Ceramides in peripheral arterial plaque lead to endothelial cell dysfunction

Abstract

Background: Peripheral arterial atheroprogression is increasingly prevalent, and is a risk factor for major limb amputations in individuals with risk factors such as diabetes. We previously demonstrated that bioactive lipids are significantly altered in arterial tissue of individuals with diabetes and advanced peripheral arterial disease.

Methods: Here we evaluated whether sphingolipid ceramide 18:1/16:0 (C16) is a cellular regulator in endothelial cells and peripheral tibial arterial tissue in individuals with diabetes.

Results: We observed that C16 is the single most elevated ceramide in peripheral arterial tissue from below the knee in individuals with diabetes (11% increase, P < .05). C16 content in tibial arterial tissue positively correlates with sphingomyelin (SPM) content in patients with and without diabetes (r² = 0.5, P < .005; r² = 0.17, P < .05; respectively). Tibial arteries of individuals with diabetes demonstrated no difference in CERS6 expression (encoding ceramide synthase 6; the predominate ceramide synthesis enzyme), but higher SMPD expression (encoding sphingomyelin phosphodiesterase that catalyzes ceramide synthesis from sphingomyelins; P < .05). SMPD4, but not SMPD2, was particularly elevated in maximally diseased (Max) tibial arterial segments (P < .05). In vitro, exogenous C16 caused endothelial cells (HUVECs) to have decreased proliferation (P < .03), increased apoptosis (P < .003), and decreased autophagy (P < .008). Selective knockdown of SMPD2 and SMPD4 decreased native production of C16 (P < .01 and P < .001, respectively), but only knockdown of SMPD4 rescued cellular proliferation (P < .005) following exogenous supplementation with C16.

Conclusions: Our findings suggest that C16 is a tissue biomarker for peripheral arterial disease severity in the setting of diabetes, and can impact endothelial cell viability and function.

Clinical relevance: Peripheral arterial disease and its end-stage manifestation known as chronic limb-threatening ischemia (CLTI) represent ongoing prevalent and intricate medical challenges. Individuals with diabetes have a heightened risk of developing CLTI and experiencing its complications, including wounds, ulcers, and major amputations. In the present study, we conducted a comprehensive examination of the molecular lipid composition within arterial segments from individuals with CLTI, and with and without diabetes. Our investigations unveiled a striking revelation: the sphingolipid ceramide 18:1/16:0 emerged as the predominant ceramide species that was significantly elevated in the peripheral arterial intima below the knee in patients with diabetes. Moreover, this heightened ceramide presence is associated with a marked impairment of endothelial cell function and viability. Additionally, our study revealed a concurrent elevation in the expression of sphingomyelin phosphodiesterases, enzymes responsible for catalyzing ceramide synthesis from sphingomyelins, within maximally diseased arterial segments. These findings underscore the pivotal role of ceramides and their biosynthesis enzymes in the context of CLTI, offering new insights into potential therapeutic avenues for managing this challenging disease process.

Keywords: Ceramide; Diabetes; Endothelial dysfunction; Peripheral arterial disease; Sphingolipid; Sphingomyelinase.

Fig 1

Ceramide 18:1/16:0 (C16) is elevated in diseased peripheral arterial intima. A,B, Lipidomic correlation of total ceramide and total sphingomyelin (SPM) content in minimally diseased tissue (MIN), maximally diseased tissue (MAX), and cumulatively (total) in arterial segments from patients without diabetes (n = 7) and with diabetes (n = 14). C, Total SPM abundance in diseased arterial segments separated by acetyl chain length (n = 10). D, Total ceramide abundance of diseased arterial segments separated by acetyl chain length (n = 28). E, C16 content in minimally and maximally arterial segments from patients without and with diabetes (n = 28; P < .05). F, Sphingomyelin 34:1/1:0 (SPM42) abundance stratified by diabetes complication severity index (DCSI) category (n = 14). G, C16 abundance correlated with DCSI category (n = 28).

Fig 2

Expression profiles of ceramide synthesis enzymes in human tibial arterial tissue. A, Real-time (RT) polymerase chain reaction (PCR) of CERS6 ratio with the RPL32 housekeeping gene in the maximally (n = 5) and minimally (n = 4) diseased tissue of patients without diabetes vs maximally (n = 7) and minimally (n = 6) diseased tissue of patients with diabetes. B, RT-PCR of CERS6 of total minimally (n = 10) and maximally (n = 12) diseased arterial intima segments. C, RT-PCR of CERS6 in patients without diabetes (n = 9) and with diabetes (n = 13). D, Correlation of ceramide 18:1/16:0 (C16) and sphingomyelin 34:1/1:0 (SPM34) content in arterial intima segments of patients without diabetes (n = 14). E, Correlation of C16 and SPM34 in arterial intima segments of patients with diabetes (n = 28). F, RT-PCR of genes encoding for sphingomyelin phosphodiesterase enzymes 2 and 4 (SMPD2 and SMPD4) and normalized to RPL32. This was evaluated in maximally (n = 5) and minimally (n = 4) arterial intima segments of patients without diabetes and maximally (n = 7) and minimally (n = 6) arterial intima segments from patients with diabetes. G,SMPD2 and SMPD4 normalized to RPL32 in patients without diabetes (n = 9) and patients with diabetes (n = 13). H,SMPD2 and SMPD4 normalized to RPL32 of maximally (n =12) and minimally (n = 10) in patients with and without diabetes.

Fig 3

Ceramide 18:1/16:0 (C16) impairs human umbilical vein endothelial cell (HUVEC) viability and function. A, Proliferation of HUVEC cultures evaluated after treatment with C16 or C2 (P = .03). B,C, HUVEC apoptosis analysis with caspase 3/7 immunostaining (CellEvent caspase-3/7 green detection reagent) compared with vehicle (ethanol) or solvent (ethanol in growth media). Relative to C2 controls, HUVECs treated with C16 demonstrated higher levels of apoptosis (P = .003). D, Representative Western blot demonstrating decreased LC3II/I in HUVECs treated with C16. E, LC3II/I ratio is decreased in HUVECs treated with C16 (P = .008). HUVECs treated with C2 demonstrated no significant differences in LC3II/I ratio relative to the positive vehicle control dimethyl sulfoxide (DMSO) and showed a significant difference relative to the negative control MCF7 breast cancer cell line (P = .006).

Fig 4

Effect of selective knockdown of sphingomyelin phosphodiesterases (SMPDs) in human umbilical vein endothelial cells (HUVECs). A, Small interfering RNA (siRNA) knockdown of SMPD2 and SMPD4 caused a decrease in ceramide 18:1/16:0 (C16) content in HUVECs compared with enhanced green fluorescent protein (GFP) siRNA control (P < .0001, P = .006, and P < .0001, respectively). B, Selective knockdown of SMPDs in HUVECs followed by C2, C16, or vehicle (ethanol) supplementation. Each group is expressed relative to the vehicle treatment for each siRNA knockdown condition. Selective knockdown of SMPD2 and SMPD4 caused a decrease in LC3 ratio (P = .03). C, Selective knockdown of SMPD4 led to rescue of HUVEC proliferation after C16 treatment.

Fig 5

Ceramide 18:1/16:0 (C16) potentiates endothelial cell (EC) dysfunction and vascular disease. Sphingomyelin phosphodiesterases (SMPDs) can convert sphingomyelin substrate to ceramides such as C16. Increased peripheral arterial C16 leads to EC dysfunction, which ultimately contributes to atheroprogression and advanced forms of peripheral arterial disease (PAD) such as chronic limb-threatening ischemia (CLTI).