Deep Vein Thrombosis Resolution Is Modulated by Monocyte CXCR2-Mediated Activity in a Mouse Model

Abstract

Objective-To determine the role of CXCR2, the receptor for cysteine-X-cysteine (CXC) chemokines, and its primary effector cell, the neutrophil (PMN), on deep venous thrombosis (DVT) resolution. Methods and Results-DVT in BALB/c, anti-CXCR2 antibody-treated, and BALB/c CXCR2 Ϫ/Ϫ mice were created by infrarenal inferior vena cava (IVC) ligation and the thrombus harvested at various time points over 21 days. The CXCR2 Ϫ/Ϫ mice had significantly larger thrombi at early time points (days 2 to 8), and significantly decreased intrathrombus PMNs, monocytes, and neovascularization as compared with controls. Thrombus KC/CXCL1 was significantly higher at 2 days in CXCR2 Ϫ/Ϫ thrombi as measured by enzyme-linked immunosorbent assay. Fibrin content was significantly higher, with less uPA gene expression at 4 days in CXCR2 Ϫ/Ϫ thrombi. Late fibrotic maturation of the thrombus was delayed in the CXCR2Ϫ/Ϫ mice, with significantly decreased 8 day MMP-2 activity, whereas MMP-9 activity was elevated as compared with controls. Similar impairment in DVT resolution was found at 8 days with anti-CXCR2 inhibition. However, systemic neutropenia, unlike CXCR2 deletion, did not increase the thrombus size as compared with controls. Conclusions-Normal DVT resolution involves CXCR2-mediated neovascularization, collagen turnover, and fibrinolysis, and it is probably primarily monocyte-dependent.

Targeted Deletion of CCR2 Impairs Deep Vein Thombosis Resolution in a Mouse Model

“…Monocytes are particularly important, and MCP-1 drives monocyte chemotaxis via CCR2 signaling (12)(13)(14). Similarly, monocytes expressing CXCR2 were also shown to be essential for early thrombus resolution, possibly by promoting fibrinolysis (5).…”

“…The rodent model of stasis-induced DVT has been well described (4,5,7,30) and used B6/129 (wild-type (WT) controls, F2 hybrids; The Jackson Laboraory) and B6/129 CCR2 Ϫ/Ϫ mice (10) weighing 20 -30 g, between 4 and 6 wk old. The mice underwent general inhalational anesthesia and, via a laparotomy, the inferior vena cava (IVC) was ligated with a 6-0 polypropylene suture.…”

“…At harvest, the thrombosed IVC was weighed and measured, snap frozen, and stored at Ϫ70°C or placed in formalin, followed by ethanol, for histological analysis. Baseline comparisons of serum clotting factors with measurement of activated partial thromboplastin time and total clotting time of WT and CCR2 Ϫ/Ϫ mice was done, as was peripheral differential leukocyte counts, using an automated system as described (5).…”

“…Neovessels were counted only if a nucleus and cellular morphology were present (5). Other sections were processed per standard protocol for Trichrome staining (5,7,19).…”

“…Quantification of peptide mediators was normalized to total protein in the sample, by a modified Bradford assay per manufacturer's instructions (Pierce) with serial dilutions of BSA (Sigma-Aldrich) as standards. Tissue homogenate ELISAs for mouse MIP-1␣, RANTES, MCP-1, TGF␤, IFN-␥, IL-2, IL-4, IL-5, and IL-13 were performed with speciesspecific primary Abs quantified using a double ligand technique, as has been described for similar chemokines (4,5,35). Mouse bFGF and VEGF were analyzed by using a commercial ELISA according to the manufacturer's instructions (R&D Systems).…”