Blood Vessel Tone Research Articles

When an enemy becomes a friend. On the benefits of alcohol consumption

While use of high doses of alcohol is a proven risk factor for various organs and systems, use of small and moderate doses bears evidence of the opposite data. Thus, regularly drinking a small amount of alcoholic drinks could reduce a person’s chances of developing coronary heart disease, stroke, type 2 diabetes, non-alcoholic fatty liver disease, osteoporosis and some other diseases, and also reduce overall mortality. Small doses of alcohol suppress the inflammation and peroxidation, normalize hemocoagulation, improve the lipid profile, favourably affect blood vessel tone and the insulin-signaling pathways. Positive effects of wine are the most studied, which is usually explained by the fact that it contains polyphenols, while positive effects of beer are studied to a lesser extent; however, it should be borne in mind that the studies were aimed at different types of alcoholic beverages. Thus, it can be considered inappropriate to completely prohibit the use of alcohol to persons without dependence and specific organ damage.

The effect of intra-renal administration of L-NAME on renal medullary and cortical blood flow of stroke-prone spontaneously hypertensive rats and wistar rats

Background: Autocrine and paracrine factors produced within the kidney regulate the cortical and medullary blood perfusion (CBP and MBP, respectively), which include endothelins, prostaglandins, reactive oxygen species, and nitric oxide (NO). This study investigated the role of NO in regulating the CBP and MBP of a kidney in both stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar rats. Materials and Methods: Groups (n = 10 for each) of male SHRSP and Wistar rats (250–300 g) were prepared for experiments. Two laser Doppler microprobes were inserted, 1.5 and 4.0 mm, into their kidneys to measure CBP and MBP before and after the intrarenal infusion of L-NAME, the NO synthase inhibitor at dose of 10 μg/kg/min. At the end of the experiments, the animals were killed with an anesthetic overdose. Data ± standard error of the mean were subjected to Student's t-test and significance taken at P < 0.05. Results: Interstitial infusion of L-NAME into the corticomedullary border (CMB) causes significant reduction in MBP in both SHRSP and Wistar rats by 18% ±5% and 12% ±4%, respectively. The magnitude of reduction is closely similar in both strains. Acute infusion of L-NAME into CMB has no effect on CBP but increases the blood pressure (BP) in both strains equally (P < 0.05). Conclusion: These results suggest that NO plays an important role in regulating the tone of medullary blood vessels in both hypertensive and normotensive states with similar extent. L-NAME can easily spread into the systemic circulation, which is evidenced by the increase of BP.

Оцінювання функціонального стану мікроциркуляції крові в тканинах людини методами спекл-метрії і допплерівської флоуметрії

Based on an analytical method for estimating parameters of a speckle structure formed by multiple scattered light in multilayer biological tissue such as human skin at visible wavelengths - the near-IR range, using known solutions of the radiation transfer equation in biological tissue and linking the theory of light propagation in a scattering medium to coherence theory built a mathematical model of the propagation of laser radiation in biological tissue. Using the mechanics of multiphase media, blood for mathematical modeling was initially considered a two-phase viscous suspension consisting of two phases: a parietal and an axial plasma layer with erythrocytes. The blood flow through vessels was modeled taking into account a number of anomalous effects (rheological properties) of blood flow: the Farus effect, the non-erythrocyte parietal plasma layer, the Farus — Lindquist effect, and the blunt blood velocity profile. The characteristics of microcirculation in human tissues were investigated by a non-invasive speckle-optical method using the Speckle-Scan device. In the frequency range of 40-1000 Hz, the power of the spectrum, the average frequency of the spectrum, and the root-mean-square velocity of the particles were determined. At the same time, skin microhemodynamics was investigated using Doppler ultrasound (USDG) using the Minimax-Doppler-K device. The mean linear and average volumetric blood flow rates were determined from the average velocity curve. The data obtained by the USDG and the Speckle-Scan devices were compared with each other and with a mathematical model of the propagation of laser radiation in the microcirculation channel. It was established that the parameter “average frequency of the spectrum” largely reflects the perfusion, and the area under the spectral curve reflects the capacity of the capillary bed. It has been established that the average power of the spectrum of fluctuations of the intensity of scattered radiation after decompression of a vessel increases by about 15 % in comparison with the normal state. The autocorrelation functions of the field fluctuations are obtained when the particles are scattered back at different pressures after decompression of the brachial artery into a difference in the time periods for registering changes. The slope of the autocorrelation function, depending on pressure, can be used to diagnose the tone (elasticity) of blood vessels. Methodical approaches are proposed for evaluating the obtained data in order to verify speckle measurements using the widely used Doppler flowmetry technique.

Type 2 Diabetes and Vascular Endothelial Dysfunction

Vascular endothelial function is important for maintaining the homeostasis of the living body. Especially, nitric oxide (NO) produced in vascular endothelial cells regulates blood vessel tone and has an antiatherosclerotic effect. Type 2 diabetes is a typical disease that causes impaired vascular endothelial function, resulting in various vascular complications and damage to organs. Cardiovascular disease associated with type 2 diabetes is a chronic inflammatory disease that starts with endothelial dysfunction (ED), and vascular ED is important as an initial change in arteriosclerotic lesions. Vascular ED in type 2 diabetes is thought to be caused by hyperglycemia, hyperinsulinemia associated with insulin resistance, and hypoglycemia, in which elevated oxidative stress accompanying postprandial hyperglycemia and blood glucose fluctuation are involved. Vascular ED is also caused by postprandial metabolic abnormalities, so correcting postprandial metabolic abnormalities is also important. Meanwhile, Glucagon-like peptide-1 (GLP-1) receptor agonist, thiazolidine, biguanide and Dipeptidyl peptidase-4 (DPP-4) inhibitor have an effect of protecting vascular endothelial function beyond glycemic control. In order to promote a healthy lifestyle for diabetes patients, it is important not only to lower HbA1c but also to avoid postprandial hyperglycemia, blood glucose fluctuation, and hypoglycemia. It is also important to conduct treatment with a view to suppressing vascular complications, such as the selection of antiarteriosclerosis medications.

Low Immunogenic Endothelial Cells Maintain Morphological and Functional Properties Required for Vascular Tissue Engineering.

The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue-engineered vascular grafts containing autologous cells are considered ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC, but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leukocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood and umbilical cord blood and vein. EC from all three sources were transduced by 93.1% ± 4.8% and effectively, HLA I-silenced by up to 67% compared to nontransduced (NT) cells or transduced with a nonspecific short hairpin RNA, respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier, as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low-density lipoprotein, and to form capillary-like tube structures in three-dimensional fibrin gels similar to NT cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC toward long and widely branched vascular networks necessary for prevascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological and functional properties required for vascular tissue engineering. This extends the spectrum of available cell sources from autologous to allogeneic sources, thereby accelerating the generation of tissue-engineered vascular grafts in acute clinical cases.

The anthelmintic praziquantel is a human serotoninergic G-protein-coupled receptor ligand

Schistosomiasis is a debilitating tropical disease caused by infection with parasitic blood flukes. Approximately 260 million people are infected worldwide, underscoring the clinical and socioeconomic impact of this chronic infection. Schistosomiasis is treated with the drug praziquantel (PZQ), which has proved the therapeutic mainstay for over three decades of clinical use. However, the molecular target(s) of PZQ remain undefined. Here we identify a molecular target for the antischistosomal eutomer — (R)-PZQ — which functions as a partial agonist of the human serotoninergic 5HT2B receptor. (R)-PZQ modulation of serotoninergic signaling occurs over a concentration range sufficient to regulate vascular tone of the mesenteric blood vessels where the adult parasites reside within their host. These data establish (R)-PZQ as a G-protein-coupled receptor ligand and suggest that the efficacy of this clinically important anthelmintic is supported by a broad, cross species polypharmacology with PZQ modulating signaling events in both host and parasite.

Deep tissue analysis of distal aqueous drainage structures and contractile features

Outflow resistance in the aqueous drainage tract distal to trabecular meshwork is potentially an important determinant of intraocular pressure and success of trabecular bypass glaucoma surgeries. It is unclear how distal resistance is modulated. We sought to establish: (a) multimodal 2-photon deep tissue imaging and 3-dimensional analysis of the distal aqueous drainage tract (DT) in transgenic mice in vivo and ex vivo; (b) criteria for distinguishing the DT from blood and lymphatic vessels; and (c) presence of a DT wall organization capable of contractility. DT lumen appeared as scleral collagen second harmonic generation signal voids that could be traced back to Schlemm’s canal. DT endothelium was Prox1-positive, CD31-positive and LYVE-1-negative, bearing a different molecular signature from blood and true lymphatic vessels. DT walls showed prominent filamentous actin (F-actin) labeling reflecting cells in a contracted state. F-actin co-localized with mesenchymal smooth muscle epitopes of alpha-smooth muscle actin, caldesmon and calponin, which localized adjacent and external to the endothelium. Our findings support a DT wall organization resembling that of blood vessels. This reflects a capacity to contract and support dynamic alteration of DT caliber and resistance analogous to the role of blood vessel tone in regulating blood flow.

Inflow of oxygen and glucose in brain tissue induced by intravenous norepinephrine: relationships with central metabolic and peripheral vascular responses.

As an essential part of sympathetic activation that prepares the organism for "fight or flight," peripheral norepinephrine (NE) plays an important role in regulating cardiac activity and the tone of blood vessels, increasing blood flow to the heart and the brain and decreasing blood flow to the organs not as necessary for immediate survival. To assess whether this effect is applicable to the brain, we used high-speed amperometry to measure the changes in nucleus accumbens (NAc) levels of oxygen and glucose induced by intravenous injections of NE in awake freely moving rats. We found that NE at low doses (2-18 μg/kg) induces correlative increases in NAc oxygen and glucose, suggesting local vasodilation and enhanced entry of these substances in brain tissue from the arterial blood. By using temperature recordings from the NAc, temporal muscle, and skin, we show that this central effect is associated with strong skin vasoconstriction and phasic increases in intrabrain heat production, indicative of metabolic neural activation. A tight direct correlation between NE-induced changes in metabolic activity and NAc levels of oxygen and glucose levels suggests that local cerebral vasodilation is triggered via a neurovascular coupling mechanism. Our data suggest that NE, by changing vascular tone and cardiac activity, triggers a visceral sensory signal that rapidly reaches the central nervous system via sensory nerves and induces neural activation. This neural activation leads to a chain of neurovascular events that promote entry of oxygen and glucose in brain tissue, thus preventing any possible metabolic deficit during functional activation. NEW & NOTEWORTHY Using high-speed amperometry and thermorecording in freely moving rats, we demonstrate that intravenous norepinephrine at physiological doses induces rapid correlative increases in nucleus accumbens oxygen and glucose levels coupled with increased intrabrain heat production. Although norepinephrine cannot cross the blood-brain barrier, by changing cardiac activity and vascular tone, it creates a sensory signal that reaches the central nervous system via sensory nerves, induces neural activation, and triggers a chain of neurovascular events that promotes intrabrain entry of oxygen and glucose.

Sex differences in the role of phospholipase A2 -dependent arachidonic acid pathway in the perivascular adipose tissue function in pigs.

The fat surrounding blood vessels (perivascular adipose tissue or PVAT) releases vasoactive compounds that regulate vascular smooth muscle tone. There are sex differences in the regulation of vascular tone, but, to date, no study has investigated whether there are sex differences in the regulation of blood vessel tone by PVAT. This study has identified that the cyclooxygenase products thromboxane and PGF2α are released from coronary artery PVAT from pigs. Thromboxane appears to mediate the PVAT-induced contraction in arteries from females, whereas PGF2α appears to mediate the contraction in arteries from males. These sex differences in the role of these prostanoids in the PVAT-induced contraction can be explained by a greater release of thromboxane from PVAT from female animals and greater sensitivity to PGF2α in the porcine coronary artery from males. Previous studies have demonstrated that perivascular adipose tissue (PVAT) causes vasoconstriction. In this present study, we determined the role of cyclooxygenase-derived prostanoids in this contractile response and determined whether there were any sex differences in the regulation of vascular tone by PVAT. Contractions in isolated segments of coronary arteries were determined using isolated tissue baths and isometric tension recording. Segments were initially cleaned of PVAT, which was then re-added to the tissue bath and changes in tone measured over 1h. Levels of PGF2α and thromboxane B2 (TXB2 ) were quantified by ELISA, and PGF2α (FP) and thromboxane A2 (TP) receptor expression determined by Western blotting. In arteries from both male and female pigs, re-addition of PVAT caused a contraction, which was partially inhibited by the cyclooxygenase inhibitors indomethacin and flurbiprofen. The FP receptor antagonist AL8810 attenuated the PVAT-induced contraction in arteries from males, whereas the TP receptor antagonist GR32191B inhibited the PVAT-induced contraction in arteries from females. Although there was no difference in PGF2α levels in PVAT between females and males, PGF2α produced a larger contraction in arteries from males, correlating with a higher FP receptor expression. In contrast, release of TXB2 from PVAT from females was greater than from males, but there was no difference in the contraction by the TXA2 agonist U46619, or TP receptor expression in arteries from different sexes. These findings demonstrate clear sex differences in PVAT function in which PGF2α and TXA2 antagonists can inhibit the PVAT-induced vasoconstriction in male and female PCAs, respectively.

Micro-anatomical changes in major blood vessel caused by dengue virus (serotype 2) infection

Dengue virus (DENV) has emerged as a major economic concern in developing countries, with 2.5 billion people believed to be at risk. Vascular endothelial cells (ECs) lining the circulatory system from heart to end vessels perform crucial functions in the human body, by aiding gas exchange in lungs, gaseous, nutritional and its waste exchange in all tissues, including the blood brain barrier, filtration of fluid in the glomeruli, neutrophil recruitment, hormone trafficking, as well as maintenance of blood vessel tone and hemostasis. These functions can be deregulated during DENV infection. In this study, BALB/c mice infected with DENV serotype 2 were analyzed histologically for changes in major blood vessels in response to DENV infection. In the uninfected mouse model, blood vessels showed normal architecture with intact endothelial monolayer, tunica media, and tunica adventitia. In the infected mouse model, DENV distorted the endothelium lining and disturbed the smooth muscle, elastic laminae and their supporting tissues causing vascular structural disarrangement. This may explain the severe pathological illness in DENV-infected individuals. The overall DENV-induced damages on the endothelial and it’s supporting tissues and the dysregulated immune reactions initiated by the host were discussed.

Effects of endogenous nitric oxide on adrenergic nerve-mediated vasoconstriction and calcitonin gene-related peptide-containing nerve-mediated vasodilation in pithed rats

Vascular adrenergic nerves mainly regulate the tone of blood vessels. Calcitonin gene-related peptide-containing (CGRPergic) vasodilator nerves also participate in the regulation of vascular tone. Furthermore, there are nitric oxide (NO)-containing (nitrergic) nerves, which include NO in blood vessels as vasodilator nerves, but it remains unclear whether nitrergic nerves participate in vascular regulation. The present study investigated the role of nitrergic nerves in vascular responses to spinal cord stimulation (SCS) and vasoactive agents in pithed rats. Wistar rats were anesthetized and pithed, and vasopressor responses to SCS and injections of norepinephrine were observed. To evaluate vasorelaxant responses, the BP was increased by a continuous infusion of methoxamine with hexamethonium to block autonomic outflow. After the elevated BP stabilized, SCS and injections of acetylcholine (ACh), sodium nitroprusside (SNP), and CGRP were intravenously administered. We then evaluated the effects of the NO synthase (NOS) inhibitor, N-ω-nitro-L-arginine methylester hydrochloride (L-NAME), on these vascular responses. Pressor responses to SCS and norepinephrine in pithed rats were enhanced by L-NAME, while the combined infusion of L-NAME and L-arginine had no effect on these responses. L-NAME infusion significantly increased the release of norepinephrine evoked by SCS. In pithed rats with artificially increased BP and L-NAME infusion, depressor response to ACh (except for 0.05nmol/kg) was suppressed and SNP (only 2nmol/kg) was enhanced. However, depressor responses to SCS and CGRP were similar to control responses. The present results suggest endogenous NO regulates vascular tone through endothelium function and inhibition of adrenergic neurotransmission, but not through CGRPergic nerves.

Selective venous vasodilator properties of the analgesic metamizole (dipyrone) in a human ex vivo model-implications for postoperative pain management.

Metamizole (dipyrone) is a first-line, non-opioid analgesic used for postoperative pain management. Clinical data and animal experiments indicate a possible vasodilator action of this drug. We investigated the effects of metamizole on human artery and vein tone in an ex vivo model to assess potential contributions to venous pooling. Excess segments of bypass grafts were harvested during coronary artery bypass grafting procedures. Tensions were measured in an organ bath for 120min after adding metamizole to the preconstricted vessels. Contribution of endothelium was assessed in endothelium-denuded vessels, and indometacin was used to identify cyclooxygenase-mediated effects. Internal mammary arteries (n=6) constricted after addition of 1, 3, and 10μM metamizole and remained constricted at the lower doses. Transient constrictions also occurred in saphenous veins (n=20), but veins relaxed below solvent controls after 20min at all concentrations. Endothelium removal (n=12) and cyclooxygenase inhibition (n=12) suppressed the vasoconstrictor effect but not the vasodilator effect. Metamizole and its metabolites display counteracting effects on blood vessel tone ex vivo. The vasoconstrictor effect is mediated by cyclooxygenase-derived products. The net effect is site-specific, resulting in a selective venous vasodilator action. This may exacerbate unwanted venous pooling during postoperative pain therapy.

Endothelial Cell-Expressed α Hemoglobin and Its Molecular Chaperone Ahsp Modulate Arterial Vascular Reactivity

We are studying a novel mechanism by which prototypical erythrocyte proteins also act in endothelial cells to regulate vascular tone. Previously, we reported that the alpha subunit of hemoglobin (αHb) is expressed in the myoendothelial junction of endothelial cells in resistance arterioles where it binds endothelial nitric oxide (NO) synthase (eNOS) and degrades NO, thereby stimulating vasoconstriction. To extend this observation, we examined arterioles of mice lacking 2 out of 4 α globin genes [HbA1 knockout *α2/*α2]. Compared to wild type (wt) controls, the thoracodorsal artery (TDA, a resistance arteriole) from mutant animals exhibited abnormal morphology with thinned internal elastic lamina, excessive dilation, and reduced contractility after treatment with the vasoconstrictor phenylephrine (EC50 8.2e-6M-1 n=6 in wt vs 2.3e-5 M-1 n=6 in mutant; p<0.0001).Wild type TDAs expressed αHb (Fig. 1A), but not βHb mRNA and protein. However, free αHb is unstable and not expected to exist as an isolated monomer. Alpha hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds free αHb, stabilizes its structure and facilitates assembly of HbA tetramers (α2β2) in red blood cells. Immunohistochemistry showed that AHSP and αHb co-localize in endothelial cells lining the TDA. In the TDA of Ahsp knockout (KO) mice, αHb immunostaining was disorganized and reduced in intensity (Fig. 1A), and Western blotting showed reduced αHb protein compared to wt controls. Moreover, TDAs from Ahsp KO mice exhibited abnormal thinning of the internal elastic lamina, excessive dilation (lumen diameter 7500 μM2 in Ahsp KO vs 4100 μM2 in controls; n=5 mice, p=0.0037) and reduced constriction after phenylephrine treatment (EC50 9.6e-6 M-1 in wt n=7 vs 5.6e-6 M-1 in mutant n=6; p<0.0001) (Fig. 1B-C), similar to what we observed in HbA1-/- mice.To examine physical and functional interactions between AHSP, αHb and eNOS, we expressed fluorescent-tagged proteins in cultured human coronary ECs. αHb-GFP alone was expressed at relatively low level (mean fluorescent intensity (MFI) = 631) (Fig. 1D). Coexpressed mCherry-AHSP colocalized with αHb-GFP and increased its expression level (MFI 5312) (Fig. 1D). Similarly, coexpressed mCherry-eNOS colocalized with αHb-GFP and enhanced its expression (MFI 1519) (Fig. 1D). Purified αHb co-immunoprecipitated with AHSP or eNOS, but not both, suggesting mutually exclusive interactions.Overall, our studies provide genetic evidence that αHb expressed in arteriolar endothelial cells regulates blood vessel tone in vivo. Moreover, biochemical studies show that AHSP stabilizes endothelial-expressed αHb and facilitates its assembly with eNOS. Thus, AHSP acts as a molecular chaperone for αHb in erythrocytes and endothelial cells to promote the formation of HbA (α2b2) for O2 transport, and αHb-eNOS for NO degradation. We hypothesize that the αHb-AHSP-eNOS axis functions to fine-tune systemic blood pressure and/or regional blood flow to specific vascular beds. Defining the magnitude of these effects in mice and humans during specific circulatory stresses and in α thalassemia is an important and interesting topic for future investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ДИНАМИКА МОРФОЛОГИЧЕСКИХ ПОКАЗАТЕЛЕЙ КРОВИ СОБАК ПРИ ДОБАВЛЕНИИ ДИГИДРОКВЕРЦЕТИНА

The purpose of research is to increase protective and adaptive reactions, the service and the working potential of an organism of dogs through the use of DHQ. The article presents the results of an experiment to the effect of DHQ on the dynamics of hematological parameters of dogs blood. Dihydroquercetin – an active antioxidant, a natural scavenger of oxygen free radicals, hepatoprotective, has anti-inflammatory action due to limitations of the formalin edema and histamine, and inhibits the formation of serous fluid, pain killers, immunocorrectional properties. Due to the high complexing properties it displays the body of heavy metals, including radionuclides, it helps to restore the tone of blood vessels, normalization of the lipid spectrum of the blood and slows the development of atherosclerotic plaques. Investigations were carried out in Dogs State Service Ministry of I A of Russia zonal center in Samara Region by clinically healthy of dog breed German Shepherd, age 2-4 years with an average body weight of 30 kg on the background conditions and feeding, adopted by the enterprise. Dogs experimental group received Dihydroquercetin to 0.001 g/kg body weight dose once a day with food. When added to the basic diet Dihydroquercetin dog experimental group show an increase in indicators such as red blood cells – by 18.3% (p&amp;#60;0.01), hemoglobin – by 11.7% (p &amp;#60;0.01) hematocrit – 7.1% (p&amp;#60;0.01), white blood cells – by 9.1% (p&amp;#60;0.05) compared to control data. The research results give reason to believe that the use of dietary supplements in Dihydroquercetin in dogs diet dose 0.001 g/kg allows significantly increase the morphological and physiological indicators of animal organism. In this case a significant increase erythrocytes and hemoglobin and hematocrit indicates increased oxidative metabolism and function of the intensity of the blood in dogs treated with the drug, and increase in the blood indicates an increase in immune status.

Abstract 378: Cytochrome b5 Reductase 3 Regulates Vascular Smooth Muscle Phenotypic Switching

Contractile vascular smooth muscle cells (VSMC) play a key role in the regulation of arterial blood vessel tone and cardiovascular health. However, in many vascular diseases, VSMCs undergo a phenotypic switch from a contractile state to a synthetic phenotype, where loss of the contractile markers myosin heavy chain 11 (Myh11), smooth muscle alpha actin (ACTA2) and transgelin (SM22) are observed and proliferation is increased. Recent evidence from our lab demonstrates that cytochrome b5 reductase 3 (Cyb5R3) regulates the redox state of soluble guanylate cyclase to control cGMP levels in VSMCs. Because cGMP modulates protein kinase G activity, a critical kinase that maintains VSMCs in a contractile state, we tested the hypothesis that Cyb5R3 is critical for maintenance of the contractile phenotype. To test this hypothesis, we transduced primary rat aortic smooth muscle cells with non-targeting (NT) or Cyb5R3 shRNA followed by serum starvation for 24, 48, and 72 hours to induce phenotypic switching. After each time point, mRNA measurements of Cyb5R3, Myh11, ACTA2, and SM22 were conducted using RT-PCR. In NT shRNA transduced VSMCs, we observed a significant increase in Cyb5R3, Myh11, ACTA2, and SM22 mRNA, but not in Cyb5R3 knockdown VSMCs. Next, we conducted proliferation studies by serum starving NT shRNA and Cyb5R3 shRNA treated VSMCs for 24 hours followed by stimulation of platelet growth factor BB (PDGF-BB, 40 ng/mL). After 24 hours of PDGF-BB treatment, Cyb5R3 deficient cells showed augmented proliferation compared to control cells measured by 3 H-thymidine incorporation. Together, our data suggest that Cyb5R3 is essential for VSMC phenotypic switching and proliferation, which may unravel a new therapeutic target for treating individuals with cardiovascular disease.

CaV2.3 Mediate ATP Release from Rat Tail Artery Sympathetic Nerves

Peripheral sympathetic nerves are essential for regulating homeostatic control of target organs, such as the tone of blood vessels, and many details of the molecular organization of peripheral sympathetic nerve varicosities remain to be defined. Unlike central synapses, peripheral varicosities typically exhibit co-transmission of multiple neurotransmitters. Several lines of evidence show co-transmission of the sympathetic transmitters norepinephrine (NE) or ATP is mediated by the preferential association of separate pools of vesicles with CaV2.1 and CaV2.2 isoforms that provide the Ca2+ entry for vesicle release. CaV2.3 is a closely related channel that has been studied in several central synapses, but its role in peripheral sympathetic nerves is largely unknown. Using confocal microscopy to study perivascular nerves, we previously found that ATP and NE are stored in segregated pools of vesicles. Further, we found that CaV2.3 are expressed in rat tail perivascular sympathetic nerves and that they co-localize with vesicles expressing the vesicular nucleotide transporter (VNUT) that loads ATP into vesicles rather than vesicle harbouring the NE-transporting vesicular monoamine translocase 2 (VMAT2). We now report one of the first functional characterizations of CaV2.3 in perivascular nerves. We assessed the functional role of CaV2.3 in field-stimulated isometric force generated by rat tail arteries in response to pulse-number ramps (3-100 pulses, 20 Hz). Inhibition of vasoconstriction with the CaV2.3 blocker SNX-482 (250 nM) mimicked the effect of blocking smooth muscle P2X receptors with suramin while blocking α-adrenergic receptors with phentolamine productive a distinct inhibitory profile. These functional tests were corroborated by immuno-fluorescence microscopy showing that CaV2.3 immunoreactivity correlated with the sensitivity of individual blood vessels to suramin and SNX-482.

Cryo-EM Structure of the BK Ion Channel in a Lipid Membrane

The large-conductance voltage- and Ca2+-activated potassium (BK) channel has many physiological roles including the control of firing patterns in neurons, the modulation of the tone of blood vessels and the regulation of heart rate. Among ion channels, it has served as a model system because of its remarkable ion-permeation properties and its accessibility for studies of allosteric control of gating.As shown in both structural and functional studies, the lipid membrane environment has played an essential role for membrane proteins. To restore the lipid membrane environment, BK channels were reconstituted into liposomes (lipid vesicles) for both functional and structural studies. In 2009, we have reported the full-length BK structure in lipid environment at a resolution of 1.7nm using “random spherically constrained” (RSC) single-particle Cryo-EM method. Recent breakthrough in hardware and the optimization of image collection and processing techniques make it possible to achieve a higher resolution. Here, we present the BK structure in a lipid membrane in the closed state with a greatly improved resolution. The helices in the transmembrane region can be clearly identified, and the helix S0 is identified to be next to helices S2 and S3.

Endothelium dependent hyperpolarization-type relaxation compensates for attenuated nitric oxide-mediated responses in subcutaneous arteries of diabetic patients

Diabetes impairs endothelium-dependent relaxations. The present study evaluated the contribution of different endothelium-dependent relaxing mechanisms to the regulation of vascular tone in subcutaneous blood vessels of humans with Type 2 diabetes mellitus. Subcutaneous arteries were isolated from tissues of healthy controls and diabetics. Vascular function was determined using wire myography. Expressions of proteins were measured by Western blotting and immunostaining. Endothelium-dependent relaxations to acetylcholine were impaired in arteries from diabetics compared to controls (P = 0.009). Acetylcholine-induced nitric oxide (NO)-mediated relaxations [in the presence of an inhibitor of cyclooxygenases (COX; indomethacin) and small and intermediate conductance calcium-activated potassium channel blockers (UCL1684 and TRAM 34, respectively)] were attenuated in arteries from diabetics compared to controls (P < 0.001). However, endothelium-dependent hyperpolarization (EDH)-type relaxations [in the presence of indomethacin and the NO synthase blocker, l-NAME] were augmented in arteries from diabetics compared to controls (P = 0.003). Endothelium-independent relaxations to sodium nitroprusside (NO donor) and salbutamol (β-adrenoceptor agonist) were preserved, but those to prostacyclin were attenuated in diabetics compared to controls (P = 0.017). In arteries of diabetics, protein expressions of endothelial NO synthase, prostacyclin synthase and prostacyclin receptors were decreased, but those of COX-2 were increased. These findings suggest that in human diabetes, the impairment of endothelium-dependent relaxations is caused by a diminished NO bioavailability; however, EDH appears to compensate, at least in part, for this dysfunction.

Nitric Oxide: From the Molecule of the Year to an Important Signaling Molecule

Nitric oxide (NO) is a colorless, ubiquitous, free radical gas because of having an unpaired electron. Nitric oxide is synthesized from an amino acid called L-arginine by the action of the enzyme nitric oxide synthase (NOS) which has three important isoforms. NO has been molecule of attention because of its small size, short duration of action and variety of physiological functions. NO plays many important and critical physiological functions such as cell signaling, regulation of tone of blood vessels, neurotransmission, inflammatory processes (inhibition of leukocytic adhesion to vascular endothelium), antithrombotic effects by inhibiting platelet adhesion to vascular endothelium and the defense mechanism of the body against microorganisms. Alterations in the regulation of NO metabolism play key role in many pathological processes like development of the cancer, hypertension, obesity, atherosclerosis, preeclampsia and aging. This review will focus on its role in physiological functions in the body and alterations in some important pathological states. An insight into the functioning of this molecule will help the clinicians as well as researchers to understand the different dimensions in the pathogenesis of many diseases.

The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.