Endothelial Cept1 Promotes Post-Ischemic Angiogenesis in a Pparα -Dependent Fashion

Background

Cept1 is essential for de novo phopholipogenesis and is impacted by diabetes. We previously demonstrated that conditional knockdown of Cept1 in the endothelium leads to reduced hindlimb angiogenesis and tissue recovery. We hypothesized that Cept1 may also be sufficient in promoting post-ischemic angiogenesis and recovery in the setting of diabetes.

Methods

CEPT1 content was evaluated in peripheral arteries of patients with peripheral arterial disease (PAD), and with or without diabetes. An endothelial cell (EC)-specific Cept1 overexpression mouse model was developed ( Cept1 ^fl/fl Cre ⁺ ) in adult C57BL6 mice. Murine aortae were harvested, for single-cell RNA sequencing (scRNA-seq), and unilateral hindlimb ischemia was used to evaluate angiogenesis in Cept1 ^fl/fl Cre ⁺ mice. Primary ECs were isolated and HUVECs transduced with Cept1 cDNA were developed, and evaluated using molecular assays, in vitro functional assays, and mass spectrometry.

Results

In humans, arterial intima CEPT1 was elevated in the setting of PAD and diabetes, along with ACOX1, VEGF2R, p-Akt, and p-eNOS. In mice, scRNA-seq demonstrated that ECs with Cept1 overexpression were enriched with wound healing, angiogenesis, sprouting, and cell migration pathways. Diabetic Cept1 ^fl/fl Cre ⁺ mice had improved hind-limb perfusion and angiogenesis, and their aortic rings had increased ex vivo capillary sprouting. Cept1 overexpression in ECs significantly increased migration, tubule formation, and proliferation as predicted by scRNA-seq. Cept1 overexpression in ECs led to increased Pparα, Acox1, Vegfa, and Vegf2r . Similarly, treatment with si Pparα , and inhibitors for PPARα (GW6471), VEGFR2 (ZM323881), Akt (LY294002), and eNOS (L- NAME ) abrogated CEPT1-induced EC migration.

Conclusions

Cept1 overexpression promotes EC function and post-ischemic recovery. The impact of CEPT1 on ECs is at least in part dependent on p-Akt/p-eNOS angiogenic signaling and PPARα. Since CEPT1 is elevated in diseased human peripheral arterial tissue, these findings suggest that CEPT1 may be playing an important compensatory role in vascular recovery and reperfusion following ischemic injury in the setting diabetes.

Highlights

• CEPT1 content is higher in the peripheral arteries of individuals with peripheral arterial disease (PAD) and type 2 diabetes.

• Cept1 over expression induces endothelial cell activation and function and enhances post-ischemia angiogenesis in vivo .

• CEPT1 induces endothelial pAkt/p-eNOS signaling and VEGF-A production in a PPARα dependent fashion.

• CEPT1 may be an important regenerative signal that is increased in the peripheral arteries in the setting of PAD.

Graphical Abstract