Modulation of Small Artery Function by Insulin in Young Women: Role of Adiposity.

OBJECTIVES: Vascular dysfunction is common in obesity. Insulin can directly modulate arterial function, but its role is unclear in obesity. We examined the influence of adiposity on direct effects of insulin on human artery responses. METHODS: 22 healthy women were stratified by median BMI into lower (LA) (n=11) and higher adiposity (HA) (n=11). Small arteries from gluteal biopsies were tested for contractile responses to Noradrenaline (NA), the endothelium-dependent dilator Carbachol and the endothelium-independent dilator sodium nitroprusside were examined before and after incubation with 100 mU/ml human insulin. RESULTS: Contractile responses were similar in the two groups. Insulin reduced NA-induced contraction in HA [3.5 (2.4-4.6) vs. 2.4 (1.4-3.4) mN/mm: p=0.004] but not those from LA [4.1 (2.8-5.3) vs. 3.7 (2.5-5.0) mN/mm: p=0.33]. Endothelium-dependent dilation (EDD) was significantly reduced in arteries from women in the HA (34.7 (18.8-50.6%)) compared to those from women in the LA (62.3 (46.2- 78.4); p=0.013). Insulin improved EDD (change in maximal dilation before/after insulin (%)) in arteries from the HA (37.7 (18.0 to 57.3) but not the LA (6.3 (-6.5 to 19.1), p=0.007. CONCLUSION: Reduced EDD evident in arteries from HA subjects improve by incubating in insulin. Hyperinsulinaemia may be necessary in maintaining endothelial function in obesity.

Differential effects of resveratrol on the dilator responses of femoral arteries, ex vivo.

Resveratrol is a plant-derived phytoalexin with antioxidant, anti-inflammatory and cardio-protective properties and may be a promising therapeutic intervention strategy in cardiovascular disease. Here, we investigated the acute direct effects of trans-resveratrol (RV), on acetylcholine (ACh)-induced and flow-mediated dilation (FMD) of isolated pressurized femoral arteries of young (4-month-old) and old (26-month-old) mice. Vessel exposure to RV enhanced ACh (0.01-1.0 mM)-induced dilation (p < 0.05), but not FMD (@ 5-10 µL⋅min-1) (p < 0.05) in both young and old mice. After RV incubation, acute nitric oxide (NO) production by cultured endothelial cells was increased in response to 0.01 mM ACh, but reduced by flow (5-10 µL⋅min-1; p < 0.05). In isolated femoral arteries from endothelial nitric oxide synthase knockout (eNOS-/-) mice, RV had no overall effect on FMD, but potentiated ACh induced dilation, that was completely abolished by potassium channel blockers, Apamin and Tram 34 (p < 0.01). We demonstrate that the non-metabolised form of RV stimulates ACh-induced dilation via the NO and EDHF pathways, but not FMD by interaction with the cyclo-oxygenase pathway. Our findings have important implications in the use of RV (for both young and aged) under 'normal' non-diseased physiological states.

Exploring Gender Differences within Forest Schools as a Physical Activity Intervention.

This study investigated whether children engaged in more physical activity (PA) on school days that included Forest School (FS) sessions than a regular school day or a school day with a Physical Education (PE) lesson. How FS sessions influenced children's general levels of PA and wellbeing was also explored across gender. A mixed-methods study followed a sample of 59 child participants aged 7 to 9 years old, from four primary schools, whilst taking part in twelve weekly FS sessions. Measures included the PA Questionnaire for Older Children and accelerometry data together with an individual Write and Draw task to inform focus groups. Children had significantly greater levels of light PA on a FS day and a PE school day compared to a regular school day and children reported feeling both happier and relaxed as a consequence of the intervention. From the qualitative data, boys and girls reported different likes of the FS interventions, whereas their dislikes of FS were comparable. Findings from this research provide evidence for such outdoor, nature-based learning within the school curriculum contributing to daily PA in children.

Acute inhibition of PMCA4, but not global ablation, reduces blood pressure and arterial contractility via a nNOS-dependent mechanism.

Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure (BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild-type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild-type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl-l-NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4.

Reduced expression of PMCA1 is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries.

Hypertension is a well-established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1Ht ) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age-matched wild-type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.

The Role of O-GlcNAcylation in Perivascular Adipose Tissue Dysfunction of Offspring of High-Fat Diet-Fed Rats.

Perivascular adipose tissue (PVAT), which reduces vascular contractility, is dysfunctional in the male offspring of rats fed a high-fat diet (HFD), partially due to a reduced NO bioavailability. O-GlcNAcylation of eNOS decreases its activity, thus we investigated the role of O-GlcNAcylation in the prenatal programming of PVAT dysfunction. Female Sprague-Dawley rats were fed either a control (10% fat) or an obesogenic HFD (45% fat) diet for 12 weeks prior to mating, and throughout pregnancy and lactation. Offspring were weaned onto the control diet and were killed at 12 and 24 weeks of age. Mesenteric arteries from the 12-week-old offspring of HFD dams (HFDO) contracted less to U46619; these effects were mimicked by glucosamine in control arteries. PVAT from 12- and 24-week-old controls, but not from HFDO, exerted an anticontractile effect. Glucosamine attenuated the anticontractile effect of PVAT in the vessels from controls but not from HFDO. AMP-activated protein kinase (AMPK) activation (with A769662) partially restored an anticontractile effect in glucosamine-treated controls and HFDO PVAT. Glucosamine decreased AMPK activity and expression in HFDO PVAT, although phosphorylated eNOS expression was only reduced in that from males. The loss of anticontractile effect of HFDO PVAT is likely to result from increased O-GlcNAcylation, which decreased AMPK activity and, in males, decreased NO bioavailability.

The effects of resveratrol on aging vessels.

Aging is a major risk factor for the development of cardiovascular disease. Despite a significant reduction in the mortality and morbidity rates over the last decade, the socio-economic burden of cardiovascular disease is still substantial. Consequently, there is a considerable need for alternative strategies, such as nutraceutical supplementation, that delay the functional vascular decline present in the elderly. Compromised autophagy and oxidative stress (OS) are considered major causes of the age-related endothelial dysfunction. OS reduces the bioavailability of nitric oxide (NO), which has been associated with hypertension, arteriosclerosis, and a reduced vasodilatory response. High levels of free radicals and the low bioavailability of NO lead to a positive feedback loop of further OS, organelle damage, poor repair, and endothelial dysfunction. Here we draw attention to the relationship between OS and autophagy in the aged vasculature. We have reviewed the published literature and provided arguments that support that treatment with resveratrol stimulates autophagy and thereby has the potential to restore oxidative balance in the endothelium, which indicates that treatment with resveratrol might have therapeutic potential to restore endothelial function in the elderly.

Plasma membrane calcium ATPases (PMCAs) as potential targets for the treatment of essential hypertension.

The incidence of hypertension, the major modifiable risk factor for cardiovascular disease, is increasing. Thus, there is a pressing need for the development of new and more effective strategies to prevent and treat hypertension. Development of these relies on a continued evolution of our understanding of the mechanisms which control blood pressure (BP). Resistance arteries are important in the regulation of total peripheral resistance and BP; changes in their structure and function are strongly associated with hypertension. Anti-hypertensives which both reduce BP and reverse changes in resistance arterial structure reduce cardiovascular risk more than therapies which reduce BP alone. Hence, identification of novel potential vascular targets which modify BP is important. Hypertension is a multifactorial disorder which may include a genetic component. Genome wide association studies have identified ATP2B1, encoding the calcium pump plasma membrane calcium ATPase 1 (PMCA1), as having a strong association with BP and hypertension. Knockdown or reduced PMCA1 expression in mice has confirmed a physiological role for PMCA1 in BP and resistance arterial regulation. Altered expression or inhibition of PMCA4 has also been shown to modulate these parameters. The mechanisms whereby PMCA1 and 4 can modulate vascular function remain to be fully elucidated but may involve regulation of intracellular calcium homeostasis and/or comprise a structural role. However, clear physiological links between PMCA and BP, coupled with experimental studies directly linking PMCA1 and 4 to changes in BP and arterial function, suggest that they may be important targets for the development of new pharmacological modulators of BP.

Morphological Characterisation of Unstained and Intact Tissue Micro-architecture by X-ray Computed Micro- and Nano-Tomography.

Characterisation and quantification of tissue structures is limited by sectioning-induced artefacts and by the difficulties of visualising and segmenting 3D volumes. Here we demonstrate that, even in the absence of X-ray contrast agents, X-ray computed microtomography (microCT) and nanotomography (nanoCT) can circumvent these problems by rapidly resolving compositionally discrete 3D tissue regions (such as the collagen-rich adventitia and elastin-rich lamellae in intact rat arteries) which in turn can be segmented due to their different X-ray opacities and morphologies. We then establish, using X-ray tomograms of both unpressurised and pressurised arteries that intra-luminal pressure not only increases lumen cross-sectional area and straightens medial elastic lamellae but also induces profound remodelling of the adventitial layer. Finally we apply microCT to another human organ (skin) to visualise the cell-rich epidermis and extracellular matrix-rich dermis and to show that conventional histological and immunohistochemical staining protocols are compatible with prior X-ray exposure. As a consequence we suggest that microCT could be combined with optical microscopy to characterise the 3D structure and composition of archival paraffin embedded biological materials and of mechanically stressed dynamic tissues such as the heart, lungs and tendons.

Effects of diabetes and hypertension on structure and distensibilty of human small coronary arteries.

OBJECTIVES: Previous studies have demonstrated that hypertension and diabetes induce significant structural remodelling of resistance arteries from various vascular beds. The hypothesis of this study is that structural alterations of small coronary arteries may occur during hypertension and diabetes. This study is the first to compare human coronary small resistance artery structure from normotensive and hypertensive patients, with and without diabetes undergoing coronary arterial bypass graft surgery. METHODS: Small arteries were dissected from the atrial appendage removed from nondiabetic normotensive patients, nondiabetic hypertension and diabetic normotensive patients and hypertensive diabetic patients. Arteries were mounted in a pressure myograph and lumen diameter and wall thickness were measured across the pressure range of 3-100 mmHg to assess vessel structure and distensibility. RESULTS: There were no significant differences in the lumen diameter, wall thickness, wall-to-lumen ratio and cross-sectional area of arteries in all groups. Arteries from nondiabetic patients with hypertension demonstrated decreased distensibility compared with nondiabetic normotensive patients. There is no difference in distensibility between vessels from diabetic hypertensive patients and either diabetic or nondiabetic normotensive patients. CONCLUSION: Neither diabetes nor hypertension appears to have influenced arterial structure which may indicate that successful treatment of hypertension is associated with normal vascular structure in coronary small arteries.

Small artery function 2 years postpartum in women with altered glycaemic distributions in their preceding pregnancy.

GDM (gestational diabetes mellitus) is associated with later adverse cardiovascular risk. The present study examined the relationship between glycaemia during pregnancy and small artery function and structures approx. 2 years postpartum. Women were originally enrolled in the HAPO (Hyperglycaemia and Adverse Pregnancy Outcome) study from which they were classified by their glycaemic distribution during pregnancy as controls (in the lower half of the distribution), UQ (upper quartile; in the UQ of the glycaemic distribution) or having had overt GDM. Subcutaneous arteries from a gluteal fat biopsy taken at follow-up 2 years later were examined using wire myography. Small artery structure, stiffness and vasoconstrictor responses were similar across groups. Maximal endothelium-dependent dilation in response to carbachol was impaired in arteries from both GDM (43.3%, n=8 and P=0.01) and UQ (51.7%, n=13 and P=0.04) women despite generally 'normal' current glycaemia (controls, 72.7% and n=8). Inhibition of NOS (nitric oxide synthase) significantly reduced maximum endothelium-dependent dilation in controls but had no effect on arteries from UQ and GDM women, suggesting impaired NOS activity in these groups. Endothelium-independent dilation was unaffected in arteries from previous GDM and UQ women when compared with the control group. Multiple regression analysis suggested that BMI (body mass index) at biopsy was the most potent factor independently associated with small artery function, with no effect of current glycaemia. Overweight women with either GDM or marginally raised glycaemia during pregnancy (our UQ group) had normal vascular structure and stiffness, but clearly detectable progressively impaired endothelium-dependent function at 2 years follow-up. These results suggest that vascular pathology, which may still be reversible, is detectable very early in women at risk of decline into Type 2 diabetes mellitus.

Age-related changes in the contractile and passive arterial properties of murine mesenteric small arteries are altered by caveolin-1 knockout.

Caveolin-1, an integral protein of caveolae, is associated with multiple cardiovascular signalling pathways. Caveolin-1 knockout (KO) mice have a reduced lifespan. As changes in artery structure and function are associated with ageing we have investigated the role of caveolin-1 ablation on age-related changes of small artery contractility and passive mechanical properties. Mesenteric small arteries isolated from 3 and 12-month wild-type (WT) and caveolin-1 KO mice were mounted on a pressure myograph and changes in passive and functional arterial properties were continuously monitored. In WT mice ageing was associated with a reduction in arterial contractility to noradrenaline which was reversed by inhibition of nitric oxide synthase with L-NNA. Similarly, in 3-month-old mice, caveolin-1 KO reduced contractility to noradrenaline by an L-NNA-sensitive mechanism. However, ageing in caveolin-1 KO mice was not associated with any further change in contractility. In WT mice ageing was associated with an increased passive arterial diameter and cross-sectional area (CSA), consistent with outward remodelling of the arterial wall, and a reduced arterial distensibility. Caveolin-1 ablation at 3 months of age resulted in similar changes in passive arterial properties to those observed with ageing in WT animals. However, ageing in caveolin-1 KO mice resulted in a reduced arterial CSA indicating different effects on passive structural characteristics from that observed in WT mice. Thus, caveolin-1 mice show abnormalities of small mesenteric artery function and passive mechanical characteristics indicative of premature vascular ageing. Moreover, caveolin-1 ablation modulates the age-related changes usually observed in mesenteric arteries of WT mice.

Ca2+ signalling in cardiovascular disease: the role of the plasma membrane calcium pumps.

The plasma membrane calcium ATPases (PMCA) are a family of genes which extrude Ca(2+) from the cell and are involved in the maintenance of intracellular free calcium levels and/or with Ca(2+) signalling, depending on the cell type. In the cardiovascular system, Ca(2+) is not only essential for contraction and relaxation but also has a vital role as a second messenger in signal transduction pathways. A complex array of mechanisms regulate intracellular free calcium levels in the heart and vasculature and a failure in these systems to maintain normal Ca(2+) homeostasis has been linked to both heart failure and hypertension. This article focuses on the functions of PMCA, in particular isoform 4 (PMCA4), in the heart and vasculature and the reported links between PMCAs and contractile function, cardiac hypertrophy, cardiac rhythm and sudden cardiac death, and blood pressure control and hypertension. It is becoming clear that this family of calcium extrusion pumps have essential roles in both cardiovascular health and disease.

Acute simvastatin increases endothelial nitric oxide synthase phosphorylation via AMP-activated protein kinase and reduces contractility of isolated rat mesenteric resistance arteries.

Statins can have beneficial cholesterol-independent effects on vascular contractility, which may involve increases in the bioavailability of NO (nitric oxide) as a result of phosphorylation of eNOS (endothelial NO synthase). Although this has been attributed to phosphorylation of Akt (also known as protein kinase B), studies in cultured cells have shown that statins can phosphorylate AMPK (AMP-activated protein kinase); it is unknown whether this has functional effects in intact arteries. Thus we investigated the acute effects of simvastatin on resistance arterial contractile function, evaluating the involvement of NO, Akt and AMPK. Isolated rat mesenteric resistance arteries were mounted on a wire myograph. The effects of incubation (1 and 2 h) with simvastatin (0.1 or 1 µM) on contractile responses were examined in the presence and absence of L-NNA (N-nitro-L-arginine; 10 µM) or mevalonate (1 mM). Effects on eNOS, phospho-eNOS (Ser1177), and total and phospho-Akt and -AMPK protein expression were investigated using Western blotting. The effect of AMPK inhibition (compound C, 10 µM) on eNOS phosphorylation and contractile responses were also studied. Simvastatin (1 µM, 2 h) significantly reduced constriction to U46619 and phenylephrine and enhanced dilations to ACh (acetylcholine) in depolarized, but not in U46619-pre-constricted arteries. These effects were completely and partially prevented by L-NNA and mevalonate respectively. Simvastatin increased eNOS and AMPKα phosphorylation, but had no effect on Akt protein expression and phosphorylation after 2 h incubation. Compound C prevented the effects of simvastatin on eNOS phosphorylation and contractility. Thus simvastain can acutely modulate resistance arterial contractile function via mechanisms that involve the AMPK/phospho-eNOS (Ser1177)/NO-dependent pathway.

Support for portfolio in the initial years of the undergraduate medical school curriculum: what do the tutors think?

BACKGROUND: Personal and Professional Development portfolios have recently been introduced into phase 1 (years 1 and 2) of the undergraduate medical curriculum in the School of Medicine in Manchester. As the large student numbers at Manchester precluded the use of an individual mentoring system, students are supported in their portfolio development by tutor facilitators, in groups of 10-12. AIMS: The aims of this study were to investigate the views of these tutor facilitators on the delivery and support of portfolios in this way. METHODS: A mixed method approach was used with data collected from both questionnaires and a focus group meeting. RESULTS: Tutor facilitators were generally positive about their roles in the support of portfolio development in group sessions and identified several advantages to this type of tutoring - namely the value of group discussion and discussion between peers to encourage reflection, and the practical ability to integrate portfolio sessions more closely with clinical experiences - which would not be possible in one to one tutor/student meetings. With the training and guidance notes provided tutors were confident that they understood the support they should give students and felt that they could encourage reflection. CONCLUSIONS: Thus, tutors perceived that the support of portfolio in groups is an effective way of developing portfolios for large numbers of students.

2'-hydroxy-fendiline analogues as potent relaxers of isolated arteries.

Novel, 2'-hydroxy derivatives of fendiline have been synthesised and their ability to induce relaxation of isolated rat small mesenteric and coronary arteries were determined. Both derivatives examined were significantly more potent as vasodilators than fendiline itself. Similar effects were observed on both mesenteric and coronary arteries.

The effects of endothelial factor inhibition on the time course of responses of isolated rat coronary arteries to intraluminal flow.

The aims of this study were to investigate, for the first time, the effects of endothelial factor inhibition on both the magnitude and dynamics of the response of isolated small coronary arteries to intraluminal flow. Isolated rat coronary arteries were mounted on a pressure myograph and left to develop myogenic tone. Flow was introduced and maintained until stable diameters were attained. Dilatory responses were observed which were maximal at low flow rates (5-10 microl/min) and thus shear stresses (1-2 dyn/cm(2)). These responses were transient in nature. Transient dilations were also observed upon cessation of flow. All responses (to 5 microl/min) were endothelium dependent and were completely abolished by addition of charybdotoxin (100 nM) and apamin (100-500 nM) suggesting an important role for a hyperpolarizing mechanism most likely involving an endothelium-derived hyperpolarizing factor. However, inhibitors of nitric oxide synthase (L-NNA; 100 microM) or cyclo-oxygenase (indomethacin; 10 microM) also modulated the response causing an increase and decrease in maximum vasodilation, respectively. By examining the time course we showed that both agents also made the response significantly more transient in nature. These results show that inhibition of endothelial factor pathways can influence both the magnitude and dynamics of the response of isolated rat coronary arteries to flow.

Caveolae and sarcoplasmic reticular coupling in smooth muscle cells of pressurised arteries: the relevance for Ca2+ oscillations and tone.

OBJECTIVE: A close association of caveolae and sarcoplasmic reticulum (SR) has been suggested to be important for contractile activation of smooth muscle. Here, we investigate the presence of such arrangements in pressurised resistance arteries and examine the influence of two agents purported to disrupt caveolae and/or SR conformations by different mechanisms of action. METHODS: Rat mesenteric small arteries (RMSA) were mounted on a pressure myograph and the functional (lumen diameter and Ca(2+) oscillations) and ultrastructural effects of the phosphatase inhibitor calyculin-A (cal-A), or the cholesterol binding agent methyl-beta-cyclodextrin (mbetacd), examined by light and electron microscopy. RESULTS: Smooth muscle cells of RMSA exhibited a prominent peripheral SR that often encircled individual caveolae. The peripheral SR on occasion was observed to make contact with centrally located SR allowing for a structural association of caveoale-SR-myofilaments. Cal-A maximally constricted RMSA and disrupted the regular SR-caveolae appearance such that concentrated swirls of SR not enveloping caveolae were evident. Mbetacd treatment, in contrast, inhibited agonist contractility and reduced the appearance of caveolae whilst peripheral SR apposition to the plasmalemma could still be observed. Treatment with either agent inhibited agonist-mediated smooth muscle Ca(2+) oscillations. CONCLUSION: We present data that supports a structural arrangement of caveolae and underlying peripheral SR in smooth muscle cells of pressurised resistance arteries that serves to regulate Ca(2+) oscillations and contractile activation.

alphaV integrins are necessary for eutrophic inward remodeling of small arteries in hypertension.

Human essential hypertension is characterized by eutrophic remodeling of small arteries, with little evidence of hypertrophy. Likewise, vessels of young hypertensive TGR(mRen2)27 animals have undergone similar structural alterations. The role of integrins in resistance arteries of TGR(mRen2)27 during the eutrophic-remodeling process was examined as blood pressure rose. Initially, 8 alpha and 3 beta integrins were identified and levels of expression investigated using RT-PCR. As pressure increased and remodeling advanced, integrin expression profiles revealed that only alphaV was significantly raised. In conjunction, we confirmed elevated integrin alphaV protein levels in TGR(mRen2)27 rat arteries and localization to the media using immunofluorescence. beta1 and beta3, but not beta5 integrin subunits were coprecipitated with integrin alphaV and are implicated in the eutrophic remodeling process. Administration of a peptide antagonist of alphaVbeta3 abolished remodeling but enhanced growth, indicating that hypertrophy supervened as a response to hypertension-induced increases in wall stress. We have established that the only upregulated integrin, the alphaV subunit of integrin alphaVbeta3, has a crucial role in the hypertensive remodeling process of TGR(mRen2)27 rat resistance arteries. During hypertensive remodeling, functions of specific alphaVbeta3-extracellular matrix interactions are likely to allow vascular smooth muscle cell-length autoregulation, which includes a migratory process, to maintain a narrowed lumen after a prolonged constricted state.