Plasma levels of soluble Tie2 and vascular endothelial growth factor distinguish critical limb ischemia from intermittent claudication in patients with peripheral arterial disease

Abstract

Objectives: Our purpose was to determine whether factors that regulate angiogenesis are altered in peripheral arterial disease (PAD) and whether these factors are associated with the severity of PAD.

Background: Alterations in angiogenic growth factors occur in cardiovascular disease (CVD), but whether these factors are altered in PAD or correlate with disease severity is unknown.

Methods: Plasma was collected from patients with PAD (n = 46) and healthy control subjects (n = 23). Peripheral arterial disease patients included those with intermittent claudication (IC) (n = 23) and critical limb ischemia (CLI) (n = 23). Plasma angiopoietin-2 (Ang2), soluble Tie2 (sTie2), vascular endothelial growth factor (VEGF), soluble VEGF receptor 1 (sVEGFR-1), and placenta growth factor (PlGF) were measured by enzyme-linked immunoadsorbent assay. In vitro, endothelial cells (ECs) were treated with recombinant VEGF to investigate effects on sTie2 production.

Results: Plasma concentrations of sTie2 (p < 0.01), Ang2 (p < 0.001), and VEGF (p < 0.01), but not PlGF or sVEGFR-1, were significantly greater in PAD patients compared with control subjects. Plasma Ang2 was significantly increased in both IC and CLI compared with control subjects (p < 0.0001), but there was no difference between IC and CLI. Plasma VEGF and sTie2 were similar in control subjects and IC but were significantly increased in CLI (p < 0.001 vs. control or IC). Increased sTie2 and VEGF were independent of CVD risk factors or the ankle-brachial index, and VEGF treatment of ECs in vitro significantly increased sTie2 shedding.

Conclusions: Levels of VEGF and sTie2 are significantly increased in CLI, and sTie2 production is induced by VEGF. These proteins may provide novel biomarkers for CLI, and sTie2 may be both a marker and a cause of CLI.

Figure 1. Plasma concentrations of Ang2, VEGF, and sTie2 are increased in PAD

Plasma concentrations of sVEGFR-1 (A), PlGF (B), Ang2 (C), VEGF (D), and Tie2 (E) were measured in control subjects (n=23) and patients with PAD (n=46). Concentrations of sVEGFR-1 and PlGF were not significantly different between the two populations, however Ang2 (*, P<0.0001), VEGF (*, P<0.01), and sTie2 (*, P<0.01) were significantly greater in PAD patients.

Figure 2. Plasma VEGF and sTie2 levels distinguish patients with CLI from controls or patients with IC

Plasma concentrations of Ang2, VEGF, and sTie2 were measured and compared in control subjects (n=23) and those with IC (n=23) or CLI (n=23). (A) Plasma concentrations of Ang2 were significantly increased in both IC (**, P<0.01) and CLI patients (***, P<0.0001) compared to controls, but there was no significant difference between IC and CLI. (B) VEGF levels were significantly increased in CLI patients compared to controls (**, P<0.01) and IC (*, P<0.05), but there was no difference between IC and controls. (C) sTie2 concentrations were significantly greater in CLI patients compared to controls (***, P<0.0001) and IC (***, P<0.0001), but there was no difference between IC and controls.

Figure 3. sTie2 production is induced by VEGF in vitro

HUVECs were stimulated with VEGF-A₁₆₅ (25 ng/mL) for 24 hours. Concentration of sTie2 in the cell conditioned media was quantified by ELISA. VEGF treatment significantly increased release of sTie2 (*, P<0.01).