of the present study point to the benefits of early initiation of treatment with eplerenone in reducing experimental ATS.

Background: Epidemiological and clinical studies have shown that traffic-related air pollution and, particularly, diesel exhaust particles (DEP) are strongly linked to cardiovascular mortality., Methods: Vascular toxicity was studied by assessing vasomotor responses of aortas isolated from normotensive Wistar rats exposed in vitro to DEP (DEP suspension and aqueous DEP extract). In vivo experiments were performed on Wistar rats and spontaneously hypertensive rats (SHRs) exposed for 4 weeks via intratracheal instillation to either DEP or saline vehicle. After killing, vascular responses to acetylcholine (ACh) or sodium nitroprusside were assessed in vitro and the expression of p22phox, a major nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit, was studied by real-time quantitative polymerase chain reaction., Results: In aortas from Wistar rats, in vitro DEP incubation (both preparations) markedly inhibited the relaxations to ACh and slightly to sodium nitroprusside; this effect was reversed in the presence of superoxide dismutase. In contrast, in aortas from in vivo-exposed animals, ACh-induced relaxations were only significantly impaired in the SHR group, accompanied with a significant upregulation of p22phox and no change in systolic blood pressure., Conclusions: Although in vitro exposure to DEP produces a vascular oxidative stress, repeated in vivo exposures to DEP only impair vascular function in SHR, via an upregulation of p22phox. This suggests a synergistic effect on endothelial dysfunction between particulate air pollution and hypertension.

Background: Kinin B1 receptor (B1R) intervenes in a positive feedback loop to amplify and perpetuate the vascular oxidative stress in glucose-fed rats, a model of insulin resistance. This study aims at determining whether B1R blockade could reverse vascular inflammation in this model., Methods/results: Young male Sprague-Dawley rats were fed with 10% D-glucose or tap water (controls) for 8 weeks, and during the last week, rats were administered the B1R antagonist SSR240612 (10 mg/kg/day, gavage) or the vehicle. The outcome was determined on glycemia, insulinemia, insulin resistance (homeostasis model assessment index), and on protein or mRNA expression of the following target genes in the aorta (by Western blot and real-time quantitative polymerase chain reaction): B1R, endothelial nitric oxide synthase, inducible nitric oxide synthase, macrophage CD68, macrophage/monocyte CD11b, interleukin (IL) -1β, tumor necrosis factor-α, IL-6, macrophage migration inhibitory factor, intercellular adhesion molecule-1, and E-selectin (endothelial adhesion molecule). Data showed increased expression of all these markers in the aorta of glucose-fed rats except endothelial nitric oxide synthase and tumor necrosis factor-α, which were not affected. SSR240612 reversed hyperglycemia, hyperinsulinemia, insulin resistance, and the upregulation of B1R, inducible nitric oxide synthase, macrophage CD68, and CD11b, IL-1β, inter-cellular adhesion molecule-1, macrophage migration inhibitory factor, and E-selectin in glucose-fed rats, yet it had no significant effect on IL-6 and in control rats., Conclusions: Kinin B1R antagonism reversed the upregulation of its own receptor and several pro-inflammatory markers in the aorta of glucose-fed rats. These data provide the first evidence that B1R may contribute to the low-grade vascular inflammation in insulin resistance, an early event in the development of type-2 diabetes.

This study was conducted to explore the vascular reactivity of angiotensin II and noradrenaline and their relationship with endothelial function in rabbits fed a high-fat diet (HFD). The animals were fed either an HFD or regular chow [control diet (CD)]. After 12 weeks, the rabbits fed the HFD showed higher blood pressure, body weight, and insulin levels. Glucose tolerance was impaired and positively related to blood pressure. An endothelium-independent decrease of the sensitivity to angiotensin II [pD2 endothelium-intact aortic rings (E+) in CD: 8.02 ± 0.07 vs. HFD: 7.60 ± 0.01; pD2 endothelium-removed aortic rings (E-) in CD: 8.16 ± 0.11 vs. HFD: 7.83 ± 0.16] and noradrenaline (pD2 E+ in CD: 6.36 ± 0.06 vs. HFD: 5.29 ± 0.06; pD2 E- in CD: 6.11 ± 0.08 vs. HFD: 5.80 ± 0.08) was found. Noradrenaline desensitized the angiotensin II response (pD2 with noradrenaline pretreatment in E+: 7.03 ± 0.16; in E-: 7.10 ± 0.02), but angiotensin II did not change the noradrenaline response. Acetylcholine maximal relaxation and basal nitric oxide (NO) release were comparable in both diet groups. The efficacy of angiotensin II (Rmax CD: 4604 ± 574 mg vs. HFD: 3251 ± 533 mg) and noradrenaline (Rmax CD: 11,675 ± 804 mg vs. HFD: 7975 ± 960 mg) was reduced in E+. L-N-nitroarginine methyl ester (L-NAME) recovered the efficacy of noradrenaline (Rmax L-NAME: 12,015 ± 317 mg). In contrast, L-NAME had no effect on the angiotensin II response. Noradrenaline enhanced NO levels, but angiotensin II did not. Therefore, NO was associated with hyporeactivity to noradrenaline. The resting potential was more negative in E+, and the endothelium diminished the angiotensin II-induced depolarization. These findings demonstrated that the crosstalk and the endothelium may induce hyporeactivity to angiotensin II and noradrenaline as a mechanism to compensate the increase in the blood pressure in HFD-induced obesity.

The vascular effects of tamoxifen (Tam) and its metabolites are poorly known. We compared the vasorelaxation induced by Tam and its metabolites (N-desmethyl-Tam, 4-hydroxy-Tam, and endoxifen) in aortic rings from rats using standardized organ bath procedures, and we investigated the mechanisms involved in this effect. Tam and its metabolite-induced vasorelaxation in a concentration-dependent manner. Although 4-hydroxy-Tam and Tam had similar potency (pD2 = 8.5 ± 0.1 vs. 8.8 ± 0.1, respectively) and maximum effect (Emax = 88.5% ± 1.3% vs. 92.6% ± 1.3%, respectively), N-desmethyl-Tam and endoxifen were more potent and showed higher Emax than Tam did (pD2 = 9.0 ± 0.1 and 8.9 ± 0.1; Emax = 101.1% ± 1.8% and 101.0% ± 1.8% for N-desmethyl-Tam and endoxifen, respectively). Although preincubation of aortic rings with the estrogen receptor antagonist ICI 182780 or with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride induced no changes in the vasorelaxation induced by Tam or 4-hydroxy-Tam, both drugs significantly reduced Emax in response to N-desmethyl-Tam or to endoxifen. Inhibition of cyclooxygenase with indomethacin or the incubation with the prostaglandin D2 and E2 receptor antagonist AH6809 reduced the vasorelaxation-induced Tam and its metabolites by approximately 50%. Preincubation with Nω-nitro-L-arginine methyl ester hydrochloride combined with indomethacin abolished the vasorelaxation-induced Tam and its metabolites. These results show that Tam and its metabolites cause acute vasorelaxation by inducing vasodilator prostanoids synthesis.

Oxidative stress is well known to lead to vascular dysfunction. Thioredoxin reductase (TrxR) catalyzes the reduction of oxidized thioredoxin. Reduced thioredoxin plays a role in cellular antioxidative defense and in decreasing S-nitrosylation. It is not known whether TrxR affects vascular reactivity. We hypothesized that TrxR inhibition decreases vascular relaxation via increased oxidative stress and S-nitrosylation. Aortic rings from C57BL/6 mice were treated with the TrxR inhibitor, 1-chloro-2,4-dinitrobenzene (DNCB), or auranofin for 30 minutes. Vascular relaxation to acetylcholine was measured in the rings contracted with phenylephrine. DNCB and auranofin reduced relaxation compared with vehicle (vehicle Emax = 71 ± 3%, DNCB Emax = 53 ± 3%; P < 0.05). The antioxidants, apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor), and tempol (superoxide dismutase mimetic) normalized impaired relaxation by DNCB in aorta (DNCB Emax = 53 ± 3%, DNCB + tempol Emax = 66 ± 3%; P < 0.05). In addition, DNCB reduced sodium nitroprusside-induced relaxation. DNCB increased soluble guanylyl cyclase (sGC) S-nitrosylation and decreased sGC activity. These data suggest that TrxR regulates vascular relaxation via antioxidant defense and sGC S-nitrosylation. TrxR may be an enzyme to approach for treatment of vascular dysfunction and arterial hypertension.

Thienopyridines (ticlopidine, clopidogrel, and prasugrel) require in vivo metabolism to exhibit a critical thiol group in the active form that binds to the P2Y12 platelet receptor to inhibit platelet activation. We hypothesized that formation of thienopyridine-derived nitrosothiols (ticlopidine-SNO, clopidogrel-SNO, and prasugrel-SNO) occurs directly from the respective parent drug. Pharmaceutical-grade thienopyridine (ticlopidine, clopidogrel chloride, clopidogrel sulfate, clopidogrel besylate, or prasugrel) was added to nitrite in aqueous solution to form the respective thienopyridine-SNO (Th-SNO). An isolated aortic ring preparation was used to test vasoactivity of the Th-SNO derivatives. Increasing nitrite availability resulted in increased Th-SNO formation for all drugs (other than ticlopidine). Th-SNO induced significant endothelium-independent relaxation of preconstricted aortic rings. Clopidogrel-chloride-SNO displayed rapid-release kinetics in a chemical environment, which was reflected by immediate and transient vasorelaxation when compared with the SNO derivatives of the other thienopyridines. Accounting for differences in yield, clopidogrel-chloride-SNO exhibited the greatest propensity to immediately relax vascular tissue. Th-SNO derivatives exhibit nitrovasodilator properties by supplying NO that can directly activate vascular soluble guanylate cyclase to induce vasorelaxation. Differences in SNO yield and vasoactivity exist between thienopyridine preparations that might be important to our understanding of the direct pharmacological effectiveness of thienopyridines on vascular and platelet function.

The anaphylatoxin C5a generated by activation of the innate immunity complement system is a potent inflammatory peptide mediator through the G-protein-coupled receptor C5aR (CD88) present in immune-inflammatory cells, including monocytes, macrophages, neutrophils, T cells, and mast cells. Inflammatory cells infiltrate and initiate the development of fibrosis in the chronically hypertensive heart. In this study, we have investigated whether treatment with a selective C5aR antagonist prevents cardiovascular remodeling in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Control and DOCA-salt rats were treated with PMX53 (AcF-[OPdChaWR], 1 mg·kg·d oral gavage) for 32 days; structural and functional changes in cardiovascular system were determined. DOCA-salt hypertension increased leukocyte extravasation into ventricular tissue, increasing collagen deposition and ventricular stiffness; PMX53 treatment attenuated these changes, thereby improving cardiac function. Further, treatment with PMX53 suppressed an increased expression of C5aR in the left ventricle from DOCA-salt rats, consistent with the reduced infiltration of inflammatory cells. Vascular endothelial dysfunction in thoracic aortic rings was attenuated by PMX53 treatment, but systolic blood pressure was unchanged in DOCA-salt rats. In the heart, PMX53 treatment attenuated inflammatory cell infiltration, fibrosis, and ventricular stiffness, indicating that C5aR is critically involved in ventricular remodeling by regulating inflammatory responses in the hypertensive heart.

In contrast to the intact wine, cardiovascular effects of the thermally treated wine have not been studied, despite widespread habits of cooking with wine and consumption of mulled wine. Vasodilatory effects of the red wine heated at 75 and 125°C were examined in the isolated rat and guinea pig aorta and compared with the intact and wine dealcoholized without thermal stress. Samples were analyzed for their phenolic content, antioxidant capacity, resveratrol and ethanol contents. Heating-induced degradation of individual phenolic fraction was observed only in the samples treated at 125°C, although total phenolic concentration and related antioxidant activity increased in the thermally treated samples due to the reduction in their volume. All wine samples regardless of treatment caused similar maximal relaxation in both species, but the response was stronger in aortas from guinea pigs. At the lowest concentrations up to 1‰, dealcoholized wine produced vasodilation greater than that produced by intact wine and wines treated at 75 and 125°C, which showed similar vasodilating activity at all concentrations. Our results indicate that wine thermally treated under heating conditions applicable to the preparation of a mulled wine and cooking with wine largely retains vasodilatory activity in vitro despite significant heat-induced changes in its composition.

Inflammation contributes to atherosclerotic plaque initiation and progression. Recent studies suggest that nicotinic acid has anti-inflammatory effects independent of its lipid-modifying capabilities. We assessed the hypothesis that administration of nicotinic acid to older apolipoprotein E (apoE)-deficient mice with established lesions will reduce lesion size and plaque inflammation independent of its lipid-modifying effects. Therefore nicotinic acid was administered to 27-week-old apo E-deficient mice exhibiting advanced atherosclerotic lesions within the innominate artery. After 27 weeks of treatment both animal groups had no significant changes in plasma lipid levels. Mice treated with nicotinic acid (n = 22) demonstrated a 30% reduction in total lesion area compared with controls (n = 20). Furthermore, they revealed a more stable plaque composition with an increase in fibrous cap thickness and a reduction in the size of the necrotic core. Immunohistochemistry demonstrated a reduced accumulation of macrophages and a reduced expression of vascular cell adhesion molecule-1 and tissue factor. Additionally, administration of nicotinic acid significantly reduced tumor necrosis factor alpha expression in the thoracic aorta as demonstrated by real-time PCR. In conclusion, these data suggest that long-term administration of nicotinic acid has anti-atherogenic and anti-inflammatory properties on advanced atherosclerotic lesions, which are independent of its lipid-modifying actions.

Salvia miltiorrhiza (Danshen) has been widely used in China and other Asian countries for treating various cardiovascular diseases resulting from its ability to improve coronary microcirculation and increase coronary blood flow. Tanshinone IIA (Tan IIA), the major active lipophilic ingredient responsible for the beneficial actions of Salvia miltiorrhiza, has been shown to induce vasodilation in coronary arteries. Because our recent study identified Tan IIA as a new member of the phytoestrogens, we hypothesized that its action might be mediated by estrogen receptor (ER) in vascular endothelial cells. The aim of the present study was to assess whether cardiovascular protection exerted by Tan IIA is mediated by the ER signal pathway and whether the genomic or nongenomic action of ER is involved within arteries and vascular endothelial cells. The effect of Tan IIA on blood vessels was investigated by vascular ring assay using endothelium-intact and endothelium-denuded rat aortas. Similar to estrogen, Tan IIA caused an nitric oxide- and endothelium-dependent relaxation, which was blocked by ER antagonist ICI 182,780. Primary cardiac microvascular endothelial cells were used as a model to study the cellular and molecular mechanisms of Tan IIA-induced vasorelaxation. We demonstrate that Tan IIA is capable of activating the estrogen receptor signal pathway, leading to increased endothelial nitric oxide synthase gene expression, nitric oxide production, ERK1/2 phosphorylation, and Ca mobilization. Collectively, these effects contribute to Tan IIA's vasodilative activity effects of y ER antagonist Cnt of cardiovascular diseases. Our findings support a continued effort in discovering and developing novel phytoestrogens as an alternative hormone replacement therapy for safer and more effective treatment of cardiovascular diseases.

Endothelial damage triggers platelet adhesion and platelet-associated prothrombinase formation at the point of injury, resulting in the progression of thrombus formation. The present study compared the inhibitory effects of fondaparinux, an indirect factor Xa (FXa) inhibitor, and TAK-442, a direct FXa inhibitor, on platelet-associated prothrombinase activity in the balloon-injured rat artery. TAK-442 and fondaparinux inhibited endogenous FXa activity in platelet-poor human [half-maximal inhibitory concentration (IC(50)): 53 nM, TAK-442; 11 nM, fondaparinux] and rat (IC(50): 32 nM, TAK-442; 19 nM, fondaparinux) plasma. TAK-442 inhibited in vitro reconstituted human prothrombinase (system included FXa, calcium, and washed platelets) with an IC(50) value of 51 nM, whereas fondaparinux exhibited only weak inhibition (IC(50): 1700 nM). In a rat model of balloon injury, thrombin activity on the surface of injured vessels increased to 3.2-, 22-, and 5.8-fold the activity on the surface of the intact aorta at 5 minutes, 1 hour, and 24 hours after the injury, respectively. At approximately 1 hour after the injury, TAK-442 blocked platelet-associated thrombin generation on the surface of injured aortas with an IC(50) value of 19 nM, whereas fondaparinux showed no significant inhibition at the highest concentration tested (IC(50): >300 nM). These results suggest a possible limitation of fondaparinux in inhibiting platelet-associated prothrombinase activity and resultant thrombus formation as compared with TAK-442.

To investigate the effect of chronic cannabinoid 1 antagonism on vascular prostanoid production, obese Zucker rats were treated with rimonabant (10 mg/kg per day) during 20 weeks and then vascular and endothelial reactivity were assessed in aortic rings by analyzing response to phenylephrine and acetylcholine. The presence of cyclo-oxygenase-1 and cyclo-oxygenase-2 selective inhibitors (SC-560 and NS-398, respectively) and the enzyme immunoassay revealed lower PGI2 production by aortic rings from obese rats with rimonabant able to restore such response toward levels found in the lean animals. The treatment also reduced TXB2 but did not alter its participation on acetylcholine-induced relaxation as the TP receptor antagonist ICI-192,605 revealed. Those effects were associated with an enhancement of cyclo-oxygenase-2 expression without affecting p38MAPK phosphorylation. Obese rats also exhibited higher nitric oxide plasma concentrations and greater inducible nitric oxide synthase participation on vascular phenylephrine-induced response without changes in inducible nitric oxide synthase protein expression. Although rimonabant reduced such alteration, the values were still higher than those found in lean rats. Finally, rimonabant was also able to reduce tumor necrosis factor-α produced by adipose tissue of obese Zucker rats. These results highlight a crosstalk among cannabinoids and cyclo-oxygenase-derived products in the vasculature of obese animals.

Pulmonary hypertension is a kind of disease associated with a very high rate of mortality, and there are not many effective drugs for the treatment. Today, endothelin (ET)-1 receptor antagonists were proved to be effective in the treatment of pulmonary hypertension. Aiming at developing new endothelin-A receptor (ETA) antagonist for treatment of pulmonary hypertension, di-n-butylaminocarbamyl-L-leucyl-D-tryptophanyl-D-4-chloro-Phe, named GF063, was synthesized at base of selective ETA receptor antagonist BQ485 and selected for the further pharmacological characterization. The preliminary pharmacodynamics of GF063 was evaluated by radioligand receptor binding assay and test of antivasoconstriction effects in vitro and in vivo. The integrative pharmacodynamics was evaluated in hypoxia-induced rat pulmonary hypertension. In vitro, GF063 bound to ETA receptor with 100,000-fold higher affinity than to ETB receptor. GF063 concentration dependently inhibited contraction of isolated rat aortic ring induced by ET-1 and shifted the cumulative concentration-contraction response curve to right with no change in the maximal response. In vivo, GF063 inhibited the increase of mean systemic arterial pressure induced by ET-1 in anesthetized rat. In hypoxia-induced rat pulmonary hypertension model, pretreatment with GF063 (40 mg/kg, s.c.) significantly decreased pulmonary artery pressure and right ventricular hypertrophy, also significantly inhibited the increase of ET-1 level in lung, improved hemodynamics, and alleviated the wall thickness of pulmonary vessels. This study indicated that GF063, as a selective ETA receptor antagonist, could inhibit vasoconstriction effects in vivo and in vitro, could prevent pulmonary hypertension induced by hypoxia, and may have great potential to be developed as a new drug of antipulmonary hypertension.

Genistein is a phytoestrogen that is known to have a protective effect on the vascular endothelial wall. However, it exhibits poor bioavailability, which limits the use of genistein to treat cardiovascular and cerebrovascular diseases. A novel genistein derivative, 7-difluoromethyl-5,4'-dimethoxygenistein (dFMGEN), has shown a better protective effect on vascular endothelial damage in vitro than genistein. In this study, we further evaluated therapeutic effects of dFMGEN on the vascular endothelial wall and atherosclerosis in a rabbit model in vivo. There were 5 groups: the GEN group (genistein 5 mg/kg per day), lovastatin group (lovastatin 5 mg/kg per day), dFMGEN group (dFMGEN 5 mg/kg per day), model control group (the same amount of vehicle solvent), and the normal control group; all feedings administered via intragastric administration. We demonstrated that dFMGEN (1) attenuated the development of atherosclerosis, (2) reduced serum total cholesterol and low-density lipoprotein cholesterol concentrations, (3) decreased lipid peroxidation in the rabbit atherosclerosis model, and (4) increased smooth muscle cell and collagen content in atheroma of thoracic aortas. These results provide an experimental foundation for dFMGEN's potential effects in preventing and treating atherosclerosis, acute coronary syndromes, and potentially ischemia-reperfusion injury during acute myocardial infarction and cerebral infarction.

Oxidative stress produced through reactive oxygen species (ROS) enhancement is considered to play a key role in the development and maintenance of hypertension. In the vasculature, the most important source of ROS is the reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase enzyme. The principal stimulus of this enzyme is angiotensin II (Ang II). However, oxidative stress seems to be present in virtually all forms of hypertension including low-renin hypertension, where the levels of Ang II are reduced. For this reason, the question is if ROS generation is induced by Ang II or it is a consequence of hypertension. We used as hypertensive model the aortic coarctated rats, which were treated with losartan or minoxidil for 7 days. Thoracic aortic segments were excised, and the NAD(P)H oxidase subunits expression, oxidative stress parameters, and heme oxygenase-1 abundance were evaluated. Hypertensive animals had an increase in the activity and expression of NAD(P)H oxidase and, as a consequence, in the oxidative stress parameters. Interestingly, either losartan or minoxidil administration blunted those parameters, indicating that arterial pressure is the key factor in the development of oxidative stress in the hypertensive aorta. We suggest that antihypertensive drug administration at the beginning of this pathology delays the oxidative stress generation, thus preventing the aggravation of this disease.

The mechanism by which insulin causes vasodilatation remains unclear, so we explored this in aortic rings from normal Wistar Kyoto and streptozotocin-induced diabetic rats. Insulin-induced relaxation of phenylephrine-contracted [endothelium (ED) intact or denuded] aortic rings was recorded in the presence or absence of various drug probes. Insulin relaxant effect was more in ED-intact than in-denuded tissues from normal or diabetic rats. l-NAME or methylene blue partially inhibited insulin effect in ED-intact but not the ED-denuded tissues, whereas indomethacin (cyclooxygenase inhibitor) had no effect on any of the tissues, indicating that insulin induces relaxation by ED-dependent and -independent mechanisms, the former via the NOS-cyclic guanosine monophosphate but not the cyclooxygenase pathway. The voltage-dependent K channel (KV) blocker (4-aminopyridine) inhibited insulin action in all the tissues (normal or diabetic, with or without ED), whereas the selective BKCa blocker, tetraethylammonium, inhibited it in normal (ED intact or denuded) but not in diabetic tissues, indicating that KV mediates insulin action in normal and diabetic tissues, whereas the BKCa mediates it only in normal tissues, with possible pathophysiologic absence in diabetic tissues. The inward rectifier K channel (Kir) blocker (barium chloride) significantly inhibited insulin effect only in ED-intact or -denuded diabetic tissues, whereas the KATP channel blocker, glibenclamide, inhibited it only in the ED-denuded diabetic tissues, suggesting that Kir channels mediate insulin-induced relaxation in ED-intact or -denuded diabetic tissues, whereas the KATP channel mediates it in ED-denuded diabetic tissues. All the agents combined did not abolish insulin action, suggestive of a direct vasodilatory effect. In conclusion, insulin causes vasodilatation in normal and diabetic tissues via ED-dependent and -independent mechanisms differentially modulated by K channels, some of which functions are altered in diabetes and thus are potential therapeutic targets.

We examined and compared mechanisms of the red wine (RW)-induced vasorelaxation in guinea pig (GP) and rat aorta. Acetylcholine-induced relaxation of norepinephrine-precontracted aortic rings was stronger in rat aorta than in GP aorta, whereas RW-induced vasorelaxation was stronger in GP aorta. L-nitro-arginine methyl ester (L-NAME) abolished RW-induced vasorelaxation in rat aorta, whereas in GP aorta, it was only reduced by 50%. To examine mechanisms of the L-NAME-resistant relaxation, GP aortic rings were additionally exposed to indomethacin, clotrimazole, and their combination. Indomethacin insignificantly reduced RW-induced relaxation, but in combination with L-NAME, the relaxation was synergistically decreased (80%). After clotrimazole exposure, the relaxation was reduced by 25%, and addition of indomethacin caused no further reduction. Only the combination of L-NAME, indomethacin, and clotrimazole prevented RW-induced vasorelaxation. RW-induced vasorelaxation in KCl-precontracted GP rings was significantly smaller (Emax 78.31% +/- 6.09%) than the RW-induced relaxation in norepinephrine-precontracted rings (Emax 126.01% +/- 2.11%). L-NAME in KCl-precontracted GP rings prevented RW-induced vasorelaxation. In conclusion, different pathways are involved in the RW-induced vasorelaxation in GP aorta, in contrast to rat aorta, in which NO plays main role. Therefore, the uncritical extrapolation of the results from one species to another could be misleading.