CEPT1-Mediated Phospholipogenesis Regulates Endothelial Cell Function and Ischemia-Induced Angiogenesis Through PPARα

Abstract

De novo phospholipogenesis, mediated by choline-ethanolamine phosphotransferase 1 (CEPT1), is essential for phospholipid activation of transcription factors such as peroxisome proliferator-activated receptor α (PPARα) in the liver. Fenofibrate, a PPARα agonist and lipid-lowering agent, decreases amputation incidence in patients with diabetes. Because we previously observed that CEPT1 is elevated in carotid plaque of patients with diabetes, we evaluated the role of CEPT1 in peripheral arteries and PPARα phosphorylation (Ser12). CEPT1 was found to be elevated in diseased lower-extremity arterial intima of individuals with peripheral arterial disease and diabetes. To evaluate the role of Cept1 in the endothelium, we engineered a conditional endothelial cell (EC)-specific deletion of Cept1 via induced VE-cadherin-CreERT2-mediated recombination (Cept1Lp/LpCre ⁺). Cept1Lp/LpCre ⁺ ECs demonstrated decreased proliferation, migration, and tubule formation, and Cept1Lp/LpCre ⁺ mice had reduced perfusion and angiogenesis in ischemic hind limbs. Peripheral ischemic recovery and PPARα signaling were further compromised by streptozotocin-induced diabetes and ameliorated by feeding fenofibrate. Cept1 endoribonuclease-prepared siRNA decreased PPARα phosphorylation in ECs, which was rescued with fenofibrate but not PC16:0/18:1. Unlike Cept1Lp/LpCre ⁺ mice, Cept1Lp/LpCre ⁺ Ppara ^-/- mice did not demonstrate hind-paw perfusion recovery after feeding fenofibrate. Therefore, we demonstrate that CEPT1 is essential for EC function and tissue recovery after ischemia and that fenofibrate rescues CEPT1-mediated activation of PPARα.

Figure 1

CEPT1 is localized to endothelium and is elevated in the intima of diseased peripheral arteries. A: CEPT1 colocalizes with CD31 along the intima endothelial layer of nondiseased aorta. CEPT1 also colocalizes with CD31 in microvascular structures in skeletal muscle tissue (inserts original magnification 1.5×). Arterial intima staining control with IgG secondary antibody demonstrates minimal background staining. B: Min and Max diseased peripheral arterial intima segments were procured from lower extremities of patients with severe PAD, with or without T2D, who were undergoing amputation. C: Real-time PCR analysis demonstrates higher Cept1 expression in Max and Min diseased arterial segments of patients with diabetes (n = 7 with T2D; n = 15 with no T2D). D: Representative Western blot demonstrates higher CEPT1 content in Min and Max arterial intima segments of a patient with diabetes. Caveolin-1 blot is a representative loading control. E: CEPT1 content is higher in Max diseased arterial intima segments (n = 12 patients). F: CEPT1 content is also higher in patients with known clinical history of T2D (16 with T2D; 8 with no T2D). G and H: Mass spectrometry phospholipidomic analysis demonstrated higher aPC content (G) and pPE content (H) in the arterial intima of patients with T2D (n = 14 with T2D; n = 14 with no T2D). *P < 0.05; **P < 0.01. WB, Western blot.

Figure 2

Conditional knockout of Cept1 in murine endothelium alters phospholipid profile. A: Representative Western blot (WB) demonstrates dramatically decreased CEPT1 content in MHECs and MLECs isolated from Cept1Lp/LpCre⁺ mice. B: Quantitative of three independent Western blots demonstrates significant decrease in CEPT1 protein content in MHECs and MLECs isolated from Cept1Lp/LpCre⁺ mice. C: Immunostaining of MLECs demonstrates decreased cytoplasmic CEPT1 content after TMX treatment to facilitate Cre-medicated recombination and knockout of Cept1. D and E: Mass spectrometry phospholipidomic analysis demonstrated lower PC content (D) and PE content (E) in Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ MLECs (n = 3 per analysis). *P < 0.05; ***P < 0.001. RU, relative unit.

Figure 3

Reduced function and altered gene expression in Cept1Lp/LpCre⁺ ECs. A: Proliferation of MHECs and MLECs isolated from Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice over 24 h (n = 3 per condition). B: Cell death of isolated MHECs and MLECs over 24 h (n = 3 per condition). C: Isolated MHEC monolayer migration over 16 h (n = 3 per condition). D: Representative images of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ MLEC formation of tubules after 6 h of incubation on Matrigel. E: Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ MLEC tubule formation over 6 h (n = 3 per condition). F–I: Cept1Lp/LpCre⁺ MLECs demonstrate robust Cre expression (F), reduced Cept1 (G), reduced Acox1 (H), and reduced Cpt1a (I) (n = 3 per condition). OD, optical density. *P < 0.05; **P < 0.01.

Figure 4

Effect of STZ treatment and fenofibrate diet on HLI recovery in Cept1Lp/LpCre⁺ mice. A: Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice underwent one of four outlined treatment courses. B: Representative Doppler perfusion of ischemic hind limbs of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice treated with or without STZ and maintained on a regular diet. C: Cept1Lp/LpCre⁺ mice treated with or without STZ have decreased hind-limb Doppler perfusion (ischemic to nonischemic limb perfusion ratio; n = 4–5 mice per group). D: Representative Doppler perfusion of ischemic hind limbs of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice treated with or without STZ and receiving a fenofibrate diet at 6 weeks of age. E: Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice treated with or without STZ and receiving a fenofibrate diet demonstrated equivalent hind-limb Doppler perfusion (n = 4–15 mice per group). F: Representative gastrocnemius microcapillary staining of ischemic hind limbs of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice treated with or without STZ and maintained on a regular or fenofibrate diet. G: Representative gastrocnemius microcapillary staining of ischemic hind limbs of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice treated with or without STZ and receiving a fenofibrate diet at 6 weeks of age. H: Relative microvessel density in ischemic limbs of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice that received either a regular or fenofibrate diet (n = 3–5 mice per group). I: Relative microvessel density in ischemic hind limbs of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice treated with or without STZ and maintained on a regular or fenofibrate diet (n = 3–5 mice per group). J: Representativehematoxylin-eosin–stained gastrocnemius muscle fiber sections of ischemic hind limbs of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice treated with or without STZ and maintained on a regular diet. K: Representative gastrocnemius muscle fiber sections of ischemic hind limbs of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice treated with or without STZ and receiving a fenofibrate diet at 6 weeks of age. L: Relative muscle fiber size in ischemic limbs of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ that received either a regular or fenofibrate diet (n = 3 mice per group). M: Relative muscle fiber size in ischemic limbs of Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice that received STZ and a regular or fenofibrate diet (n = 3 mice per group). N–P: Quantified Western blot analysis of PPARα (N), CPT1a (O), and ACOX1 (P) in Cept1Lp/LpCre⁻ and Cept1Lp/LpCre⁺ mice that received STZ treatment (n = 3 per sample). *P < 0.05; **P < 0.01. I/NI, ischemic/non-ischemic; RU, relative unit.

Figure 5

Fenofibrate rescues PPARα phosphorylation after acute knockdown of Cept1, and CEPT1 is essential for 16:0/18:1 activation of PPARα. A: Immunostaining of HUVECs treated with Gfp or Cept1 esiRNA. B: Representative Western blot (WB) of CEPT1 and caveolin-1 loading control in HUVECs treated with Gfp or Cept1 esiRNA. C: Relative content of CEPT1 in HUVECs treated with Gfp or Cept1 esiRNA (n = 3 per condition). D: Representative WB of phospho-PPARα, total PPARα, and caveolin-1 loading control in HUVECs treated with Gfp or Cept1 esiRNA for 36 h (n = 3 shown per condition). E: Ratio of phospho-PPARα to PPARα in HUVECs treated with Gfp or Cept1 esiRNA (n = 3 per condition). F: Representative WB of phospho-PPARα, total PPARα, and caveolin-1 loading control in HUVECs treated with Gfp or Cept1 esiRNA and with or without fenofibrate for 90 min. G: Ratio of phospho-PPARα to cavolin-1 in HUVECs treated with Gfp or Cept1 esiRNA (n = 5 per condition). H: Representative immunostains of phospho-PPARα in HUVECs treated with Gfp or Cept1 esiRNA and maintained in growth media. I: Quantification of phospho-PPARα intensity in HUVECs treated with Gfp or Cept1 esiRNA and maintained in growth media (n = 4–5 per condition). J: Representative immunostains of phospho-PPARα in HUVECs treated with Gfp or Cept1 esiRNA and maintained in growth media supplemented with 50 μmol/L fenofibrate. K: Quantification of phospho-PPARα intensity in HUVECs treated with Gfp or Cept1 esiRNA and maintained in growth media supplemented with 50 μmol/L fenofibrate (n = 5–6 per condition). L: HUVECs treated with Gfp or Cept1 esiRNA were also treated with PC18:0/18:2 or PC16:0/18:1 and relative content of phospho-PPARα and CPT1a evaluated. M and N: Cept1 (M) and Ppara (N) expression in HUVECs treated with different PC and esiRNA conditions (n = 3 per condition). O and P: Relative to PC14:0 treatments, the ratios of phospho-PPARα to total PPARα (O) and CPT1a to caveolin-1 loading control (P) are demonstrated in HUVECs treated with Gfp or Cept1 esiRNA and PC18:0/18:2 or PC16:0/18:1. *P < 0.05; **P < 0.01; ***P < 0.001. AU, arbitrary unit.

Figure 6

Ppara is essential for fenofibrate rescue of ischemic hind-paw Doppler perfusion. A–C: Expression of Ppara, (A) Acox1, (B) and Cpt1a (C) in MLECs isolated from Ppara^+/+ and Ppara^−/− mice (n = 3 per condition from ECs pooled from three mice per genotype). D: Representative hind-paw perfusion at baseline pre-HLI (day 0) and post-HLI days 3 and 7 in Cept1Lp/LpCre⁻, Cept1Lp/LpCre⁺, Ppara^−/−, and Cept1Lp/LpCre⁺Ppara^−/− mice maintained on a regular diet. E: Quantitative assessment of relative hind-paw Doppler perfusion across the different mouse genotypes maintained on a regular diet (n = 4–10 mice per genotype). F: Representative hind-paw perfusion of different genotypes of mice that received a fenofibrate diet. G: Quantitative assessment of relative hind-paw Doppler perfusion across the different genotypes of mice that received a fenofibrate diet (n = 10–15 mice per genotype). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Role of CEPT1 and fenofibrate in PPARα activation. In endothelium, CEPT1 generates PCs and PEs from fatty acid precursors via phospholipogenesis. PCs and PEs are essential for various cellular functions, including membrane integrity and activation of transcription factors such as PPARα. PPARα phosphorylation is dependent on CEPT1 and is also induced by fenofibrate treatment. PPARα activation leads to increased β-fatty acid oxidation enzymes, such as ACOX1 and CPT1a. PPARα expression seems to be essential for EC activation and appropriate recovery response to peripheral-limb ischemia and microvascular angiogenesis. FAS, fatty acid synthase.