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Background Endothelial dysfunction is an early pathogenic event in the progression of cardiovascular disease in patients with Type 2 Diabetes (T2D). Endothelial KCa2.3 and KCa3.1 K+ channels are important regulators of arterial diameter, and we thus hypothesized that SKA-31, a small molecule activator of KCa2.3 and KCa3.1, would positively influence agonist-evoked dilation in myogenically active resistance arteries in T2D. Methodology Arterial pressure myography was utilized to investigate endothelium-dependent vasodilation in isolated cremaster skeletal muscle resistance arteries from 22 to 24 week old T2D Goto-Kakizaki rats, age-matched Wistar controls, and small human intra-thoracic resistance arteries from T2D subjects. Agonist stimulated changes in cytosolic free Ca2+ in acutely isolated, single endothelial cells from Wistar and T2D Goto-Kakizaki cremaster and cerebral arteries were examined using Fura-2 fluorescence imaging. Main findings Endothelium-dependent vasodilation in response to acetylcholine (ACh) or bradykinin (BK) was significantly impaired in isolated cremaster arteries from T2D Goto-Kakizaki rats compared with Wistar controls, and similar results were observed in human intra-thoracic arteries. In contrast, inhibition of myogenic tone by sodium nitroprusside, a direct smooth muscle relaxant, was unaltered in both rat and human T2D arteries. Treatment with a threshold concentration of SKA-31 (0.3 μM) significantly enhanced vasodilatory responses to ACh and BK in arteries from T2D Goto-Kakizaki rats and human subjects, whereas only modest effects were observed in non-diabetic arteries of both species. Mechanistically, SKA-31 enhancement of evoked dilation was independent of vascular NO synthase and COX activities. Remarkably, SKA-31 treatment improved agonist-stimulated Ca2+ elevation in acutely isolated endothelial cells from T2D Goto-Kakizaki cremaster and cerebral arteries, but not from Wistar control vessels. In contrast, SKA-31 treatment did not affect intracellular Ca2+ release by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid. Conclusions Collectively, our data demonstrate that KCa channel modulation can acutely restore endothelium-dependent vasodilatory responses in T2D resistance arteries from rats and humans, which appears to involve improved endothelial Ca2+ mobilization.

Matching oxygen supply to consumption across different tissues is a critical function of the vasculature. The remarkably wide range of metabolic demand between resting and active skeletal muscle renders control of blood flow to skeletal muscle tissues especially important and challenging. Mechanisms of exercise hyperemia are believed to be multifaceted and redundant, reflecting both the complexity of this response as well as its importance. We explored two candidate mechanisms of exercise hyperemia, examining ATP release from red blood cells and hemoglobin alpha in the endothelium of resistance arteries. We first studied the participation of Pannexin 1 in a red blood cell pathway reported to link hypoxia to elevated cyclic AMP and finally ATP release via Pannexin 1 to promote vasodilatory signaling in the endothelium. Our findings contradict previous claims that these components participate in the same signaling pathway. Furthermore, our work highlights a number of methodological considerations that we present as best practices for future in vitro work studying ATP release from red blood cells. Finally, we report exercise data that suggests more broadly a lack of significant role for Pannexin 1 in exercise performance. Subsequently, we hypothesized a novel role for endothelial hemoglobin alpha as a nitrite reductase during hypoxia. This mechanism has received significant attention in red blood cell hemoglobin, but has not previously been examined in the endothelium. Creating novel genetic models of Hba1 deletion, we demonstrated a reduction in exercise performance upon deletion of endothelial Hba1 in mice. These effects appear to be independent of the endothelial Hbα-eNOS interaction previously reported by our laboratory. Preliminary myography data shows impaired dilation of EC Hba1Δ/Δ skeletal muscle resistance arteries in response to hypoxia. We identify further experiments planned to clarify the mechanism underlying these results. Taken together, our findings point to a novel role for endothelial hemoglobin alpha as a critical regulator of skeletal muscle blood flow during exercise.

Chemical signals from perivascular adipose tissue (PVAT) modulate the structure and function of blood vessels. This may be partially under sympathetic neural control, however, little is known about the density of sympathetic innervation of PVAT. We hypothesized sympathetic nerve (SN) density in PVAT surrounding mesenteric resistance arteries (MRAs) is similar to that in the adventitia of the MRAs, and that high-fat feeding (HFF, 16 weeks) would decrease the density of SNs on MRAs and in PVAT. To test these hypotheses, we labeled SNs on MRAs and in PVAT with rabbit anti-tyrosine hydroxylase (TH) antibodies/AlexaFluor488-conjugated donkey-anti-rabbit antibodies, ex vivo. TH staining density (fraction occupied by TH-labeled structures) was computed from thresholded, background subtracted, maximum intensity z-projections of image stacks. TH staining density was higher at the surface of MRAs (0.21±0.03, n = 14) than within PVAT (0.013±0.003, n = 42, p16 fold lower. HFF does not affect SN density on MRAs or in PVAT. However, given the larger size of adipocytes in HFF rats, this may mean an increase in SN density/adipocyte. Nonetheless, the low density of SNs in mesenteric PVAT suggests limited direct SN control of PVAT function in the bulk of PVAT adipocytes.

In the present review, microvascular remodelling refers to alterations in the structure of resistance vessels contributing to elevated systemic vascular resistance in hypertension. We start with some historical aspects, underscoring the importance of Folkow's contribution made half a century ago. We then move to some basic concepts on the biomechanics of blood vessels, and explicit the definitions proposed by Mulvany for specific forms of remodelling, especially inward eutrophic and inward hypertrophic. The available evidence for the existence of remodelled resistance vessels in hypertension comes next, with relatively more weight given to human, in comparison with animal data. Mechanisms are discussed. The impact of antihypertensive drug treatment on remodelling is described, again with emphasis on human data. Some details are given on the three studies to date which point to remodelling of subcutaneous resistance arteries as an independent predictor of cardiovascular risk in hypertensive patients. We terminate by considering the potential role of remodelling in the pathogenesis of end-organ damage and in the perpetuation of hypertension.

The aorta has limited ability to accommodate increasing body size by remodeling. The dramatic rise in pediatric obesity threatens to overwhelm this intrinsic remodeling program and lead to abnormal aortic function. As hypothesized, pulse pressure, as an index of aortic function, has indeed risen dramatically in parallel with the rise of pediatric obesity, while at the same time mean arterial pressure, as an index of small resistance artery function, has fallen. These divergent large-artery-versus-small-artery indices may combine to explain the counterintuitive decrease in systolic blood pressure in children and adults during the global obesity pandemic. The pathophysiologic mechanisms underpinning these contrasting trends are not yet known.

The effect of intraluminal hyperglycaemia was investigated in the iliac artery of 11 anaesthetised pigs. Following isolation of a test segment, hyperglycaemic blood (40 mmol·L–1) caused a significant dilatation of the artery of 167 ± 208 μm (mean ± SD; n = 6, P = 0.031). Dilatations were reduced by N(G)-nitro-l-arginine methyl esther (250 μg·mL–1) from 145 ± 199 to 38 ± 5 μm), but this was not statistically significant (n = 6, P = 0.18). Intra-arterial infusions of d-glucose (20–40 mmol·L–1·min–1), during graded constrictions, caused statistically significant increases in blood flow (n = 11, P = 0.0013). Vasodilatation was confirmed by measurements of the ratio of immediate pressure steps to flow steps (∂P/∂F) during the graded obstruction experiments, showing a decrease in instantaneous vascular resistance from a control of 0.62 ± 0.30 to 0.33 ± 0.34 mm Hg·mL–1·min–1 (n = 7, P = 0.016). Autoregulation was assessed from the slopes of the plots of steady-state flow versus pressure. There were significant increases in the slope from 2.32 ± 1.03 to 5.88 ± 5.60 mL·min–1·(mm Hg)–1 (n = 7, P = 0.0078), indicating significant impairment of autoregulation. In conclusion, luminal hyperglycaemia relaxes both arterial and resistance vessel smooth muscle.

BACKGROUND: Patients with chronic kidney disease have a markedly increased cardiovascular mortality compared with the general population. Long chain n-3 polyunsaturated fatty acids have been suggested to possess cardioprotective properties. This cross-sectional and comparative study evaluated correlations between hemodynamic measurements, resistance artery function and fish consumption to the content of long chain n-3 polyunsaturated fatty acids in adipose tissue, a long-term marker of seafood intake.METHODS: Seventeen patients with chronic kidney disease stage 5 + 5d and 27 healthy kidney donors were evaluated with hemodynamic measurements before surgery; from these subjects, 11 patients and 11 healthy subjects had vasodilator properties of subcutaneous resistance arteries examined. The measurements were correlated to adipose tissue n-3 polyunsaturated fatty acids. Information on fish intake was obtained from a dietary questionnaire and compared with adipose tissue n-3 polyunsaturated fatty acids.RESULTS: Fish intake and the content of n-3 polyunsaturated fatty acids in adipose tissue did not differ between patients and controls. n-3 polyunsaturated fatty acid levels in adipose tissue were positively correlated to systemic vascular resistance index; (r = 0.44; p = 0.07 and r = 0.62; p CONCLUSIONS: The correlations found, suggest a role for n-3 polyunsaturated fatty acids in hemodynamic properties. However, this is apparently not due to changes in intrinsic properties of the resistance arteries as no correlation was found to n-3 polyunsaturated fatty acids.

Arteries from young healthy animals respond to chronic changes in blood flow and blood pressure by structural remodeling. We tested whether the ability to respond to decreased (-90%) or increased (+100%) blood flow is impaired during the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats, a model for an upregulated endothelin-1 system. Mesenteric small arteries (MrA) were exposed to low blood flow (LF) or high blood flow (HF) for 4 or 7 weeks. The bioavailability of vasoactive peptides was modified by chronic treatment of the rats with the dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor SOL1. After 3 or 6 weeks of hypertension, the MrA showed hypertrophic arterial remodeling (3 weeks: media cross-sectional area (mCSA): 10 +/- 1 x 10(3) to 17 +/- 2 x 10(3) mu m(2); 6 weeks: 13 +/- 2 x 10(3) to 24 +/- 3 x 10(3) mu m(2)). After 3, but not 6, weeks of hypertension, the arterial diameter was increased (empty set: 385 +/- 13 to 463 +/- 14 mu m). SOL1 reduced hypertrophy after 3 weeks of hypertension (mCSA: 6 x 10(3) +/- 1 x 10(3) mu m(2)). The diameter of the HF arteries of normotensive rats increased (empty set: 463 +/- 22 mu m) but no expansion occurred in the HF arteries of hypertensive rats (empty set: 471 +/- 16 mu m). MrA from SOL1-treated hypertensive rats did show a significant diameter increase (empty set: 419 +/- 13 to 475 +/- 16 mu m). Arteries exposed to LF showed inward remodeling in normotensive and hypertensive rats (mean empty set between 235 and 290 mu m), and infiltration of monocyte/ macrophages. SOL1 treatment did not affect the arterial diameter of LF arteries but reduced the infiltration of monocyte/ macrophages. We show for the first time that flow-induced remodeling is impaired during the development of DOCA-salt hypertension and that this can be prevented by chronic NEP/ECE inhibition. Hypertension Research (2012) 35, 1093-1101; doi:10.1038/hr.2012.94; published online 12 July 2012

Hypertension is associated with altered structure of the resistance vessels, a process known as remodeling. This review summarizes current concepts concerning the structure of a subgroup of the resistance vessels, the small arteries, and the modes of remodeling, some of the determinants of remodeling, and some signaling pathways for remodeling. It is shown that the available evidence points to important roles for blood flow and growth factors, in addition to blood pressure, as causes of resistance artery remodeling. Finally, the relationship between vascular structure and blood pressure is discussed, in particular with regard to the effects of antihypertensive therapy. Here again, it appears that blood flow plays an important role in allowing the correction of the abnormal resistance vessel structure seen in hypertension.

Aims Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs). Methods and results ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCδ1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca2+]i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ETAR) antagonist, BQ123. To characterize ETAR desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ETAR responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ETAR desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive D110A,K220RGRK2, D110A,K220RGRK3, K215RGRK5, or K215RGRK6 constructs. D110A,K220RGRK2 expression significantly attenuated ETAR desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ETAR desensitization. Finally, immunocyotchemical data showed that ETAR activation recruited endogenous GRK2 from cytoplasm to membrane. Conclusion These studies identify GRK2 as a key regulator of ETAR responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

The diameter of resistance arteries has a profound effect on the distribution of microvascular blood flow and the control of systemic blood pressure. Here, we review mechanisms that contribute to the regulation of resistance artery diameter, both acutely and chronically, their temporal characteristics, and their interdependence. Furthermore, we hypothesize the existence of a remodeling continuum that allows for the vascular wall to rapidly modify its structural characteristics, specifically through the re-positioning of vascular smooth muscle cells. Importantly, the concepts presented more closely link acute vasoregulatory responses with adaptive changes in vessel wall structure. These rapid structural adaptations provide resistance vessels the ability to maintain a desired diameter under presumed optimal energetic and mechanical conditions.

This is the first study to elucidate and quantify the microstructural bases of the mechanical properties of human resistance arteries. The geometrically accurate mechanical analysis provides new insights into strain fields existing in the walls of small arteries, and raises questions about the mechanobiology of vascular remodeling., Vascular diseases such as diabetes and hypertension cause changes to the vasculature that can lead to vessel stiffening and the loss of vasoactivity. The microstructural bases of these changes are not presently fully understood. We present a new methodology for stain-free visualization, at a microscopic scale, of the morphology of the main passive components of the walls of unfixed resistance arteries and their response to changes in transmural pressure. Human resistance arteries were dissected from subcutaneous fat biopsies, mounted on a perfusion myograph, and imaged at varying transmural pressures using a multimodal nonlinear microscope. High-resolution three-dimensional images of elastic fibers, collagen, and cell nuclei were constructed. The honeycomb structure of the elastic fibers comprising the internal elastic layer became visible at a transmural pressure of 30 mmHg. The adventitia, comprising wavy collagen fibers punctuated by straight elastic fibers, thinned under pressure as the collagen network straightened and pulled taut. Quantitative measurements of fiber orientation were made as a function of pressure. A multilayer analytical model was used to calculate the stiffness and stress in each layer. The adventitia was calculated to be up to 10 times as stiff as the media and experienced up to 8 times the stress, depending on lumen diameter. This work reveals that pressure-induced reorganization of fibrous proteins gives rise to very high local strain fields and highlights the unique mechanical roles of both fibrous networks. It thereby provides a basis for understanding the micromechanical significance of structural changes that occur with age and disease.

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.

This work aimed to establish the lineage of cells similar to the interstitial cells of Cajal (ICC), the arterial ICC-like (AIL) cells, which have recently been described in resistance arteries, and to study their location in the artery wall. Segments of guinea-pig mesenteric arteries and single AIL cells freshly isolated from them were used. Confocal imaging of immunostained cells or segments and electron microscopy of artery segments were used to test for the presence and cellular localization of selected markers, and to localize AIL cells in intact artery segments. AIL cells were negative for PGP9.5, a neural marker, and for von Willebrand factor (vWF), an endothelial cell marker. They were positive for smooth muscle α-actin and smooth muscle myosin heavy chain (SM-MHC), but expressed only a small amount of smoothelin, a marker of contractile smooth muscle cells (SMC), and of myosin light chain kinase (MLCK), a critical enzyme in the regulation of smooth muscle contraction. Cell isolation in the presence of latrunculin B, an actin polymerization inhibitor, did not cause the disappearance of AIL cells from cell suspension. The fluorescence of basal lamina protein collagen IV was comparable between the AIL cells and the vascular SMCs and the fluorescence of laminin was higher in AIL cells compared to vascular SMCs. Moreover, cells with thin processes were found in the tunica media of small resistance arteries using transmis-sion electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries.

Objectives Our goal was to investigate whether the association between established cardiovascular risk factors and arterial reactivity differs between the lower and upper extremities. Background Resistance artery reactivity in the arm is associated with cardiovascular risk factors, coronary disease, and events. However, the relationship of lower versus upper extremity vasoreactivity to increasing cardiovascular risk factors has not been determined. Methods We studied 82 subjects in 3 groups: 33 young healthy (YH) (21 to 41 years), 30 older healthy (OH) (>50 years), and 19 older type 2 diabetic subjects (OD). We directly measured systolic shear rate, flow, and radius in brachial and femoral arteries at rest and during post-occlusion hyperemia using magnetic resonance imaging. Results Brachial and femoral systolic shear rate, flow, and radius were similar among the groups at rest. Brachial hyperemic shear rate and hyperemic flow normalized as a function of baseline radius were not statistically different when YH were compared with OH and OH with OD. In contrast, femoral hyperemic shear rate and hyperemic flow normalized to baseline radius were lower in OH than YH (680 ± 236 s−1 vs. 843 ± 157 s−1, p = 0.001, and 0.84 ± 0.25 mm1.27/s vs. 1.01 ± 0.16 mm1.27/s, p = 0.001) and lower in OD than OH (549 ± 183 s−1, p = 0.02, and 0.74 ± 0.19 mm1.27/s, p = 0.046). Conclusions Persons with increasing cardiovascular risk factor burden had progressively reduced arterial reactivity in lower but not upper extremities. This may help to explain why atherosclerosis usually develops more severely in legs than in arms, and suggests that legs may be more sensitive than arms for assessing early global atherosclerotic risk.

Resistance artery narrowing and stiffening are key elements in the pathogenesis of essential hypertension, but their origin is not completely understood. In mesenteric resistance arteries (MRA) from spontaneously hypertensive rats (SHR), we have shown that inward remodeling is associated with abnormal elastic fiber organization, leading to smaller fenestrae in the internal elastic lamina. Our current aim is to determine whether this alteration is an early event that precedes vessel narrowing, or if elastic fiber reorganization in SHR arteries occurs because of the remodeling process itself. Using MRA from 10-day-old, 30-day-old, and 6-mo-old SHR and normotensive Wistar Kyoto rats, we investigated the time course of the development of structural and mechanical alterations (pressure myography), elastic fiber organization (confocal microscopy), and amount of elastin (radioimmunoassay for desmosine) and collagen (picrosirius red). SHR MRA had an impairment of fenestrae enlargement during the first month of life. In 30-day-old SHR, smaller fenestrae and more packed elastic fibers in the internal elastic lamina were paralleled by increased wall stiffness. Collagen and elastin levels were unaltered at this age. MRA from 6-mo-old SHR also had smaller fenestrae and a denser network of adventitial elastic fibers, accompanied by increased collagen content and vessel narrowing. At this age, elastase digestion was less effective in SHR MRA, suggesting a lower susceptibility of elastic fibers to enzymatic degradation. These data suggest that abnormal elastic fiber deposition in SHR increases resistance artery stiffness at an early age, which might participate in vessel narrowing later in life.

Prolonged vasoconstrictor-stimulated phospholipase C activity can induce arterial constriction, hypertension, and smooth muscle hypertrophy/hyperplasia. Arrestin proteins are recruited by agonist-occupied G protein-coupled receptors to terminate signaling and counteract changes in vascular tone. Here we determine whether the development of hypertension affects arrestin expression in resistance arteries and how such changes alter arterial contractile signaling and function. Arrestin2/3 expression was increased in mesenteric arteries of 12-wk-old spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) controls, while no differences in arrestin expression were observed between 6-wk-old SHR and WKY animals. In mesenteric artery myography experiments, high extracellular K(+)-stimulated contractions were increased in both 6- and 12-wk-old SHR animals. Concentration-response experiments for uridine 5'-triphosphate (UTP) acting through P2Y receptors displayed a leftward shift in 12-wk, but not 6-wk-old animals. Desensitization of UTP-stimulated vessel contractions was increased in 12-wk-old (but not 6-wk-old) SHR animals. Dual IP3/Ca(2+) imaging in mesenteric arterial cells showed that desensitization of UTP and endothelin-1 (ET1) responses was enhanced in 12-wk-old (but not 6-wk-old) SHR compared with WKY rats. siRNA-mediated depletion of arrestin2 for UTP and arrestin3 for ET1, reversed the desensitization of PLC signaling. In conclusion, arrestin2 and 3 expression is elevated in resistance arteries during the emergence of the early hypertensive phenotype, which underlies an enhanced ability to desensitize vasoconstrictor signaling and vessel contraction. Such regulatory changes may act to compensate for increased vasoconstrictor-induced vessel contraction.

The effect of maturation on collateral development of resistance arteries was investigated. Three to four sequential mesenteric arteries were ligated to create collateral pathways in anesthetized young (approximately 200 g) and mature (approximately 600 g) rats. Blood flow was similarly elevated in collaterals of young and mature animals. In vivo inner arterial diameter was increased only within young collaterals (33 +/- 7%, P0.001). Increases in number of intimal nuclei (57 +/- 10% vs. 52 +/- 14%) and cross-sectional medial area (33 +/- 13% vs. 38 +/- 5%) were similar between young and mature collaterals. Relative to the same animal controls, collateral endothelial nitric oxide synthase mRNA was increased as much in mature as in young rats. Proteomic analysis revealed significant differences in protein expression with maturation between control arteries as well as flow-loaded collateral vessels. The results indicate that, whereas intimal and medial remodeling events were similar in collaterals of young and mature rats, luminal expansion occurred only in young rats. Alteration in arterial protein expression with maturation and altered responses to stimuli for collateral development may contribute to this impairment.

Background: Chronic kidney disease (CKD) is associated with cardiovascular (CV) complications. However, interventional trials targeting classical CV risks factors have been often unsuccessful in advanced stage CKD, which emphasizes the need to better understand CKD-associated vascular disorders. Resistance arteries are a key determinant of blood pressure (BP) and their changes in different CV conditions contribute to target organ damage. The aim of the present study was to characterize resistance artery remodeling and function in CKD patients, compared to vessels from hypertensive (HTN) subjects. Method: Twenty-two stage 4 CKD patients (aged 63.6±3.1 years) and 16 HTN subjects (45.6±16.1 years) were included in the present study. They all underwent a subcutaneous biopsy under local anaesthesia. Small artery remodeling and function were studied on a pressurized myograph, and subcutaneous fat CD3 infiltration and media fibronectin expression by immunostaining. Vascular smooth muscle cells (VSMCs) were counted after hematoxilin-eosin staining. Results: CKD systolic BP was similar to HTN (133±18 vs. 143±10 mmHg, respectively). Vasodilatory responses to acetylcholine were lower in CKD compared to HTN (maximal relaxation (%), 74.3±3.4 vs. 87.5±2.7, P Conclusion: Despite higher levels of BP, resistance arteries isolated from CKD patients exhibited no vascular remodeling and lower arterial stiffness compared with HTN patients. These results are in line with the maladaptive hypotrophic remodeling observed in large vessels in CKD, suggesting a generalized vascular defect in mechanotransduction in CKD.

Fasted adipose tissue blood flow (ATBF) supplies energy via transport of stored free fatty acids, whereas after feeding ATBF increases to reduce circulating free fatty acids. Although high fat diet...

Intraluminal pressure has a significant impact on vascular adaptability, phenotype and regulation of blood flow and pressure. On one hand, increased pressure/stretch for a prolonged time can cause structural changes in vessel wall; on the other hand, lack of pressure/stretch can promote a phenotype shift. This thesis investigates novel roles of microRNAs and protein tyrosine kinase 2 in pressure/stretch-induced signaling mechanisms in the vascular wall. Using two different knockout mouse models, we uncovered a novel role of microRNAs in the pressure-induced myogenic response. We demonstrated that global deletion of smooth muscle-specific microRNAs causes a loss of pressure-induced contraction and that this likely involves diminished calcium influx due to reduced stretch-induced activation of the PI3K/Akt pathway. Similarly, global deletion of the smooth muscle enriched miRNA-143/145 also depleted myogenic responses but this effect could be due to several combined factors including loss of calcium influx and decreased expression of myosin light chain kinase. Furthermore portal veins of miRNA-143/145 KO mice exhibit lack of stretch-induced contractile differentiation, which may in part be due to a reduced expression of L-type calcium channels caused by an increased expression of the transcriptional repressor DREAM. Using a novel small molecule inhibitor of PYK2, we demonstrated that PYK2 could distinguish between non-voltage and voltage-dependent calcium pools to initiate signal transduction in the smooth muscle of portal vein. Inhibition of PYK2 can reduce phenotype modulation and apoptosis in balloon injured carotid arteries. In conclusion, we have established an indispensable role of microRNAs in the presssure-induced myogenic response and maintainance of stretch-induced conctractile differentiation. Morover we have established that PYK2 is involved in stretch-induced calcium handling in spontaneously active portal vein and in phenotypic shift of smooth muscle cells following vascular injury.

Background Recent studies have raised concern about the safety of erythropoiesis‐stimulating agents because of evidence of increased risk of hypertension and cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients. In the present study, we investigated the effects of recombinant human erythropoietin (EPO) on endothelial function of gluteal subcutaneous resistance arteries isolated from 17 stage 4 patients (estimated glomerular filtration rate 21.9±7.4 mL/min per 1.73 m2) aged 63±13 years. Methods and Results Arteries were mounted on a pressurized myograph. EPO impaired endothelium‐dependent relaxation in a concentration‐dependent manner. The maximal response to acetylcholine with EPO at 1, 10, and 20 IU/mL was reduced by 12%, 34%, and 43%, respectively, compared with the absence of EPO (P

BACKGROUND: Endothelin-1 (ET1) is a potent vasoconstrictor peptide with pro-mitogenic and pro-inflammatory properties and is therefore of interest in the development of endothelial dysfunction, endothelium-dependent flow-related remodeling, and hypertension-related remodeling. ET1 can be formed through cleavage of big ET1 by endothelin-converting enzyme (ECE) and neutral endopeptidase (NEP).METHOD: We investigated whether the dual NEP/ECE inhibitor SOL1 improves resistance artery function and structure in 12 weeks old spontaneously hypertensive rats (SHRs) and whether arterial structural responses to decreased (-90%) or increased (+100%) blood flow are impaired in young SHRs. To this end two groups of SHRs received chronic 4-week treatment at two different time points (4-8 and 8-12 weeks) prior to the experiment. We compared in-vitro effects of cyclo-oxygenase inhibition (1 μmol/l indomethacine), nitric oxide synthase inhibition (100 μmol/l N(ω)-L-nitro arginine methyl ester), and stimulation of the endothelium by 0.001-10 μmol/l acetylcholine (ACh) in isolated third-order mesenteric arteries of SHRs and aged-matched Wistar-Kyoto (WKY) rats.RESULTS: SOL1 had no effect on blood pressure in SHRs or WKY rats. ACh caused biphasic effects in mesenteric arteries of SHRs. The contractile component (endothelium-derived contractile factor) was absent in WKY and abolished by acute indomethacin administration or chronic SOL1 treatment. Endothelium-derived nitric oxide-type responses did not differ in both strains and were not influenced by SOL1 treatment. Endothelium-derived hyperpolarizing factor-type responses were severely impaired in SHRs as compared to WKY rats and were normalized by chronic SOL1 treatment. In first-order mesenteric arteries, outward flow-induced remodeling was impaired in SHRs. Chronic SOL1 treatment did not restore this response.CONCLUSION: Thus chronic SOL1 treatment during the development of hypertension in SHRs has no effect on blood pressure but improves several aspects of endothelium-dependent vasomotor responses but not arterial remodeling.

The purpose of this study was to determine whether the increased sensitivity of uterine resistance arteries from late pregnant (LP) rats to alpha-adrenergic stimulation is due to an alteration in the fundamental relationship between cytosolic calcium (Ca2+) and arterial lumen diameter. Uterine arcuate arteries were permeabilized with Staphylococcus aureus alpha-toxin under optimal conditions and constricted to varying degrees with discrete Ca2+ concentrations at a distending pressure of 50 mmHg. Arterial segments from nonpregnant (NP) and LP rats exhibited similar Ca2+/lumen diameter characteristics. Ca2+ (0.1 microM) produced appreciable constriction, and lumen diameter decreased steeply between 0.175 and 0.25 microM Ca2+; maximal responses were attained with 0.5 microM Ca2+. Activation of guanine nucleotide binding proteins (G proteins) with guanosine 5'-triphosphate (GTP; 1-100 microM), as reportedly occurs during alpha-adrenergic stimulation, potentiated the Ca(2+)-induced constriction by 121 and 79% in arteries from LP and NP rats, respectively. No significant differences between the two animal groups were noted. Guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S; 0.1-10 microM), a nonhydrolyzable analogue of GTP, effected a larger potentiating effect over that maximal response caused by GTP in arteries from NP rats. Ca(2+)- and Ca2+/GTP-induced constrictions were more potently reversed by guanosine 5'-O-(beta-thiodiphosphate) (GDP beta S)., a competitive inhibitor of GTP, in arteries from NP rats. These data suggest that pregnancy-induced increases in sensitivity to alpha-adrenergic stimulation may be related to altered G protein cycling rates, such that G proteins in smooth muscle cells in arcuate arteries from NP rats are more susceptible to deactivation. Alternatively, consistent with the model of G protein-mediated inhibition of myosin light chain phosphatase, myosin light chain phosphatase activity may be enhanced in uterine vascular smooth muscle from NP rats relative to that from LP rats.

We have investigated the effect of the novel hypotensive agent, flosequinan, on human small artery constrictor function. Concentration-dependent tension development to both noradrenaline and K+ was reduced in the presence of flosequinan. Arteries pre-contracted with noradrenaline demonstrated a greater concentration-dependent relaxation to flosequinan than did those pre-contracted with K+. Flosequinan depressed the sensitivity to extracellular Ca2+ of arteries pre-contracted with noradrenaline, but this effect was less marked in arteries pre-contracted with K+. These data support previous studies which suggest that flosequinan may affect receptor-mediated contraction by reducing Ca2+ sensitivity.

Aims The lipid and protein kinase phosphoinositide 3-kinase γ (PI3Kγ) is abundantly expressed in inflammatory cells and in the cardiovascular tissue. In recent years, its role in inflammation and in cardiac function and remodelling has been unravelled, highlighting the beneficial effects of its pharmacological inhibition. Furthermore, a role for PI3Kγ in the regulation of vascular tone has been emphasized. However, the impact of this signalling in the control of blood pressure is still poorly understood. Our study investigated the effect of a selective inhibition of PI3Kγ, obtained by using two independent small molecules, on blood pressure. Moreover, we dissected the molecular mechanisms involved in control of contraction of resistance arteries by PI3Kγ. Methods and results We showed that inhibition of PI3Kγ reduced blood pressure in normotensive and hypertensive mice in a concentration-dependent fashion. This effect was dependent on enhanced vasodilatation, documented in vivo by decreased peripheral vascular resistance, and ex vivo by vasorelaxing effects on isolated resistance vessels. The vasorelaxation induced by PI3Kγ inhibition relied on blunted pressure-induced Akt phosphorylation and a myogenic contractile response. Molecular insights revealed that PI3Kγ inhibition affected smooth muscle L-type calcium channel current density and calcium influx by impairing plasma membrane translocation of the α1C L-type calcium channel subunit responsible for channel open-state probability. Conclusion Overall our findings suggest that PI3Kγ inhibition could be a novel tool to modulate calcium influx in vascular smooth muscle cells, thus relaxing resistance arteries and lowering blood pressure.

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.