Reductions in flow-induced dilation (FID; vasodilation in response to increased blood flow from shear stress) is predictive of future adverse cardiac events. Vasodilatory capacity to flow is maintained in arterioles from healthy adults following exposure to high intraluminal pressure (150mmHg, 30min) by utilizing hydrogen peroxide (H2O2) to compensate for decreased bioavailability of nitric oxide (NO). Here, we tested the hypothesis that knockdown of NAD-dependent deacetylase sirtuin-3 (SIRT3), a mitochondrial protein activated during stress and known to stimulate cellular survival pathways, would prevent compensatory signaling during FID and reduce overall vasodilatory capacity following high intraluminal pressure. Adipose arterioles (100-200μm) from otherwise healthy adults (0-1 risk factor for cardiovascular disease) were treated intraluminally with siSIRT3 or negative control siRNA for 16-20hrs and cannulated for videomicroscopy prior to pre-constriction with endothelin-1. Changes in internal microvessel diameter in response to graded increases in flow were measured. Maximal dilation to flow was maintained both pre- and post-pressure in arterioles treated with negative control siRNA (93.3%±2.3 of maximal dilator capacity±SEM, n=3 versus 69.9%±11.5, n=3, respectively), however, vasodilatory capacity was significantly reduced post-pressure in arterioles treated with siSIRT3 (19.8%±5.7, n=3) compared to pre-pressure (76.0%±10.6, n=3, p<0.01, one-way ANOVA). These data suggest that SIRT3 plays a key mechanistic component in compensatory signaling during flow in human arterioles following stress. We conclude that SIRT3 may be a potential therapeutic target to increase vascular resilience and protect from stress in the human microcirculation thereby preventing microvascular dysfunction and future cardiovascular disease. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Microvascular dysfunction is an independent predictor of adverse cardiac events; however, the effect of estrogen on the human microcirculation represents a critical knowledge gap. To our knowledge, this is the first study to report sex-specific detrimental effects of chronic estrogen on human microvascular reactivity. These findings may offer insight into the increased CVD risk associated with estrogen use in both cis- and trans-females.

BACKGROUND:Elevated plasma ceramides and microvascular dysfunction both independently predict adverse cardiac events. Despite the known detrimental effects of ceramide on the microvasculature, evidence suggests that activation of the shear-sensitive, ceramide-forming enzyme NSmase (neutral sphingomyelinase) elicits formation of vasoprotective nitric oxide (NO). Here, we explore a novel hypothesis that acute ceramide formation through NSmase is necessary for maintaining NO signaling within the human microvascular endothelium. We further define the mechanism through which ceramide exerts beneficial effects and discern key mechanistic differences between arterioles from otherwise healthy adults (non-coronary artery disease [CAD]) and patients diagnosed with CAD. METHODS:Human arterioles were dissected from discarded surgical adipose tissue (n=166), and vascular reactivity to flow and C2-ceramide was assessed. Shear-induced NO and mitochondrial hydrogen peroxide (H2O2) production were measured in arterioles using fluorescence microscopy. H2O2 fluorescence was assessed in isolated human umbilical vein endothelial cells. RESULTS:Inhibition of NSmase in arterioles from otherwise healthy adults induced a switch from NO to NOX-2 (NADPH-oxidase 2)-dependent H2O2-mediated flow-induced dilation. Endothelial dysfunction was prevented by treatment with sphingosine-1-phosphate (S1P) and partially prevented by C2-ceramide and an agonist of S1P-receptor 1 (S1PR1); the inhibition of the S1P/S1PR1 signaling axis induced endothelial dysfunction via NOX-2. Ceramide increased NO production in arterioles from non-CAD adults, an effect that was diminished with inhibition of S1P/S1PR1/S1P-receptor 3 signaling. In arterioles from patients with CAD, inhibition of NSmase impaired the overall ability to induce mitochondrial H2O2 production and subsequently dilate to flow, an effect not restored with exogenous S1P. Acute ceramide administration to arterioles from patients with CAD promoted H2O2 as opposed to NO production, an effect dependent on S1P-receptor 3 signaling. CONCLUSION:These data suggest that despite differential downstream signaling between health and disease, NSmase-mediated ceramide formation is necessary for proper functioning of the human microvascular endothelium. Therapeutic strategies that aim to significantly lower ceramide formation may prove detrimental to the microvasculature.

The loss of nitric oxide (NO)-mediated flow-induced dilation (FID) in the microvasculature, or microvascular endothelial dysfunction, is strongly linked to future cardiovascular events. Our lab has previously shown that chronic inhibition of sphingosine-1-phosphate (S1P) formation induces the change in FID mediator from the vasoprotective NO to the pro-inflammatory, pro-atherosclerotic hydrogen peroxide. The same transition is observed in arterioles from patients with coronary artery disease (CAD). Since S1P promotes NO-mediated FID, we hypothesized that formation of S1P and activation of S1P receptor 1 (S1PR1) is critical for FID in microvessels from healthy adults as opposed to arterioles from patients with CAD. Videomicroscopy was used to perform vascular function studies on human resistance arterioles (100-250μm) dissected from discarded surgical adipose tissue. The vessels were pre-constricted with endothelin-1 and changes in internal diameter in response to flow was measured. In arterioles from healthy adults, treatment with a sphingosine kinase (SpK) inhibitor for 30 minutes abolished FID (7.7±8.0% of maximal dilator capacity, n=3; mean±SEM) compared to control (73.1±8.2% n=9, p<0.05; *two-way ANOVA), whereas inhibition of S1PR1 appeared to delay the response to dilation (19.1±10.5% vs. 52.8±9.0% at 20mmHg pressure gradient, p<0.05*). In microvessels from patients with disease, both inhibition of SpK (91.8±2.8%, n=5) and S1PR1 (72.5±6.4%, n=6) did not affect FID compared to control (90.9±2.4%, n=4). Interestingly, S1PR1 expression was similar in CAD and nonCAD arterioles. Together these data suggest that formation of S1P and potentially the activation of S1PR1 is critical for FID during health, however this pathway does not contribute to FID during disease. We conclude that while arterioles can compensate for loss of this pathway in disease to maintain tissue perfusion, formation of S1P and activation of its receptors may be necessary to promote NO formation during shear and may serve as potential targets to prevent future cardiovascular disease.

Background Hypotension that is resistant to phenylephrine is a complication that occurs in anesthetized patients treated with angiotensin converting enzyme (ACE) inhibitors. We tested the hypothesis that Ang 1–7 and the endothelial Mas receptor contribute to vasodilation produced by propofol in the presence of captopril. Methods The internal diameters of human adipose resistance arterioles were measured before and after administration of phenylephrine (10 –9 to 10 –5 M) in the presence and absence of propofol (10 –6 M; added 10 min before the phenylephrine) or the Mas receptor antagonist A779 (10 –5 M; added 30 min before phenylephrine) in separate experimental groups. Additional groups of arterioles were incubated for 16 to 20 h with captopril (10 –2 M) or Ang 1–7 (10 –9 M) before experimentation with phenylephrine, propofol, and A779. Results Propofol blunted phenylephrine-induced vasoconstriction in normal vessels. Captopril pretreatment alone did not affect vasoconstriction, but the addition of propofol markedly attenuated the vasomotor response to phenylephrine. A779 alone did not affect vasoconstriction in normal vessels, but it restored vasoreactivity in arterioles pretreated with captopril and exposed to propofol. Ang 1–7 reduced the vasoconstriction in response to phenylephrine. Addition of propofol to Ang 1–7-pretreated vessels further depressed phenylephrine-induced vasoconstriction to an equivalent degree as the combination of captopril and propofol, but A779 partially reversed this effect. Conclusions Mas receptor activation by Ang 1–7 contributes to phenylephrine-resistant vasodilation in resistance arterioles pretreated with captopril and exposed to propofol. These data suggest an alternative mechanism by which refractory hypotension may occur in anesthetized patients treated with ACE inhibitors.

Background: Preclinical studies suggest that S1P (sphingosine-1-phosphate) influences blood pressure regulation primarily through NO-induced vasodilation. Because microvascular tone significantly contributes to mean arterial pressure, the mechanism of S1P on human resistance arterioles was investigated. We hypothesized that S1P induces NO-mediated vasodilation in human arterioles from adults without coronary artery disease (non–coronary artery disease) through activation of 2 receptors, S1PR 1 (S1P receptor 1) and S1PR 3 (S1P receptor 3). Furthermore, we tested whether this mechanism is altered in vessels from patients diagnosed with coronary artery disease. methods: Human arterioles (50–200 µm in luminal diameter) were dissected from otherwise discarded surgical adipose tissue, cannulated, and pressurized. Following equilibration, resistance vessels were preconstricted with ET-1 (endothelin-1) and changes in internal diameter to increasing concentrations of S1P (10-12 to 10-7 M) in the presence or absence of various inhibitors were measured. Results: S1P resulted in significant dilation that was abolished in vessels treated with S1PR 1 and S1PR 3 inhibitors and in vessels with reduced expression of each receptor. Dilation to S1P was significantly reduced in the presence of the NOS (NO synthase) inhibitor Nω-nitro-L-arginine methyl ester and the NO scavenger 2-4-(carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Interestingly, dilation was also significantly impaired in the presence of PEG-catalase (polyethylene glycol–catalase), apocynin, and specific inhibitors of NOX (NADPH oxidases) 2 and 4. Dilation in vessels from patients diagnosed with coronary artery disease was dependent on H 2 O 2 alone which was only dependent on S1PR 3 activation. Conclusions: These translational studies highlight the inter-species variation observed in vascular signaling and provide insight into the mechanism by which S1P regulates microvascular resistance and ultimately blood pressure in humans.

Chronic administration of exogenous adiponectin restores nitric oxide (NO) as the mediator of flow-induced dilation (FID) in arterioles collected from patients with coronary artery disease (CAD). Here we hypothesize that this effect as well as NO signaling during flow during health relies on activation of Adiponectin Receptor 1 (AdipoR1). We further posit that osmotin, a plant-derived protein and AdipoR1 activator, is capable of eliciting similar effects as adiponectin. Human arterioles (80–200 μm) collected from discarded surgical adipose specimens were cannulated, pressurized, and pre-constricted with endothelin-1 (ET-1). Changes in vessel internal diameters were measured during flow using videomicroscopy. Immunofluorescence was utilized to compare expression of AdipoR1 during both health and disease. Administration of exogenous adiponectin failed to restore NO-mediated FID in CAD arterioles treated with siRNA against AdipoR1 (siAdipoR1), compared to vessels treated with negative control siRNA. Osmotin treatment of arterioles from patients with CAD resulted in a partial restoration of NO as the mediator of FID, which was inhibited in arterioles with decreased expression of AdipoR1. Together these data highlight the critical role of AdipoR1 in adiponectin-induced NO signaling during shear. Further, osmotin may serve as a potential therapy to prevent microvascular endothelial dysfunction as well as restore endothelial homeostasis in patients with cardiovascular disease.

Endothelial dysfunction, or the loss of nitric oxide (NO)‐mediated dilation to shear stress (flow‐induced dilation; FID), is observed in human adipose arterioles from patients with coronary artery disease (CAD). FID is maintained during CAD by release of the mitochondrial‐derived, pro‐inflammatory dilator, H2O2. Ceramide, a sphingolipid that when elevated in plasma is an independent risk factor for future cardiac events, also induces this transition in human arterioles (10µM, 16‐20hrs). The initial events leading to ceramide‐induced endothelial dysfunction are unknown. Here we hypothesize that activation of NADPH oxidase 2 (NOX2), an endothelial membrane enzyme activated by acute stress, occurs prior to the formation of mitochondrial H2O2 in arterioles exposed to chronic ceramide. Human arterioles (100‐250µm in diameter) were dissected from discarded surgical adipose tissues and prepared for videomicroscopy. Following preconstriction with endothelin‐1, internal diameters were measured in response to increased flow. To determine the minimum exposure time necessary to convert to H2O2‐dependent FID in arterioles exposed to ceramide, microvessels from healthy nonCAD adults were incubated with C2 ceramide for 30 min, 2 hrs, and 4 hrs. FID remained mediated by NO at 30min and 2hrs, however, following a 4hr incubation, FID was significantly impaired in the presence of PEG‐catalase (44.6% of maximal dilator capacity ±13.8 (SEM), n=8, p<0.05, one‐way ANOVA*) compared to vehicle control (83.5%±5.0, n=8). The source of H2O2 generated during FID in arterioles treated for 4hr with ceramide was not mitochondrial as increases in fluorescent intensity of mito peroxy yellow 1 (mitoPY1) were not observed during maximal flow. To test whether NOX2 is the initial source of H2O2 during FID in arterioles exposed to 4hr ceramide, nonCAD arterioles were first treated with the NOX2 inhibitor GSK2795039 (NOX2i, 10‐6 M) or underwent intraluminal administration of siRNA to decrease NOX2 expression prior to treatment with ceramide. FID was reduced in vessels treated with both ceramide and the NOX2 inhibitor in the presence of cPTIO (NO scavenger) (50.2%±30.1, n=4* vs. NOX2 inhibitor and ceramide alone 80.8%±6.4, n=7). L‐NAME also impaired FID in vessels with reduced expression of NOX2 and subsequently treated with ceramide (10.8%±38.9, n=3* compared to siNOX2 and ceramide alone 85.5%±5.8, n=3). This suggests that activation of NOX2 may be the initial event in the conversion of NO‐ to H2O2‐dependent FID due to ceramide and may serve as a potential therapeutic target in those at risk for developing cardiovascular disease due to increased plasma ceramide.

The loss of nitric oxide (NO)-mediated flow-induced dilation (FID) in the microvasculature, or microvascular endothelial dysfunction, is strongly linked to future cardiovascular events. Our lab has previously shown that chronic inhibition of sphingosine-1-phosphate (S1P) formation induces the change in FID mediator from the vasoprotective NO to the pro-inflammatory, pro-atherosclerotic hydrogen peroxide. The same transition is observed in arterioles from patients with coronary artery disease (CAD). Since S1P promotes NO-mediated FID, we hypothesized that formation of S1P and activation of S1P receptor 1 (S1PR1) is critical for FID in microvessels from healthy adults as opposed to arterioles from patients with CAD. Videomicroscopy was used to perform vascular function studies on human resistance arterioles (100-250μm) dissected from discarded surgical adipose tissue. The vessels were pre-constricted with endothelin-1 and changes in internal diameter in response to flow was measured. In arterioles from healthy adults, treatment with a sphingosine kinase (SpK) inhibitor for 30 minutes abolished FID (7.7±8.0% of maximal dilator capacity, n=3; mean±SEM) compared to control (73.1±8.2% n=9, p<0.05; *two-way ANOVA), whereas inhibition of S1PR1 appeared to delay the response to dilation (19.1±10.5% vs. 52.8±9.0% at 20mmHg pressure gradient, p<0.05*). In microvessels from patients with disease, both inhibition of SpK (91.8±2.8%, n=5) and S1PR1 (72.5±6.4%, n=6) did not affect FID compared to control (90.9±2.4%, n=4). Interestingly, S1PR1 expression was similar in CAD and nonCAD arterioles. Together these data suggest that formation of S1P and potentially the activation of S1PR1 is critical for FID during health, however this pathway does not contribute to FID during disease. We conclude that while arterioles can compensate for loss of this pathway in disease to maintain tissue perfusion, formation of S1P and activation of its receptors may be necessary to promote NO formation during shear and may serve as potential targets to prevent future cardiovascular disease.

The sphingolipid sphingosine-1-phosphate (S1P) has emerged as a regulator of microvascular tone in animals, however its role in the human microcirculation remains unknown. Pre-clinical studies suggest that S1P-induced activation of two endothelial-specific receptors, S1PR1 and S1PR3,elicits nitric-oxide (NO)-mediated vasodilation. As opposed to S1PR1, the S1PR3 pathway also activates NADPH oxidase (NOX) to produce reactive oxygen species (ROS). We therefore hypothesized that S1P induces vasodilation in the human microvasculature through activation of S1PR1 and S1PR3 in both a ROS-independent and ROS-dependent manner, respectively. Human microvessels (100-200µm in diameter) were prepared for videomicroscopy. Following equilibration, arterioles were pre-constricted with endothelin-1 to 30-70% of their passive diameters. Luminal diameter was measured and recorded in regular intervals (1 min) in response to increasing concentrations of S1P (10-12 to 10-6 M) in the presence or absence of the S1PR1 receptor antagonist W146 (10-5M), S1PR3 antagonist CAY10444 (10-5 M), nitric oxide synthase inhibitor Nω-nitro-ʟ-arginine (L-NAME, 10-4 M), NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO, 10-4 M), polyethylene glycol-catalase (peg-Cat, 500U/ml), NADPH oxidase inhibitor apocynin (3x10-4 M), NOX-2 inhibitor GSK2795039 (10-6 M) or the NOX-4 inhibitor GKT137831 (10−6 M). S1P induced vasodilation in a dose-responsive manner with a maximum dilation of 56.5%±4.9, n=12 (mean±SEM). Dilation was abolished during inhibition of S1PR1 (1.5%±5.2, n=4) and was reduced during inhibition of S1PR3 (19.7%±8.7, n=6). Both L-NAME and c-PTIO inhibited S1P-induced dilation (13.0%±7.5, n=4 and 11.2%±3.3, n=4, respectively). Interestingly, dilation was nearly completely inhibited by peg-Cat (11%±6.6, n=4), apocynin (12.5%±4.4, n=3) and the NOX-4 inhibitor (7.2%±3.1, n=4). Dilation was also partly reduced during inhibition of NOX-2 (25.6%±7.8, n=4). These data suggest that S1P-induced dilation occurs through activation of S1PR1 and S1PR3 through formation of NO and NOX-4-generated H2O2. These translational studies highlight the inter-species variation observed in vascular signaling and provide insight into the mechanism by which S1P regulates microvascular resistance in humans.

An important measure of microvascular endothelial function is flow-induced dilation (FID), the ability of an arteriole to dilate in response to increased flow. In healthy adults, FID is predominantly mediated by endothelial-derived nitric oxide (NO), a vasoactive compound with anti-inflammatory properties. In microvessels from patients with coronary artery disease (CAD), FID occurs via formation and release of hydrogen peroxide (H2O2), a pro-inflammatory vasoactive mediator. We have previously shown that exogenous adiponectin restores NO-mediated FID in CAD arterioles, however the mechanism remains unknown. Because AMP kinase (AMPK), a downstream target of adiponectin receptors, increases cellular NO, we hypothesized that inhibition of AMPK would promote H2O2 as the mediator of FID in arterioles from healthy, nonCAD subjects. Human microvessels (80-200µm) were dissected from discarded surgical adipose specimens, cannulated onto micropipettes and pressurized. Arterioles were pre-constricted with endothelin-1 and changes in vessel wall diameter were measured during graded increases in flow using videomicroscopy. FID was observed in healthy arterioles treated with the AMPK inhibitor, Compound C (1µM, 16hrs) in the presence of the NO scavenger c-PTIO (100µM) (77.9 % max dilation ± 6, n=3), as well as with the H2O2 degradation enzyme PEG-Catalase (500U), (62.4% max dilation ±15, n=3). Interestingly, FID was suppressed only in the presence of both c-PTIO and PEG-Catalase (42.1% max dilation ±16, n=3) compared to controls (69.4% max dilation ±6, n=3). Together, these data may suggest that NO and H2O2 compensate for loss of one another during inhibition of AMPK. Future studies will investigate whether adiponectin-induced restoration of NO requires activation of AMPK as thorough understanding of this pathway during FID is critical for understanding the mechanism by which adiponectin improves microvascular endothelial function during disease.

Increased plasma ceramide levels and microvascular dysfunction are both independent risk factors for major adverse cardiac events (MACE). We have previously shown that chronic exposure to exogenous ceramide promotes microvascular endothelial dysfunction, defined as hydrogen peroxide (H2O2)‐mediated flow‐induced dilation (FID) as opposed to dilation due to formation and release of endothelial nitric oxide (NO). Interestingly, ceramide and its metabolites (e.g. sphingosine‐1‐phosphate; S1P) have also been shown to stimulate production of NO. Our previous studies indicate that activation of the ceramide‐producing enzyme neutral sphingomyelinase (NSmase) in necessary for NO‐mediated FID. We therefore hypothesized that formation of S1P is responsible for cellular increases in NO from both acute exogenous administration as well as endogenous formation of ceramide from shear. Human resistance arterioles (100‐200µm) were dissected from adipose tissue collected from healthy patients and were prepared for videomicroscopy. Increasing doses of exogenous C2 ceramide (10‐9 to 10‐5 M) were administered in the absence or presence of the NO scavenger c‐PTIO (1µM) and a sphingosine kinase inhibitor (SpKi, 5µM). We observed a dose‐response increase in arteriolar dilation from ceramide (46.9%±10.3 of maximal dilator capacity±SEM, n=3) that was impaired by c‐PTIO (15%±6.0, n=3). Ceramide‐induced dilation was also decreased in the presence of the SpKi (20.8%±4.0, n=3). To examine the role of S1P formation in NO‐mediated FID, SpKi was administered to healthy arterioles (5µM, 30 min) prior to initiating flow (pressure gradient 5‐100cm H2O) and a dramatic decrease in overall dilation was observed (7.7%±8.0, n=3). Together these findings suggest that NO generated from ceramide is primarily due to the formation of S1P, a process also critical to maintain FID in arterioles from healthy individuals.

The microvasculature is increasingly recognized as a major contributor to many cardiovascular diseases including atherosclerosis and heart failure with preserved ejection fraction (HFpEF). Endothelial microvascular dysfunction, the inability to vasodilate to endothelial-dependent agonists, precedes the development of large artery disease. While microvascular (dys)function can be assessed using invasive techniques (e.g., cardiac catheterization) there are limited strategies in which to assess microvascular function in a non-invasive and cost-effective manner. Recent advances in hand-held vital microscopy allow for reliable direct imaging of the sublingual microcirculation at bedside. One of the latest advancements (Cytocam, Braedius Medical) utilizes state of the art incident dark field (IDF) technology to allow for quantification of total vascular density (TVD). We therefore hypothesized an increase in TVD in response to both an endothelial-dependent, and -independent vasodilator. Cytocam-IDF was optimized to measure in vivo sublingual microvascular response to topical application of the endothelial-independent vasodilator nitroglycerin (NTG) (0.3mg) and the endothelial-dependent dilator acetylcholine (5.5 ·10-2 M). Five images were taken at baseline. Immediately following topical administration of acetylcholine, an additional 5 images were taken to compare TVD pre- and post-treatment. The area was washed and after 30 min of rest, a NTG tablet was administered sublingually and additional 5 images were obtained. Massey's microcirculation image quality score was applied, and TVD was measured using the De Backer score manual analysis by two separate investigators to account for interobserver variability, with consensus achieved. Both NTG (12.6 mm/mm2± 0.5, n=7 compared to baseline 9.9 mm/mm2± 0.42, n=7) (Mean±SEM) and acetylcholine (13.1 mm/mm2± 0.3, n=3 compared to baseline 10.4 mm/mm2± 0.5, n=3) increased TVD. These data suggest that measurement of TVD in response to endothelium-specific pharmacological agonists may allow for assessment of the human systemic microvasculature in vivo. This is a promising strategy that may allow for early detection of endothelial microvascular dysfunction prior to the onset of large artery disease or heart failure.

Flow induced dilation (FID) is a critical physiological mechanism to maintain tissue perfusion and modulate vascular homeostasis. Recently our laboratory has shown that sphingolipids can influence the endothelial‐derived mediator that is generated in response to flow. Exposure to elevated levels of ceramide, or, inhibition of ceramide metabolism, induces a transition in endothelial‐derived FID mediator from nitric oxide (NO) to hydrogen peroxide (H2O2), the same change in mechanism that occurs with presence of CAD. Interestingly, studies have suggested that endothelial nitric oxide synthase (eNOS) is regulated by ceramide metabolites, such as sphingosine‐1‐phosphate (S1P), suggesting that ceramide is necessary for NO formation due to shear stress. We therefore hypothesized that activation of the shear sensitive, ceramide‐producing enzyme, neutral sphingomyelinase (NSmase) is required for NO‐dependent FID. Resistance arterioles (100–200μm) were dissected from adipose tissue collected from healthy patients and were prepared for videomicroscopy. Following equilibration and preconstriction with endothelin‐1, changes in internal diameter to flow were recorded. L‐NAME (100mM) did not affect FID in healthy arterioles pre‐treated with the NSmase inhibitor, GW4869 (4mM, 16–20hrs), (69.1%±4.9 of maximal dilator capacity, n=4), however dilation was decreased in the presence of PEG‐Catalase (500 Units/ml) (15.2%±9.4, n=3). A similar effect was observed with acute inhibition (30 min) of NSmase. Vasodilation due to increased flow was maintained in the presence of L‐NAME (88.0%±3.5, n=3), however was impaired when exposed to PEG‐Catalase (−5.3%±1.3, n=3). GW4869 alone did not impair the overall magnitude of dilation due to flow (86.1%±3.9, n=2). These findings suggest that while elevated levels of ceramide promote H2O2‐dependent FID, NSmase‐derived products of ceramide are a key component of NO‐mediated signaling in the human microvasculature.Support or Funding InformationK08HL141562

Precise regulation of vascular resistance is critical to maintain proper tissue perfusion. Recent data in animal models have shown that sphingolipids, a ubiquitous class of bioactive lipid messengers, can influence vasomotor tone via dilation or constriction. It has been suggested that sphingosine‐1‐phosphate (S1P) elicits vasodilation through generation of endothelial cell nitric oxide (NO) via activation of sphingosine‐1‐phosphate receptor 1 (S1PR1). We therefore examined the hypothesis that S1P is a regulator of human microvascular tone and elicits dilation via S1PR1‐induced increase in NO. Arterioles from healthy human adipose tissue were dissected and prepared for videomicroscopy, equilibrated and pre‐constricted with endothelin 1. Changes in arteriolar luminal diameter were recorded in response to increasing concentrations of S1P (10−12 to 10−6M) in the presence or absence of the S1PR1 receptor antagonist W146 (10−5M), nitric oxide synthase (NOS) inhibitor Nω‐nitro‐ l‐arginine (L‐NAME, 10−4M) or hydrogen peroxide (H2O2) scavenger polyethylene glycol‐catalase (peg‐Cat, 500U/ml). Administration of exogenous S1P induced vasodilation in healthy vessels to a maximal dilation of 63.7%±5.0, n=8 (mean±SEM). As with previous animal studies, vascular constriction was observed with higher, but still physiological levels of S1P (10−6 M). Vasodilation due to S1P was reduced following inhibition of S1PR1 compared to S1P alone (1.8%±4, n=5). In the presence of L‐NAME, S1P‐induced dilation decreased (9%±7.9, n=6). Interestingly, addition of peg‐Cat, an H2O2 scavenger, also decreased S1P‐induced dilation (11.1%±6.6, n=3). Together these data suggest that S1PR1 is a critical mechanistic component of S1P‐induced vasodilation in the human vasculature. Further, the S1P vasodilatory pathway may require formation of H2O2 in addition to NO.Support or Funding InformationThis research was supported by National Institute of Health (NHLBI) K08 HL141562‐02 (JKF)

Flow‐induced dilation (FID), the ability of an arteriole to dilate in response to increased flow, is primarily mediated by endothelial‐derived nitric oxide (NO) in healthy individuals, however is replaced by hydrogen peroxide (H2O2) in patients with coronary artery disease (CAD). Our laboratory has shown that adiponectin restores NO‐mediated FID in microvessels from patients with CAD. Adiponectin activates multiple signaling pathways that promote NO formation, including activation of neutral ceramidase to hydrolyze ceramide, a sphingolipid that in elevated amounts promotes FID mediated by H2O2. Our prior work has shown that both NO and H2O2 contribute as mediators of FID in diseased vessels treated with adiponectin and ceranib‐1, an inhibitor of neutral ceramidase. As adiponectin is also capable of activating AMP kinase (AMPK) to increase cellular NO levels, we tested the hypothesis that AMPK activation is required to restore NO‐mediated FID in diseased arterioles. Human microvessels (80–200μm) were dissected from adipose tissue collected from patients diagnosed with CAD. Following cannulation and equilibration, vessels were constricted with endothelin‐1. Using videomicroscopy, vessel diameters were measured in response to graded increases in intraluminal flow. To investigate whether activation of AMPK is a key mechanistic step in adiponectin‐induced restoration of NO‐mediated signaling, vessels from patients diagnosed with CAD were first treated with the AMPK inhibitor Compound C (1μM, 4hrs) followed by adiponectin treatment (2μg/mL, 16–20hrs). Dilation to flow was maintained during exposure to the nitric oxide synthase (NOS) inhibitor L‐NAME (100μM) (69.8% max dilation±2.7, n=2) as well as in the presence of the PEG‐Catalase (500U) (64.5%±21.2, n=4) compared to compound C and adiponectin alone (61.0%±9.4, n=4). To determine whether ceramide hydrolysis via neutral ceramidase contributes to the maintained dilation despite NOS inhibition, FID was also assessed in diseased arterioles treated with both compound C and ceranib‐1 prior to treatment with adiponectin. Both NO and H2O2 contributed to dilation under both AMPK and neutral ceramidase inhibition as dilation was observed in the presence of L‐NAME (33.6%±15.9, n=3) or PEG‐Catalase (51.7%±23.9, n=3). However, FID was decreased during exposure to both L‐NAME and PEG‐Catalase (11.6%±12.4, n=2). These preliminary data suggest that adiponectin promotes NO formation through multiple pathways and contributes to the redundancy of endothelial‐derived mediators in order to maintain FID.Support or Funding InformationThis research was supported by National Institute of Health (NHLBI) K08HL141562 (J.K.F.) and National Institute of Health 5R38HL143561‐02 (M.E.W.).

The membrane deforming dynamin family members MxA and MxB are large GTPases that convey resistance to a variety of infectious viruses. During viral infection, Mx proteins are known to show markedly increased expression via an interferon-responsive promoter to associate with nuclear pores. In this study we report that MxB is an inner mitochondrial membrane GTPase that plays an important role in the morphology and function of this organelle. Expression of mutant MxB or siRNA knockdown of MxB leads to fragmented mitochondria with disrupted inner membranes that are unable to maintain a proton gradient, while expelling their nucleoid-based genome into the cytoplasm. These findings implicate a dynamin family member in mitochondrial-based changes frequently observed during an interferon-based, anti-viral response.

Background-Elevated 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 Results-Using 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. Conclusions-Sphingolipid 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.