Sensitivity Of Vascular Smooth Muscle Research Articles

Nadir blood pressure responses to longer consecutive cardiac cycle sequences absent of sympathetic bursts are associated with popliteal endothelial-dependent dilation

PurposeThe nadir pressure responses to cardiac cycles absent of muscle sympathetic nerve activity (MSNA) bursts (or non-bursts) are typically reported in studies quantifying sympathetic transduction, but the information gained by studying non-bursts is unclear. We tested the hypothesis that longer sequences of non-bursts (≥8 cardiac cycles) would be associated with a greater nadir diastolic blood pressure (DBP) and that better popliteal artery function would be associated with an augmented reduction in DBP. MethodsResting beat-by-beat DBP (via finger photoplethysmography) and common peroneal nerve MSNA (via microneurography) were recorded in 39 healthy, adults (age 23.4 ± 5.3 years; 19 females). For each cardiac cycle absent of MSNA bursts, the mean nadir DBP (ΔDBP) during the 12 cardiac cycles following were determined, and separate analyses were conducted for ≥8 or < 8 cardiac cycle sequences. Popliteal artery endothelial-dependent (via flow-mediated dilation; FMD) and endothelial-independent vasodilation (via nitroglycerin-mediated dilation; NMD) were determined. ResultsThe nadir DBP responses to sequences ≥8 cardiac cycles were larger (−1.40 ± 1.27 mmHg) than sequences <8 (−0.38 ± 0.46 mmHg; p < 0.001). In adjusting for sex and burst frequency (14 ± 8 bursts/min), larger absolute or relative FMD (p < 0.01), but not NMD (p > 0.53) was associated with an augmented nadir DBP. This overall DBP-FMD relationship was similar in sequences ≥8 (p = 0.04–0.05), but not <8 (p > 0.72). ConclusionThe DBP responses to non-bursts, particularly longer sequences, were inversely associated with popliteal endothelial function, but not vascular smooth muscle sensitivity. This study provides insight into the information gained by quantifying the DBP responses to cardiac cycles absent of MSNA.

Does actin polymerization contribute to closure of the avian ductus arteriosus (Gallus gallus)?

The ductus arteriosus (DA) is a cardiovascular embryonic shunt present in all developing land vertebrates. This oxygen-sensitive blood vessel plays the important role of connecting the pulmonary artery to the aorta during development and directs blood flow toward the fetal respiratory organ (placenta in mammals or chorioallantois in birds and reptiles). Permanent vessel closure upon birth or hatch is necessary to establish proper circulation in the neonate. Increased arterial PO2 at birth or hatch stimulates DA closure via smooth muscle contraction. Smooth muscle contraction, such as in the DA, is governed by myosin light chain kinase (MLCK) stimulating increased cross-bridge cycling, and through increases in actin polymerization. The Ras homologous protein family (Rho) of GTPases are involved in calcium sensitization and contraction of vascular smooth muscle but have mostly been studied for their role in cell migration and signaling. Rho GTPases activate the Rho activated kinase (ROCK) pathway, which works in conjunction with MLCK to generate smooth muscle contraction as well as activating LIM kinase which inhibits the actin depolymerizer cofilin. Rac and Cdc42 GTPase pathways increase contractile strength in smooth muscle cells by activating the nucleator Arp2/3, leading to increased actin polymerization. The role of these GTPase pathways during closure of the DA is unknown. We examined the role of the Rho, Rac, and Cdc42 GTPase pathways leading to actin polymerization for their role in the closure of the avian DA in late-term chicken ( Gallus gallus) embryos. We hypothesized that inhibiting the pathways that lead to actin polymerization will inhibit tension development in the DA. Using a Danish Myo Technology wire myograph, DA physiology was examined when exposed to 25% oxygen followed by activators and inhibitors of the Rho, Rac, and Cdc42 GTPase pathways. Activation of Rho A pathways with CN01 (0.2 units/ml) produced contraction of the DA. Blocking ROCK pathways with Y-27632 (10 mM) removed the O2-sensitivity of the vessel, while the vessel was still able to respond to phenylephrine (100 mM). This suggests the Rho pathway is involved in maintaining baseline tension and contractile response to O2. Exposure to inhibitors of Rac (10 uM EHT 1864) and Cdc42 (via ML141) GTPases blocked the ability of vessels to contract in response to 25% oxygen, even after 3 hours of constant O2. Similarly, inhibition of the Arp2/3 complex (100 uM CK666) reduced the ability of the vessel to respond to oxygen. These findings suggest that Rho A, Rac, and Cdc42 pathways play a role in active tension of the chicken DA during development by potentially increasing actin polymerization. This research was funded by Grant R15HL14887 from NIH-NHLBI. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sodium valproate treatment reverses endothelial dysfunction in aorta from rabbits with acute myocardial infarction

Sodium valproate (VPA), a histone deacetylase (HDAC) inhibitor, could be a promising candidate to treat acute myocardial infarction (AMI). In this study, AMI was induced in New Zealand White rabbits by occluding the left circumflex coronary artery for 1 h, followed by reperfusion. The animals were distributed into three experimental groups: the sham-operated group (SHAM), the AMI group and the AMI + VPA group (AMI treated with VPA 500 mg/kg/day). After 5 weeks, abdominal aorta was removed and used for isometric recording of tension in organ baths or protein expression by Western blot, and plasma for the determination of nitrate/nitrite (NOx) levels by colorimetric assay. Our results indicated that AMI induced a reduction of the endothelium-dependent response to acetylcholine without modifying the endothelium-independent response to sodium nitroprusside, leading to endothelial dysfunction. VPA treatment reversed AMI-induced endothelial dysfunction and even increased NO sensitivity in vascular smooth muscle. This response was consistent with an antioxidant effect of VPA, as it was able to reverse the superoxide dismutase 1 (SOD 1) down-regulation induced by AMI. Our experiments also ruled out that the VPA mechanism was related to eNOS, iNOS, sGC and arginase expression or changes in NOx plasma levels. Therefore, we conclude that VPA improves vasodilation by increasing NO bioavailability, likely due to its antioxidant effect. Since endothelial dysfunction was closely related to AMI, VPA treatment could increase aortic blood flow, making it a potential agent in reperfusion therapy that can prevent the vascular damage.

Calponin 1 inhibits agonist-induced ERK activation and decreases calcium sensitization in vascular smooth muscle.

Smooth muscle cell (SMC) contraction and vascular tone are modulated by phosphorylation and multiple modifications of the thick filament, and thin filament regulation of SMC contraction has been reported to involve extracellular regulated kinase (ERK). Previous studies in ferrets suggest that the actin-binding protein, calponin 1 (CNN1), acts as a scaffold linking protein kinase C (PKC), Raf, MEK and ERK, promoting PKC-dependent ERK activation. To gain further insight into this function of CNN1 in ERK activation and the regulation of SMC contractility in mice, we generated a novel Calponin 1 knockout mouse (Cnn1 KO) by a single base substitution in an intronic CArG box that preferentially abolishes expression of CNN1 in vascular SMCs. Using this new Cnn1 KO mouse, we show that ablation of CNN1 has two effects, depending on the cytosolic free calcium level: (1) in the presence of elevated intracellular calcium caused by agonist stimulation, Cnn1 KO mice display a reduced amplitude of stress and stiffness but an increase in agonist-induced ERK activation; and (2) during intracellular calcium depletion, in the presence of an agonist, Cnn1 KO mice exhibit increased duration of SM tone maintenance. Together, these results suggest that CNN1 plays an important and complex modulatory role in SMC contractile tone amplitude and maintenance.

Endothelial dysfunction of the mesenteric arteries of rats in the early obesity induced by high-fat diet

Background: Disturbance of the morphological and functional properties of the vascular bed in obesity are a serious clinical problem. Basis to their development is endothelial dysfunction. The developed models of obesity in animals using various diets indicate a change in vascular reactivity, however, questions about the stage at which this occurs and what mechanisms are involved in this process remain open, while they are decisive for choosing the correct tactics for correcting dysfunctions.Aim: The aim of the present study is to determine the changes in acetylcholine (ACh)-induced vasodilation of isolated arteries from rats after six weeks of administration of a high-fat diet (HFD).Materials and methods: The experiments were performed on Sprague-Dawley males, which at the age of 8 weeks were divided into 2 experimental groups that were treated for the next 6 weeks in the following manner: 1 - control) with standard dry food; 2 - a group fed with a HFD, the total amount of fat in which was 50%. At finish of the diet, the degree of obesity, biochemical parameters in the blood, and blood pressure were measured. Intravital microscopy of the rat mesentery with video recording was used to study the reactivity of the vessels. The contractile and relaxant responses of the vessels were determined by changes in their diameter.Results: The rats after treatment with the HFD (n=15) had higher body weight and amount of visceral fat, significantly increased blood triglycerides, moderate increases in glucose level in blood and systolic pressure compared with the control (n=15). Relaxation responses of mesenteric arteries, having a diameter of 140 to 300 μm in PSS, were recorded after precontraction by phenylephrine. A decrease in ACh-induced vasorelaxation was obtained, which manifests itself before the development of significant changes in carbohydrate metabolism. Incubation of drugs with the inhibitor of endothelial NO synthase L-NAME led to a pronounced weakening of relaxation in animals on a standard diet, and had little effect on vasodilation in the arteries of rats with the HFD. Vasodilation induced by the administration of sodium nitroprusside (NO donor) did not differ significantly in control and experimental animals, which indicates that the sensitivity of vascular smooth muscle to NO remained practically unchanged. ACh-induced relaxation of arteries in dietary rats did not change when the cyclooxygenase pathway was blocked by diclofenac.Conclusion: Functional changes in the contractile activity of the mesenteric arteries, manifested in the form of a decrease in ACh-induced vasorelaxation, occur after treatment with the HFD when animals had an early stage of obesity development before the onset of pronounced disorders of carbohydrate metabolism. This decrease is mainly due to the disruption of the NO-dependent mechanism underlying ACh-induced relaxation in the norm.

Characterization of Endothelium-Dependent Relaxation in the Saphenous Artery and Its Caudal Branches in Young and Old Adult Sprague Dawley Rats.

Ageing is associated with reduced endothelium-derived nitric oxide (NO) production in the femoral artery of Sprague Dawley (SD) rats. In the current study, we examined endothelium-dependent relaxation (EDR) in the saphenous artery and its caudal branches. We used acetylcholine and the Proteinase-Activated receptor-2 (PAR2)-specific agonist (2fLIGRLO) with nitroarginine methylester (L-NAME) to assess EDR in two groups of male SD rats (age in weeks: young, 10–12; old, 27–29). Acetylcholine and 2fLIGRLO were potent NO-dependent relaxant agents in all arteries. For all arteries, EDR by acetylcholine decreased significantly in old compared to young SD rats. Interestingly, PAR2-induced EDR of proximal saphenous artery segments and caudal branches decreased significantly in old compared to young, but did not differ for the in-between middle and distal ends of the saphenous artery. L-NAME treatment increased subsequent contractions of proximal and middle segments of saphenous arteries by phenylephrine and U46619 in young, but not in old, SD rats. We conclude the SD saphenous artery and caudal branches exhibit regional characteristics that differ in response to specific EDR agonists, endothelial NO synthase inhibitor, and changes to endothelium function with increased age, which are, in part, attributed to decreased sensitivity of vascular smooth muscle to the gaseous transmitter NO.

Hesperetin Inhibits Sphingosylphosphorylcholine-Induced Vascular Smooth Muscle Contraction by Regulating the Fyn/Rho-Kinase Pathway.

Cardiovascular diseases are the leading cause of mortality and disability worldwide. We have previously found that sphingosylphosphorylcholine (SPC) is the key molecule leading to vasospasm. We have also identified the SPC/Src family protein tyrosine kinase Fyn/Rho-kinase (ROK) pathway as a novel signaling pathway for Ca2+ sensitization of vascular smooth muscle (VSM) contraction. This study aimed to investigate whether hesperetin can inhibit the SPC-induced contraction with little effect on 40 mM K+-induced Ca2+-dependent contraction and to elucidate the underlying mechanisms. Hesperetin significantly inhibited the SPC-induced contraction of porcine coronary artery smooth muscle strips with little effect on 40 mM K+-induced contraction. Hesperetin blocked the SPC-induced translocation of Fyn and ROK from the cytosol to the membrane in human coronary artery smooth muscle cells (HCASMCs). SPC decreased the phosphorylation level of Fyn at Y531 in both VSMs and HCASMCs and increased the phosphorylation levels of Fyn at Y420, myosin phosphatase target subunit 1 at T853, and myosin light chain (MLC) at S19 in both VSMs and HCASMCs, which were significantly suppressed by hesperetin. Our results indicate that hesperetin inhibits the SPC-induced contraction at least in part by suppressing the Fyn/ROK pathway, suggesting that hesperetin can be a novel drug to prevent and treat vasospasm.

Testosterone and Exercise in Middle-to-Older Aged Men: Combined and Independent Effects on Vascular Function.

[Figure: see text].

The impact of age and sex on popliteal artery endothelial-dependent vasodilator and vasoconstrictor function.

Lower-limb arteries, such as the popliteal artery, are a common site of atherosclerosis. These arteries are habitually exposed to large fluctuations in blood flow during physical and sedentary activities. Low-flow-mediated constriction (L-FMC) and flow-mediated dilation (FMD) provide indices of endothelial-dependent vasoconstriction and vasodilation, respectively. Age and sex both impact upper-limb FMD. However, it is unclear whether these factors also influence popliteal endothelial-dependent function. Popliteal endothelial function was compared between younger and older males and females (n=14 per group) matched for age- and sex-specific relative aerobic fitness levels (each group's normative percentile: ~45%). Nitroglycerin-mediated dilation (NMD) was also assessed as a measure of endothelial-independent vasodilation. Ageing reduced relative popliteal FMD in both males (older: 4.3±1.8% versus younger 5.7±1.9%) and females (older: 2.9±1.8% versus younger: 6.1±1.6%, both: P<0.046). FMD was also lower in older females versus older males (P=0.04). Popliteal NMD findings followed the same pattern as FMD. Compared to younger adults, relative L-FMC responses were blunted among older males (older: -1.2±1.1% versus younger: -2.2±1.0%) and females (older: -1.0±1.2% versus younger: -2.1±1.3%, both P<0.03) with no sex-differences observed in either age group (all, P>0.60). The adverse age- and sex-related (older adults only) declines in popliteal FMD were mediated, in part, by reduced vascular smooth muscle sensitivity to nitric oxide. Endothelial-dependent vasoconstriction was also attenuated with age, but unaffected by sex. Despite similar normative aerobic fitness percentiles (~45%), older adults exhibited attenuated popliteal endothelial function than their younger counterparts. This was particularly evident in older females who exhibited the lowest endothelial-dependent vasodilatory responses.

Exposure to maternal diabetes induces endothelial dysfunction and hypertension in adult male rat offspring

The adverse environment in early life can modulate adult phenotype, including blood pressure. Our previous study shows, in a rat streptozotocin (STZ)-induced maternal diabetes model, fetal exposure to maternal diabetes is characterized by established hypertension in the offspring. However, the exact mechanisms are not known. Our present study found, as compared with male control mother offspring (CMO), male diabetic mother offspring (DMO) had higher blood pressure with arterial dysfunction, i.e., decreased acetylcholine (Ach)-induced vasodilation. But there is no difference in blood pressure between female CMO and DMO. The decreased Ach-induced vasodilation was related to decreased nitric oxide (NO) production in the endothelium, not NO sensitivity in vascular smooth muscle because sodium nitroprusside (SNP)-mediated vasodilation was preserved; there was decreased NO production and lower eNOS phosphorylation in male DMO. The reactive oxygen species (ROS) level was increased in male DMO than CMO; normalized ROS levels with tempol increased NO production, normalized Ach-mediated vasodilation, and lowered blood pressure in male DMO rats. It indicates that diabetic programming hypertension is related to arterial dysfunction; normalizing ROS might be a potential strategy for the prevention of hypertension in the offspring.

Sex and light physical activity impact popliteal, but not brachial artery flow-mediated dilation in physically active young adults.

When controlling for baseline diameter, males have greater brachial flow-mediated dilation (BA-FMD) responses than females. It is unclear whether sex differences in baseline diameter also influences popliteal FMD (POP-FMD), which may be impacted by cardiorespiratory fitness and physical activity levels. We hypothesized that males would exhibit greater BA-FMD and POP-FMD when allometrically scaled to baseline diameter. FMD (ultrasonography), cardiorespiratory fitness (indirect calorimetry), and objectively measured physical activity were assessed in males (n = 13; age, 23 ± 3 years; peak oxygen consumption, 48.0 ± 7.1mL·kg-1·min-1) and females (n = 13; age, 24 ± 2 years; peak oxygen consumption, 36.8 ± 6.0mL·kg-1·min-1). Both groups had similarly high levels of moderate-to-vigorous intensity physical activity (503 ± 174 vs. 430 ± 142min·week-1, p= 0.25). However, males were more aerobically fit (p < 0.001) and females accumulated more light-intensity physical activity (182 ± 67 vs. 127 ± 53min·week-1, p= 0.03). Relative and allometrically scaled BA-FMD were similar (both, p≥ 0.09) between sexes. In contrast, relative (6.2% ± 1.0% vs. 4.6% ± 1.4%, p= 0.001) and scaled (6.8% ± 1.7% vs. 4.7% ± 1.7%, p= 0.03) POP-FMD were greater in females. Relative POP-FMD was related to light-intensity physical activity in the pooled sample (r = 0.43; p= 0.04). However, the enhanced relative POP-FMD in females remained after adjusting for higher light-intensity physical activity levels (p= 0.01). Young females have enhanced popliteal, but not brachial, endothelial health than males with similar moderate-to-vigorous intensity physical activity levels and higher cardiorespiratory fitness. Novelty In physically active adults, females had greater POP-FMD but not BA-FMD than males. The enhanced POP-FMD in females was not related to greater vascular smooth muscle sensitivity to nitric oxide or their smaller baseline diameters. POP-FMD was associated with light physical activity levels in the pooled sample.

Cigarette smoke extract increases expression and plasma membrane trafficking of α2C ‐ adrenoceptors in human microvascular smooth muscle cells

Vasoconstriction of cutaneous arterioles in response to cold temperatures or emotion is a normal adaptive process in which the body diverts blood flow from the superficial circulation to internal organs to prevent heat loss. This is mediated by increased sympathetic activation as well as increased sensitivity of the digital arteriolar vascular smooth muscle to vasoconstrictors. When cold‐induced constriction is exacerbated, Raynaud’s phenomenon (RP) is precipitated. Remarkably, the entirety of this augmented cold‐induced vasoconstriction is mediated by α2Cadrenoceptors (α2C‐ARs) . We have previously established that cooling induces translocation of α2C‐ARs from the Golgi compartment, where they are trapped, to the plasma membrane of microvascular smooth muscle cells.Epidemiological studies show that cigarette smoking is a significant risk factor for RP. Here, we provide exciting evidence which shows that cigarette smoke extract (CSE) increases α2C‐AR protein expression in a concentration and time‐dependent manner in human arteriolar smooth muscle cells. This expression appears to be due to increased transcriptional activity of the α2C‐AR promoter since pretreatment with actinomcyin D, a transcription inhibitor, abolished CSE‐induced α2C‐AR protein expression. Moreover, CSE increased transcriptional activity of α2C‐AR promoter: reporter construct. Interestingly, apocynin, a ROS scavenger, abolished CSE‐induced α2C‐AR protein expression, indicative of a ROS‐mediated pathway. CSE also increased RhoA activation, a pathway that we have previously established to drive trafficking of α2C‐ARs to the membrane. Indeed, CSE evoked mobilization of α2C‐ARs to the cell membrane. Taken together, these results may explain the underlying mechanism of CSE‐exacerbated cold‐induced vasoconstriction and provide an alternative avenue for treatment of patients who have RP and are smokers.Support or Funding InformationThis publication was made possible by an MPP fund (#320133) from the American University of Beirut to Ali Eid

The commonly‐used anthracycline chemotherapy drug Doxorubicin impairs vascular endothelial function via stimulation of mitochondrial superoxide

Doxorubicin (DOXO), a commonly used anthracycline chemotherapeutic agent, increases the risk of cardiovascular (CV) related morbidity and mortality. A key mechanism by which DOXO increases CV disease risk is by inducing endothelial dysfunction (impaired endothelium‐dependent dilation [EDD]) but: 1) the underlying mechanisms are unknown; 2) there are no established treatments for preventing these side effects.PurposeWe tested the hypotheses that: 1) DOXO impairs EDD via excess mitochondrial (mito) superoxide‐induced reductions in nitric oxide (NO) bioavailability; 2) these effects can be prevented with a mito‐targeted antioxidant.MethodsC57BL6 male mice (4mo of age) received one injection of either sham (PBS; n=4) or DOXO (10mg/ml; n=8). After 4 weeks, we assessed endothelial function (ex vivo carotid artery EDD to increasing doses of acetylcholine [ACh]) and potential underlying mechanisms: NO bioavailability (ACh dose response in the presence of the NO‐synthase inhibitor, L‐NAME); aortic total and mito‐specific superoxide production (electron paramagnetic resonance spectroscopy); aortic cytosolic, extracellular and mito isoforms of superoxide dismutase (SOD) (immunoblot); and the roles of excess total and mito‐specific superoxide in regulating EDD (ACh dose response in the presence of the SOD mimetic, TEMPOL, and the mito‐targeted antioxidant, MitoQ, respectively).ResultsMechanisms of ActionDOXO reduced maximal EDD by 41% (95±3 vs. 54±3 %; P=0.003). This reduction was mediated by a decrease in NO bioavailability, as EDD was not different between groups following incubation with L‐NAME. Vascular smooth muscle sensitivity to NO (dilatory response to sodium nitroprusside) was unaltered. DOXO increased aortic total superoxide production by 38% (5.3±0.7 to 7.3±0.5 amplitude units; P=0.003), without compensatory upregulation in aortic cytosolic or extracellular SOD. Reduced EDD with DOXO was mediated by excess superoxide production, as indicated by restoration of maximal EDD with TEMPOL (97±2%). DOXO increased aortic mito superoxide production by 50% (1.0±0.1 to 1.5±0.1 amplitude units; P=0.01) and induced a 60% reduction in aortic mito SOD (1.0±0.2 vs. 0.4±0.1 absorbance units; P=0.01). Reduced EDD with DOXO was mediated by increased mito superoxide production, as indicated by restoration of maximal EDD with MitoQ (91±8%).PreventionNext, we supplemented mice with MitoQ (250μM in drinking water; n=5) for 4 weeks following DOXO and showed that MitoQ prevented the reduction in EDD (97±1%, P=0.002 vs. DOXO alone) by preserving NO bioavailability via reduced mito superoxide (no added improvement with MitoQ incubation).ConclusionThese results suggest DOXO‐induced vascular endothelial dysfunction is mediated by excess mito superoxide‐induced reduction in NO bioavailability. Our results also demonstrate that MitoQ supplementation prevented DOXO‐induced endothelial dysfunction by suppressing mito superoxide and preserving NO bioavailability. Because MitoQ is available and safe for human use, there may be translational potential for reducing CV‐related morbidity and mortality in patients that have received DOXO chemotherapy.Support or Funding InformationNIH T32 DK007135‐44 (Z.S.C.)

P4477Editing of myosin phosphatase as a novel approach for sensitization of vascular smooth muscle to vasodilators (NO/cGMP/ROS) and lowering of blood pressure in hypertension

Abstract Background Despite the many drugs for treatment of hypertension, it remains inadequately treated in &gt;50% of patients and the number one contributor to cardiovascular mortality world-wide. Thus new targets and treatment strategies are badly needed. Myosin Phosphatase (MP) is a viable target: it is the primary effector of vascular smooth muscle relaxation and a critical mediator of signaling pathways regulating vessel tone. Purpose We are using complementary/ translatable approaches to test the hypothesis: editing of the Myosin Phosphatase Regulatory (Targeting) subunit (MYPT1), by shifting the expression of naturally occurring isoforms, will sensitize vascular smooth muscle to NO/cGMP/ROS mediated vasorelaxation and thereby lower BP in models of hypertension. A further goal is to determine mechanisms by which these signals activate MP thereby causing vasorelaxation. Methods LoxP sites were inserted in introns flanking alternative Exon24 (E24) of Mypt1. Mice were crossed with smMHCCreER mice and treated with Tamoxifen for smooth muscle specific deletion of E24 (SMcKO E24).Skipping E24 codes for a Mypt1 isoform that contains a C-terminal leucine zipper (LZ) motif required for cGMP-dependent protein kinase (cGK1) binding and NO/cGMP/ROS activation of MP. Second, we developed and tested guide RNAs for the purpose of AAV-CRISPR/CAS9 editing of Mypt1 E24 as a treatment for hypertension. Effect of editing is tested in otherwise normal mice and in the AngII sub-pressor model of hypertension. Results SMcKO E24 mice had mean BP that was 15+3 mmHg lower than control (n=5; p&lt;0.05). Mesenteric arteries from these mice were significantly more sensitive to DEA/NO mediated relaxation (EC50: 2.1+0.5 nM vs 18.2+5.6 mM; n=5–6, p&lt;0.05). Experiments testing response to AngII infusion are in progress and will be presented at the meeting. Preliminary biochemical assays support a 2-pool model, in which NO/cGMP/ROS activates the LZ+ pool, while contractile agonists inhibit the LZ- pool of MP, in the control of BP/ blood flow. We have generated a number of AAV Crispr/Cas9 gRNAs and validated their efficacy of editing of Mypt1 E24 in vitro. Experiments are in progress to test their efficacy and effect on BP in vivo. Conclusion These studies support that editing of Mypt1 E24 could be a novel strategy for vasodilator sensitization and effective lowering of blood pressure in humans with hypertension, thereby having a substantial impact on CV mortality world-wide. Acknowledgement/Funding NIH

Deletion of Rap1 disrupts redox balance and impairs endothelium-dependent relaxations

AimsRepressor activator protein 1 (Rap1) is conventionally known as a static structural component of the telomere, but recent evidence indicates that it exerts functions within and outside the nucleus taking part in metabolic regulation and promoting inflammatory responses. The present study investigated whether or not Rap1 deletion affects oxidative stress and nitric oxide (NO) bioavailability in the vascular wall, thus modulating endothelial function. Methods and resultsVascular responsiveness was studied in wire myographs in aortae from Rap1 wildtype and knockout mice. Deletion of Rap1 impaired endothelium-dependent relaxations elicited by acetylcholine. Rap1 deficiency did not affect the activation of endothelial NO synthase or the sensitivity of vascular smooth muscle to NO donors. The blunted acetylcholine-mediated relaxations in Rap1 deficient aortae were restored with nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, apocynin or VAS2870. Rap1 deletion lowered cellular thiol-redox status and diminished activities of thiol-redox enzymes, thioredoxin 1 and glutaredoxin 1. ConclusionsThe capacity of thioredoxin 1 and glutaredoxin 1 to reduce intra-protein disulfide bridges is weakened in Rap1 deficient mice, resulting in hyper-activation of NADPH oxidase and greater reactive oxygen species generation. The high oxidative stress in Rap1 deficient mice is implicated with greater oxidative breakdown of NO, explaining the blunted acetylcholine-mediated relaxations in this animal. These findings imply that Rap1 plays an unanticipated role in regulating the fate of NO (a pivotal determinant of vascular homeostasis) and thus identify a new physiological importance of the telomere-associated protein.

The AMP-Related Kinase (AMPK) Induces Ca 2+ -Independent Dilation of Resistance Arteries by Interfering With Actin Filament Formation

Decreasing Ca2+ sensitivity of vascular smooth muscle (VSM) allows for vasodilation without lowering of cytosolic Ca2+. This may be particularly important in states requiring maintained dilation, such as hypoxia. AMP-related kinase (AMPK) is an important cellular energy sensor in VSM. Regulation of Ca2+ sensitivity usually is attributed to myosin light chain phosphatase activity, but findings in non-VSM identified changes in the actin cytoskeleton. The potential role of AMPK in this setting is widely unknown. To assess the influence of AMPK on the actin cytoskeleton in VSM of resistance arteries with regard to potential Ca2+ desensitization of VSM contractile apparatus. AMPK induced a slowly developing dilation at unchanged cytosolic Ca2+ levels in potassium chloride-constricted intact arteries isolated from mouse mesenteric tissue. This dilation was not associated with changes in phosphorylation of myosin light chain or of myosin light chain phosphatase regulatory subunit. Using ultracentrifugation and confocal microscopy, we found that AMPK induced depolymerization of F-actin (filamentous actin). Imaging of arteries from LifeAct mice showed F-actin rarefaction in the midcellular portion of VSM. Immunoblotting revealed that this was associated with activation of the actin severing factor cofilin. Coimmunoprecipitation experiments indicated that AMPK leads to the liberation of cofilin from 14-3-3 protein. AMPK induces actin depolymerization, which reduces vascular tone and the response to vasoconstrictors. Our findings demonstrate a new role of AMPK in the control of actin cytoskeletal dynamics, potentially allowing for long-term dilation of microvessels without substantial changes in cytosolic Ca2+.

Omega-3 and omega-6 DPA equally inhibit the sphingosylphosphorylcholine-induced Ca2+-sensitization of vascular smooth muscle contraction via inhibiting Rho-kinase activation and translocation

We previously reported that eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid (n-3 PUFA), effectively inhibits sphingosylphosphorylcholine (SPC)-induced Ca2+-sensitization of vascular smooth muscle (VSM) contraction which is a major cause of cardiovascular and cerebrovascular vasospasm, and EPA is utilized clinically to prevent cerebrovascular vasospasm. In this study, we clearly demonstrate that docosapentaenoic acid (DPA), which exists in two forms as omega-3 (n-3) and omega-6 (n-6) PUFA, strongly inhibits SPC-induced contraction in VSM tissue and human coronary artery smooth muscle cells (CASMCs), with little effect on Ca2+-dependent contraction. Furthermore, n-3 and n-6 DPA inhibited the activation and translocation of Rho-kinase from cytosol to cell membrane. Additionally, SPC-induced phosphorylation of myosin light chain (MLC) was inhibited in n-3 and n-6 DPA pretreated smooth muscleVSM cells and tissues. In summary, we provide direct evidence that n-3 and n-6 DPA effectively equally inhibits SPC-induced contraction by inhibiting Rho-kinase activation and translocation to the cell membrane.

34 Decreased dilation of hippocampal arterioles in a rat model of preeclampsia

Introduction Women with preeclampsia (PE) and eclampsia have increased incidence of white matter lesions in the brain and self-report cognitive impairment later in life, suggesting seizures during PE may lead to long-lasting brain injury. The hippocampus, a brain region central to cognition, is more susceptible to seizure-induced injury due to less effective functional hyperemia. How PE affects the function and structure of the hippocampal vasculature remains unknown, but could further limit functional hyperemia and potentiate seizure-induced injury. Objective We tested the hypothesis that hippocampal arterioles (HpAs) in PE have impaired responses to mediators of dilation generated during seizure including extracellular potassium (K+) and nitric oxide (NO) and undergo inward remodeling that limits blood flow to the hippocampus during seizure. Methods Sprague Dawley rats were nonpregnant (NP; n = 6), pregnant (P; d20; n = 6) or with PE (induced by a high cholesterol diet gestational days 7–20; n = 6). HpAs were isolated and pressurized to 60 mmHg in an arteriograph chamber. The function of HpAs was investigated by measuring changes in lumen diameter in response to the small- and intermediate-conductance calcium-activated potassium (SK/IK) channel agonist NS309, and in response to increasing K+ concentration from 3–15 mM to activate inward rectifier potassium (KIR) channels. Basal production of NO and the sensitivity of vascular smooth muscle to NO were investigated using the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10-3M) and the NO donor sodium nitroprusside (SNP; 10-5M), respectively. HpA structure and distensibility were determined by measuring lumen diameter and wall thickness of fully relaxed arterioles. Data are mean ± SEM and comparisons made using a one-way ANOVA with a post-hoc Bonferroni test. Results HpAs from PE rats dilated less when SK/IK channels were activated with NS309 compared to P and NP rats (55 ± 18% vs. 88 ± 2% and 90 ± 4%; p = 0.053). Activation of KIR with 15 mM K+ caused less dilation in HpAs from PE rats compared to P and NP rats, although this did not reach statistical significance (9 ± 8% vs. 61 ± 27% and 20 ± 11%; p = 0.13). NOS inhibition with L-NAME did not constrict any HpAs, suggesting there was no basal NO production. HpAs from PE rats were less sensitive to NO, with less reactivity to SNP compared to P and NP rats (22 ± 10% vs. 30 ± 10% and 38 ± 8%; p > 0.05). Structurally, lumen diameters were smaller in fully relaxed HpAs from PE rats than NP rats (Fig. 1), and vessel walls were thinner in PE and P rats compared to NP rats (7.7 ± 0.6 μm and 8.3 ± 0.7 μm vs. 11.5 ± 0.7 μm at 60 mmHg; p p Conclusion HpAs from PE rats had impaired dilatory responses, were less distensible, and underwent inward, hypotrophic remodeling making them structurally smaller than the NP and P states. This combination of impaired vasodilation and structural remodeling of HAs in PE could limit blood flow to the hippocampus during seizure, resulting in ischemic neuronal injury.

Mechanisms Involved in Thromboxane A2 -induced Vasoconstriction of Rat Intracavernous Small Penile Arteries.

Diabetes is associated with erectile dysfunction and with hypercontractility in erectile tissue and this is in part ascribed to increased formation of thromboxane. Rho kinase (ROCK) is a key regulator of calcium sensitization and contraction in vascular smooth muscle. This study investigated the role of calcium and ROCK in contraction evoked by activation of the thromboxane receptors. Rat intracavernous penile arteries were mounted for isometric tension and intracellular calcium ([Ca2+ ]i ) recording and corpus cavernosum for measurements of MYPT1 phosphorylation. In penile arteries, U46619 by activation of thromboxane receptors concentration dependently increased calcium and contraction. U46619-induced calcium influx was blocked by nifedipine, a blocker of L-type calcium channels, and by 2-aminoethoxydiphenyl borate, a blocker of transient receptor potential (TRP) channels. Inhibitors of ROCK, Y27632 and glycyl-H1152P, concentration dependently reduced U46619-induced contraction, but only Y27632 reduced [Ca2+ ]i levels in the penile arteries activated with either high extracellular potassium or U46619. MYPT-Thr850 phosphorylation in corpus cavernous strips was increased in response to U46619 through activation of TP receptors and was found to be a direct result of phosphorylation by ROCK. Y27632 induced less relaxation in mesenteric arteries, H1152P induced equipotent relaxations, and a protein kinase C inhibitor, Ro-318220, failed to relax intracavernous penile arteries, but induced full relaxation in rat mesenteric arteries. Our findings suggest that U46619 contraction depends on Ca2+ influx through L-type and TRP channels, and ROCK-dependent mechanisms in penile arteries. Inhibition of the ROCK pathway is a potential approach for the treatment of erectile dysfunction associated with hypertension and diabetes.

Protein Kinase C Isoforms Distinctly Regulate Propofol-induced Endothelium-dependent and Endothelium-independent Vasodilation

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.