Abstract Body: Background
Chronic Limb-Threatening Ischemia (CLTI), the most severe form of peripheral arterial disease (PAD), results in ischemic rest pain, non-healing ulcers, or gangrene. Pro-angiogenic VEGF-based therapies have failed clinically. VEGF drives endothelial cell (EC) proliferation in PAD while increasing vascular permeability. In PAD conditions, we showed VEGF drives glycolytic metabolism in EC by increasing phosphofructokinase-2/fructose-2,6-bisphosphatase-3 (PFKFB3). Whether EC permeability is driven by increased glycolytic flux or by PFKFB3 itself remains unknown.
Objective
To determine whether increased EC PFKFB3 promotes vascular permeability in PAD-relevant angiogenesis independent of glycolysis.
Methods
HUVECs were exposed to PAD-relevant conditions of hypoxia and serum starvation (HSS) following PFKFB3 overexpression (OE) or empty vector (EV) transfection. Glycolysis was suppressed using citrate, 3-PO or kinase-inactive PFKFB3 mutant plasmid. Barrier function was assessed by VE-cadherin expression, FITC-dextran, and trans-endothelial resistance. PFKFB3-interacting proteins were identified by immunoprecipitation followed by mass spectrometry. AHNAK knockdown and rescue experiments were performed. Immunofluorescence of human skeletal muscle sections assessed EC PFKFB3 and VE-cadherin in control versus CLTI.
Results
In HUVECs, PFKFB3 OE increased endothelial permeability under HSS. Glycolysis inhibition reduced flux but failed to restore barrier integrity, indicating a glycolysis-independent effect. Proteomic analysis of ECs with and without PFKFB3 OE revealed reduced association of AHNAK, a 700-kDa endothelial scaffolding protein critical for junctional stability, and Annexin A2 with PFKFB3 OE. Validation of the proteomics showed that PFKFB3 OE reduced AHNAK abundance and AHNAK knockdown vs. control recapitulated VE-cadherin loss, increased permeability, and impaired barrier resistance. AHNAK OE rescued PFKFB3-induced barrier dysfunction. In CLTI muscle, ECs showed increased PFKFB3 with reduced VE-cadherin.
Conclusions
In-vitro and in-vivo, ECs show greater PFKFB3 in PAD conditions, further enhanced by VEGF. These conditions promote vascular barrier dysfunction through suppression of AHNAK, independent of glycolytic flux. These findings identify a non-metabolic role of PFKFB3 in PAD EC permeability and suggest that targeting EC barrier stability, rather than angiogenic stimulation alone, may be needed to improve therapeutic revascularization in PAD/CLTI.