Activation Of Vascular Smooth Muscle Research Articles

Active and passive mechanical characterization of a human descending thoracic aorta with Klippel-Trenaunay syndrome

A human aorta from a female donor affected by Klippel-Trenaunay syndrome was retrieved during a surgery for organ donation for transplant. The aorta was preserved in refrigerated Belzer UW organ preservation solution and tested within a few hours for mechanical characterization with and without vascular smooth muscle activation. KCl and Noradrenaline were used as vasoactive agents in bubbled Krebs-Henseleit buffer solution at 37 °C. A quasi-static and a dynamic mechanical characterization of the full wall and the three individual layers were carried out for strips taken in longitudinal and circumferential directions. The full wall in the descending portion of the aorta underwent mechanical tests with and without smooth muscle activation. Results were compared to data obtained from healthy aortas and show a reduced stiffness of the full wall in circumferential direction. Also, a significant reduction of the response to vasoactive agents in circumferential direction was observed, while the longitudinal response was similar to healthy cases.

Organ-specific off-target effects of Pim/ZIP kinase inhibitors suggest lack of contractile Pim kinase activity in prostate, bladder, and vascular smooth muscle.

Smooth muscle contraction by Pim kinases and ZIPK has been suggested, but evidence for lower urinary tract organs or using Pim-selective inhibitor concentrations is not yet available. Here, we assessed effects of the Pim inhibitors AZD1208 and TCS PIM-1 and the dual ZIPK/Pim inhibitor HS38 on contractions of human prostate and bladder tissues and of porcine interlobar arteries. Human tissues were obtained from radical prostatectomy and radical cystectomy and renal interlobar arteries from pigs. Contractions were studied in an organ bath. Noradrenaline-, phenylephrine- and methoxamine-induced contractions were reduced (up to > 50%) with 500-nM AZD1208 in prostate tissues and to lesser degree and not consistently with all agonists in interlobar arteries. A total of 100-nM AZD1208 or 500-nM TCS PIM-1 did not affect agonist-induced contractions in prostate tissues. Decreases in agonist-induced contractions with 3-µM HS38 in prostate tissues and interlobar arteries were of small extent and did not occur with each agonist. Carbachol-induced contractions in detrusor tissues were unchanged with AZD1208 (500nM) or HS38. Electric field stimulation-induced contractions were not affected with AZD1208 or HS38 in any tissue, but slightly reduced with 500-nM TCS PIM-1 in prostate tissues. Concentration-dependent effects of Pim inhibitors suggest lacking Pim-driven smooth muscle contraction in the prostate, bladder, and interlobar arteries but point to organ-specific functions of off-targets. Procontractile functions of ZIPK in the prostate and interlobar arteries may be limited and are lacking in the detrusor.

Differential Functional Contribution of BK Channel Subunits to Aldosterone-Induced Channel Activation in Vascular Smooth Muscle and Eventual Cerebral Artery Dilation.

Calcium/voltage-activated potassium channels (BK) control smooth muscle (SM) tone and cerebral artery diameter. They include channel-forming α and regulatory β1 subunits, the latter being highly expressed in SM. Both subunits participate in steroid-induced modification of BK activity: β1 provides recognition for estradiol and cholanes, resulting in BK potentiation, whereas α suffices for BK inhibition by cholesterol or pregnenolone. Aldosterone can modify cerebral artery function independently of its effects outside the brain, yet BK involvement in aldosterone's cerebrovascular action and identification of channel subunits, possibly involved in steroid action, remains uninvestigated. Using microscale thermophoresis, we demonstrated that each subunit type presents two recognition sites for aldosterone: at 0.3 and ≥10 µM for α and at 0.3-1 µM and ≥100 µM for β1. Next, we probed aldosterone on SM BK activity and diameter of middle cerebral artery (MCA) isolated from β1-/- vs. wt mice. Data showed that β1 leftward-shifted aldosterone-induced BK activation, rendering EC50~3 μM and ECMAX ≥ 10 μM, at which BK activity increased by 20%. At similar concentrations, aldosterone mildly yet significantly dilated MCA independently of circulating and endothelial factors. Lastly, aldosterone-induced MCA dilation was lost in β1-/- mice. Therefore, β1 enables BK activation and MCA dilation by low µM aldosterone.

A Low Concentration of Citreoviridin Prevents Both Intracellular Calcium Deposition in Vascular Smooth Muscle Cell and Osteoclast Activation In Vitro

Vascular calcification (VC) and osteoporosis are age-related diseases and significant risk factors for the mortality of elderly. VC and osteoporosis may share common risk factors such as renin-angiotensin system (RAS)-related hypertension. In fact, inhibitors of RAS pathway, such as angiotensin type 1 receptor blockers (ARBs), improved both vascular calcification and hip fracture in elderly. However, a sex-dependent discrepancy in the responsiveness to ARB treatment in hip fracture was observed, possibly due to the estrogen deficiency in older women, suggesting that blocking the angiotensin signaling pathway may not be effective to suppress bone resorption, especially if an individual has underlying osteoclast activating conditions such as estrogen deficiency. Therefore, it has its own significance to find alternative modality for inhibiting both vascular calcification and osteoporosis by directly targeting osteoclast activation to circumvent the shortcoming of ARBs in preventing bone resorption in estrogen deficient individuals. In the present study, a natural compound library was screened to find chemical agents that are effective in preventing both calcium deposition in vascular smooth muscle cells (vSMCs) and activation of osteoclast using experimental methods such as Alizarin red staining and Tartrate-resistant acid phosphatase staining. According to our data, citreoviridin (CIT) has both an anti-VC effect and anti-osteoclastic effect in vSMCs and in Raw 264.7 cells, respectively, suggesting its potential as an effective therapeutic agent for both VC and osteoporosis.

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.

MULTIVESSEL CORONARY VASOSPASM WITH ABORTED SUDDEN DEATH AND REFRACTORY ANGINA: EFFICACY OF ALPHA-BLOCKERS AND INDICATION OF IMPLANTABLE CARDIOVERTER-DEFIBRILLATOR

Vasospasm of the coronary arteries is an uncommon and underdiagnosed cause of angina pectoris. There is a myriad of clinical presentations with variable prognoses. The typical presentation in the scenario of chronic coronary syndrome includes angina at rest or with variable threshold, from late evening to early morning, including transient ischemia responsive to nitrates, detectable by 12-lead electrocardiogram or Holter. Concurrent multivessel vasospasm is rare and with worse prognosis, given the potential for acute ST-elevation myocardial infarction, ventricular arrhythmias and subsequent sudden cardiac death. The presence of refractory angina is a sign of persistent vasospasm, indicating severe manifestations. The complex physiopathology, including hypercontractility of the vascular smooth muscle, endothelial dysfunction and adrenergic receptor activation has not been fully elucidated, which makes it difficult to define a targeted therapy. We present a case of refractory angina after multivessel vasospasm with aborted sudden death, which responded to alpha-blocker and had implantable cardioverter-defibrillator implantation postponed.

Loss of Cyb5R3 Function Silences Fetal Hemoglobin and Hematocrit Responses to Hydroxyurea in Patients with Sickle Cell Anemia

BACKGROUND: Hydroxyurea (HU) is the first FDA-approved and most widely prescribed drug for sickle cell (SS) patients due to its beneficial effects (decreased Hb polymerization, pain crises and hospitalization) and relative safety profile. While the clinical benefits of HU have been found to associate with increased nitric oxide (NO) and fetal hemoglobin (HbF) in most SS patients, there has been considerable interpatient variability in HbF response to the drug. From this standpoint, HU is imperfectly understood mechanistically. Cytochrome b5 reductase 3 (Cyb5R3), also known as methemoglobin reductase, is an enzyme known to modulate redox signaling in erythrocytes and vascular cells by catalyzing one electron transfer reactions using reduced nicotinamide adenine dinucleotide (NADH) as an electron donor. Functionally, membrane-associated CYB5R3 exerts antioxidant properties by modulating heme iron reduction, plasma membrane coenzyme Q reduction, elongation and unsaturation of fatty acids, cholesterol biosynthesis and drug metabolism, while the soluble form of CYB5R3 reduces red blood cell methemoglobin. Unremitting oxidation of the soluble guanylate cyclase (sGC) heme iron in the absence of sufficient Cyb5R3 activity in vascular smooth muscle has been shown to explain some NO resistance that characterizes pulmonary arterial hypertension and other vasculopathy associated with SCD. More than 40 variants of Cyb5R3 have been identified. Of particular interest is a Cyb5R3 variant (T117S) that contains a threonine to serine substitution in the flavoprotein, which lessens the reductase's activity by roughly 50% compared to wild-type. This variant occurs with high frequency (0.23 minor allele) in persons of African ancestry but is rare (<1%) in all other races. Nothing is known about the role of Cyb5R3 in the anemia of sickle cell disease. We hypothesized that Cyb5R3 may act as a modifier of HbF induction by HU in sickle cell anemia. METHODS: Male and female SS patients (n=633, 12-70 years of age) from the Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (walk-PHaSST) trial were recruited for genetic analysis (University of Pittsburgh IRB Protocol Study 19060255). Genotyping of rs1800457 (T117S SNP) was performed using Taqman genotyping assay. One-way ANOVA and Holms Sidak were used for multiple comparisons of HbF levels by Cyb5R3 genotype and HU treatment status. Intrapatient correlation analyses of HbF and hematocrit (HCT) were conducted using Pearsons r. RESULTS: Comparisons of HbF levels by Cyb5R3 genotype (CC, wild-type; CG, heterozygous wild-type and T117S; GG, homozygous T117S) and HU treatment status (treated vs. untreated) at time of study enrollment (baseline) show that HU is a less potent HbF inducer in the GG group (4.32 mean diff, P=0.344) versus the CC and CG patient groups (CC: 6.027 mean diff, P<0.0001 and CG: 6.914 mean diff, P<0.0001). Correlation analyses showed that as HbF levels increased among HU-untreated patients, the trend was for HCT to decrease. This was true for all SS patients, irrespective of Cyb5R3 genotype (CC: r = -0.336, P<0.0001, n=320; CG: r = -0.437 P<0.0001, n=120; GG: r = -0.253, P=0.136, n=36). However, with HU therapy, the inverse relationship between HbF and HCT was less negative in SS patients carrying at least one copy of the wild-type Cyb5R3 gene (CC or CG). In these individuals, increasing levels of HbF (up to 10%) correlated with positive gains in HCT at HbF greater than 5-6% (CC: r = -0.161, P=0.097, n=107) and 6-7% (CG: r = -0.186, P=0.25, n=40). In patients homozygous for T117S, HCT continued trending downward as HbF increased (GG: r = -0.419, P=0.228, n=10). These data indicate that the positive relationship between HbF and HCT is subject to uncoupling by loss of Cyb5R3 function. In vitro studies with human erythroleukemic K562 cells reveal loss of gamma globin induction by HU treatment when wild-type Cyb5R3 is knocked down by siRNA, indicating a requirement for Cyb5R3 in HU-stimulated HbF induction. SUMMARY andCONCLUSION: Loss of Cyb5R3 activity negatively impacts HbF and HCT in HU treated SS patients. We conclude that the T117S variant may be a useful marker for predicting which SS patients will incur beneficial HbF and HCT responses to HU, thereby improving clinical decision-making about best drug choices for preventing HbS polymerization and treating anemia in SS patients.

Abstract P136: Exploring The Interactions Between Rho-related BTB Domain Containing 1 (RhoBTB1) And Phosphodiesterase 5 (PDE5) To Identify Novel Therapeutic Candidates To Counter Hypertension

Protein-protein interaction studies have provided valuable insights to understand fundamental mechanisms in cells. We previously identified that PDE5 is a binding partner of RhoBTB1 and stringently regulates PDE5 activity in vascular smooth muscle (VSM). This in turn regulates the nitric oxide response of VSM and consequently blood pressure. We showed that RhoBTB1 can function as an adapter for Cullin-3 (CUL3) E3 ring ubiquitin ligase complex delivering PDE5 for ubiquitination and proteasomal degradation. In order to identify other RhoBTB1 binding partners, we assessed the binding interface of RhoBTB1 which binds to PDE5 with the notion that the same region will bind to other RhoBTB1-CUL3 substrates. Full-length RhoBTB1 was truncated into its component domains, separately (GTPase, BTB1, BTB2, and C-terminal (CT)) and in combination (e.g. B1B2C) domains. HEK293 cells were co-transfected with epitope-tagged PDE5 and RhoBTB1 domain constructs, and their interaction was analyzed by co-immunoprecipitation (Co-IP). Interestingly, the CT domain itself was essential for the interaction between RhoBTB1 and PDE5 but was not sufficient on its own to bind to PDE5. This was supported by our finding that the domains lacking CT region could not bind with PDE5. The Co-IP data was further corroborated by immunofluorescence and proximity labeling assay (PLA). Our data suggest that RhoBTB1 could bind to PDE5 utilizing its BTB1-BTB2-CT domains. Moreover, protein stability and ubiquitination studies revealed that the BTB1-BTB2-CT domain is sufficient for CUL3-dependent proteasomal degradation. Molecular approaches including site-directed mutagenesis are being explored to investigate potential “hotspots” underlying the RhoBTB1-PDE5 interaction. We are currently utilizing ascorbate peroxidase (APEX2) fusion system involving BTB1-BTB2-CT domain as “bait” to identify unknown interacting partner in RhoBTB1-Cullin3 axis. Preliminary experiments have been performed to validate the system. For example, BTB1-BTB2-CT-APEX2 can bind to PDE5. These studies are designed to assess if there are other proteins which are involved in the regulation of blood pressure in VSM which may become novel therapeutic candidates against hypertension.

AICAR promotes endothelium-independent vasorelaxation by activating AMP-activated protein kinase via increased ZMP and decreased ATP/ADP ratio in aortic smooth muscle.

AICAR, an adenosine analog, has been shown to exhibit vascular protective effects through activation of AMP-activated protein kinase (AMPK). However, it remains unclear as to whether adenosine kinase-mediated ZMP formation or adenosine receptor activation contributes to AICAR-mediated AMPK activation and/or vasorelaxant response in vascular smooth muscle. In the present study using endothelium-denuded rat aortic ring preparations, isometric tension measurements revealed that exposure to 1mM AICAR for 30min resulted in inhibition of phenylephrine (1μM)-induced smooth muscle contractility by ∼35%. Importantly, this vasorelaxant response by AICAR was prevented after pretreatment of aortic rings with an AMPK inhibitor (compound C, 40µM) and adenosine kinase inhibitor (5-iodotubercidin, 1µM), but not with an adenosine receptor blocker (8-sulfophenyltheophylline, 100µM). Immunoblot analysis of respective aortic tissues showed that AMPK activation seen during vasorelaxant response by AICAR was abolished by compound C and 5-iodotubercidin, but not by 8-sulfophenyltheophylline, suggesting ZMP involvement in AMPK activation. Furthermore, LC-MS/MS MRM analysis revealed that exposure of aortic smooth muscle cells to 1mM AICAR for 30min enhanced ZMP level to 2014.9±179.4picomoles/mg protein (vs. control value of 8.5±0.6; p<0.01), which was accompanied by a significant decrease in ATP/ADP ratio (1.08±0.02 vs. 2.08±0.06; p<0.01). Together, the present findings demonstrate that AICAR-mediated ZMP elevation and the resultant AMPK activation in vascular smooth muscle contribute to vasorelaxation.

Role of smooth muscle activation in the static and dynamic mechanical characterization of human aortas

Experimental data and a suitable material model for human aortas with smooth muscle activation are not available in the literature despite the need for developing advanced grafts; the present study closes this gap. Mechanical characterization of human descending thoracic aortas was performed with and without vascular smooth muscle (VSM) activation. Specimens were taken from 13 heart-beating donors. The aortic segments were cooled in Belzer UW solution during transport and tested within a few hours after explantation. VSM activation was achieved through the use of potassium depolarization and noradrenaline as vasoactive agents. In addition to isometric activation experiments, the quasistatic passive and active stress-strain curves were obtained for circumferential and longitudinal strips of the aortic material. This characterization made it possible to create an original mechanical model of the active aortic material that accurately fits the experimental data. The dynamic mechanical characterization was executed using cyclic strain at different frequencies of physiological interest. An initial prestretch, which corresponded to the physiological conditions, was applied before cyclic loading. Dynamic tests made it possible to identify the differences in the viscoelastic behavior of the passive and active tissue. This work illustrates the importance of VSM activation for the static and dynamic mechanical response of human aortas. Most importantly, this study provides material data and a material model for the development of a future generation of active aortic grafts that mimic natural behavior and help regulate blood pressure.

Abstract 11720: Loss of Vascular Smooth-Muscle Activity During Moderate Physical Activity as a Hallmark of Aging in Human Subjects

Studies have shown a higher risk of adverse outcomes after cardiac surgery in patients with reduced complexity of arterial-pressure dynamics (APD). We hypothesized that this effect is caused by age-related loss of vascular smooth-muscle activity (VSMA). This hypothesis was tested using data from 30 humans and a validated, large-scale theoretical model of circulation with VSMA blocked (T-model). The results were also verified in a realistic physical model of circulation (P-model, Fig.1). Methods: ECG and APD data were recorded using a high-fidelity system (APEX, PinMed, Inc.) during moderate physical activity (handgrip followed by leg lifts) in 30 subjects (age: 46±10y, 53% female; pressure ranges for systolic, diastolic, and mean: 90-210, 49-116, and 67-147mm, respectively). Results: T-model accurately predicted APD in older subjects ≥48y (n=13, r 2 =0.74; Fig.2); this effect was preserved in both females and males, but not in younger subjects (n=17, r 2 =0.13). The difference between the older and younger subjects was pronounced (40%) and significant (p=0.01, F-test). In P-model, changes in cardiac force produced changes in central arterial pressure that were in excellent agreement with those observed in humans ≥48y (r 2 =0.93, Fig.3). Conclusions: Loss of VSMA during physical activity could serve as an important prognostic/diagnostic indicator of cardiovascular aging and disease.

Myogenic Tone in Peripheral Resistance Arteries and Arterioles: The Pressure Is On!

Resistance arteries and downstream arterioles in the peripheral microcirculation contribute substantially to peripheral vascular resistance, control of blood pressure, the distribution of blood flow to and within tissues, capillary pressure, and microvascular fluid exchange. A hall-mark feature of these vessels is myogenic tone. This pressure-induced, steady-state level of vascular smooth muscle activity maintains arteriolar and resistance artery internal diameter at 50–80% of their maximum passive diameter providing these vessels with the ability to dilate, reducing vascular resistance, and increasing blood flow, or constrict to produce the opposite effect. Despite the central importance of resistance artery and arteriolar myogenic tone in cardiovascular physiology and pathophysiology, our understanding of signaling pathways underlying this key microvascular property remains incomplete. This brief review will present our current understanding of the multiple mechanisms that appear to underlie myogenic tone, including the roles played by G-protein-coupled receptors, a variety of ion channels, and several kinases that have been linked to pressure-induced, steady-state activity of vascular smooth muscle cells (VSMCs) in the wall of resistance arteries and arterioles. Emphasis will be placed on the portions of the signaling pathways underlying myogenic tone for which there is lack of consensus in the literature and areas where our understanding is clearly incomplete.

Endothelium-Dependent Hyperpolarization: The Evolution of Myoendothelial Microdomains.

Endothelium-derived hyperpolarizing factor (EDHF) was envisaged as a chemical entity causing vasodilation by hyperpolarizing vascular smooth muscle (VSM) cells and distinct from nitric oxide (NO) ([aka endothelium-derived relaxing factor (EDRF)]) and prostacyclin. The search for an identity for EDHF unraveled the complexity of signaling within small arteries. Hyperpolarization originates within endothelial cells (ECs), spreading to the VSM by 2 branches, 1 chemical and 1 electrical, with the relative contribution varying with artery location, branch order, and prevailing profile of VSM activation. Chemical signals vary likewise and can involve potassium ion, lipid mediators, and hydrogen peroxide, whereas electrical signaling depends on physical contacts formed by homocellular and heterocellular (myoendothelial; MEJ) gap junctions, both able to conduct hyperpolarizing current. The discovery that chemical and electrical signals each arise within ECs resulted in an evolution of the single EDHF concept into the more inclusive, EDH signaling. Recognition of the importance of MEJs and particularly the fact they can support bidirectional signaling also informed the discovery that Ca2+ signals can pass from VSM to ECs during vasoconstriction. This signaling activates negative feedback mediated by NO and EDH forming a myoendothelial feedback circuit, which may also be responsible for basal or constitutive release of NO and EDH activity. The MEJs are housed in endothelial projections, and another spin-off from investigating EDH signaling was the discovery these fine structures contain clusters of signaling proteins to regulate both hyperpolarization and NO release. So, these tiny membrane bridges serve as a signaling superhighway or infobahn, which controls vasoreactivity by responding to signals flowing back and forth between the endothelium and VSM. By allowing bidirectional signaling, MEJs enable sinusoidal vasomotion, co-ordinated cycles of widespread vasoconstriction/vasodilation that optimize time-averaged blood flow. Cardiovascular disease disrupts EC signaling and as a result vasomotion changes to vasospasm.

PPARγ and RhoBTB1 in hypertension.

This review provides an up-to-date understanding of how peroxisome proliferator activated receptor γ (PPARγ) exerts its cardioprotective effect in the vasculature through its activation of novel PPARγ target genes in endothelium and vascular smooth muscle. In vascular endothelial cells, PPARγ plays a protective role by increasing nitric oxide bioavailability and preventing oxidative stress. RBP7 is a PPARγ target gene enriched in vascular endothelial cells, which is likely to form a positive feedback loop with PPARγ. In vascular smooth muscle cells, PPARγ antagonizes the renin-angiotensin system, maintains vascular integrity, suppresses vasoconstriction, and promotes vasodilation through distinct pathways. Rho-related BTB domain containing protein 1 (RhoBTB1) is a novel PPARγ gene target in vascular smooth muscle cells that mediates the protective effect of PPARγ by serving as a substrate adaptor between the Cullin-3 RING ubiquitin ligase and phosphodiesterase 5, thus restraining its activity through ubiquitination and proteasomal degradation. In the vasculature, PPARγ exerts its cardioprotective effect through its transcriptional activity in endothelium and vascular smooth muscle. From the understanding of PPARγ's transcription targets in those pathways, novel hypertension therapy target(s) will emerge.

Soluble adenylyl cyclase links Ca2+ entry to Ca2+/cAMP-response element binding protein (CREB) activation in vascular smooth muscle

Ca2+-transcription coupling controls gene expression patterns that define vascular smooth muscle cell (VSMC) phenotype. Although not well understood this allows normally contractile VSMCs to become proliferative following vessel injury, a process essential for repair but which also contributes to vascular remodelling, atherogenesis and restenosis. Here we show that the Ca2+/HCO3−-sensitive enzyme, soluble adenylyl cyclase (sAC), links Ca2+ influx in human coronary artery smooth muscle cells (hCASMCs) to 3′,5′-cyclic adenosine monophosphate (cAMP) generation and phosphorylation of the transcription factor Ca2+/cAMP response element binding protein (CREB). Store-operated Ca2+ entry (SOCE) into hCASMCs expressing the FRET-based cAMP biosensor H187 induced a rise in cAMP that mirrored cytosolic [Ca2+]. SOCE also activated the cAMP effector, protein kinase A (PKA), as determined by the PKA reporter, AKAR4-NES, and induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and CREB. Transmembrane adenylyl cyclase inhibition had no effect on the SOCE-induced rise in cAMP, while sAC inhibition abolished SOCE-generated cAMP and significantly reduced SOCE-induced VASP and CREB phosphorylation. This suggests that SOCE in hCASMCs activates sAC which in turn activates the cAMP/PKA/CREB axis. sAC, which is insensitive to G-protein modulation but responsive to Ca2+, pH and ATP, may therefore act as an overlooked regulatory node in vascular Ca2+-transcription coupling.

P38-MAP Kinase Negatively Regulates the Slow Force Response to Stretch in Rat Myocardium through the Up-Regulation of Dual Specificity Phosphatase 6 (DUSP6).

Myocardial stretch increases cardiac force in two consecutive phases: The first one due to Frank-Starling mechanism, followed by the gradually developed slow force response (SFR). The latter is the mechanical counterpart of an autocrine/paracrine mechanism involving the release of angiotensin II (Ang II) and endothelin (ET) leading to Na⁺/H⁺ exchanger 1 (NHE-1) phosphorylation and activation. Since previous evidence indicates that p38-MAP kinase (p38-MAPK) negatively regulates the Ang II-induced NHE1 activation in vascular smooth muscle and the positive inotropic effect of ET in the heart, we hypothesized that this kinase might modulate the magnitude of the SFR to stretch. Experiments were performed in isolated rat papillary muscles subjected to sudden stretch from 92 to 98% of its maximal length, in the absence or presence of the p38-MAPK inhibitor SB202190, or its inactive analogous SB202474. Western blot technique was used to determine phosphorylation level of p38-MAPK, ERK1/2, p90RSK and NHE-1 (previously immunoprecipitated with NHE-1 polyclonal antibody). Dual specificity phosphatase 6 (DUSP6) expression was evaluated by RT-PCR and western blot. Additionally, the Na⁺-dependent intracellular pH recovery from an ammonium prepulse-induced acid load was used to asses NHE-1 activity. The SFR was larger under p38-MAPK inhibition (SB202190), effect that was not observed in the presence of an inactive analogous (SB202474). Myocardial stretch activated p38-MAPK, while pre-treatment with SB202190 precluded this effect. Inhibition of p38-MAPK increased stretched-induced NHE-1 phosphorylation and activity, key event in the SFR development. Consistently, p38-MAPK inhibition promoted a greater increase in ERK1/2-p90RSK phosphorylation/activation after myocardial stretch, effect that may certainly be responsible for the observed increase in NHE-1 phosphorylation under this condition. Myocardial stretch induced up-regulation of the DUSP6, which specifically dephosphorylates ERK1/2, effect that was blunted by SB202190. Taken together, our data support the notion that p38-MAPK activation after myocardial stretch restricts the SFR by limiting ERK1/2-p90RSK phosphorylation, and consequently NHE-1 phosphorylation/activity, through a mechanism that involves DUSP6 up-regulation.

131 - Disrupted regulation of Nox1 by protein disulfide isomerase-A1 in colon cancer cells with KRas overactivity

Tumor cells, e.g., colorectal cancer cells (CRC) often upregulate Nox NADPH oxidases, but underlying mechanisms are unclear. Thiol isomerases such as protein disulfide isomerase-A1 (PDIA1) are thioredoxin superfamily redox chaperones overexpressed in cancer and associated with tumoral progression. We previously showed an essential requirement of PDIA1 for agonist-triggered Nox1 activation in vascular smooth muscle . We thus investigated PDIA1/Nox1 crosstalk in CRC, known to highly express Nox1 and mutated overactive KRas. Our analysis of RNAseq databanks and direct validation in CRC cell lines indicated significant positive correlation between PDIA1 expression and Ras activity. We questioned whether Ras activity might influence PDIA1/Nox1 crosstalk and studied CRC with constitutive high (HCT116) vs. moderate (HKE3) or basal (CaCo2) Ras activity. In Caco2 and HKE3, PDIA1 siRNA silencing promoted decreased superoxide generation (HPLC/2OH-ethidium). Unexpectedly, in HCT116, PDIA1 silencing promoted increased superoxide output. Effects of NoxA1ds peptide inhibitor confirmed the Nox1 source. Thus, analogous to vascular cells, PDIA1 supports Nox1 in CRC with basal or moderate Ras activity, while PDIA1 silencing with overactive Ras unleashes Nox1 activity. Since vascular PDIA1 effects involve Rac1 RhoGTPase, a known Nox1 subunit, we hypothesized that direct Ras-mediated Rac1 activation could bypass PDIA1 regulation. Indeed, in HCT116 treated with Rac1 inhibitor, PDIA1 became supportive of superoxide production; conversely, Rac1-G12V active mutant transfection into HKE3 induced PDIA1 to restrict Nox1-dependent superoxide. PDIA1 silencing impaired cell proliferation and migration in HKE3, an effect lost in HCT116. Also, E-cadherin expression enhanced in HKE3 and decreased in HCT116 upon PDIA1 silencing, consistent with PDIA1 supporting epithelial-mesenchymal transition. Thus, PDIA1 may be a servomechanism of Nox1 regulation, supporting it within its operational range but capping its overactivation. Ras upregulation, unleash Nox1 activation possibly via Rac1, inducing PDIA1 capping mechanism switch. PDIA1 may crucially regulate redox-dependent adaptive processes related to cancer progression.

Calcium movement in smooth muscle and evaluation of graded functional intercellular coupling.

Spontaneous activity of vascular smooth muscle is present in small arteries and some venous tissues like the hepatic portal vein. Whereas the ability to generate rhythmic membrane potential changes is expressed in a high number of primary oscillators, the generation of physiological tone and phasic activity requires synchronization of specialized pacemaker activity (Interstitial Cajal-like cells) by intercellular propagation and regeneration of excitation or a strong coupling mechanism of smooth muscle cells. The aim of this study was to deduce oscillator coupling by analyzing the spatiotemporal homogeneity of calcium oscillations within a native tissue preparation. Portal vein tissue was loaded with a calcium-sensitive dye (Fluo-3). By combining confocal microscopy and computation of spatial auto- and cross-correlation of the calcium signals, temporal and spatial coupling between cells was characterized. Spontaneous oscillations of calcium signals were measured at different predefined regions of interest. Cross-correlation analysis of these signals revealed that their damping was very similar in all directions of the investigated z-plane. In single experiments, improved cell-to-cell coupling was seen when noradrenaline (1-10 μM) was added to the bath-solution. With the chosen parameters of frame refresh, the velocity of signal propagation was faster than the maximum detectable velocity, but it could be estimated to exceed 0.1 mm/s. Correlative Network Analysis is a new and very useful tool to determine the functional coupling parameters of quasi-homogenous biological networks and their temporal changes. The action and significance of pharmacological modulators can be well studied on cellular and functional aspects with this newly introduced technique in biological sciences.

The large-conductance voltage- and Ca2+ -activated K+ channel and its γ1-subunit modulate mouse uterine artery function during pregnancy.

The uterine artery (UA) markedly vasodilates during pregnancy to direct blood flow to the developing fetus. Inadequate UA vasodilatation leads to intrauterine growth restriction and fetal death. The large-conductance voltage- and Ca2+ -activated K+ (BKCa ) channel promotes UA vasodilatation during pregnancy. We report that BKCa channel activation increases the UA diameter at late pregnancy stages in mice. Additionally, a BKCa channel auxiliary subunit, γ1, participates in this process by increasing channel activation and inducing UA vasodilatation at late pregnancy stages. Our results highlight the importance of the BKCa channel and its γ1-subunit for UA functional changes during pregnancy. Insufficient vasodilatation of the uterine artery (UA) during pregnancy leads to poor utero-placental perfusion, contributing to intrauterine growth restriction and fetal loss. Activity of the large-conductance Ca2+ -activated K+ (BKCa ) channel increases in the UA during pregnancy, and its inhibition reduces uterine blood flow, highlighting a role of this channel in UA adaptation to pregnancy. The auxiliary γ1-subunit increases BKCa activation in vascular smooth muscle, but its role in pregnancy-associated UA remodelling is unknown. We explored whether the BKCa and itsγ1-subunit contribute to UA remodelling during pregnancy. Doppler imaging revealed that, compared to UAs from wild-type (WT) mice, UAs from BKCa knockout (BKCa-/- ) mice had lower resistance at pregnancy day 14 (P14) but not at P18. Lumen diameters were twofold larger in pressurized UAs from P18 WT mice than in those from non-pregnant mice, but this difference was not seen in UAs from BKCa-/- mice. UAs from pregnant WT mice constricted 20-50% in response to the BKCa blocker iberiotoxin (IbTX), whereas UAs from non-pregnant WT mice only constricted 15%. Patch-clamp analysis of WT UA smooth muscle cells confirmed that BKCa activity increased over pregnancy, showing three distinct voltage sensitivities. The γ1-subunit transcript increased 7- to 10-fold during pregnancy. Furthermore, γ1-subunit knockdown reduced IbTX sensitivity in UAs from pregnant mice, whereas γ1-subunit overexpression increased IbTX sensitivity in UAs from non-pregnant mice. Finally, at P18, γ1-knockout (γ1-/- ) mice had smaller UA diameters than WT mice, and IbTX-mediated vasoconstriction was prevented in UAsfrom γ1-/- mice. Our results suggest that the γ1-subunit increases BKCa activation in UAs during pregnancy.

Treatment of β-thujaplicin counteracts di(2-ethylhexyl)phthalate (DEHP)-exposed vascular smooth muscle activation, inflammation and atherosclerosis progression

The initiation of atherosclerosis involves up-regulation of molecules such as E-selectin, VCAM-1, and ICAM-1. The progression of atherosclerosis is linked to proliferation and migration of vascular smooth muscle cell via MMP-2 and MMP-9 activities. However, the etiology of atherosclerosis concerning plasticizers is unknown. We evaluated β-thujaplicin in preventing the development of atherosclerosis in a model induced by pro-inflammatory cytokines. Moreover, we established a new atherosclerosis model in vascular smooth muscle cells (VSMC) exposed to a common contact plasticizer, di(2-ethylhexyl)phthalate (DEHP). SEVC4-10 endothelial cells were treated with 50% RAW conditioned medium and A7r5 VSMC was treated with the plasticizer, with/without β-thujaplicin (4 or 12 μM). Production of E-selectin, ICAM-1, and VCAM-1 in SEVC4-10 cells as well as MMP-2/MMP-9 (both expression and activity) in VSMC were monitored. Results showed that the conditioned medium induced E-selectin and ICAM were significantly prevented by β-thujaplicin. However, inhibition on the production of VCAM by β-thujaplicin was only seen in a concentration of 12 μM. Both concentrations of β-thujaplicin also significantly prevented DEHP-induced MMP-2 and MMP-9 expression and activities. Evidence uncovers that β-thujaplicin has additional factors in amelioration of atherosclerosis and corroborates that β-thujaplicin is a strong candidate in preventing the initiation and progression of atherosclerosis.