Ceramides, a group of biologically active sphingolipids, have been described as the new cholesterol given strong evidence linking high plasma ceramide with endothelial damage, risk for early adverse cardiovascular events, and development of cardiometabolic disease. This relationship has sparked great interest in investigating therapeutic targets with the goal of suppressing ceramide formation. However, the growing data challenge this paradigm of ceramide as solely eliciting detrimental effects to the cardiovascular system. Studies show that ceramides are necessary for maintaining proper endothelial redox states, mechanosensation, and membrane integrity. Recent work in preclinical models and isolated human microvessels highlights that the loss of ceramide formation can in fact propagate vascular endothelial dysfunction. Here, we delve into these conflicting findings to evaluate how ceramide may be capable of exerting both beneficial and damaging effects within the vascular endothelium. We propose a unifying theory that while basal levels of ceramide in response to physiological stimuli are required for the production of vasoprotective metabolites such as S1P (sphingosine-1-phosphate), the chronic accumulation of ceramide can promote activation of pro-oxidative stress pathways in endothelial cells. Clinically, the evidence discussed here highlights the potential challenges associated with therapeutic suppression of ceramide formation as a means of reducing cardiovascular disease risk.

To our knowledge, this is the first study to comprehensively characterize in vivo sublingual microvascular structure and function (endothelium-dependent and -independent) in healthy patients and those with CVD. Importantly, we used an easy-to-use handheld device that can be easily translated to clinical settings. Our results indicate that baseline microvascular impairments in structure and function can be detected using the CytoCam technology, although reactivity to acetylcholine may be maintained even during disease in the peripheral microcirculation.

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.

Premenopausal women have a lower incidence of cardiovascular disease (CVD) compared with their age-matched male counterparts; however, this discrepancy is abolished following the transition to menopause or during low estrogen states. This, combined with a large amount of basic and preclinical data indicating that estrogen is vasculoprotective, supports the concept that hormone therapy could improve cardiovascular health. However, clinical outcomes in individuals undergoing estrogen treatment have been highly variable, challenging the current paradigm regarding the role of estrogen in the fight against heart disease. Increased risk for CVD correlates with long-term oral contraceptive use, hormone replacement therapy in older, postmenopausal cisgender females, and gender affirmation treatment for transgender females. Vascular endothelial dysfunction serves as a nidus for the development of many cardiovascular diseases and is highly predictive of future CVD risk. Despite preclinical studies indicating that estrogen promotes a quiescent, functional endothelium, it still remains unclear why these observations do not translate to improved CVD outcomes. The goal of this review is to explore our current understanding of the effect of estrogen on the vasculature, with a focus on endothelial health. Following a discussion regarding the influence of estrogen on large and small artery function, critical knowledge gaps are identified. Finally, novel mechanisms and hypotheses are presented that may explain the lack of cardiovascular benefit in unique patient populations.

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.

Radiation therapy (RT) is a commonly used treatment for thoracic cancers. However, the risk of coronary events increase by 4-16% per Gy of heart dose. Our prior studies show that localized heart RT leads to radiation-induced heart disease (RIHD) and increased mortality in Dahl Salt Sensitive (SS) rats starting at 3mo post-RT. We have also shown that whole thoracic RT increases susceptibility to ischemia-reperfusion (IR) injury in Wistar rats. Taken together, we hypothesized that 10weeks after targeted cardiac RT (prior to RIHD) SS rat hearts would have worsened cardiac function as measured by echocardiography and be more susceptible to IR injury. Echocardiograms were performed 10weeks after 24Gy dose of CT-guided localized cardiac RT and in age-matched controls (C). Next, hearts were isolated and perfused ex vivo using the Langendorf method - without ischemia reperfusion (time controls; TC; n=3 in C and RT) or exposed to 25min of global ischemia followed by 60min reperfusion (IR; n=6 in C and RT). During TC and IR, cardiac function, mitochondrial redox state, and vascular reactivity were assessed; the hearts were then sectioned and stained to assess infarct size. Echocardiography of the RT group when compared to C showed an increase in interventricular septal thickness (1.32±0.1 vs 0.78±0.07 cm, p<0.0001; mean±SD) and left ventricular posterior wall thickness (1.03±0.08 vs 0.75±0.06 cm, p<0.005). Accordingly, a decrease in end diastolic (1.79±0.46 vs 2.38±0.35 ml/kg, p<0.05) and end systolic (0.10±0.04 vs 0.23±0.08 ml/kg, p<0.05) volume was also seen, with no significant differences in ejection fraction (94.5±2.1 vs 90.3±3.9 %) or stroke volume (1.69±0.44 vs 2.15±0.27 ml; p>0.05). Interestingly, in the ex vivo perfused hearts, TC RT had a 2 times higher rate-pressure product - a measure of cardiac work - compared to C (p<0.05), and following IR, the RT group recovered to 2.5x higher levels to C (p<0.0001). Following IR, the RT group compared to C also showed a greater recovery of coronary flow rate (1.6x, p<0.05) and rates of contractility (2.25x) and relaxation (lusitropy; 2.05x) (p<0.0001). No difference in mitochondrial redox state, infarct size, and endothelium-dependent and independent vascular reactivity were seen between the groups. In conclusion, our results show for the first time that 10weeks post-targeted cardiac RT, ex vivo hearts show better recovery and cardiac function compared to C after IR through mechanisms other than differences in redox state and vascular reactivity.

Our previous work has shown that chronic exposure to ceramide, a sphingolipid that when elevated in plasma is an independent risk factor major adverse cardiac events, causes microvascular endothelial dysfunction in arterioles collected from healthy adult patients. This presents as a change in the mediator of flow-induced dilation (FID) from the vasoprotective nitric oxide (NO) to the pro-atherosclerotic hydrogen peroxide (H 2 O 2 ). Despite the known detrimental effects of ceramide, its metabolite sphingosine-1-phosphate (S1P) can promote NO formation. However, shear-induced ceramide formation is also necessary for maintaining NO-mediated FID, as arterioles from healthy individuals transition to H 2 O 2 -mediated FID during inhibition of the ceramide-forming enzyme neutral sphingomyelinase (NSmase). We hypothesize that the transition in mediator is due to the loss of acute S1P production, and thus addition of exogenous S1P can prevent microvascular endothelial dysfunction during inhibition of NSmase. Human arterioles (100-250μm) were dissected from otherwise discarded adipose tissue from healthy patients undergoing surgery. Videomicroscopy was used to assess vascular function in vitro . Microvessels were pre-constricted with endothelin-1, and changes in internal diameter were measured following exposure to increased levels of flow. Dilation to flow was significantly impaired in the presence of the NO-synthase inhibitor L-NAME (100μM, 30 min) when healthy human arterioles were treated acutely with S1P (1μM, 30 min) in the presence of the NSmase inhibitor, GW4869 (10μM, 30 min), compared to GW4859 alone (% maximal diameter±SEM, 5.1±8.7, n=4 vs 70.1±5.3, n=4; p=0.001, 2-way ANOVA). Whereas, the presence of PEG-catalase, an enzyme that breaks down H 2 O 2 , had no effect (85.0±4.3; n=3). These data highlight the importance of S1P in maintaining NO signaling during exposure to shear and strengthens the concept that the conversion of ceramide to S1P is critical in promoting a quiescent endothelium.

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.

Cardiovascular disease (CVD) risk increases with estrogen (E2) deficiency as well as during the transition to menopause, suggesting that estrogen is protective against CVD. On the contrary, increases in plasma E2 from contraception in cis‐females or hormone therapy (HT) in trans‐females (male‐to‐female transition) is also associated with increased CVD. Microvascular endothelial dysfunction, or the loss of nitric oxide (NO)‐mediated dilation in response to flow (flow‐induced dilation; FID), precedes large artery disease and is predictive of future cardiac events. We hypothesize that the effect of E2 on human microvascular endothelial function is hormetic with low or elevated E2 promoting dysfunction. We further posit that the effect of estrogen on the microvascular endothelium is influenced by both age and sex. Small adipose arterioles (100‐350µm) from healthy adults (0‐1 risk factors for coronary artery disease) were prepared for videomicroscopy and constricted with endothelin‐1 prior to measuring changes in internal diameters in response to flow. Historical data was analyzed to examine changes in FID over time in healthy women, and it showed that FID is maintained over time (75.4%±4.2 of maximal dilator capacity±SEM, n=26 age<40yrs; 75.39%±3.0 age>40yrs). L‐NAME significantly inhibited FID in healthy women <40yrs of age (30.6%±9.9 n=17; p<0.0005, two‐way ANOVA*) compared to control (75.4%±4.2, n=26), however this effect was abolished when incubated with E2 (100nM, 16‐20hrs) (71.2%±10.3, n=5 vs. control 84.7%±3.8, n=5). L‐NAME also decreased FID in arterioles from women 40‐60yrs of age (46.9%±9.8, n=19) compared to control (81.3%±2.6, n=37, p<0.005), however there was a greater amount of residual dilation compared to women <40yrs. E2 also resulted in a loss of NO‐mediated FID in women >40yrs as L‐NAME had no effect (82.9%±4.3, n=4 vs. control 72.6%±8.8, n=5). In biological males, FID appears to be reduced in microvessels after E2 treatment regardless of age (52.1%±5.1 n=2, age<40yrs; ‐2.7%±9.5, n=3, age>40yrs). Together these data suggest that exposure to elevated levels of E2 may induce microvascular endothelial dysfunction in adult women regardless of age and appears to be more damaging in biological males. We conclude that elevated levels of E2 are detrimental to the microvasculature and may increase CVD risk in young women taking oral contraceptives as well as older women and trans‐females undergoing HT.

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.

Cardiovascular disease risk increases with age regardless of sex. Some of this risk is attributable to alterations in natural hormones throughout the life span. The quintessential example of this being the dramatic increase in cardiovascular disease following the transition to menopause. Plasma levels of adiponectin, a "cardioprotective" adipokine released primarily by adipose tissue and regulated by hormones, also fluctuate throughout one's life. Plasma adiponectin levels increase with age in both men and women, with higher levels in both pre- and postmenopausal women compared with men. Younger cohorts seem to confer cardioprotective benefits from increased adiponectin levels yet elevated levels in the elderly and those with existing heart disease are associated with poor cardiovascular outcomes. Here, we review the most recent data regarding adiponectin signaling in the vasculature, highlight the differences observed between the sexes, and shed light on the apparent paradox regarding increased cardiovascular disease risk despite rising plasma adiponectin levels over time.

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.

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.