Human Small Arteries Research Articles

A correlated light and electron microscopy approach to study the subcellular localization of phosphorylated vimentin in human lung tissue.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500μm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.

Abstract 17276: NOTCH3-BMPR Interactions Drive Smooth Muscle Cell Proliferation and Medial Hyperplasia in Pulmonary Arterial Hypertension

Background: One of the characteristics of pulmonary arterial hypertension (PAH) is the proliferation of smooth muscle cells. In heritable PAH, aberrant BMPR2 signaling results in low levels of intracellular ID1. An important pathway involved in vascular smooth muscle cell proliferation and the development of non-heritable PAH is the NOTCH3-HES5 signaling pathway. Previous studies have demonstrated that HES-5 and ID-1 are peptides belonging to the basic helix-loop-helix family, and they act as transcriptional enhancers of cell cycle genes. Transcriptional enhancement or repression requires the heterodimerization of different HES and ID proteins. Hypothesis: We hypothesize that HES-5 binds to and degrades IDs in non-heritable cases of PAH. Methods: The half-life of ID proteins was assessed in human small pulmonary artery smooth muscle cells that either constitutively expressed or minimally expressed HES-5. In order to understand whether this effect was due to direct binding between HES-5 and ID1, co-immunoprecipitation experiments were performed. Immunofluorescent staining was used to detect the expression level of ID1 and HES-5. Proximity ligation assay (PLA) was utilized to examine of endogenous in-situ interaction of ID1-HES5. Results: We found that overexpression of HES-5 significantly diminished the half-life of ID1 and ID3, and had little effect on ID2. All three proteins localized to the nucleus of small pulmonary artery smooth muscle cells. Immunofluorescent staining confirmed elevated levels of HES and reduced levels of ID1 and ID3 in the medial layer of PAH vessels. By PLA assay, we show that HES-5 and ID1 as well as HES-5 and ID-3 physically associate with each other to the level of 40 nanometers. Conclusions: Constitutive NOTCH3 signaling and high intracellular HES-5 levels drive vascular smooth muscle cell proliferation in non-familial PAH by binding to and degrading ID intracellular proteins. Our study demonstrates cross-talk between the NOTCH3-pathway and the BMPR2 pathway in this disease, at the level of their downstream effectors: ID1 and HES-5. Further studies should shed light on how these two pathways control the PAH disease process.

Neuropeptide Y: Direct vasoconstrictor and facilitatory effects on P2X1 receptor-dependent vasoconstriction in human small abdominal arteries

Neuropeptide Y (NPY) is co-released with norepinephrine and ATP by sympathetic nerves innervating arteries. Circulating NPY is elevated during exercise and cardiovascular disease, though information regarding the vasomotor function of NPY in human blood vessels is limited. Wire myography revealed NPY directly stimulated vasoconstriction (EC50 10.3 ± 0.4 nM; N = 5) in human small abdominal arteries. Maximum vasoconstriction was antagonised by both BIBO03304 (60.7 ± 6%; N = 6) and BIIE0246 (54.6 ± 5%; N = 6), suggesting contributions of both Y1 and Y2 receptor activation, respectively. Y1 and Y2 receptor expression in arterial smooth muscle cells was confirmed by immunocytochemistry, and western blotting of artery lysates. α,β-meATP evoked vasoconstrictions (EC50 282 ± 32 nM; N = 6) were abolished by suramin (IC50 825 ± 45 nM; N = 5) and NF449 (IC50 24 ± 5 nM; N = 5), suggesting P2X1 mediates vasoconstriction in these arteries. P2X1, P2X4 and P2X7 were detectable by RT-PCR. Significant facilitation (1.6-fold) of α,β-meATP-evoked vasoconstrictions was observed when submaximal NPY (10 nM) was applied between α,β-meATP applications. Facilitation was antagonised by either BIBO03304 or BIIE0246. These data reveal NPY causes direct vasoconstriction in human arteries which is dependent upon both Y1 and Y2 receptor activation. NPY also acts as a modulator, facilitating P2X1-dependent vasoconstriction. Though in contrast to the direct vasoconstrictor effects of NPY, there is redundancy between Y1 and Y2 receptor activation to achieve the facilitatory effect.

ROK and RSK2-kinase pathways differ between senescent human renal and mesenteric arteries.

Small arteries from different organs vary with regard to the mechanisms that regulate vasoconstriction. This study investigated the impact of advanced age on the regulation of vasoconstriction in isolated human small arteries from kidney cortex and periintestinal mesenteric tissue. Renal and mesenteric tissues were obtained from patients (mean age 71 ± 9 years) undergoing elective surgery. Furthermore, intrarenal and mesenteric arteries from young and aged mice were studied. Arteries were investigated by small vessel myography and western blot. Human intrarenal arteries (h-RA) showed higher stretch-induced tone and higher reactivity to α 1 adrenergic receptor stimulation than human mesenteric arteries (h-MA). Rho-kinase (ROK) inhibition resulted in a greater decrease in Ca 2+ and depolarization-induced tone in h-RA than in h-MA. Basal and α 1 adrenergic receptor stimulation-induced phosphorylation of the regulatory light chain of myosin (MLC 20 ) was higher in h-RA than in h-MA. This was associated with higher ROK-dependent phosphorylation of the regulatory subunit of myosin light-chain-phosphatase (MLCP), MYPT1-T853. In h-RA phosphorylation of ribosomal S6-kinase II (RSK2-S227) was significantly higher than in h-MA. Stretch-induced tone and RSK2 phosphorylation was also higher in interlobar arteries (m-IAs) from aged mice than in respective vessels from young mice and in murine mesenteric arteries (m-MA) from both age groups. Vasoconstriction in human intrarenal arteries shows a greater ROK-dependence than in mesenteric arteries. Activation of RSK2 may contribute to intrarenal artery tone dysregulation associated with aging. Compared with h-RA, h-MA undergo age-related remodeling leading to a reduction of the contractile response to α 1 adrenergic stimulation.

Validation and Feasibility of an Automated System for the Assessment of Vascular Structure and Mechanical Properties in the Digital Arteries: An Ultrahigh-Frequency Ultrasound Study.

Ultrahigh-frequency ultrasound (UHFUS) allows sharp visualization of human small muscular arteries. This may help in elucidating some aspects of the pathophysiology of arterial aging, such as the stiffness gradient between large and small conduit arteries and its consequences on the microcirculation, as well as vascular diseases affecting medium-sized arteries. However, UHFUS use is still limited, partly because of the lack of validated tools to quantify vascular structure and mechanical properties of small muscular arteries. In this validation study, scans of digital arteries were obtained with UHFUS (VevoMD, Visualsonics-Fujifilm, Toronto, ON, Canada), analyzed using Carotid Studio software (Quipu, Pisa, Italy) and compared with the manual measurement. Agreement between the two techniques on measures of diameter, distension and intima-media thickness (IMT) was evaluated using Bland-Altman analyses; inter- and intra-operator reproducibility was evaluated using coefficients of variation (CVs). Overall, no trend or significant bias was observed between Carotid Studio and manual analysis. All limits of agreement were acceptable. The intra-observer CV of diastolic diameter and IMT were 4.1% and 4.2%, respectively. The inter-observer CV for diastolic diameter and IMT were 7.3% and 5.4%, respectively. Intra- and inter-observer CVs for distension were higher (25.7% and 26.7%, respectively). These results suggest that the Carotid Studio software is a valid and reproducible tool to study UHFUS scans of digital arteries, with potential utility both in rare vascular diseases of medium-sized arteries and in the study of the pathophysiology of arterial aging in general.

Dipyridamole Potentiates the Endothelium-Dependent and -Independent Vasomotion in Isolated Human Small Arteries.

BACKGROUND: To investigate the effects of dipyridamole, a drug with phosphodiesterase-, adenosine reuptake-inhibiting, and prostacyclin-stimulating activity on the biological actions of nitric oxide, 30 norepinephrine-precontracted subcutaneous arterioles were prepared from specimens removed during surgery. METHODS AND RESULTS: Specimens were mounted on a myograph and relaxes through either acetylcholine, a muscarinic agonist that stimulates endothelial nitric oxide production, or sodium nitroprusside, an endothelium-independent vasodilator. Studies were performed under control conditions and after dipyridamole which potentiated in a concentration-dependent manner the vasorelaxation induced both by acetylcholine and sodium nitroprusside, indicating an endothelium-independent mechanism of action. The contribution of nitric oxide to the relaxation produced by acetylcholine was confirmed by N-monomethyl-L-arginine, a nitric oxide synthase inhibitor. In contrast, indomethacin, a cyclo-oxygenase inhibitor, was ineffective, indicating that prostacyclin stimulation could not explain the effect of dipyridamole. CGS 21680 C, an A(2)-selective adenosine receptor agonist insensitive to tissue deaminase, did not influence the relaxations induced by acetylcholine, suggesting that interference with adenosine metabolism was not implicated in the potentiating action of dipyridamole. CONCLUSIONS: Dipyridamole potentiated the vasorelaxing effect of acetylcholine and sodium nitroprusside in human subcutaneous arterioles; neither prostacyclin stimulation nor A(2) adenosine receptor stimulation could explain this effect. The data are consistent with an increase in intracellular cyclic 3' 5'-guanosine monophosphate levels secondary to the phosphodiesterase-inhibiting properties of the drug.

LIMK (LIM Kinase) Inhibition Prevents Vasoconstriction- and Hypertension-Induced Arterial Stiffening and Remodeling.

Increased arterial stiffness and vascular remodeling precede and are consequences of hypertension. They also contribute to the development and progression of life-threatening cardiovascular diseases. Yet, there are currently no agents specifically aimed at preventing or treating arterial stiffening and remodeling. Previous research indicates that vascular smooth muscle actin polymerization participates in the initial stages of arterial stiffening and remodeling and that LIMK (LIM kinase) promotes F-actin formation and stabilization via cofilin phosphorylation and consequent inactivation. Herein, we hypothesize that LIMK inhibition is able to prevent vasoconstriction- and hypertension-associated arterial stiffening and inward remodeling. We found that small visceral arteries isolated from hypertensive subjects are stiffer and have greater cofilin phosphorylation than those from nonhypertensives. We also show that LIMK inhibition prevents arterial stiffening and inward remodeling in isolated human small visceral arteries exposed to prolonged vasoconstriction. Using cultured vascular smooth muscle cells, we determined that LIMK inhibition prevents vasoconstrictor agonists from increasing cofilin phosphorylation, F-actin volume, and cell cortex stiffness. We further show that localized LIMK inhibition prevents arteriolar inward remodeling in hypertensive mice. This indicates that hypertension is associated with increased vascular smooth muscle cofilin phosphorylation, cytoskeletal stress fiber formation, and heightened arterial stiffness. Our data further suggest that pharmacological inhibition of LIMK prevents vasoconstriction-induced arterial stiffening, in part, via reductions in vascular smooth muscle F-actin content and cellular stiffness. Accordingly, LIMK inhibition should represent a promising therapeutic means to stop the progression of arterial stiffening and remodeling in hypertension.

Expression and Localization of Alpha Globin in the Vascular Endothelium

RationaleAlpha globin was recently discovered in the endothelium of murine resistance arteries, where it regulates diffusion of nitric oxide. In humans, alpha globin is expressed by two distinct loci, HBA1 and HBA2, with HBA2 expression predominating in red cell progenitors. However, gene expression of each specific locus in the vasculature, as well as the cellular localization and abundance of alpha globin in human vascular endothelium, is currently unknown.MethodsSmall arteries from the omentum and subcutaneous adipose were collected from patients during clinically indicated surgery at NIH Clinical Center (protocol 13‐C‐0176) and perfused to remove residual blood. To determine locus specific gene expression, RNA was extracted from 8–10 small arteries dissected from omentum and subcutaneous adipose of each patient. Expression of HBA1, HBA2, NOS3 (endothelial Nitric Oxide Synthase, eNOS), and AE1 (an erythrocyte gene) was quantified by digital droplet PCR. To interrogate vascular localization and abundance, dissected and perfused arteries were stained with fluorescent antibodies for alpha globin and eNOS and were imaged by multiphoton microscopy (Leica DMi8). Nuclear staining was performed with DAPI. The abundance of alpha globin was calculated as the ratio between alpha globin positive punctates and the number of endothelial nuclei measured in each vessel.ResultsRobust expression of HBA1 and HBA2 was observed in freshly dissected small arteries from both the omentum and subcutaneous adipose of human research subjects. While HBA2 is expressed 10‐fold higher than HBA1 in whole blood, HBA2 and HBA1 are expressed only 1.6:1 in the vasculature. AE1 was highly expressed in blood but not in arteries, confirming complete perfusion of vessels. NOS3 was expressed in all vessels, but not blood. These data indicate that alpha globin is expressed in human small arteries, but locus‐specific expression is significantly different in vessels than in blood, suggesting tissue‐specific regulation of HBA transcription. Multiphoton microscopy of intact small arteries from the omentum showed robust localization of alpha globin on the basolateral side of the vascular endothelium, co‐localizing with eNOS. Structural analysis shows alpha globin primarily localized to the junctions of the endothelium and vascular smooth muscle in small protrusions through the internal elastic lamina. In both omentum and subcutaneous adipose arteries, imaging revealed a ratio of 1.2 alpha globin positive punctates per nucleus, suggesting that the alpha globin within each cell localizes in a single point along the internal elastic lamina.ConclusionWe found strong expression of HBA1 and HBA2 in human arterial tissue, with an expression ratio distinct from expression in whole blood. We also found that alpha globin protein localizes with eNOS at junctures between the vascular endothelium and smooth muscle. Building on these results, we plan to investigate the mechanisms of tissue‐specific enhancement of HBA expression, as well as to further elucidate the function of endothelial alpha globin in arterial vasoregulation.Support or Funding InformationFunding from NIH Division of Intramural Research.

ADAM17 Cleaves the Insulin Receptor Ectodomain on Endothelial Cells and Induces Vascular Insulin Resistance

Despite efforts to reduce the incidence of type 2 diabetes (T2D) and its cardiovascular consequences, T2D‐associated cardiovascular mortality continues to rise in the US and worldwide. The presence of a pro‐inflammatory state and microvascular insulin resistance are hallmarks of T2D that contribute to cardiovascular disease. The former is associated with increased activity of ADAM17, an enzyme that cleaves the ectodomain of multiple transmembrane proteins, while the latter is characterized by decreased insulin‐induced vasodilatory responses. However, the specific mechanisms, including the potential role(s) of ADAM17, underlying vascular insulin resistance in T2D, remain unknown. We hypothesize that in T2D, increased expression and activation of ADAM17 sheds the insulin receptor ectodomain (IRα) from endothelial cells and thus impairs insulin‐induced vasodilation. We tested this hypothesis using isolated small visceral arteries from T2D and non‐T2D subjects undergoing bariatric surgery and cultured human endothelial cells. Results indicate that arteries from subjects with T2D exhibit increased ADAM17 expression, reduced presence of TIMP3 (the endogenous inhibitor of ADAM17), decreased extracellular IRα, and impaired insulin‐induced vasodilation in comparison with arteries from non‐T2D subjects (P&lt;0.05). In vitro, ADAM17 cleaved insulin receptor recombinant proteins at a specific extracellular site of the IRβ subunit (p&lt;0.05). Exposure of endothelial cells to the PKC activator, PMA, increased the shedding activity of ADAM17 (P&lt;0.05) and decreased the presence of IRα on cell surfaces occupied by ADAM17, as observed using super‐resolution microscopy. Moreover, pharmacological inhibition of ADAM17 with TAPI‐0 rescued PMA‐induced impairments in insulin signaling in endothelial cells and insulin‐stimulated vasodilation in human small visceral arteries (P&lt;0.05). Collectively, these findings suggest that ADAM17‐mediated shedding of IR ectodomains from the endothelial surface impairs insulin‐mediated vasodilation. Therefore, inhibition of ADAM17 sheddase activity should be considered a potential new therapeutic strategy to restore vascular insulin sensitivity in T2D.Support or Funding InformationResearch support: National Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).

Vasodilatory effects of cannabidiol in human pulmonary and rat small mesenteric arteries: modification by hypertension and the potential pharmacological opportunities.

Objective:Cannabidiol (CBD) has been suggested as a potential antihypertensive drug. The aim of our study was to investigate its vasodilatory effect in isolated human pulmonary arteries (hPAs) and rat small mesenteric arteries (sMAs).Methods:Vascular effects of CBD were examined in hPAs obtained from patients during resection of lung carcinoma and sMAs isolated from spontaneously hypertensive (SHR); 11-deoxycorticosterone acetate (DOCA-salt) hypertensive rats or their appropriate normotensive controls using organ bath and wire myography, respectively.Results:CBD induced almost full concentration-dependent vasorelaxation in hPAs and rat sMAs. In hPAs, it was insensitive to antagonists of CB1 (AM251) and CB2 (AM630) receptors but it was reduced by endothelium denudation, cyclooxygenase inhibitors (indomethacin and nimesulide), antagonists of prostanoid EP4 (L161982), IP (Cay10441), vanilloid TRPV1 (capsazepine) receptors and was less potent under KCl-induced tone and calcium-activated potassium channel (KCa) inhibitors (iberiotoxin, UCL1684 and TRAM-34) and in hypertensive, overweight and hypercholesteremic patients. The time-dependent effect of CBD was sensitive to the PPARγ receptor antagonist GW9662. In rats, the CBD potency was enhanced in DOCA-salt and attenuated in SHR. The CBD-induced relaxation was inhibited in SHR and DOCA-salt by AM251 and only in DOCA-salt by AM630 and endothelium denudation.Conclusion:The CBD-induced relaxation in hPAs that was reduced in hypertensive, obese and hypercholesteremic patients was endothelium-dependent and mediated via KCa and IP, EP4, TRPV1 receptors. The CBD effect in rats was CB1-sensitive and dependent on the hypertension model. Thus, modification of CBD-mediated responses in disease should be considered when CBD is used for therapeutic purposes.

Abnormal Remodeling of Subcutaneous Small Arteries Is Associated With Early Diastolic Impairment in Metabolic Syndrome

BackgroundSmall artery pathophysiology is frequently invoked as a cause of obesity‐related diastolic heart failure. However, evidence to support this hypothesis is scant, particularly in humans.Methods and ResultsTo address this, we studied human small artery structure and function in obesity and looked for correlations between vascular parameters and diastolic function. Seventeen obese patients with metabolic syndrome and 5 control participants underwent echocardiography and subcutaneous gluteal fat biopsy. Small arteries were isolated from the biopsy and pressure myography was used to study endothelial function and wall structure. In comparison with the control group, small arteries from obese participants exhibited significant endothelial dysfunction, assessed as the vasodilatory response to acetylcholine and also pathological growth of the wall. For the obese participants, multiple regression analysis revealed an association between left atrial volume and both the small artery wall thickness (β=0.718, P=0.02) and wall‐to‐lumen ratio (β=0.605, P=0.02). Furthermore, the E:E′ ratio was associated with wall‐to‐lumen ratio (β=0.596, P=0.02) and inversely associated with interleukin‐6 (β=−0.868, P=0.03). By contrast, endothelial function did not correlate with any of the echocardiographic parameters studied.ConclusionsAlthough the small arteries studied were not cardiac in origin, our results support a role for small artery remodeling in the development of diastolic dysfunction in humans. Further direct examination of the structure and function of the myocardial resistance vasculature is now warranted, to elucidate the temporal association between metabolic risk factors, small artery injury, and diastolic impairment.

Abstract P124: Human Small Artery MicroRNA Expression Profiles: Changes in Type 2 Diabetes and Associations with Endothelial Function

Background: Studies of experimental models and human blood samples support an important role of microRNAs (miRNAs) in the development of vascular dysfunction in hypertension and diabetes mellitus (DM). Information on miRNA expression in clinically directly relevant tissues such as human small arteries and its relationship with impaired vascular endothelial function is currently lacking. Methods and Results: 38 subjects (18 type 2 DM, 20 controls) underwent gluteal adipose pad biopsy to obtain small arteries for miRNA expression profiling by small RNA deep sequencing. In vivo conduit artery endothelial function was measured by brachial artery reactivity. In vitro microvascular endothelium dependent vasodilation was measured by videomicroscopy. Correlations between miRNA expression and measurements of endothelial function were calculated using generalized linear models. Several miRNAs correlated with measurements of vascular structure and function. Endothelium dependent vasodilation was impaired in type 2 DM subjects compared to controls based on both the vasodilatory response to peak dose acetylcholine (44±25 vs. 69±18 %, P=0.04) and by analyses of the entire acetylcholine dose-response curve. Several miRNAs were differentially expressed in small arteries from type 2 DM subjects, two of which were verified by real-time PCR. Cross-referencing the top 30 miRNAs (P&lt;0.015) with prior studies of plasma miRNA expression in DM subjects identified 7 miRNAs differentially expressed in both human small arteries and plasma, all of which have some reported role in vascular regulation. Conclusions: Multiple miRNAs are differentially expressed in human small arteries in DM patients and correlated with in vivo or in vitro measurements of endothelial function, suggesting an important role of microvascular miRNAs in the development of endothelial dysfunction in humans.

Activation of KV7 channels stimulates vasodilatation of human placental chorionic plate arteries

IntroductionPotassium (K+) channels are key regulators of vascular smooth muscle cell (VSMC) excitability. In systemic small arteries, Kv7 channel expression/activity has been noted and a role in vascular tone regulation demonstrated. We aimed to demonstrate functional Kv7 channels in human fetoplacental small arteries. MethodsHuman placental chorionic plate arteries (CPAs) were obtained at term. CPA responses to Kv7 channel modulators was determined by wire myography. Presence of Kv7 channel mRNA (encoded by KCNQ1-5) and protein expression were assessed by RT-PCR and immunohistochemistry/immunofluorescence, respectively. ResultsKv7 channel blockade with linopirdine increased CPA basal tone and AVP-induced contraction. Pre-contracted CPAs (AVP; 80 mM K+ depolarization solution) exhibited significant relaxation to flupirtine, retigabine, the acrylamide (S)-1, and (S) BMS-204352, differential activators of Kv7.1 – Kv7.5 channels. All CPAs assessed expressed KCNQ1 and KCNQ3-5 mRNA; KCNQ2 was expressed only in a subset of CPAs. Kv7 protein expression was confirmed in intact CPAs and isolated VSMCs. DiscussionKv7 channels are present and active in fetoplacental vessels, contributing to vascular tone regulation in normal pregnancy. Targeting these channels may represent a therapeutic intervention in pregnancies complicated by increased vascular resistance.

Effects of Bariatric Surgery on Human Small Artery Function: Evidence for Reduction in Perivascular Adipocyte Inflammation, and the Restoration of Normal Anticontractile Activity Despite Persistent Obesity

ObjectivesThe aim of this study was to investigate the effects of bariatric surgery on small artery function and the mechanisms underlying this.BackgroundIn lean healthy humans, perivascular adipose tissue (PVAT) exerts an anticontractile effect on adjacent small arteries, but this is lost in obesity-associated conditions such as the metabolic syndrome and type II diabetes where there is evidence of adipocyte inflammation and increased oxidative stress.MethodsSegments of small subcutaneous artery and perivascular fat were harvested from severely obese individuals before (n = 20) and 6 months after bariatric surgery (n = 15). Small artery contractile function was examined in vitro with wire myography, and perivascular adipose tissue (PVAT) morphology was assessed with immunohistochemistry.ResultsThe anticontractile activity of PVAT was lost in obese patients before surgery when compared with healthy volunteers and was restored 6 months after bariatric surgery. In vitro protocols with superoxide dismutase and catalase rescued PVAT anticontractile function in tissue from obese individuals before surgery. The improvement in anticontractile function after surgery was accompanied by improvements in insulin sensitivity, serum glycemic indexes, inflammatory cytokines, adipokine profile, and systolic blood pressure together with increased PVAT adiponectin and nitric oxide bioavailability and reduced macrophage infiltration and inflammation. These changes were observed despite the patients remaining severely obese.ConclusionsBariatric surgery and its attendant improvements in weight, blood pressure, inflammation, and metabolism collectively reverse the obesity-induced alteration to PVAT anticontractile function. This reversal is attributable to reductions in local adipose inflammation and oxidative stress with improved adiponectin and nitric oxide bioavailability.

Involvement of Large‐Conductance Ca2+‐Activated K+ Channels in both Nitric Oxide and Endothelium‐Derived Hyperpolarization‐Type Relaxation in Human Penile Small Arteries

Large-conductance Ca(2+) -activated K(+) channels (BKC a ), located on the vascular smooth muscle, play an important role in regulation of vascular tone. In penile corpus cavernosum tissue, opening of BKC a channels leads to relaxation of corporal smooth muscle, which is essential during erection; however, there is little information on the role of BKC a channels located in penile vascular smooth muscle. This study was designed to investigate the involvement of BKC a channels in endothelium-dependent and endothelium-independent relaxation of human intracavernous penile arteries. In human intracavernous arteries obtained in connection with transsexual operations, change in isometric force was recorded in microvascular myographs, and endothelium-dependent [nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-type] and endothelium-independent (NO-donor) relaxations were measured in contracted arteries. In penile small arteries contracted with phenylephrine, acetylcholine evoked NO- and EDH-type relaxations, which were sensitive to iberiotoxin (IbTX), a selective blocker of BKC a channels. Iberiotoxin also inhibited relaxations induced by a NO-donor, sodium nitroprusside. NS11021, a selective opener of BKC a channels, evoked pronounced relaxations that were inhibited in the presence of IbTX. NS13558, a BKC a -inactive analogue of NS11021, failed to relax human penile small arteries. Our results show that BKC a channels are involved in both NO- and EDH-type relaxation of intracavernous penile arteries obtained from healthy men. The effect of a selective opener of BKC a channels also suggests that direct activation of the channel may be an advantageous approach for treatment of impaired endothelium-dependent relaxation often associated with erectile dysfunction.

Effects of bariatric surgery on human small artery function: evidence for reduction in perivascular adipocyte inflammation, and restoration of normal anticontractile activity despite persistent obesity

BackgroundIn lean healthy human beings, perivascular adipose tissue (PVAT) exerts an anticontractile effect on adjacent small arteries, but this is lost in obesity where there is evidence of adipocyte inflammation and increased oxidative stress. The aim of this study was to investigate the effects of bariatric surgery on small artery function and the underlying mechanisms. MethodsSmall arteries were isolated from subcutaneous adipose tissue samples obtained by gluteal biopsies from morbidly obese patients (n=15). Vessels were prepared as segments with and without intact PVAT. Response of vessel segments to cumulative doses of norepinephrine was recorded with wire myography. Biopsy samples were re-taken 6 months after bariatric surgery and effect of PVAT on vessel contractility was re-assessed. Vessel segments with intact PVAT were also incubated with blocking peptide for adiponectin receptor 1 (1·6 × 10−4 mol/L, n=5). PVAT adiponectin levels were assessed by proteomic analysis of samples from obese (n=13) and control (n=10) individuals. Inflammatory profile of PVAT was assessed by staining for macrophages and tumour necrosis factor α. FindingsObese patients had a mean body-mass index of 52 kg/m2; 6 months following surgery and significant weight loss (p<0·05), patients remained clinically obese. There were significant improvements in serum biomarkers including adiponectin (p=0·009), as well as inflammatory markers and measures of insulin sensitivity. Proteomic analysis of PVAT showed that adiponectin levels were low in the obese group compared with controls (fold change 2·1, p<0·05). Presence of PVAT made no difference in contractility of vessels to noradrenaline in samples from obese patients (p=0·81). However, post weight loss, PVAT had a significant anticontractile effect (p<0·001) which was lost after incubation with adiponectin receptor 1 blocking peptide. There was a significant reduction in inflammation post surgery. InterpretationDamage to the anticontractile property of PVAT in obesity is in part due to a reduction in adiponectin levels within the PVAT and is rescued by weight loss following bariatric surgery. FundingBritish Heart Foundation.

Abstract 662: Perivascular Adipose Tissue-derived Prostaglandins Constrict Vessel

Background: Perivascular adipose tissue (PVAT) is associated with reduced response to vasoconstricting agents in the murine vasculature and also in human small arteries, and this anti-contractile effect is reduced in subjects with metabolic syndrome. However, it was suggested that other vasoconstrictors released from PVAT such as leptin or Angiotensin II causes vasoconstriction, suggesting that PVAT has contractile properties as well. Here we show that prostaglandins and adrenalin signaling derived from PVAT contribute to contractile properties of PVAT. Results: The effect of donor PVAT on the vascular tone was evaluated in carotid, thoracic and mesenteric arteries using a myograph chamber in which the PVAT surrounding the tested vessel was completely removed. Fifteen mg of minced PVAT (size of ∼1 mm 3 ) from donor C57BL/6J mice into the myograph chamber results in mild vasoconstriction (1.52±0.6mN) on carotid artery rings. This contractile property of PVAT is effective on vessel rings obtained from the thoracic aorta and mesenteric artery as well. PVAT from leptin knockout mice used as a donor causes vasoconstriction similar to that of wild-type mice (1.55±0.5mN leptin knockout vs. 1.52±0.6mN C57BL/6J mice). Pre-incubation of vessel rings with increasing concentrations of nonspecific COX inhibitor (10 -9 M to 10 -6 M indomethacin) inhibits PVAT-induced constriction in a dose-dependent manner (1.55±0.7mN, 1.41±0.6mN, 1.23±0.5mN, 0.95±0.4mN, 0.56±0.6mN, respectively), suggesting that PVAT-derived prostaglandin are responsible for vascular constriction. Similarly, pre-incubation with alpha-adrenergic receptor antagonists blocks PVAT-dependent vasoconstriction. Carotid artery responses to a series of 10 -7 M phenylephrine (PE) addition/wash out (5x) experiments in the myograph chamber cause alpha-adrenergic desensitization. However, in the presence of donor PVAT, carotid arteries have preserved maximal contraction after 5 rounds of PE addition/wash out experiments. Conclusion: Our studies indicate that PVAT-derived prostaglandins rather than leptin or other adipokines cause vasoconstriction and reduce vascular alpha-adrenergic desensitization.

Activation of endothelial and epithelial KCa2.3 calcium‐activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles

Small (K(Ca) 2) and intermediate (K(Ca) 3.1) conductance calcium-activated potassium channels (K(Ca) ) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we investigated the expression of K(Ca) 2.3 and K(Ca) 3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries. Channel expression and functional studies were conducted in human isolated small pulmonary arteries and bronchioles. K(Ca) 2 and K(Ca) 3.1 currents were examined in human small airways epithelial (HSAEpi) cells by whole-cell patch clamp techniques. While K(Ca) 2.3 expression was similar, K(Ca) 3.1 protein was more highly expressed in pulmonary arteries than bronchioles. Immunoreactive K(Ca) 2.3 and K(Ca) 3.1 proteins were found in both endothelium and epithelium. K(Ca) currents were present in HSAEpi cells and sensitive to the K(Ca) 2.3 blocker UCL1684 and the K(Ca) 3.1 blocker TRAM-34. In pulmonary arteries contracted by U46619 and in bronchioles contracted by histamine, the K(Ca) 2.3/ K(Ca) 3.1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the K(Ca) 2 channel blocker apamin, while the K(Ca) 3.1 channel blocker, charybdotoxin failed to reduce relaxation to NS309 (0.01-1 µM). K(Ca) 2.3 and K(Ca) 3.1 channels are expressed in the endothelium of human pulmonary arteries and epithelium of bronchioles. K(Ca) 2.3 channels contributed to endo- and epithelium-dependent relaxations suggesting that these channels are potential targets for treatment of pulmonary hypertension and chronic obstructive pulmonary disease.

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

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. 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. 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. 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.

Vascular Remodeling in Hypertension

Over the past few years noninvasive and invasive techniques have allowed a better appreciation of vascular changes in hypertensive humans and experimental animals.1 Large arteries undergo outward hypertrophic remodeling and increased stiffness with aging,2–4 and in hypertension there may be an acceleration of this process leading to enhanced pulse pressure. A reduced aortic diameter in middle-aged hypertensive subjects may also play a role in increases in pulse pressure through increased specific impedance,5 contradicting the classic hypertensive aortic phenotype characterized by vascular wall degeneration and calcification and increased aortic diameter. In advanced hypertension, however, the elastic laminae undergo duplication and fragmentation, with increased deposition of collagen and fetal (EIIIA) fibronectin, contributing to increased stiffness. Classically the remodeling of small arteries in hypertension has been associated with increased media thickness, but recent studies have demonstrated that 2 types of remodeling are found, inward eutrophic or inward hypertrophic remodeling, depending on whether the media cross-sectional area is enlarged (true hypertrophy).6–9 Although eutrophic remodeling is usually found in essential (primary) hypertension in humans and spontaneously hypertensive rats (SHRs); in secondary hypertension such as in renovascular hypertension, primary aldosteronism, or in pheochromocytoma10; in hypertension associated with diabetes mellitus11,12; and in acromegaly,13 hypertrophic remodeling has been described. In mineralocorticoid hypertension in rodents14,15 and in salt-sensitive Dahl rats,16 in both of which the endothelin (ET) system is activated, remodeling of small arteries is also hypertrophic (see below). Thus, when the renin-angiotensin system is even mildly activated (primary hypertension and SHRs), remodeling is usually eutrophic. In salt-dependent hypertension, diabetes mellitus, and malignant hypertension, all conditions in which the ET system is activated, remodeling is hypertrophic.17 Hyperplasia of vascular smooth muscle cells (VSMCs) is found in small arteries of …