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

Abstract

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.

Keywords: adiponectin; flow‐induced dilation; microvascular dysfunction; sphingosine‐1‐phosphate.

Figure 1

Inhibition of neutral ceramidase (NCdase) drives dilation towards hydrogen peroxide in non–coronary artery disease (non‐CAD) arterioles. A, Flow‐induced dilation is impaired in non‐CAD arterioles exposed to N^ω‐nitro‐ʟ‐arginine (l‐NAME) (n=7), whereas incubation with the NCdase inhibitor Ceranib‐1 (10⁻⁵ mol/L, 16–20 hours) did not affect the magnitude of dilation compared with vehicle (n=6 and n=7, respectively). B, The response to flow was impaired in non‐CAD vessels incubated with Ceranib‐1 in the presence of polyethylene glycol‐catalase (PEG‐catalase) (500 U) (n=7), whereas l‐NAME had no effect (n=7). *P<0.05 vs vehicle for curve mean (A) or at specific pressure gradients (B).

Figure 2

Neutral ceramidase (NCdase) expression in human arterioles. Representative images from 3 patients (3 non–coronary artery disease [non‐CAD], 3 coronary artery disease [CAD], and 3 CAD+adenovirus with promoters for GFP and NCdase [Ad‐GFP‐NCdase]). Areas of the endothelium (box) are magnified to show staining specifically within the endothelial layer (red arrow). The area of staining is increased in non‐CAD (A) compared with CAD arterioles (B). Following overnight administration of Ad‐GFP‐NCdase, the total amount of staining is increased (C) compared with a CAD control vessel (B). Primary antibody was removed to determine specificity (D). Bar=40 μm.

Figure 3

Overexpression of neutral ceramidase (NCdase) restores nitric oxide–dependent flow‐induced dilation (FID) in diseased arterioles. A, Treatment with either adenovirus with promoters for GFP (Ad‐GFP) (n=7), or Ad‐NCdase‐GFP (n=8), has no effect on dilation in response to flow compared with vehicle (n=7). B, Exposure to N^ω‐nitro‐ʟ‐arginine (l‐NAME) has no effect on FID in arterioles collected from patients with coronary artery disease (CAD) that have been treated with Ad‐GFP alone (n=7) compared with vehicle (n=7), whereas the response to flow is impaired following exposure to polyethylene glycol‐catalase (PEG‐catalase) (n=10). C, FID is impaired in CAD microvessels treated with Ad‐NCdase‐GFP following incubation with l‐NAME (n=7), whereas PEG‐catalase has no effect (n=7) compared with Ad‐NCdase‐GFP alone (n=8). *P<0.05 for response curve averages.

Figure 4

Effect of sphingosine‐1‐phosphate (S1P) on the mediator of flow‐induced dilation (FID). A, Non–coronary artery disease (non‐CAD) arterioles incubated with the sphingosine kinase (SpK) inhibitor (5×10⁻⁶ mol/L, 16–20 hours) reached normal dilation capacity compared with vehicle (n=7 and n=7, respectively). B, The response to flow was inhibited in non‐CAD arterioles incubated with SpK inhibitor overnight in the presence of polyethylene glycol‐catalase (PEG‐catalase) (500 U) (n=7) compared with SpK inhibitor alone (n=7), whereas N^ω‐nitro‐ʟ‐arginine (l‐NAME) had no effect (n=7). C, Dilation was abolished in arterioles from diseased patients following exposure to PEG‐catalase (n=6) compared with vehicle (n=6). CAD arterioles incubated with S1P (10⁻⁶ mol/L, 16–20 hours) had no effect on FID compared with vehicle (n=6 and n=8, respectively). D, CAD arterioles incubated with S1P in the presence of l‐NAME had impaired dilation, whereas PEG‐catalase had no effect (n=8 and n=7, respectively) compared with S1P alone (n=8). *P<0.05 for curve averages (A) or at specific pressure gradients (B through D).

Figure 5

Exogenous adiponectin or activation of adiponectin receptors restores nitric oxide–dependent flow‐induced dilation in microvessels from diseased patients. A, Coronary artery disease (CAD) arterioles treated with adiponectin (2 μg/mL, 16–20 hours) did not alter the dilation capacity of the vessel in response to flow compared with vehicle (n=9 and n=6, respectively). B, Dilation to flow was inhibited in CAD vessels incubated with adiponectin in the presence of N^ω‐nitro‐ʟ‐arginine (l‐NAME) (n=6) compared with adiponectin alone (n=6), whereas polyethylene glycol‐catalase (PEG‐catalase) had no effect. C, Vessels from patients with CAD treated with AdipoRon (5×10⁻⁶ mol/L, 16–20 hours) dilated normally in response to flow compared with vehicle (n=7 and n=6, respectively). D, Dilation to flow was impaired in CAD vessels first treated with AdipoRon in the presence of l‐NAME, whereas PEG‐catalase had no effect (n=6 and n=7, respectively) compared with AdipoRon alone (n=7). *P<0.05 for specific pressure gradients.

Figure 6

Effect of adiponectin administration with concurrent neutral ceramidase (NCdase) inhibition on the mediator of flow‐induced dilation (FID). A, Microvessels from patients with coronary artery disease (CAD) maintained dilator capacity following exposure to Ceranib‐1 for 4 hours followed by administration of adiponectin (n=6) compared with vehicle (n=6). B, Neither N^ω‐nitro‐ʟ‐arginine (l‐NAME) (n=8) nor polyethylene glycol‐catalase (PEG‐catalase) (n=6) impaired FID in CAD arterioles treated with both Ceranib‐1 and adiponectin; however, dilation was significantly decreased in the presence of both drugs (n=6) compared with vehicle. *P<0.05 at specific pressure gradients.

Figure 7

Schematic illustration highlighting the effect of the sphingolipid pathway on the mediator of flow‐induced dilation (FID). Shear stress from increased blood flow results in the formation and release of nitric oxide (NO) or hydrogen peroxide (H₂O₂) in adults without coronary artery disease (CAD) versus those with CAD, respectively. Inhibition of neutral ceramidase (NCdase) favors ceramide accumulation favoring FID mediated by H₂O_2. Likewise, preventing formation of intracellular sphingosine‐1‐phosphate (S1P) through inhibition of sphingosine kinase (SpK) also results in H₂O₂‐dependent dilation in vessels from patients without CAD. Overexpression of NCdase, exogenous S1P, or adiponectin, or activation of adiponectin receptors, restores NO‐dependent dilation in arterioles from patients with CAD. Adiponectin, or the nonselective adiponectin receptors 1 and 2 (AdipoR1/R2) agonist AdipoRon, is capable of either activating NCdase or has intrinsic ceramidase activity allowing for the hydrolysis of ceramide.