Human Arterioles Research Articles

P1238EVALUATION OF AN IN VITRO CO-CULTURE MODEL FOR TESTING EFFECTS OF CYTOPROTECTIVE ADDITIVES IN PERITONEAL DIALYSIS FLUIDS ON CARDIOVASCULAR OUTCOME

Abstract Background and Aims The composition of all currently available peritoneal dialysis (PD) fluids triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization, and diabetes-like damage of vessels, eventually leading to failure of the technique. Patients undergoing dialysis generally, have a high risk of cardiovascular events. It is currently unclear if there is a mechanistic link between peritoneal membrane failure and cardiovascular risk. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PDF reduce peritoneal damage. Here, we developed an experimental model for investigating effects of these cytoprotective additives in PDF in the cardiovascular context. Method For modelling the peritoneal membrane in vitro, mesothelial and endothelial cells were co-cultured in transwell plates. Mesothelial cells were grown in the upper compartment and primary human umbilical vein endothelial cells (HUVEc) or primary microvascular cells were grown in the lower compartment. PDF with or without cytoprotective compounds, was added to the upper compartment to only expose mesothelial cells directly to different dilutions of the fluid. Effects on cell damage was assessed by quantification of lactate-dehydrogenase (LDH) release and live-dead staining of cells. Proteome profiles were analysed for both cell-types separately and in combination using two-dimensional difference gel electrophoresis (2D-DiGE) and liquid chromatography coupled to mass spectrometry (LC-MS). In vitro findings were related to PD-induced arteriolar changes based on abundance profiles of micro-dissected omental arterioles of children treated with conventional PD-fluids and age-matched controls with normal renal function. Results Marked cellular injury of HUVEc after PD-fluid exposure was associated with a molecular landscape of the enriched biological process clusters ‘glucose catabolic process’, ‘cell redox homeostasis’, ‘RNA metabolic process’, ‘protein folding’, ‘regulation of cell death’, and ‘actin cytoskeleton reorganization’ that characterize PD-fluid cytotoxicity and counteracting cellular repair process respectively. PDF-induced cell damage was reduced by AlaGln and LiCl both in mesothelial and endothelial cells. Proteome analysis revealed perturbation of major cellular processes including regulation of cell death and cytoskeleton reorganization. Selected markers of angiogenesis, oxidative stress, cell junctions and transdifferentiation were counter-regulated by the additives. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to separate culture. Comparison to human arterioles confirmed overlapping protein regulation between endothelial cells in vitro and in vivo, proving harmful effects of PD-fluids on endothelial cells leading to drastic changes of the cellular process landscape. Conclusion In summary, this study shows harmful effects of PD-fluids also effecting endothelial cells and elucidates potential mechanisms by which cytoprotective additives may counteract the signalling axis between local peritoneal damage and systemic vasculopathy. An in vitro co-culture system may be an attractive approach to simulate the peritoneal membrane for testing direct and indirect effects of cytoprotective additives in PDF. When cultured and stressed in close proximity cells may respond differently. Characterisation of PD-induced perturbations will allow identifying molecular mechanisms linking the peritoneal and cardiovascular context, offering therapeutic targets to reduce current limitations of PD and ultimately decreasing cardiovascular risk of dialysis patients.

NADPH Oxidase 2 and 4 Contribute to Endothelium‐Dependent Dilation in Healthy Human Arterioles

NADPH oxidases (NOXs) are a major source of reactive oxygen species (ROS) in the vasculature and play diverse roles in vascular health and disease. We have recently shown that NOX‐derived ROS are involved in flow‐induced dilation of human arterioles from subjects with coronary artery disease (CAD). However, the role of NOXs in non‐diseased human arterioles remains largely unclear. Previous studies indicate that NOXs contribute to endothelium‐dependent vasodilation in normal animal arteries. This study is designed to test whether NOX2 and NOX4 play a role in receptor and non‐receptor mediated endothelium‐dependent vasodilation in human adipose arterioles (HAA). HAAs (~150 μm ID), isolated from fat tissues from patients without coronary artery disease (non‐CAD) or <1 risk factor, were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. In arterioles constricted with endothelin‐1, acetylcholine (Ach; 10−9 to 10−5 M) elicited concentration‐dependent dilation (maximal dilation at 10−5 M: 95±3%, n=4), and this dilation was reduced in the presence of nitric oxide (NO) synthase inhibitor nitro‐L‐arginine methyl ester (L‐NAME) and cyclooxygenase inhibitor indomethacin (Indo) (maximal dilation: 63±7%, n=4). Ach‐induced dilation was inhibited by NOX2 inhibitor GSK2795039 (10−6 M; maximal dilation: 57±10%, n=5) and to a less extent by NOX4 inhibitor GKT137831 (10−6 M; maximal dilation: 86±4%, n=5). In the presence of L‐NAME and Indo, NOX2 and NOX4 inhibitors largely abolished Ach‐induced vasodilation (maximal dilation: 29±9% and 18±5%, respectively, n=4). We also examined the effect of NOX inhibition on non‐receptor‐mediated concentration‐dependent dilation by the Ca2+ ionophore A23187 (10−11 to 10−7 M). A23187‐induced dilation (maximal dilation at 10−7 M: 68±5%, n=4) was inhibited by NOX2 (maximal dilation: 6±2%, n=4) and NOX4 (maximal dilation: 15±2%, n=5). Unexpectedly, L‐NAME and Indo also inhibited A23187‐dependent dilation (maximal dilation: 7±2%, n=4). Together, these data support our hypothesis that NOX2 and NOX4 are involved in receptor and non‐receptor mediated endothelium‐dependent vasodilation in non‐CAD HAA. While A23187 dilation is largely NO/prostacyclin (PGI2)‐dependent, there is a non‐NO/PGI2 component contributing to NOX inhibitor‐sensitive vasodilator response to Ach.Support or Funding InformationSupport for this research was provided by the National Heart, Lung and Blood Institute Grant R01‐HL 096647. Tissue was supplied by Cardiovascular Surgery Associates, Midwest Heart Surgery Institute, Wisconsin Heart Group, and Froedtert Memorial Lutheran Hospital.

Estrogen Attenuates Phenotypic Switch of Human Arteriolar Smooth Muscle Cells: Role of p53 and pRb

Cardiovascular diseases (CVD) remains the leading cause of mortality worldwide. Vascular inflammation is a major contributor to the onset and the pathogenesis of this disease. In response to inflammatory stimuli, vascular smooth muscle cells (VSMCs) switch from a contractile to a dedifferentiated synthetic phenotype, characterized by decreased expression of differentiation markers and increased proliferative and migratory abilities. By virtue of its ability to attenuate these processes, estrogen (E2) is known to play a vasculoprotective role. While the vast majority of studies investigating the role of estrogen on phenotypic modulation were conducted on VSMCs isolated from large vessels, the effect of estrogen on VSMCs extracted from microvessels (microVSMCs) remains largely obscure. Thus, we sought assess the effect of estrogen on phenotypic switch of microVSMCs, and characterize the underlying molecular mechanism.We were able to extract microVSMCs from human dermal arterioles by enzymatic sprouting method. The purity of the cells was identified by their morphology and by immunostaining of two VSMC markers: smooth muscle alpha‐actin and calponin. In addition, we previously assessed the concentration‐dependent effect of estrogen on the VSMC response, with 10−10 M being the optimal physiologic concentration. Results showed that E2 (10−10 M) inhibited FBS‐induced cell proliferation and migration. This estrogen‐induced inhibition of was accompanied by decreased activation of mitogenic extracellular signal–regulated kinase (ERK1/2) and focal adhesion kinase (FAK), involved in cell proliferation and migration, respectively. Furthermore, E2 (10−10 M) increased 5′ AMP‐activated protein kinase (AMPK) phosphorylation of. In addition, E2 blocked the cells in G0/G1 phase of cell cycle. These estrogenic effects were not inhibited by ICI 280,780 (5 μM, estrogen receptor (ERα/β) antagonist). Surprisingly, ICI 280,780 mimicked estrogen, acting as an ER agonist. Furthermore, estrogen‐induced cell cycle arrest was concomitant with increased expression of p53 and decreased phosphorylation of retinoblastoma protein (pRb). The regulation of the aforementioned tumor suppressor proteins, p53 and pRb, was not mirrored by apoptosis evident by the absence of E2‐induced caspase‐3 cleavage. Alternatively, SA‐β‐gal staining showed that E2 induced senescence of VSMC. Moreover, E2 increased the expression of contractile differentiation markers: calponin and caldesmon.Taken together, this is the first study to report the effect of estrogen on phenotypic switch of VSMCs extracted from human arterioles. Our results indicate that estrogen attenuated VSMC proliferation by inducing VSMC senescence, via an ERα/β‐independent p53/pRb‐mediated mechanism. These results potentially implicate estrogen membrane receptor, GPR30, as the mediator of rapid estrogenic response in microVSMCs. In addition, due to the potential of estrogen to favorably modulate VSMC phenotype, our results may explain the lower incidence of CVDs in estrogen‐replete premenopausal females.Support or Funding InformationMPP # 320133 to A.E. and CNRS to M.F.Effect of estrogen on VSMC proliferationCells were treated with 10% FBS in the presence or absence of E2 (10‐10 M) for 1,3,5,7, and 9 days. E2 was added 30 minutes before and during treatment with FBS.Figure 1Effect of estrogen on cell senescence in VSMCs. Cells were treated with FBS, in the presence or absence of E2 (10‐10 M) or ICI 280,780 (5 μM). E2 or ICI 280,780 were added 30 minutes before and during treatment of FBS. Photography image (magnification 5 X) of SA‐β‐gal stained cells were obtained.Figure 2

Manipulation of the Sphingolipid Rheostat Influences the Mediator of Flow-Induced Dilation in the Human Microvasculature.

BackgroundElevated levels of ceramide, a sphingolipid known to cause a transition from nitric oxide (NO)– to hydrogen peroxide–dependent flow‐induced dilation (FID) in human arterioles, correlate with adverse cardiac events. However, elevations of ceramide are associated with changed concentrations of other sphingolipid metabolites. The effects of sphingolipid metabolites generated through manipulation of this lipid pathway on microvascular function are unknown. We examined the hypothesis that inhibition or activation of the ceramide pathway would determine the mediator of FID.Methods and ResultsUsing videomicroscopy, internal diameter changes were measured in human arterioles collected from discarded adipose tissue during surgery. Inhibition of neutral ceramidase, an enzyme responsible for the hydrolysis of ceramide, favored hydrogen peroxide–dependent FID in arterioles from healthy patients. Using adenoviral technology, overexpression of neutral ceramidase in microvessels from diseased patients resulted in restoration of NO‐dependent FID. Exogenous sphingosine‐1‐phosphate, a sphingolipid with opposing effects of ceramide, also restored NO as the mediator of FID in diseased arterioles. Likewise, exogenous adiponectin, a known activator of neutral ceramidase, or, activation of adiponectin receptors, favored NO‐dependent dilation in arterioles collected from patients with coronary artery disease.ConclusionsSphingolipid metabolites play a critical role in determining the mediator of FID in human resistance arterioles. Manipulating the sphingolipid balance towards ceramide versus sphingosine‐1‐phosphate favors microvascular dysfunction versus restoration of NO‐mediated FID, respectively. Multiple targets exist within this biolipid pathway to treat microvascular dysfunction and potentially improve patient outcomes.

Adenosine Kinase Inhibition Augments Conducted Vasodilation and Prevents Left Ventricle Diastolic Dysfunction in Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is often manifested as impaired cardiovascular reserve. We sought to determine if conducted vasodilation, which coordinates microvascular resistance longitudinally to match tissue metabolic demand, becomes compromised in HFpEF. We hypothesized that the metabolic vasodilator adenosine facilitates and that inhibition of ADK (adenosine kinase) augments conducted vasodilation for a more efficient myocardial perfusion and improved left ventricle (LV) diastolic function in HFpEF. We assessed conducted vasodilation in obese ZSF1 rats that develop LV diastolic dysfunction and is used to model human HFpEF. Additionally, conducted vasodilation was measured in arterioles isolated from the right atrial appendages of patients with HFpEF. We found a markedly reduced conducted vasodilation both in obese ZSF1 rats and in patients with HFpEF. Impaired conducted vasodilation was accompanied by increased vascular ADK expression. Isolated rat and human arterioles incubated with adenosine (10 nmol/L) or ADK inhibitor ABT-702 (0.1 µmol/L) both displayed augmented conducted vasodilation. Treatment of obese ZSF1 rats with ABT-702 (1.5 mg/kg, IP for 8 weeks) prevented LV diastolic dysfunction, and in a crossover design augmented conducted vasodilation and improved LV diastolic function. ABT-702 treated obese ZSF1 rats exhibited reduced expression of myocardial carbonic anhydrase 9 and collagen, surrogate markers of myocardial hypoxia. Upregulation of vascular ADK mitigates adenosine-facilitated conducted vasodilation in obese ZSF1 rats and in patients with HFpEF. We propose that pharmacological inhibition of ADK could be beneficial for therapeutic augmentation of conducted vasodilation, thereby improving tissue perfusion and LV diastolic function in HFpEF.

Integrative Effects of Autophagy and Telomerase on Arteriolar Flow‐Mediated Dilation in Health and Coronary Artery Disease

Flow‐mediated dilation, (FMD) is manifest by arteriolar vasodilation in response to shear stress in an endothelium‐dependent manner. Nitric oxide (NO) is the primary mechanism for FMD in health, while hydrogen peroxide (H2O2) is the dominant mechanisms of dilation in subjects with coronary artery disease (CAD). TERT, the catalytic subunit of telomerase, is responsible for maintenance of telomere length, and telomerase activity (TA) and is necessary to preserve physiological mitochondrial function under conditions of acute stress. Autophagy is a basic cellular recycling process, responding to unfolded proteins and damaged organelles. Both autophagy and TERT levels are downregulated in subjects with CAD. Inhibition of TERT in non‐CAD vessels reduces autophagy (Fig 1A), and switches the mechanism of dilation from NO to H2O2. Conversely, activation of TERT switches the mechanism of dilation from H2O2 to NO in CAD (Fig 1B.). We sought to examine whether autophagy and TA are part of the same pathway regulating microvascular FMD in healthy (non‐CAD) and CAD vessels. Human arterioles from discarded adipose tissue were isolated and prepared for videomicroscopy. Non‐CAD vessels were co‐incubated overnight with TA inhibitor (BIBR 1532; 10 μM) and an activator of autophagy (Trichostatin A, TSA; 100 nM). CAD vessels were co‐incubated with an inhibitor of autophagy (Bafilomycin A1, BAFA1; 10nM) and the transcriptional activator of TERT (AGS 499; 20 nM). Internal diameters were measured after graded increases in intraluminal pressure gradients (flow) in the presence/absence of a NOS inhibitor (L‐NAME; 100μM) or H2O2 scavenger (Peg‐Catalase, Peg‐Cat; 500 units/mL). Data are expressed as % maximal dilation. In vehicle treated vessels, the magnitude of FMD was similar between non‐CAD and CAD. Within CAD vessels, inhibition of autophagy prevented the activator of TERT (AGS) from switching the mediator of FMD from H2O2 to NO (AGS 499 + BAFA1: 84.4±9.4 vs. AGS499 + BAFA1 + L‐NAME: 92.6±6.1 vs. AGS499 + BAFA1 + Peg‐Cat: 19.1±14.0; Fig 2A, n = 3–5; P<0.05). Conversely, in non‐CAD vessels, activation of autophagy prevented the switch from NO to H2O2 typically seen during inhibition of TERT (BIBR + TSA: 86.4±9.2 vs. BIBR + TSA + L‐NAME: 36.3±12 vs. BIBR + TSA + Peg‐Cat: 73.8.9±9; Fig 2B, n = 3–5; P < 0.05,). Our data demonstrate that autophagy is distal to TERT in regulating the mechanism of FMD in human adipose arterioles and may serve as a final common pathway for determining the mechanism of FMD in human arterioles.Support or Funding InformationFunded by T32GM089586‐09 (WEH), R01‐HL133029‐02 (AMB), and R01‐HL135901‐02 (DDG)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms of TRPV4 channel activation in human arteriolar endothelial cells: A structure‐activity study with arachidonic acid and analogs

Previous studies from our group indicated that arachidonic acid (AA) induces dilation of human coronary arterioles (HCAs) through the activation of endothelial TRPV4 channels followed by the release of vasodilator factors or endothelial hyperpolarization. In addition, TRPV4‐mediated vasodilatory response to flow is augmented in HCAs from subjects with coronary artery disease (CAD), a condition associated with a change of vasodilators from nitric oxide to hydrogen peroxide. However, the mechanisms of TRPV4 activation by AA and its role in the regulation of TRPV4 function under normal conditions remain to be elucidated. In this study, we examined the structure‐activity relationships of AA and its congeners in human adipose arterioles from subjects without CAD (non‐CAD). Adipose arterioles (~150 μm ID), isolated from non‐CAD human subjects, were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. In adipose arterioles pre‐constricted with endothelin‐1, AA (10−11 to 10−6 M) elicited a potent concentration‐dependent dilation (maximal dilation: 84±3%, logEC50: −8.2, n=6), and this response was largely blocked by HC‐067047 (10−6 M), a TRPV4 antagonist (17±3%, n=6). Compared with AA (C20:4ω6), vasodilator effect of AA analog homo‐γ‐linolenic acid (HGLA, C20:3ω6; 10−11 to 10−6 M) was less potent (maximal dilation: 44±8%, logEC50: −7.2, n=6) but the dilation was also largely abolished by HC‐067047 (6±3%, n=4). These HC067047‐inhibitable vasodilator effects of AA and HGLA were similar as those in HCAs from CAD subjects shown previously (maximal dilation: 84±5% and 40±5%, logEC50: −8.3 and −6.8, n=5 and 3, respectively). In adipose arterioles, another AA analog 5,8,11,14‐eicosatetraynoic acid (ETYA, 10−11 to 10−6 M) demonstrated greatly reduced response (maximal dilation: 19±3%, logEC50: −7.1, n=3), which was eliminated by HC‐067047 (5±2%, n=3). Pretreatment of adipose arterioles with ETYA (10−5 M) decreased vasodilation responses to AA (25±2%, n=5). Vessel denudation markedly reduced vasodilatory responses to AA (10−11 to 10−6 M) (maximal dilation: 33±5%, n=4) as well as to HGLA (10−11 to 10−6 M) (maximal dilation: 13±3%, n=4), confirming the endothelium‐dependent effects of AA and analogs. The dilation to the smooth muscle relaxant sodium nitroprusside (10−10 to 10−4 M) was not affected by HC‐067047 (maximal dilation: 100±0% vs. 99.8±1%, n=2). Together, these data provide evidence that AA activates endothelial TRPV4 to induce comparable dilation in non‐CAD human arterioles. AA congeners also activate TRPV4 channels, however, but they exhibit reduced vasodilatory responses and can antagonize arachidonic‐acid‐induced TRPV4‐mediated vasodilation, suggesting a potential role of specific cis‐double bonds and/or their position for AA‐induced TRPV4 activation. Future studies will examine whether the mechanisms of TRPV4 activation involve potential binding of AA to the channel protein or indirect effect through AA metabolites.Support or Funding InformationThis work was supported by R01‐HL 096647This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of NADPH oxidases in TRPV4‐mediated vasodilation of human arterioles

NADPH oxidases (NOXs) are a major source of reactive oxygen species (ROS) in the vascular system and play diverse roles in cardiovascular health and disease. We recently reported that H2O2, a diffusible ROS, regulates protein phosphorylation of endothelial transient receptor potential vanilloid 4 (TRPV4) channels and TRPV4‐mediated vasodilation in human coronary arterioles (HCA) from subjects with coronary artery disease (CAD). The endogenous source of H2O2 involved in TRPV4 regulation remains unclear. In this study, we investigated the expression of different NOX isoforms in human arterioles and their potential role in the regulation of TRPV4‐mediated arteriolar dilation.Human adipose arterioles (HAA) and HCA were dissected from fresh adipose and heart tissues obtained as discarded surgical specimens. The mRNA and protein expression of NOXs were detected by RT‐PCR and Western blotting. TRPV4 agonist‐induced vasodilation was examined in cannulated arterioles (100–200 μm internal diameter at 60 mmHg) by measuring diameter changes using video microscopy.RT‐PCR analysis revealed consistent expression of NOX2 and 4 but low expression of other isoforms (NOX1, 3, and 5) in HAA and HCA. Using endothelium‐intact and denuded arterioles (n=3 tissues/each) as well as freshly isolated endothelial cells and smooth muscle cells (n=2 tissues/each), we further found that NOX2 and 4 are more abundantly expressed in endothelial cells and at a significantly higher level than other NOX isoforms. Consistent with mRNA expression, NOX2 and 4 proteins were detected in HAA and HCA using Western blot analysis. In cannulated arterioles, TRPV4 agonist GSK1016790A (10−9–10−7 M) induced potent and concentration‐dependent dilation in HAA (maximal dilation at 10−7 M, 76±6%, logEC50 −8.0±0.1, n=6) and HCA (maximal dilation, 69±8%, logEC50 −7.9±0.1, n=5). This dilation was significantly reduced by the small molecule NOX2 inhibitor GSK2795039 (1 μM) in both HAA (22±8%, n=6, p< 0.05 vs control) and HCA (22±10%, n=5, p<0.05 vs control). Interestingly, NOX4 inhibitor GKT137831 (1 μM) also significantly reduced the dilation in HAA (maximal dilation at 10−7 M, 18±8% vs. 72±8% in control, n=6, p<0.05). Both NOX2 and 4 inhibitors did not affect sodium nitroprusside (10−10–10−4 M, an endothelium‐independent vasodilator)‐induced dilation in HAA (maximal dilation at 10−4 M, 94±2% with GSK2795039 vs. 94±2% of control, and 96±3% with GKT137831 vs. 97±3% of control, n=4 and 3, respectively).These results indicate that NOX2 and NOX4 represent two main isoforms of NOXs expressed in endothelial cells of human arterioles and play a potentially important role in TRPV4‐mediated endothelium‐dependent vasodilation. Future studies will explore whether the mechanisms of TRPV4 regulation involves NOX2/4‐derived and H2O2‐mediated channel protein phosphorylation or other signaling pathways in endothelial cells.Support or Funding InformationNational Heart, Lung and Blood Institute Grant RO1‐HL 096647This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Leukotriene B4 Contributes to Development of Cardiac Allograft Vasculopathy

Purpose Cardiac allograft vasculopathy (CAV) is a highly prevalent vaso-occlusive disease that is a leading cause of graft failure and mortality after heart transplantation. While the pathogenesis of CAV remains incompletely understood, histologic evidence suggests that macrophages, which constitute an important part of the innate immune response, may play an important role. Prior studies in pulmonary hypertension have shown that macrophage-derived leukotriene B4 (LTB4) induces proliferation and hypertrophy of human pulmonary artery smooth muscle cells. We hypothesized that LTB4 plays a similar role in the development of CAV. Methods Human studies: We performed immunofluorescence staining of sections from human CAV+ and CAV- coronary arteries with CD68 (macrophage marker) and 5-LO (5-lipoxygenase, LTB4-producing enzyme), and measured serial LTB4 plasma levels in 14 patients with angiographic CAV and 14 matched CAV- controls. Rat model: We performed aortic transplants in a rat CAV model (Fischer into Lewis orthotopic aortic transplants, n=11) with allogeneic transplants as controls (n=6), and performed CD68 and 5-LO staining of aortic cross-sections of mice with and without aortic intimal hyperplasia. We then treated rat allogeneic transplants with bestatin (protease inhibitor that blocks LTB4 production) to determine whether LTB4 inhibition can prevent the intimal proliferation seen in this rat CAV model. Results Immunofluorescence staining showed that 5-LO co-localized with CD68+ macrophages in the intimal layer of CAV+ human arterioles and in allogeneic rat aortic transplant models. Plasma LTB4 levels were higher in CAV+ cases compared to CAV- controls (n=14 per group, p Conclusion Macrophage-derived leukotriene B4 may contribute to development of CAV. Blockade of LTB4 synthesis has the potential to prevent CAV development after heart transplantation.

Abstract TP557: Endothelial Cell Proinflammatory Priming by Amyloidogenic Medin via NFκB and Inflammasome Pathways and Reversal by Monosialoganglioside Nanoliposomes

Medin is a common amyloid protein that accumulates in cerebral vessels with aging and cause endothelial dysfunction in human cerebral arterioles. Studying its role in cerebrovascular disease could provide insights on cerebrovascular aging. We aim to test whether medin induces pro-inflammatory priming of endothelial cells (ECs) and probe mechanistic bases, and determine whether monosialoganglioside-containing nanoliposomes (NLGM1), previously shown to protect against AL amyloid vascular dysfunction, would abrogate this response. Methods: Human umbilical vein ECs were treated (20 hours) with physiologic dose of medin (5 μM) or vehicle, and proinflammatory markers IL-1β, caspase-1, IL-8, IL-6, ICAM-1, phospho-NFκB and pro-thrombotic marker plasminogen activator inhibitor-1 (PAI-1) gene and/or protein expressions were measured. In some replicates, medin was co-treated with either specific NFκB small molecule inhibitor RO106-9920 100 μM or NLGM1 (70:20:5% phosphatidylcholine, cholesterol, monosialoganglioside, 1:10 medin:NLGM1 mass ratio). Results: Medin increased gene expression of IL-1β (104.0±30.2x vs. control, p=0.002), IL-8 (5560±1864x, p<0.001), IL-6 (80.4±48x, p<0.001), ICAM-1 (72.0±8.5x, p<0.001) and PAI-1 (5.4±1x, p<0.001) (n=13-44). There were corresponding increases in protein expression as well as increased phospho-NFκB and caspase-1 (Figure). RO106-9920 co-treatment reduced gene expressions of IL-8, IL-6, ICAM-1 and PAI-1 but not IL-1β (versus medin treatment) whereas NLGM1 normalized gene expressions of all cytokines including IL-1β. Conclusions: Medin induced profound proinflammatory and prothrombotic responses in endothelial cells. Both NFκB and inflammasome-mediated mechanisms mediate medin’s vascular priming effects. Medin-induced pathology is a potential new mechanism of aging-related cerebrovascular inflammation. NLGM1 protects against medin’s effects and may represent a new therapeutic approach.

Lysophosphatidic acid acts on LPA1 receptor to increase H2 O2 during flow-induced dilation in human adipose arterioles.

NO produces arteriolar flow-induced dilation (FID) in healthy subjects but is replaced by mitochondria-derived hydrogen peroxide (mtH2 O2 ) in patients with coronary artery disease (CAD). Lysophosphatidic acid (LPA) is elevated in patients with risk factors for CAD, but its functional effect in arterioles is unknown. We tested whether elevated LPA changes the mediator of FID from NO to mtH2 O2 in human visceral and subcutaneous adipose arterioles. Arterioles were cannulated on glass micropipettes and pressurized to 60mmHg. We recorded lumen diameter after graded increases in flow in the presence of either NOS inhibition (L-NAME) or H2 O2 scavenging (Peg-Cat)±LPA (10μM, 30min), ±LPA1 /LPA3 receptor antagonist (Ki16425) or LPA2 receptor antagonist (H2L5186303). We analysed LPA receptor RNA and protein levels in human arterioles and human cultured endothelial cells. FID was inhibited by L-NAME but not Peg-Cat in untreated vessels. In vessels treated with LPA, FID was of similar magnitude but inhibited by Peg-Cat while L-NAME had no effect. Rotenone attenuated FID in vessels treated with LPA indicating mitochondria as a source of ROS. RNA transcripts from LPA1 and LPA2 but not LPA3 receptors were detected in arterioles. LPA1 but not LPA3 receptor protein was detected by Western blot. Pretreatment of vessels with an LPA1 /LPA3 , but not LPA2 , receptor antagonist prior to LPA preserved NO-mediated dilation. These findings suggest an LPA1 receptor-dependent pathway by which LPA increases arteriolar release of mtH2 O2 as a mediator of FMD.

Abstract 054: Tissue-Specific Effects of Targeted Mutation of Mir29b1 in Rats

miR-29 is a master regulator of extracellular matrix genes and is generally considered anti-fibrotic, and it improves nitric oxide (NO) production in human and rat arterioles by targeting Lypla1.Targeted mutation in the Mir29b1 gene exacerbates hypertension in a rat model derived from the Dahl salt-sensitive rat. The kidney, particularly the renal outer medulla, plays a key role in the development of salt-sensitive hypertension. In the present study, we examined the effect of the targeted Mir29b1 mutationon tissue fibrosis and NO levels with a focus on kidney regions. The abundance of miR-29b and the co-transcribed miR-29a was substantially lower in the renal cortex, renal outer medulla, heart and liver in mutant rats. In mutant rats on a 0.4% NaCl diet and prior to the development of overt hypertension, tissue fibrosis was significantly increased in the renal outer medulla, but not in the renal cortex, heart or liver, compared with wild-type littermates. Levels of NO metabolites were significantly lower, and Lypla1 protein abundance significantly higher, in the renal outer medulla, but not in the renal cortex. After 14 days of a 4% NaCl diet, tissue fibrosis remained significantly higher in the renal outer medulla and became higher in the heart of mutant rats compared with wild-type littermates, but not in the renal cortex. 24h urine volume was significantly lower in mutant rats on either the 0.4% or 4% NaCl diet. Microalbuminuria was exacerbated by the 4% NaCl diet, but was not significantly different between mutant rats and wild-type littermates. These findings indicate the effects of miR-29 are tissue-specific. The renal outer medulla might be particularly susceptible to the injurious effects of a miR-29 insufficiency, which might contribute to the development of hypertension in Mir29b1 mutant rats.

Opposing vasomotor roles of TRPV1 and TRPV2 channels in the Human Adipose Microcirculation

Mechanosensitive, Ca2+‐permeable channels of the transient receptor potential vanilloid (TRPV) family, especially TRPV4, have emerged as important regulators of vascular tone. Within the vasculature, TRPV channels are expressed in smooth muscle cells, endothelial cells and perivascular nerves. We have previously demonstrated that Ca2+ influx through endothelial TRPV4 channels mediates flow‐induced dilation (FID) of human coronary arterioles (HCAs) from subjects with coronary artery disease (CAD). Recent unpublished studies from our lab show that FID is not affected by TRPV4 inhibition in HCAs of subjects without CAD (non‐CAD), even though TRPV4 channels are similarly expressed in non‐CAD and CAD HCAs. Interestingly, FID of non‐CAD HCAs is inhibited by ruthenium red, a non‐selective TRPV channel blocker, suggesting the involvement of other TRPV channel subtypes. Moreover, endothelial TRPV1 channels have been previously shown to mediate FID in rodent arteries; whereas a functional role for endothelial TRPV2 channels has yet to be determined. Together, these data have led us to hypothesize that endothelial TRPV1/TRPV2 channels mediate FID of non‐CAD human coronary and adipose arterioles. The objective of the current study was to characterize the expression and vasomotor function of TRPV1 and TRPV2 channels in HCAs and human adipose arterioles (HAAs) from non‐CAD subjects. RT‐PCR analysis revealed the presence of TRPV1 and TRPV2 transcripts in HCAs and HAAs isolated from multiple subjects. Consistent with mRNA expression, both proteins were detected in membrane fractions of HCAs and HAAs using immunoblotting. We next examined the vasomotor effect of capsaicin, a TRPV1‐ selective agonist and cannabidiol, a TRPV2‐selective agonist, on freshly isolated HAAs. HAAs (100–250 μm) were cannulated under 60 mmHg and examined for diameter changes using videomicroscopy. Bath application of cannabidiol (10−7 – 10−5 M) induced dose‐dependent dilation of endothelium‐intact and surprisingly, endothelium‐denuded HAAs (% dilation at 10−5 M, 95.3 ± 1.4%, n=9 intact; 95.4 ± 2.6%, n=6 denuded HAAs), suggesting a vasomotor role for both endothelial and smooth muscle TRPV2 channels. In contrast, bath application of capsaicin (10−10 – 10−6 M) induced constriction of both endothelium‐intact and denuded HAAs at concentrations of 10−7 M (−55 ± 8%; n=5) and 10−6 M (−50.8 ± 12.9%; n=5). Moreover, capsaicin (10−6 M) induced a prolonged increase in intracellular calcium concentration ([Ca2+]i) in smooth muscle cells of HAAs, consistent with vasoconstriction observed in functional studies; whereas cannabidiol (10−7 – 10−5 M) induced a dose‐dependent increase in endothelial [Ca2+]i, consistent with vasodilation observed in functional studies. These results suggest that TRPV1 and TRPV2 channels are functionally expressed in human arterioles and upon activation, produce opposing vasomotor responses in HAAs. Further studies are required to determine the precise mechanisms responsible for such contrasting vasomotor effects of TRPV1 and TRPV2 activation.Support or Funding InformationSupport for this research was provided by the National Heart, Lung and BloodInstitute Grant R01‐HL 096647.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 µm internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Roles of NADPH oxidase and mitochondria in flow-induced vasodilation of human adipose arterioles: ROS-induced ROS release in coronary artery disease.

H2 O2 contributes to FID of human arterioles. This study is designed to examine the roles of mitochondria and NADPH oxidase in modulating the release of ROS and in mediating FID. We tested whether NADPH oxidase contributes to mitochondrial ROS generation in arterioles during CAD. Visceral adipose arterioles obtained from patients with or without CAD were cannulated and pressurized for videomicroscopic measurement of arteriolar diameters. Dilator responses and ROS production during flow were determined in the presence and absence of the NADPH oxidase inhibitor gp91ds-tat and the mitochondrial electron transport inhibitor rotenone. Both dilation and H2 O2 generation during flow were reduced in the presence of rotenone (13.5±8% vs 97±% without rotenone) or gp91ds-tat in patients with CAD, while patients without CAD exhibited H2 O2 -independent dilations. Mitochondrial superoxide production during flow was attenuated by gp91ds-tat in arterioles from CAD patients. These findings indicate that ROS produced by NADPH oxidase are an upstream component of the mitochondria-dependent pathway contributing to flow-dependent H2 O2 generation and dilation in peripheral microvessels from patients with CAD. We conclude that in CAD, both mitochondria and NADPH oxidase contribute to FID through a redox mechanism in visceral arterioles.

Amyloidogenic medin induces endothelial dysfunction and vascular inflammation through the receptor for advanced glycation endproducts.

Medin is a common amyloidogenic protein in humans that accumulates in arteries with advanced age and has been implicated in vascular degeneration. Medin's effect on endothelial function remains unknown. The aims are to assess medin's effects on human arteriole endothelial function and identify potential mechanisms underlying medin-induced vascular injury. Ex vivo human adipose and leptomeningeal arterioles were exposed (1 h) to medin (0.1, 1, or 5µM) without or with FPS-ZM1 [100µM, receptor for advanced glycation endproducts (RAGE)-specific inhibitor] and endothelium-dependent function (acetylcholine dilator response) and endothelium-independent function (dilator response to nitric oxide donor diethylenetriamine NONOate) were compared with baseline control. Human umbilical vein endothelial cells were exposed to medin without or with FPS-ZM1 and oxidative and nitrative stress, cell viability, and pro-inflammatory signaling measures were obtained. Medin caused impaired endothelial function (vs. baseline response: -45.2 ± 5.1 and -35.8 ± 7.9% in adipose and leptomeningeal arterioles, respectively, each P < 0.05). Dilator response to NONOate was not significantly changed. Medin decreased arteriole and endothelial cell nitric oxide production, increased superoxide production, reduced endothelial cell viability, proliferation, and migration. Medin increased gene and protein expression of interleukin-6 and interleukin-8 via activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Medin-induced endothelial dysfunction and oxidative stress were reversed by antioxidant polyethylene glycol superoxide dismutase and by RAGE inhibitor FPS-ZM1. Medin causes human microvascular endothelial dysfunction through oxidative and nitrative stress and promotes pro-inflammatory signaling in endothelial cells. These effects appear to be mediated via RAGE. The findings represent a potential novel mechanism of vascular injury.

Characterization of TRPV1 and TRPV2 channel expression and vasomotor function in human arterioles

Transient receptor potential vanilloid (TRPV) channels, act as sensory mediators and are activated by endogenous ligands, heat, mechanical and osmotic stress. Within the vasculature, TRPV channels are expressed in smooth muscle cells, endothelial cells and in perivascular nerves. We have previously demonstrated that Ca2+ influx through endothelial TRPV4 channels mediates flow‐induced dilation (FID) of human coronary arterioles (HCA) from subjects with coronary artery disease (CAD). Other studies have also implicated endothelial TRPV1 channels in FID, whereas a functional role for endothelial TRPV2 channels has yet to be determined. Recent unpublished studies from our lab showed that FID was not affected by TRPV4 inhibition in HCA from subjects without CAD (non‐CAD). Instead, inhibition of TRPV1 or TRPV2 reduced FID of HCA from non‐CAD subjects. Collectively, these data have led us to hypothesize that different TRPV channels contribute to FID of arteries from non‐CAD vs CAD subjects, with TRPV4 prominent in CAD vessels and TRPV1/2 more important in non‐CAD vessels. The objective of the current study was to characterize the expression and function of TRPV1 and TRPV2 channels in HCA and human adipose arterioles (HAA) from non‐CAD subjects, using molecular, immunological and vascular reactivity studies. We first examined TRPV1 and TRPV2 mRNA expression in HCA and HAA using RT‐PCR and consistently detected both transcripts in multiple samples. As a positive control, human brain tissue was found to express TRPV1 and TRPV2 transcripts. Consistent with mRNA expression, TRPV1 and TRPV2 proteins were detected in membrane fractions of HCA and HAA using western blot analysis. Antibody specificity was confirmed in TRPV1 or TRPV2 transfected versus non‐transfected HEK‐293 cells. We next examined the vasomotor effect of capsaicin, a selective TRPV1 agonist and cannabidiol, a selective TRPV2 agonist on freshly isolated HAA. HAA (100–200 μm), obtained from subjects without CAD, were cannulated under 60 mmHg and examined for diameter changes using videomicroscopy. Surprisingly, bath application of capsaicin induced constriction of HAA at concentrations of 10−7 M and 10−6 M (−46.82 ± 4.3%; n=3), with no significant effect at lower concentrations (10−10–10−8 M). In contrast, cannabidiol (10−6,10−5,3×10−5 M) induced a dose‐dependent dilation of HAA. These results suggest that TRPV1 and TRPV2 channels are expressed in human arterioles and contribute to vasomotor responses. Further studies are required to determine the specific role of the endothelium versus smooth muscle and perivascular nerves, in mediating the vascular responses to TRPV1 and TRPV2 activation.Support or Funding InformationSupport for this research was provided by the National Heart, Lung and Blood Institute Grant R01‐HL 096647

Contribution of KV1.5 Channel to Hydrogen Peroxide-Induced Human Arteriolar Dilation and Its Modulation by Coronary Artery Disease.

Hydrogen peroxide (H2O2) regulates vascular tone in the human microcirculation under physiological and pathophysiological conditions. It dilates arterioles by activating large-conductance Ca2+-activated K+ channels in subjects with coronary artery disease (CAD), but its mechanisms of action in subjects without CAD (non-CAD) when compared with those with CAD remain unknown. We hypothesize that H2O2-elicited dilation involves different K+ channels in non-CAD versus CAD, resulting in an altered capacity for vasodilation during disease. H2O2 induced endothelium-independent vasodilation in non-CAD adipose arterioles, which was reduced by paxilline, a large-conductance Ca2+-activated K+ channel blocker, and by 4-aminopyridine, a voltage-gated K+ (KV) channel blocker. Assays of mRNA transcripts, protein expression, and subcellular localization revealed that KV1.5 is the major KV1 channel expressed in vascular smooth muscle cells and is abundantly localized on the plasma membrane. The selective KV1.5 blocker diphenylphosphine oxide-1 and the KV1.3/1.5 blocker 5-(4-phenylbutoxy)psoralen reduced H2O2-elicited dilation to a similar extent as 4-aminopyridine, but the selective KV1.3 blocker phenoxyalkoxypsoralen-1 was without effect. In arterioles from CAD subjects, H2O2-induced dilation was significantly reduced, and this dilation was inhibited by paxilline but not by 4-aminopyridine, diphenylphosphine oxide-1, or 5-(4-phenylbutoxy)psoralen. KV1.5 cell membrane localization and diphenylphosphine oxide-1-sensitive K+ currents were markedly reduced in isolated vascular smooth muscle cells from CAD arterioles, although mRNA or total cellular protein expression was largely unchanged. In human arterioles, H2O2-induced dilation is impaired in CAD, which is associated with a transition from a combined large-conductance Ca2+-activated K+- and KV (KV1.5)-mediated vasodilation toward a large-conductance Ca2+-activated K+-predominant mechanism of dilation. Loss of KV1.5 vasomotor function may play an important role in microvascular dysfunction in CAD or other vascular diseases.

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Dynamin-related protein 1 mediates low glucose-induced endothelial dysfunction in human arterioles.

Intensive glycemic regulation has resulted in an increased incidence of hypoglycemia. Hypoglycemic burden correlates with adverse cardiovascular complications and contributes acutely and chronically to endothelial dysfunction. Prior data indicate that mitochondrial dysfunction contributes to hypoglycemia-induced endothelial dysfunction, but the mechanisms behind this linkage remain unknown. We attempt to determine whether clinically relevant low-glucose (LG) exposures acutely induce endothelial dysfunction through activation of the mitochondrial fission process. Characterization of mitochondrial morphology was carried out in cultured endothelial cells by using confocal microscopy. Isolated human arterioles were used to explore the effect LG-induced mitochondrial fission has on the formation of detrimental reactive oxygen species (ROS), bioavailability of nitric oxide (NO), and endothelial-dependent vascular relaxation. Fluorescence microscopy was employed to visualize changes in mitochondrial ROS and NO levels and videomicroscopy applied to measure vasodilation response. Pharmacological disruption of the profission protein Drp1 with Mdivi-1 during LG exposure reduced mitochondrial fragmentation among vascular endothelial cells (LG: 0.469; LG+Mdivi-1: 0.276; P = 0.003), prevented formation of vascular ROS (LG: 2.036; LG+Mdivi-1: 1.774; P = 0.005), increased the presence of NO (LG: 1.352; LG+Mdivi-1: 1.502; P = 0.048), and improved vascular dilation response to acetylcholine (LG: 31.6%; LG+Mdivi-1; 78.5% at maximum dose; P < 0.001). Additionally, decreased expression of Drp1 via siRNA knockdown during LG conditions also improved vascular relaxation. Exposure to LG imparts endothelial dysfunction coupled with altered mitochondrial phenotypes among isolated human arterioles. Disruption of Drp1 and subsequent mitochondrial fragmentation events prevents impaired vascular dilation, restores mitochondrial phenotype, and implicates mitochondrial fission as a primary mediator of LG-induced endothelial dysfunction.NEW & NOTEWORTHY Acute low-glucose exposure induces mitochondrial fragmentation in endothelial cells via Drp1 and is associated with impaired endothelial function in human arterioles. Targeting of Drp1 prevents fragmentation, improves vasofunction, and may provide a therapeutic target for improving cardiovascular complications among diabetics.Listen to this article's corresponding podcast @ http://ajpheart.podbean.com/e/mitochondrial-dynamics-impact-endothelial-function/.