Distinct Effects of Unfractionated Heparin versus Bivalirudin on Circulating Angiogenic Peptides

Background: Human studies of therapeutic angiogenesis, stem-cell, and progenitor-cell therapy have failed to demonstrate consistent clinical benefit. Recent studies have shown that heparin increases circulating levels of anti-angiogenic peptides. Given the widely prevalent use of heparin in percutaneous and surgical procedures including those performed as part of studies examining the benefit of therapeutic angiogenesis and cell-based therapy, we compared the effects of unfractionated heparin (UFH) on angiogenic peptides with those of bivalirudin, a relatively newer anticoagulant whose effects on angiogenic peptides have not been studied. Methodology/Principal Findings: We measured soluble fms-like tyrosine kinase-1 (sFLT1), placental growth factor (PlGF), vascular endothelial growth factor (VEGF), and soluble Endoglin (sEng) serum levels by enzyme linked immunosorbent assays (ELISA) in 16 patients undergoing elective percutaneous coronary intervention. Compared to baseline values, sFLT1 and PlGF levels increased by 26296313 % and 253654%, respectively, within 30 minutes of UFH therapy (p,0.01 for both; n = 8). VEGF levels decreased by 93.265 % in patients treated with UFH (p,0.01 versus baseline). No change in sEng levels were observed after UFH therapy. No changes in sFLT1, PlGF, VEGF, or sEng levels were observed in any patients receiving bivalirudin (n = 8). To further explore the direct effect of anticoagulation on circulating angiogenic peptides, adult, male wild-type mice received venous injections of clinically dosed UFH or bivalirudin. Compared to saline controls, sFLT1 an

Effects on angiogenic peptide levels of UFH and bivalirudin <i>in vivo</i> in wild type C57/Bl6 mice.

<p>Treatment with UFH for 30 minutes resulted in a rapid and significant increase in mouse serum sFlt1 (A) and PGF (B) levels (>500% increase, p<0.01, for both) compared to normal saline vehicle. UFH also produced a modest but significant increase in mouse VEGF₁₆₅ (C) levels (221+101%, p<0.05). Treatment with DSO4 did not influence sFlt1 or PlGF levels. VEGF₁₆₅ levels were modestly increased by DSO4 treatment. Bivalirudin treatment had no effect on sFlt1, PlGF, and VEGF₁₆₅ levels. sEng (D) levels were mildly increased with vehicle, UFH, bivalirudin and DSO4, with no significant differences between the 4 treatments.</p

Effects of heparin and bivalirudin on angiogenic peptide release from HCAECs.

<p>Immunofluorescent microscopy shows a significant reduction in cell surface expression of the sFlt1 N-terminal extracellular domain (top panel) with no change in C-terminal expression (bottom panel). Treatment with bivalirudin had no effect on N-terminal or C-terminal expression.</p

Effects on angiogenic peptides of UFH (dotted lines) and bivalirudin (dashed lines) in human subjects undergoing PCI.

<p>sFlt1 (A) and PlGF (B) were significantly increased by UFH but not bivalirudin, VEGF₁₆₅ (C) was significantly decreased by UFH but not bivalirudin, and sEng (D) was unchanged with both UFH and bivalirudin.</p

Subject Characteristics.

<p>Subject Characteristics.</p

Levels of angiogenic peptides from human coronary artery endothelial cells treated with unfractionated heparin or bivalirudin.

<p>A) Conditioned media levels of sFlt1, PlGF, VEGF and sEng 30 minutes after treatment with increasing concentrations of UFH. (p<0.05 vs control). B) Conditioned media levels of sFlt1, PlGF, VEGF and sEng 30 minutes after treatment with increasing concentrations of bivalirudin. No differences were observed after treatment with bivalirudin.</p

Time course of changes in sFlt1 (A), PlGF (B), VEGF₁₆₅ (C) and sEng (D) with UFH and bivalirudin at baseline and 1, 6 and 24 hours after administration of anticoagulant.

<p>Note that levels of all angiogenic peptides revert to baseline levels by 24 hours with UFH and levels do not change significantly over 24 hours with bivalirudin.</p