Title : Neutrophils arteriogenesis and fibrinolysis in chronic limb threatening ischemia
Abstract:
Activated neutrophils have been reported to release pro-angiogenic Hepatocyte Growth Factor (HGF), VEGF-A, MMP-9, angiopoietin 1 and CXCL-8. They also secrete proteases that degrade fibrin. Is there a role for neutrophils in promoting Neovascularization (NV) in Chronic Limb-Threatening Ischemia (CLTI) Filgrastim induced neutrophilia occurred during our study of a novel NV strategy. Filgrastim is a Granulocyte Colony Stimulating Factor FDA approved for stem cell mobilization. Filgrastim was used to increase the circulating number of pro-angiogenic progenitor cells (CD34+, VEGFR2+) typically deficient in CLTI. It’s primary indication though is to treat neutropenia following cytotoxic chemotherapy. While our study antedated knowledge of pro-angiogenic neutrophils, the proteomic data were sufficient to provide the first assessment of neutrophilia in CLTI.Pre-amputation “no-option” CLTI patients at two independent institutions (N=14) were treated with Filgrastim 8-10 mcg/kg subcutaneously every 72 hours for up to a month. An infra-geniculate programmed compression pump (PCP) was worn for 3 hours daily to increase endothelial shear stress (to initiate arteriogenesis), as well as to deliver oxygenated nutritive blood flow, clear toxic metabolic by-products, disseminate ischemic molecular signals, and help deliver salutary progenitor cells back to the activated endothelium. Blood was drawn one day after the 5th and 10th Filgrastim doses. Comparisons were made to blood drawn prior to the first dose. The expected significant (p<0.001) elevation in the circulating number of progenitor cells was measured by cytometry. The expected increase in neutrophils (6 fold, p<0.001) was measured by cell blood count. The plasma concentrations of Fibrin Degradation Products and of Plasmin, measured by ELISA, were increased >5 fold and >10 fold respectively (p<0.01). The serum concentrations of NV proteins (HGF, VEGF-A, MMP-9, PDGF, angiopoietin1 and others), also measured by ELISA, were significantly increased (p<0.05).These findings did not occur in patients treated with PCP alone (N=19), in whom blood was drawn on Day 1 and day 30. However, increases in PECAM-1, MCP-1 and serum nitrite were consistent with endothelial activation.In our evolving “no-option” CLTI experience, PCP use is continued until wounds heal or amputation. Improvement in blood flow was documented by arterial hemodynamic assessment and angiographic evidence of corkscrew collateral growth, segmental arterial recanalization, and accelerated contrast transit.Safe sustained physiologic fibrinolysis, coupled with generation of a pro-angiogenic environment, were temporally associated with a month of neutrophilia. This potent combination may accelerate NV in the race against ischemic necrosis.
