Myogenic Tone Research Articles

TET2 confers a mechanistic link of microRNA-210 and mtROS in hypoxia-suppressed spontaneous transient outward currents in uterine arteries of pregnant sheep.

Hypoxia during pregnancy impairs uterine vascular adaptation via microRNA-210 (miR-210)-mediated mitochondrial dysfunction and mitochondrial reactive oxygen species (mtROS) generation. TET methylcytosine dioxygenase 2 (TET2) participates in regulating inflammation and oxidative stress and its deficiency contributes to the pathogenesis of multiple cardiovascular diseases. Thus, we hypothesize a role of TET2 in hypoxia/miR-210-mediated mtROS suppressing spontaneous transient outward currents (STOCs) in uterine arteries. We found that gestational hypoxia downregulated TET2 in uterine arteries of pregnant sheep and TET2 was a target of miR-210. Knockdown of TET2 with small interfering RNAs suppressed mitochondrial respiration, increased mtROS, inhibited STOCs and elevated myogenic tone. By contrast, overexpression of TET2 negated hypoxia- and miR-210-induced mtROS. The effects of TET2 knockdown in uterine arteries on mtROS, STOCs and myogenic contractions were blocked by the mitochondria-targeted antioxidant MitoQ. In addition, the recovery effects of inhibiting endogenous miR-210 with miR-210-LNA on hypoxia-induced suppression of STOCs and augmentation of myogenic tone were reversed by TET2 knockdown in uterine arteries. Together, our study reveals a novel mechanistic link between the miR-210-TET2-mtROS pathway and inhibition of STOCs and provides new insights into the understanding of uterine vascular maladaptation in pregnancy complications associated with gestational hypoxia. KEY POINTS: Gestational hypoxia downregulates TET methylcytosine dioxygenase 2 (TET2) in uterine arteries of pregnant sheep. TET2 is a downstream target of microRNA-210 (miR-210) and miR-210mediates hypoxia-induced TET2 downregulation. Knockdown of TET2 in uterine arteries recapitulates the effect of hypoxia and miR-210 and impairs mitochondrial bioenergetics and increases mitochondrial reactive oxygen species (mtROS) . Overexpression of TET2 negates the effect of hypoxia and miR-210 on increasing mtROS. TET2 knockdown reiterates the effect of hypoxia and miR-210 and suppresses spontaneous transient outward currents (STOCs) and elevates myogenic tone, and these effects are blocked by MitoQ. Knockdown of TET2 reverses the miR-210-LNA-induced reversal of the effects of hypoxia on STOCs and myogenic tone in uterine arteries.

Smooth Muscle Mineralocorticoid Receptor Promotes Hypertension After Preeclampsia.

Preeclampsia is a syndrome of high blood pressure (BP) with end organ damage in late pregnancy that is associated with high circulating soluble VEGF receptor (sFlt1 [soluble Fms-like tyrosine kinase 1]). Women exposed to preeclampsia have a substantially increased risk of hypertension after pregnancy, but the mechanism remains unknown, leaving a missed interventional opportunity. After preeclampsia, women have enhanced sensitivity to hypertensive stress. Since smooth muscle cell mineralocorticoid receptors (SMC-MR) are activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure in pregnancy induces a postpartum state of enhanced SMC-MR responsiveness. Postpartum BP response to high salt intake was studied in women with prior preeclampsia. MR transcriptional activity was assessed in vitro in sFlt1-treated SMC by reporter assays and PCR. Preeclampsia was modeled by transient sFlt1 expression in pregnant mice. Two months post-partum, mice were exposed to high salt and then to AngII (angiotensin II) and BP and vasoconstriction were measured. Women exposed to preeclampsia had significantly enhanced salt sensitivity of BP verses those with a normotensive pregnancy. sFlt1 overexpression during pregnancy in mice induced elevated BP and glomerular endotheliosis, which resolved post-partum. The sFlt1 exposed post-partum mice had significantly increased BP response to 4% salt diet and to AngII infusion. In vitro, SMC-MR transcriptional activity in response to aldosterone or AngII was significantly increased after transient exposure to sFlt1 as was aldosterone-induced expression of AngII type 1 receptor. Post-partum, SMC-MR-KO mice were protected from the enhanced response to hypertensive stimuli after preeclampsia. Mechanistically, preeclampsia mice exposed to postpartum hypertensive stimuli develop enhanced aortic stiffness, microvascular myogenic tone, AngII constriction, and AngII type 1 receptor expression, all of which were prevented in SMC-MR-KO littermates. These data support that sFlt1-induced vascular injury during preeclampsia produces a persistent state of enhanced sensitivity of SMC-MR to activation. This contributes to postpartum hypertension in response to common stresses and supports testing of MR antagonism to mitigate the increased cardiovascular risk in women after PE.

Microvascular dysfunction following cardiopulmonary bypass plays a central role in postoperative organ dysfunction.

Despite significant advances in surgical technique and strategies for tissue/organ protection, cardiac surgery involving cardiopulmonary bypass is a profound stressor on the human body and is associated with numerous intraoperative and postoperative collateral effects across different tissues and organ systems. Of note, cardiopulmonary bypass has been shown to induce significant alterations in microvascular reactivity. This involves altered myogenic tone, altered microvascular responsiveness to many endogenous vasoactive agonists, and generalized endothelial dysfunction across multiple vascular beds. This review begins with a survey of in vitro studies that examine the cellular mechanisms of microvascular dysfunction following cardiac surgery involving cardiopulmonary bypass, with a focus on endothelial activation, weakened barrier integrity, altered cell surface receptor expression, and changes in the balance between vasoconstrictive and vasodilatory mediators. Microvascular dysfunction in turn influences postoperative organ dysfunction in complex, poorly understood ways. Hence the second part of this review will highlight in vivo studies examining the effects of cardiac surgery on critical organ systems, notably the heart, brain, renal system, and skin/peripheral tissue vasculature. Clinical implications and possible areas for intervention will be discussed throughout the review.

Abstract TP228: Differential Effect Of Serum From Intracerebral Hemorrhage And Small Vessel Disease Patients On Nonischemic Cerebral Arteries

Introduction: There is evidence that circulating factors disrupt vessel function in the contralateral hemisphere after stroke. However, how different stroke subtypes, that have more severe vascular disruption, affect nonischemic vessels is unknown. Here, we compared circulating factors from human patients with distinct stroke subtypes of cardioembolic (CE), small vessel disease (SVD), and intracerebral hemorrhage (ICH). We hypothesized that serum from ICH patients would cause greater vascular dysfunction of nonischemic arteries than CE, and that serum from SVD patients would have an intermediate effect. Methods: Serum was obtained 24 hours poststroke from patients with SVD (n=3), ICH (n=4), and CE (n=7) and pooled. All patients had a medical history of hypertension. Only SVD and CE patients received tPA. Pooled serum 20% v/v was perfused into the lumen of 3 rd order posterior cerebral arteries (PCA, CE & ICH n=5, SVD n=6) that were mounted on glass cannulas and pressurized in an arteriograph chamber. Blood brain barrier (BBB) permeability and vascular reactivity was studied to investigate effects on myogenic tone and endothelial function. Data are presented as mean±SEM. Results: All vessels reacted myogenically at pressures ≥40 mmHg. However, serum from ICH patients caused a significant increase in tone at 20 mmHg vs. CE and SVD groups: 37.5±1.6 vs. 9.0±1.9% and 13.0±4.8% p<0.05. There was no difference in dilation to NS309; however, constriction to nitric oxide synthase (NOS) inhibition with L-NAME was impaired with SVD and ICH serum compared to CE: 14.0±2.3% vs. 4.6±0.8% (p<0.05) and 5.8±2.1% (p=0.09). However, reactivity to sodium nitroprusside, an NO donor, was not different between groups. Serum caused an increase in BBB permeability over time that was not different with the different serums. Conclusions: Circulating factors from ICH, SVD and CE patients had differing vascular effects on nonischemic cerebral arteries. Serum from ICH patients was vasoconstrictive, potentially due to hemostatic factors, whereas serum from both SVD and ICH patients had impaired NO reactivity demonstrating endothelial dysfunction. These effects of ischemic stroke could affect the contralateral hemisphere and contribute to outcome and therapeutic responses.

Arterial Myogenic Response and Aging.

The arterial myogenic response is an inherent property of resistance arteries. Myogenic tone is crucial for maintaining a relatively constant blood flow in response to changes in intraluminal pressure and protects delicate organs from excessive blood flow. Although this fundamental physiological phenomenon has been extensively studied, the underlying molecular mechanisms are largely unknown. Recent studies identified a crucial role of mechano-activated angiotensin II type 1 receptors (AT1R) in this process. The development of myogenic response is affected by aging. In this review, we summarize recent progress made to understand the role of AT1R and other mechanosensors in the control of arterial myogenic response. We discuss age-related alterations in myogenic response and possible underlying mechanisms and implications for healthy aging.

Myosin light chain phosphorylation exhibits a gradient across the wall of cerebellar arteries under sustained ex vivo vascular tone

Small blood vessel diseases are often associated with impaired regulation of vascular tone. The current understanding of resistance arteries often focuses on how a level of vascular tone is achieved in the acute phase, while less emphasis is placed on mechanisms that maintain vascular tone. In this study, cannulated rat superior cerebellar arteries (SCA) developed spontaneous myogenic tone and showed a marked and sustained constriction in the presence of diluted serum (10%), a stimulus relevant to cerebrovascular disease. Both phosphorylated myosin light chain (MLC-p) and smooth muscle alpha actin (SM-α-actin) aligned with phalloidin-stained actin filaments in the vessel wall, while exhibiting a ‘high to low’ gradient across the layers of vascular smooth muscle cells (VSMC), peaking in the outer layer. The MLC-p distribution profile shifted towards the adventitia in serum treated vessels, while removal of the serum reversed it. Furthermore, a positive correlation between the MLC-p signal and vessel wall tension was also evident. The gradients of phosphorylated MLC and SM-α-actin are consistent with a spatial regulation of the myosin-actin apparatus in the vessel wall during the maintenance of vascular tone. Further, the changing profiles of MLC-p and SM-α-actin are consistent with SCA vasoconstriction being accompanied by VSMC cytoskeletal reorganization.

Mechanisms of Vascular CaV1.2 Channel Regulation During Diabetic Hyperglycemia.

Diabetes is a leading cause of disability and mortality worldwide. A major underlying factor in diabetes is the excessive glucose levels in the bloodstream (e.g., hyperglycemia). Vascular complications directly result from this metabolic abnormality, leading to disabling and life-threatening conditions. Dysfunction of vascular smooth muscle cells is a well-recognized factor mediating vascular complications during diabetic hyperglycemia. The function of vascular smooth muscle cells is exquisitely controlled by different ion channels. Among the ion channels, the L-type CaV1.2 channel plays a key role as it is the main Ca2+ entry pathway regulating vascular smooth muscle contractile state. The activity of CaV1.2 channels in vascular smooth muscle is altered by diabetic hyperglycemia, which may contribute to vascular complications. In this chapter, we summarize the current understanding of the regulation of CaV1.2 channels in vascular smooth muscle by different signaling pathways. We place special attention on the regulation of CaV1.2 channel activity in vascular smooth muscle by a newly uncovered AKAP5/P2Y11/AC5/PKA/CaV1.2 axis that is engaged during diabetic hyperglycemia. We further describe the pathophysiological implications of activation of this axis as it relates to myogenic tone and vascular reactivity and propose that this complex may be targeted for developing therapies to treat diabetic vascular complications.

Enhanced Cerebral Hemodynamics and Cognitive Function Via Knockout of Dual-Specificity Protein Phosphatase 5.

Alzheimer's Disease (AD) and Alzheimer's Disease-Related Dementias (ADRD) are neurodegenerative disorders. Recent studies suggest that cerebral hypoperfusion is an early symptom of AD/ADRD. Dual-specificity protein phosphatase 5 (DUSP5) has been implicated in several pathological conditions, including pulmonary hypertension and cancer, but its role in AD/ADRD remains unclear. The present study builds on our previous findings, demonstrating that inhibition of ERK and PKC leads to a dose-dependent dilation of the middle cerebral artery and penetrating arteriole, with a more pronounced effect in Dusp5 KO rats. Both ERK and PKC inhibitors resulted in a significant reduction of myogenic tone in vessels from Dusp5 KO rats. Dusp5 KO rats exhibited stronger autoregulation of the surface but not deep cortical cerebral blood flow. Inhibition of ERK and PKC significantly enhanced the contractile capacity of vascular smooth muscle cells from both strains. Finally, a significant improvement in learning and memory was observed in Dusp5 KO rats 24 hours after initial training. Our results suggest that altered vascular reactivity in Dusp5 KO rats may involve distinct mechanisms for different vascular beds, and DUSP5 deletion could be a potential therapeutic target for AD/ADRD. Further investigations are necessary to determine the effects of DUSP5 inhibition on capillary stalling, blood-brain barrier permeability, and neurodegeneration in aging and disease models.

THE MORPHOLOGICAL AND FUNCTIONAL STATE OF THE VASCULAR COMPONENT OF HEMATOPOIESIS ORGANS IN DIFFERENT TIMES OF SIMULATED HYPOXIA IN THE EXPERIMENT

Under conditions of hypoxia, a change in the morphological and functional state of the components of the microvasculature bed will be accompanied by a change in the adequacy and usefulness of the immune response and hematopoiesis. The purpose of the study was to determine the morphofunctional state of the vascular component of the red bone marrow and spleen at various times of simulated hypoxia and to identify the degree of influence of CD68-positive cells on this state. Chronic hypoxia was modeled on 246 outbred male rats using special chambers containing natural gas from the Astrakhan gas field (Russia) at a concentration not exceeding the maximum allowable. The experiment was continued for 120 days, inhalation was carried out five days a week for four hours a day, the removal of animals from the experiment was carried out every 30 days. The functional activity of the vascular component of the red bone marrow was determined using the method of laser Doppler flowmetry. The degree of expression of inducible (iNOS) and endothelial (eNOS) NO synthases and the distribution of CD68-positive cells in the spleen structures were determined by immunohistochemical method. The study showed that as the duration of chronic simulated hypoxia increases, there is a decrease in the microcirculation index, an increase in myogenic tone and shunting index, which, taken together, indicates a deterioration of organ perfusion and confirms the formation of a hypoxic state. An analysis of the functional activity, carried out using an immunohistochemical study of the expression of iNOS and eNOS in the structures of the spleen, showed that as the duration of the experiment increased, the level of endothelial synthase decreased and the level of inducible synthase increased. Perhaps this is due to the influence of biologically active substances secreted by macrophages activated during hypoxia. This is supported by an increase in the presence of CD68-positive cells in the red pulp and along the connective tissue trabeculae as the experimental exposure increases.

Соmplex Medical Rehabilitation for Cancer-related Lower Limbs Lymphedema: a Prospective Comparative Randomized Study of 60 Patients

INTRODUCTION. The management of patients with secondary lymphedema of the lower extremities associated with radical treatment of pelvic cancer (Cancer Related Lymphedema) is extremely problematic due to persistent impairment of the draining function of regional lymph nodes due to intraoperative dissection and subsequent radiation therapy. AIM. To compare the effectiveness of complex medical rehabilitation using the kinesio taping method and innovative non-stretchable compression bandages in patients with secondary lymphedema of the lower extremities associated with radical treatment of pelvic cancer. MATERIAL AND METHODS. The study involved 60 patients with stages I-III Cancer Related Lymphedema (according to the classification of Savchenko T.V., Pokrovsky A.V., 2004) who underwent radical treatment for various forms of pelvic cancer (gynecological cancer in women and prostate cancer in men), randomized into 2 groups (30 people each). A comprehensive rehabilitation was carried out in the main group (n=30), including sequential manual lymphatic drainage, skin care, kinesio taping, therapeutic gymnastics in the gym; Adjustable Non-stretchable Compression Bandages (circaid juxtafit, Germany). Meanwhile, a Complex Decongestive Therapy was performed in the comparison group (n=30), which is considered as the gold standard of conservative treatment of lymphedema of the lower extremities, including manual lymphatic drainage, skin care, Multi-layered limb banding using Inelastic compression Bandages. In order to evaluate the anti-edematous effect the dynamics of the circumferences of the lower extremities, waist circumference and hip circumference, the Ankle Range of Motion were examined using goniometry, bio-impedancemetry. Laser Doppler Flowmetry was used to evaluate microcirculatory parameters. RESULTS AND DISCUSSION. There was a decrease in swelling and thickening of the skin in the thigh, a decrease the external genitalia edema in patients with Cancer Related Lymphedema in both groups after a course of rehabilitation. There was a regression of swelling of the feet and lower legs in patients of both groups (p<0.05) after treatment, which was confirmed by a decrease in the circumferences of the lower limbs. There was an increase in the Ankle Range of Motion (dorsiflexion) from 17.50±1.26° to 19.25±1.52° (p<0.05) in the main group and from 17.90±1.44° to 18.59±1.10° (p<0.05) in the comparison group. According to the bio-impedancemetry data, there was a decrease in the extracellular fluid content both in the main group and the comparison group by 2.64% and 2.83%, (p<0.05) respectively. There was also a comparable decrease in the total fluid content by 2.67% in the main group and 1.90% (p<0.05) in the comparison group. There were a decrease in body weight by 2.49% and 1.52% (p<0.001), a decrease in fat mass by 2.62% and 4.01% (p<0.05) in both groups respectively after the course of treatment. According to the Laser Doppler Flowmetry data, there were an improvement in the indicators of myogenic tone of arterioles and secretory activity of the endothelium, as well as elimination of venular congestion in patients of both groups (p<0.05). CONCLUSION. Regression of edema in Cancer Related Lymphedema can be achieved as a result of the use of manual lymphatic drainage which stimulates the outflow of lymph through lymphatic watersheds into adjacent lymphatic territories with intact regional lymph nodes, which was achieved in both groups. The lymphatic drainage effect of manual lymphatic drainage was supported by the daily compression profile of bandages (Multi-layered Inelastic Bandages and Adjustable Non-stretchable Compression Bandages) in patients of both groups. Our study showed that the use of kinesio tapes leveled the difference in compression of approximately 10-15 mmHg between the bandages used (50 mmHg/~60 mmHg).

Increased Ca2+-dependent intrinsic tone and arterial stiffness in mesenteric microvessels of hypertensive pregnant rats

Preeclampsia is a pregnancy-related hypertensive disorder (HTN-Preg) with unclear mechanisms. We have shown increased vascular reactivity to extrinsic vasoconstrictors in HTN-Preg rats. Here, we test whether microvascular intrinsic tone and arterial stiffness could contribute to HTN-Preg, and examined the underlying cellular mechanisms. On gestational day 19, BP was recorded in normal pregnant (Preg) rats and Preg rats with reduced uterine perfusion pressure (RUPP), and mesenteric microvessels were mounted on a pressure myograph for measurement of intrinsic tone, simultaneous changes in [Ca2+]i (fura-2 340/380 ratio), and arterial stiffness. Arteries were incubated in Ca2+-containing and 0 Ca2+ (2 mM EGTA) Krebs, pressurized at 10 to 110 mmHg in 10 mmHg increments, and the % change in vessel diameter from initial diameter at 10 mmHg was analyzed for measurement of total (active + passive) intrinsic tone and passive intrinsic response, respectively. The passive response was then subtracted from the total intrinsic tone to determine the active myogenic tone. The strain–stress relationship was also constructed as a measure of arterial stiffness. BP was higher in RUPP vs Preg rats. In Ca2+-containing Krebs, increases in intraluminal pressure caused smaller increases in diameter and greater increases in [Ca2+]i in microvessels of RUPP vs Preg rats, suggesting increased Ca2+-dependent myogenic tone. In 0 Ca2+ Krebs, increases in pressure also caused less increases in diameter in microvessels of RUPP vs Preg rats, but with no changes in [Ca2+]i, suggesting changes in the structure and mechanics of the arterial wall. The total and passive strain–stress relationship was shifted to the left in microvessels of RUPP vs Preg rats, suggesting increased arterial wall stiffness. Histology and immunohistochemistry showed greater vascular wall thickness and collagen-I staining in RUPP vs Preg rats, supporting changes in the wall architecture and structural proteins. The increased active myogenic tone and underlying increases in Ca2+ signaling as well as the increased passive intrinsic response, arterial stiffness and collagen-I in the mesenteric microvessels could play a role in the regulation of blood flow to the splanchnic region and the increased vascular resistance and BP in HTN-Preg.

Disrupting circadian control of peripheral myogenic reactivity mitigates cardiac injury following myocardial infarction.

Circadian rhythms orchestrate important functions in the cardiovascular system: the contribution of microvascular rhythms to cardiovascular disease progression/severity is unknown. This study hypothesized that (i) myogenic reactivity in skeletal muscle resistance arteries is rhythmic and (ii) disrupting this rhythmicity would alter cardiac injury post-myocardial infarction (MI). Cremaster skeletal muscle resistance arteries were isolated and assessed using standard pressure myography. Circadian rhythmicity was globally disrupted with the ClockΔ19/Δ19 mutation or discretely through smooth muscle cell-specific Bmal1 deletion (Sm-Bmal1 KO). Cardiac structure and function were determined by echocardiographic, hemodynamic and histological assessments. Myogenic reactivity in cremaster muscle resistance arteries is rhythmic. This rhythm is putatively mediated by the circadian modulation of a mechanosensitive signalosome incorporating tumour necrosis factor and casein kinase 1. Following left anterior descending coronary artery ligation, myogenic responsiveness is locked at the circadian maximum, although circadian molecular clock gene expression cycles normally. Disrupting the molecular clock abolishes myogenic rhythmicity: myogenic tone is suspended at the circadian minimum and is no longer augmented by MI. The reduced myogenic tone in ClockΔ19/Δ19 mice and Sm-Bmal1 KO mice associates with reduced total peripheral resistance (TPR), improved cardiac function and reduced infarct expansion post-MI. Augmented microvascular constriction aggravates cardiac injury post-MI. Following MI, skeletal muscle resistance artery myogenic reactivity increases specifically within the rest phase, when TPR would normally decline. Disrupting the circadian clock interrupts the MI-induced augmentation in myogenic reactivity: therapeutics targeting the molecular clock, therefore, may be useful for improving MI outcomes.

High altitude differentially modulates potassium channel-evoked vasodilatation in pregnant human myometrial arteries.

High-altitude (>2500m or 8200ft) residence reduces uterine artery blood flow during pregnancy, contributing to an increased incidence of preeclampsia and intrauterine growth restriction. However, not all pregnancies are affected by the chronic hypoxic conditions of high-altitude residence. K+ channels play important roles in the uterine vascular adaptation to pregnancy, promoting a reduction in myogenic tone and an increase in blood flow. We hypothesized that, in pregnancies with normal fetal growth at high altitude, K+ channel-dependent vasodilatation of myometrial arteries is increased compared to those from healthy pregnant women at a lower altitude (∼1700m). Using pharmacological modulation of two K+ channels, ATP-sensitive (KATP ) and large-conductance Ca2+ -activated (BKCa ) K+ channels, we assessed the vasodilatation of myometrial arteries from appropriate for gestational age (AGA) pregnancies in women living at high or low altitudes. In addition, we evaluated the localization of these channels in the myometrial arteries using immunofluorescence. Our results showed an endothelium-dependent increase in KATP -dependent vasodilatation in myometrial arteries from high versus low altitude, whereas vasodilatation induced by BKCa activation was reduced in these vessels. Additionally, KATP channel co-localization with endothelial markers was reduced in the high-altitude myometrial arteries, which suggested that the functional increase in KATP activity may be by mechanisms other than regulation of channel localization. These observations highlight an important contribution of K+ channels to the human uterine vascular adaptation to pregnancy at high altitude serving to maintain normal fetal growth under conditions of chronic hypoxia. KEY POINTS: High-altitude (>2500m or 8200ft) residence reduces uterine blood flow during pregnancy and fetal growth. Animal models of high altitude/chronic hypoxia suggest that these reductions are partially due to reduced vascular K+. channel responses, such as those elicited by large conductance Ca2+ -activated (BKCa ) and ATP-sensitive (KATP ) K+ channel activation. We found that women residing at high versus low altitude during pregnancy showed diminished myometrial artery vasodilatory responses to endothelium-independent BKCa channel activation but greater responses to endothelium-dependent KATP channel activation. Our observations indicate that KATP channels play an adaptive role in maintaining myometrial artery vasodilator sensitivity under chronic hypoxic conditions during pregnancy. Thus, KATP channels represent potential therapeutic targets for augmenting uteroplacental blood flow and, in turn, preserving fetal growth in cases of uteroplacental hypoperfusion.

Non-linear dynamic processes and their correlation with indicators of microcirculation in patients with obliterating atherosclerosis of the lower extremities arteries according to laser doppler flowmetry

Objective: To study features of nonlinear dynamic and oscillatory processes in the skin microcirculatory flow in patients with obliterating atherosclerosis in lower extremities arteries (OALEA).Material and methods. 56 male patients with obliterating atherosclerosis in the arteries of lower extremities and 15 practically healthy individuals were taken into the study. Microcirculation in the skin on feet with the assessment of nonlinear dynamic processes and spectral wavelet analysis of blood fl ow fl uctuations was studied by laser Doppler flowmetry, device “LACK-M” (firm Lazma, Russia). Normalized amplitude parameters of blood flow fluctuations in frequency ranges of hemocirculation tonus-forming factors were analyzed: endothelial, neurogenic, myogenic. Myogenic microvascular tone and capillary blood flow indices were calculated. To study nonlinear dynamic processes, the following parameters were assessed: fractal dimension, entropy and phase portrait analysis.Results. Spastic-atonic microcirculation disorders were revealed in patients with OALEA. They were characterized with metarteriole constriction and with restriction of capillary blood flow, with an increased myogenic tone and dilated arterioles. Statistically significant shifts in parameters of nonlinear dynamics in this case- in particular, decrease in entropy (–10.6 %) and in dimensions of phase portrait (–9.3%) under energy deficit in oscillatory microcirculation processes (–20.8 %) – can be interpreted as a decrease in complexity of laser Doppler flowmetric signal and better ordering in regulatory mechanisms of the peripheral blood flow.Conclusion. In patients with obliterating atherosclerosis in the arteries of lower extremities, the obtained results indicate significant functional microcirculation disorders of the spastic-atonic nature leading to the restriction of nutritive blood flow and accompanied by the lack of energy in oscillatory processes. These results have also shown the decrease in the system chaos severity which indicates limitation of connections of microcirculation control factors, simplification of the mechanisms of functioning of the microvascular bed and, as a consequence, decrease in the compensatory adaptive potential.

Abstract 15590: Smooth Muscle Specific Deletion of P90 Ribosomal S6 Kinase Alters the Vasomotor Response of Resistance Arteries

Background: Smooth muscle (SM) contraction is triggered by Ca 2+ /calmodulin-dependent myosin light chain kinase (MLCK), which phosphorylates the regulatory light chain of myosin (RLC 20 ). However, additional regulatory mechanisms exist. We found that global Rsk2 KO mice had more dilated mesenteric arteries (MAs) with reduced myogenic tone and RLC 20 phosphorylation, and significantly lower blood pressure. Furthermore, RSK2-dependent activation of the Na + /H + exchanger resulted in an increase in pH i and increased Ca 2+ i transients. However, it is unclear whether our observations were due to developmental abnormalities observed in Rsk2 global KO mice. Hypothesis: RSK2 signaling in SM is a significant regulator of myogenic tone, arterial stiffness, and Ca 2+ signaling. Methods: To study the role of RSK2 signaling in vascular tone, we generated inducible SM-specific RSK2 KO mice. Detection of total and phospho proteins were determined by western blot. Pressure myography studies assessed vascular reactivity in 4 th - and 5 th -order MAs isolated from MYH11-Cre + Rsk2 fl/fl (RSK2 SM -/- ) and MYH11-Cre - Rsk2 fl/fl (RSK2 SM +/+ ) mice. We examined SM-specific Ca 2+ events in pressurized MAs by confocal fluorescent microscopy. Results: Western blot confirms knock down of RSK2 expression (~70%) in MAs and aorta. Intraluminal pressure-induced constriction of MAs is attenuated in RSK2 SM -/- . RSK2 SM -/- MAs display a delay in development of myogenic tone at 40 mmHg and decreased vascular stiffness, compared to RSK2 SM +/+ . Vasodilatory responses to Spermine NONOate (1-100 μM) in preconstricted (L-NNA; 10 μM) RSK2 SM -/- vessels were enhanced. RSK2 SM -/- MAs display fewer Ca 2+ events (97±19.6; n = 4) than RSK2 SM +/+ (400±24.5; n = 4) at 80 mmHg ( p = 0.0001). Additionally, Ca 2+ events are slower and longer-lasting in the RSK2 SM -/- . Decreased levels of p-RLC 20 are observed at 40 mmHg, but not 80 mmHg in RSK2 SM -/- vs. RSK2 SM +/+ mice, consistent with impeding tone development. Conclusion: RSK2 signaling contributes to myogenic tone and vessel stiffness by regulating sensitivity to NO, increasing Ca 2+ events, and RLC 20 activity. RSK2 signaling pathway is a potential therapeutic target for hypertension.

Spatiotemporal Control of Vascular CaV1.2 by α1C S1928 Phosphorylation.

L-type CaV1.2 channels undergo cooperative gating to regulate cell function, although mechanisms are unclear. This study tests the hypothesis that phosphorylation of the CaV1.2 pore-forming subunit α1C at S1928 mediates vascular CaV1.2 cooperativity during diabetic hyperglycemia. A multiscale approach including patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, calcium imaging' pressure myography, and Laser Speckle imaging was implemented to examine CaV1.2 cooperativity, α1C clustering, myogenic tone, and blood flow in human and mouse arterial myocytes/vessels. CaV1.2 activity and cooperative gating increase in arterial myocytes from patients with type 2 diabetes and type 1 diabetic mice, and in wild-type mouse arterial myocytes after elevating extracellular glucose. These changes were prevented in wild-type cells pre-exposed to a PKA inhibitor or cells from knock-in S1928A but not S1700A mice. In addition, α1C clustering at the surface membrane of wild-type, but not wild-type cells pre-exposed to PKA or P2Y11 inhibitors and S1928A arterial myocytes, was elevated upon hyperglycemia and diabetes. CaV1.2 spatial and gating remodeling correlated with enhanced arterial myocyte Ca2+ influx and contractility and in vivo reduction in arterial diameter and blood flow upon hyperglycemia and diabetes in wild-type but not S1928A cells/mice. These results suggest that PKA-dependent S1928 phosphorylation promotes the spatial reorganization of vascular α1C into "superclusters" upon hyperglycemia and diabetes. This triggers CaV1.2 activity and cooperativity, directly impacting vascular reactivity. The results may lay the foundation for developing therapeutics to correct CaV1.2 and arterial function during diabetic hyperglycemia.

Increased TMEM16A-Mediated Ca2+-Activated Cl- Currents in Portal Vein Smooth Muscle Cells of Caveolin 1-Deficient Mice.

Ca2+-activated Cl- (ClCa) channels regulate membrane excitability and myogenic tone in vascular smooth muscles. TMEM16A-coding proteins are mainly responsible for functional ClCa channels in vascular smooth muscles, including portal vein smooth muscles (PVSMs). Caveolae are cholesterol-rich and Ω-shaped invaginations on the plasma membrane that structurally contributes to effective signal transduction. Caveolin 1 (Cav1) accumulates in caveolae to form functional complexes among receptors, ion channels, and kinases. The present study examined the functional roles of Cav1 in the expression and activity of ClCa channels in the portal vein smooth muscle cells (PVSMCs) of wild-type (WT) and Cav1-knockout (KO) mice. Contractile experiments revealed that the amplitude of spontaneous PVSM contractions was larger in Cav1-KO mice than WT mice. Under whole-cell patch-clamp configurations, ClCa currents were markedly inhibited by 1 µM Ani9 (a selective TMEM16A ClCa channel blocker) in WT and Cav1-KO PVSMCs. However, Ani9-sensitive ClCa currents were significantly larger in Cav1-KO PVSMCs than in WT PVSMCs. Expression analyses showed that TMEM16A expression levels were higher in Cav1-KO PVSMs than in WT PVSMs. Therefore, the caveolar structure formed by Cav1 negatively regulates the expression and activity of TMEM16A-mediated ClCa channels in vascular smooth muscle cells.

The role of nitric oxide in flow-induced and myogenic responses in 1A, 2A, and 3A branches of the porcine middle cerebral artery.

Myogenic and flow-induced reactivity contribute to cerebral autoregulation, with potentially divergent roles for smaller versus larger arteries. The present study tested the hypotheses that compared with first-order (1A) branches of the middle cerebral artery, second- and third-order branches (2A and 3A, respectively) exhibit greater myogenic reactivity but reduced flow-induced constriction. Furthermore, nitric oxide synthase (NOS) inhibition may amplify myogenic reactivity and abolish instances of flow-induced dilation. Isolated porcine cerebral arteries mounted in a pressure myograph were exposed to incremental increases in intraluminal pressure (40-120 mmHg; n = 41) or flow (1-1,170 µL/min; n = 31). Intraluminal flows were adjusted to achieve 5, 10, 20, and 40 dyn/cm2 of wall shear stress at 60 mmHg. Myogenic tone was greater in 3A versus 1A arteries (P < 0.05). There was an inverse relationship between myogenic reactivity and passive arterial diameter (P < 0.01). NOS inhibition increased basal tone to a lesser extent in 3A versus 1A arteries (P < 0.01) but did not influence myogenic reactivity (P = 0.49). Increasing flow decreased luminal diameter (P ≤ 0.01), with increased vasoconstriction at 10-40 dyn/cm2 of shear stress (P < 0.01). However, relative responses were similar between 1A, 2A, and 3A arteries (P = 0.40) with and without NOS inhibition conditions (P ≥ 0.29). Whereas NOS inhibition increases basal myogenic tone, and myogenic reactivity was less in smaller versus larger arteries (range = ∼100-550 µM), neither NOS inhibition nor luminal diameter influences flow-induced constriction in porcine cerebral arteries.NEW & NOTEWORTHY This study demonstrated size-dependent heterogeneity in myogenic reactivity in porcine cerebral arteries. Smaller branches of the middle cerebral artery exhibited increased myogenic reactivity, but attenuated NOS-dependent increases in myogenic tone compared with larger branches. Flow-dependent regulation does not exhibit the same variation; diameter-independent flow-induced vasoconstrictions occur across all branch orders and are not affected by NOS inhibition. Conceptually, flow-induced vasoconstriction contributes to cerebral autoregulation, particularly in larger arteries with low myogenic tone.

Protection against angiotensin II-induced hypertension in mice lacking sphingosine kinase 1 in vascular smooth muscle cells

Abstract Background Sphingosine-1-phosphate (S1P), a lipid mediator produced by sphingosine kinases (Sphks), significantly contributes to the maintenance of cardiovascular homeostasis. Mice lacking Sphk1 globally are protected against angiotensin II-induced hypertension, yet, due to pleiotropic action of S1P, it has been challenging to identify a specific cell type responsible for this protection. Purpose The aim of this study was to investigate the role of Sphk1 expressed in vascular smooth muscle cells (VSMCs) in the development of angiotensin II (AngII)-induced hypertension in mice. Methods Male, 12-week-old C57BL/6J wild type (WT) or VSMC-specific, Cre-mediated, Sphk1 knockout (Sphk1VSMC−/−) mice underwent either Sham or AngII (490 ng/min/kg s.c.) treatment for 2 weeks, using a surgically implanted osmotic minipump. Blood pressure was measured by the tail-cuff method. Vascular structure and function were examined ex vivo using wire (vasoconstrictor/vasorelaxant responses) and pressure myography (myogenic tone, structural and elasticity parameters). Gene set enrichment analysis (GSEA) was performed using transcriptomic profiles of the isolated mesenteric arteries (MA, RNASeq). Expression of proteins was analyzed by Western blot. Results Deletion of Sphk1 in VSMCs resulted in 5x and 40x lower expression of Sphk1 mRNA in MA and aorta compared to WT mice respectively. A 2-week infusion of AngII resulted in a development of higher systolic blood pressure (SBP) in WT mice compared to Sphk1VSMC−/− mice (mean SBP ± SEM: 168±4.3 mmHg vs. 132±4.4 mmHg respectively, p&amp;lt;0.05). Studies of ex vivo mesenteric artery (MA) function revealed diminished both, depolarization-induced (in response to KCl), as well as G protein-coupled receptor-elicited (in response to phenylephrine and thromboxane A2 analogue) contraction of MA derived from hypertensive Sphk1VSMC−/− mice compared to WT mice. In the same time, significantly impaired endothelial vasorelaxation was observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice. Moreover, MA from Sphk1VSMC−/− mice were characterized by the significantly reduced myogenic response evoked by intravascular pressure elevation (maximal myogenic response ± SEM: 19.7±1.8% for WT normotensive, 26.2±1.8% for WT AngII vs. 19.0±2.5% for Sphk1VSMC−/− AngII mice), and an increased stiffness compared to WT mice. GSEA found that the lack of Sphk1 gene in VSMCs significantly downregulated molecular pathways related to smooth muscle contraction. In line with transcriptomic studies, significantly reduced level of the Rho-associated protein kinase (Rock) has been observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice using Western blot. Conclusions This study revealed that mice lacking Sphk1 gene in VSMCs are protected against the development of AngII-induced hypertension likely due to the lowered myogenic tone in resistance arteries, which may be mediated by autocrine effects of Sphk1 on Rock proteins. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The National Science Centre, Poland

Cerebral arteriolar and neurovascular dysfunction after chemically induced menopause in mice.

Cognitive decline is linked to decreased cerebral blood flow, particularly in women after menopause. Impaired cerebrovascular function precedes the onset of dementia, possibly because of reduced functional dilation in parenchymal arterioles. These vessels are bottlenecks of the cerebral microcirculation, and dysfunction can limit functional hyperemia in the brain. Large-conductance Ca2+-activated K+ channels (BKCa) are the final effectors of several pathways responsible for functional hyperemia, and their expression is modulated by estrogen. However, it remains unknown whether BKCa function is altered in cerebral parenchymal arterioles after menopause. Using a chemically induced model of menopause, the 4-vinylcyclohexene diepoxide (VCD) model, which depletes follicles while maintaining intact ovaries, we hypothesized that menopause would be associated with reduced functional vasodilatory responses in cerebral parenchymal arterioles of wild-type mice via reduced BKCa function. Using pressure myography of isolated parenchymal arterioles, we observed that menopause (Meno) induced a significant increase in spontaneous myogenic tone. Endothelial function, assessed as nitric oxide production and dilation after cholinergic stimulation or endothelium-dependent hyperpolarization pathways, was unaffected by Meno. BKCa function was significantly impaired in Meno compared with control, without changes in voltage-gated K+ channel activity. Cerebral functional hyperemia, measured by laser-speckle contrast imaging during whisker stimulation, was significantly blunted in Meno mice, without detectable changes in basal perfusion. However, behavioral testing identified no change in cognition. These findings suggest that menopause induces cerebral microvascular and neurovascular deficits.NEW & NOTEWORTHY Cerebral parenchymal arterioles from menopause mice showed increased myogenic tone. We identified an impairment in smooth muscle cell BKCa channel activity, without a reduction in endothelium-dependent dilation or nitric oxide production. Microvascular dysfunction was associated with a reduction in neurovascular responses after somatosensory stimulation. Despite the neurovascular impairment, cognitive abilities were maintained in menopausal mice.