Joseph Hockenberry

Ceramide Changes the Mediator of Flow-Induced Vasodilation from Nitric Oxide to Hydrogen Peroxide in the Human Microcirculation

Rationale: Mitochondrial-derived hydrogen peroxide (H2O2) regulates flow-induced dilation (FID) in microvessels from patients with coronary artery disease. The relationship between ceramide, an independent risk factor for coronary artery disease and a known inducer of mitochondrial reactive oxygen species, and FID is unknown. Objective: We examined the hypothesis that exogenous ceramide induces a switch in the mediator of FID from nitric oxide to H2O2. Methods and Results: Internal diameter changes of resistance arterioles from human adipose and atrial tissue were measured by video microscopy. Mitochondrial H2O2 production was assayed in arterioles using mito peroxy yellow 1. Polyethylene glycol–catalase, rotenone, and Mito-TEMPO impaired FID in healthy adipose arterioles pretreated with ceramide, whereas N&ohgr;-nitro-L-arginine methyl ester had no effect. Mitochondrial H2O2 production was induced in response to flow in healthy adipose vessels pretreated with ceramide, and this was abolished in the presence of polyethylene glycol–catalase. Immunohistochemistry demonstrated ceramide accumulation in arterioles from both healthy patients and patients with coronary artery disease. N&ohgr;-nitro-L-arginine methyl ester reduced vasodilation to flow in adipose as well as atrial vessels from patients with coronary artery disease incubated with GW4869, a neutral sphingomyelinase inhibitor, whereas polyethylene glycol–catalase had no effect. Conclusions: Our data indicate that ceramide has an integral role in the transition of the mediator of FID from nitric oxide to mitochondrial-derived H2O2 and that inhibition of ceramide production can revert the mechanism of dilation back to nitric oxide. Ceramide may be an important target for preventing and treating vascular dysfunction associated with atherosclerosis.

An acute rise in intraluminal pressure shifts the mediator of flow-mediated dilation from nitric oxide to hydrogen peroxide in human arterioles.

Endothelial nitric oxide (NO) is the primary mediator of flow-mediated dilation (FMD) in human adipose microvessels. Impaired NO-mediated vasodilation occurs after acute and chronic hypertension, possibly due to excess generation of reactive oxygen species (ROS). The direct role of pressure elevation in this impairment of human arteriolar dilation is not known. We tested the hypothesis that elevation in pressure is sufficient to impair FMD. Arterioles were isolated from human adipose tissue and cannulated, and vasodilation to graded flow gradients was measured before and after exposure to increased intraluminal pressure (IILP; 150 mmHg, 30 min). The mediator of FMD was determined using pharmacological agents to reduce NO [N(G)-nitro-l-arginine methyl ester (l-NAME), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO)], or H2O2 [polyethylene glycol (PEG)-catalase], and mitochondrial (mt) ROS was quantified using fluorescence microscopy. Exposure to IILP decreased overall FMD (max %dilation: 82.7 ± 4.9 vs. 62 ± 5.6; P < 0.05). This dilation was abolished by treatment with l-NAME prepressure and PEG-catalase after IILP (max %dilation: l-NAME: 23.8 ± 6.1 vs. 74.8 ± 8.6; PEG-catalase: 71.8 ± 5.9 vs. 24.6 ± 10.6). To examine if this change was mediated by mtROS, FMD responses were measured in the presence of the complex I inhibitor rotenone or the mitochondrial antioxidant mitoTempol. Before IILP, FMD was unaffected by either compound; however, both inhibited dilation after IILP. The fluorescence intensity of mitochondria peroxy yellow 1 (MitoPY1), a mitochondria-specific fluorescent probe for H2O2, increased during flow after IILP (%change from static: 12.3 ± 14.5 vs. 127.9 ± 57.7). These results demonstrate a novel compensatory dilator mechanism in humans that is triggered by IILP, inducing a change in the mediator of FMD from NO to mitochondria-derived H2O2.

Critical Role for Telomerase in the Mechanism of Flow Mediated Dilation in the Human Microcirculation

Supplemental Digital Content is available in the text.

PGC-1α (Peroxisome Proliferator–Activated Receptor γ Coactivator 1-α) Overexpression in Coronary Artery Disease Recruits NO and Hydrogen Peroxide During Flow-Mediated Dilation and Protects Against Increased Intraluminal Pressure

Blood flow through healthy human vessels releases NO to produce vasodilation, whereas in patients with coronary artery disease (CAD), the mediator of dilation transitions to mitochondria-derived hydrogen peroxide (mtH2O2). Excessive mtH2O2 production contributes to a proatherosclerotic vascular milieu. Loss of PGC-1&agr; (peroxisome proliferator–activated receptor &ggr; coactivator 1&agr;) is implicated in the pathogenesis of CAD. We hypothesized that PGC-1&agr; suppresses mtH2O2 production to reestablish NO-mediated dilation in isolated vessels from patients with CAD. Isolated human adipose arterioles were cannulated, and changes in lumen diameter in response to graded increases in flow were recorded in the presence of PEG (polyethylene glycol)–catalase (H2O2 scavenger) or L-NAME (NG-nitro-L-arginine methyl ester; NOS inhibitor). In contrast to the exclusively NO- or H2O2-mediated dilation seen in either non-CAD or CAD conditions, respectively, flow-mediated dilation in CAD vessels was sensitive to both L-NAME and PEG-catalase after PGC-1&agr; upregulation using ZLN005 and &agr;-lipoic acid. PGC-1&agr; overexpression in CAD vessels protected against the vascular dysfunction induced by an acute increase in intraluminal pressure. In contrast, downregulation of PGC-1&agr; in non-CAD vessels produces a CAD-like phenotype characterized by mtH2O2-mediated dilation (no contribution of NO). Loss of PGC-1&agr; may contribute to the shift toward the mtH2O2-mediated dilation observed in vessels from subjects with CAD. Strategies to boost PGC-1&agr; levels may provide a therapeutic option in patients with CAD by shifting away from mtH2O2-mediated dilation, increasing NO bioavailability, and reducing levels of mtH2O2. Furthermore, increased expression of PGC-1&agr; allows for simultaneous contributions of both NO and H2O2 to flow-mediated dilation.

Telomerase reverse transcriptase protects against angiotensin II-induced microvascular endothelial dysfunction

A rise in reactive oxygen species (ROS) may contribute to cardiovascular disease by reducing nitric oxide (NO) levels, leading to loss of NOs vasodilator and anti-inflammatory effects. Although primarily studied in larger conduit arteries, excess ROS release and a corresponding loss of NO also occur in smaller resistance arteries of the microcirculation, but the underlying mechanisms and therapeutic targets have not been fully characterized. We examined whether either of the two subunits of telomerase, telomerase reverse transcriptase (TERT) or telomerase RNA component (TERC), affect microvascular ROS production and peak vasodilation at baseline and in response to in vivo administration to angiotensin II (ANG II). We report that genetic loss of TERT [maximal dilation: 52.0 ± 6.1% with vehicle, 60.4 ± 12.9% with Nω-nitro-l-arginine methyl ester (l-NAME), and 32.2 ± 12.2% with polyethylene glycol-catalase (PEG-Cat) ( P < 0.05), means ± SD, n = 9-19] but not TERC [maximal dilation: 79 ± 5% with vehicle, 10.7 ± 9.8% with l-NAME ( P < 0.05), and 86.4 ± 8.4% with PEG-Cat, n = 4-7] promotes flow-induced ROS formation. Moreover, TERT knockout exacerbates the microvascular dysfunction resulting from in vivo ANG II treatment, whereas TERT overexpression is protective [maximal dilation: 88.22 ± 4.6% with vehicle vs. 74.0 ± 7.3% with ANG II (1,000 ng·kg-1·min-1) ( P = not significant), n = 4]. Therefore, loss of TERT but not TERC may be a key contributor to the elevated microvascular ROS levels and reduced peak dilation observed in several cardiovascular disease pathologies. NEW & NOTEWORTHY This study identifies telomerase reverse transcriptase (TERT) but not telomerase RNA component as a key factor regulating endothelium-dependent dilation in the microcirculation. Loss of TERT activity leads to microvascular dysfunction but not conduit vessel dysfunction in first-generation mice. In contrast, TERT is protective in the microcirculation in the presence of prolonged vascular stress. Understanding the mechanism of how TERT protects against vascular stress represents a novel target for the treatment of vascular disorders.

Lysophosphatidic acid acts on LPA1 receptor to increase H2O2 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 (mtH2O2) 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 mtH2O2 in human visceral and subcutaneous adipose arterioles.

5,6-δ-DHTL, a stable metabolite of arachidonic acid, is a potential EDHF that mediates microvascular dilation

Objective Prominent among the endothelium‐derived hyperpolarizing factors (EDHFs) are the Cytochrome P450 (CYP) epoxygenase‐derived arachidonic acid metabolites—the epoxyeicosatrienoic acids (EETs), that are known as vasodilators in the microcirculation. Among the EET isomers, 5,6‐EET undergoes rapid lactonization in aqueous solution to the more stable 5,6‐&dgr; DHTL (5,6‐dihydroxytrienoic lactone) isomer. It is unclear whether this metabolic transformation maintains its vasodilator potential and what is the mechanism of action. Thus, the aim of this study was to investigate the capacity of the lactone isomer, 5,6‐ &dgr; DHTL, to induce dilation of arterioles and explore the endothelial Ca2+ response mechanism. Approach and results In isolated human microvessels 5,6‐ &dgr; DHTL induced a dose dependent vasodilation, that was inhibited by mechanical denudation of the endothelial layer. This 5,6‐ &dgr; DHTL ‐dependent dilation was partially reduced in the presence of l‐NAME (NOS inhibitor) or the NO‐scavenger, cPTIO (by 19.7%, which was not statistically significantly). In human endothelial cells, 5,6‐ &dgr; DHTL induced an increase in intracellular Ca2+([Ca2+]i) in a dose dependent manner. This increase in [Ca2+]i was similar to that induced by the 5,6‐EET isomer, and significantly higher than observed by administering the hydrolytic dihydroxy isomer, 5,6‐DHET. Further experiments aimed to investigate the mechanism of action revealed, that the 5,6‐&dgr; DHTL‐mediated ([Ca2+]i elevation was reduced by IP3 and ryanodine antagonists, but not by antagonists to the TRPV4 membrane channel. Similar to their effect on the dilation response in the arteries, NO inhibitors reduced the 5,6‐&dgr; DHTL‐mediated ([Ca2+]i elevation by 20%. Subsequent 5,6‐&dgr; DHTL ‐dependent K+ ion efflux from endothelial cells, was abolished by the inhibition of small and intermediate conductance KCa. Conclusions The present study shows that 5,6‐&dgr; DHTL is a potential EDHF, that dilates microvessels through a mechanism that involves endothelial dependent Ca2+ entry, requiring endothelial hyperpolarization. These results suggest the existence of additional lactone‐containing metabolites that can be derived from the PUFA metabolism and which may function as novel EDHFs. Graphical abstract Figure. No Caption Available. Highlights5,6‐&dgr; DHTL is a potential EDHF, that dilates microvessels.5,6‐&dgr;‐DHTL initiates intracellular Ca2+ influx through channels other than TRPV4.5,6‐&dgr;‐DHTL initiates Ca2+ released from the ER.5,6‐&dgr;‐DHTL mechanism of action is mainly NO‐independent.5,6‐&dgr;‐DHTL‐induced hyperpolarization of ECs.

ISH ADA-01 Critical role of telomerase in regulating cerebral vascular function and redox environment.

Objective: Flow mediated dilation (FMD) is the most physiological relevant form of endothelial-mediated vasodilation. Our laboratory has previously shown that telomerase, a ribo-nucleoprotein that counteracts telomere shortening, has a protective effect on endothelial function under conditions of oxidative stress in the human microcirculation. In the presence of coronary artery disease, decreased telomerase activity contributes to a shift in the mediator of FMD from atheroprotective nitric oxide (NO) to pro-inflammatory and atherogenic hydrogen peroxide (H2O2). Endothelial dysfunction in the cerebral vasculature has been directly linked to cerebral microbleeds and cognitive decline in models of stroke and dementia, thus we hypothesized that TERT plays a critical role in maintaining normal NO-mediated vasodilation in the cerebral vasculature. Design and Method: Using Crisp/Cas9 we generated a novel deletion model for TERT (the catalytic subunit of telomerase) in rats on the WKY background. Middle cerebral arteries (MCA) were isolated from wildtype (WT) and TERT−/− rats, prepared for videomicroscopy, and FMD was measured. The mediator of FMD was determined using a pharmacological inhibitor of NO-synthase (L-NAME) and a scavenger of H2O2 (PEG-catalase). Results: No changes in the magnitude of FMD were observed in MCA from TERT−/− compared to WT rats (max dilation: TERT−/− 71.97 ± 14.7% vs. WT 80.9 ± 10.6%; n≥3; p > 0.05 two way ANOVA RM). In WT animals, pre-incubation with L-NAME abolished FMD while PEG-catalase had no effect on FMD (max dilation: L-NAME -5.8 ± 7%; Peg-catalase 76.6 ± 8.6%; n≥3; p < 0.05). Conversely, in TERT−/− animals FMD was not affected in the presence of L-NAME but abolished in presence of Peg-catalase (max dilation: L-NAME 57.8.0 ± 16.5%; Peg-catalase −4.3 ± 2.4%; n≥3; p < 0.05). Conclusions: Telomerase deficiency causes a switch in the vasoactive mediator of FMD from NO to H2O2, creating a pro-oxidative environment in the cerebral vasculature. Understanding the regulatory role of telomerase in mediating this mechanistic switch may provide a novel therapeutic strategy for the treatment or prevention of cerebrovascular dysfunction.