Charles G. Pearce

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

CCR2 is required for monocyte recruitment in many inflammatory processes, as well as conferring Th1 lymphokine responses. Deep vein thrombosis (DVT) resolution represents a specific inflammatory response whereby the thrombus must be dissolved for restoration of blood flow. Using a stasis model of DVT in the mouse, we investigated the role of CCR2 on DVT resolution. Genetic deletion of CCR2 (CCR2−/−) was associated with larger thrombi at early and later time points, increased thrombus collagen, fewer thrombus monocytes (F4/80), and significantly impaired neovascularization. IL-2 and IFN-γ were significantly reduced in early CCR2−/− thrombi, whereas MCP-1 was significantly increased, and Th2 lymphokines were unaffected. Supplementation of CCR2−/− mice with IFN-γ normalized early thrombus resolution without increasing monocyte influx. Neither Ab depletion of IFN-γ nor genetic deletion of IFN-γ impaired early DVT resolution. Early fibrinolysis was not impaired in CCR2−/− mice, but a significant reduction in both matrix metalloproteinase (MMP)-2 and MMP-9 activity was observed. However, only MMP-9 activity was restored with administration of IFN-γ. We conclude that an early CCR2-dependent Th1 lymphokine response predominates in normal DVT resolution, mediates this in part by MMP-9 activation, but is not solely dependent on IFN-γ.

L-Selectin-Mediated Neutrophil Recruitment in Experimental Rodent Aneurysm Formation

Background—This investigation tested the hypothesis that L-selectin is important in experimental abdominal aortic aneurysm (AAA) formation in rodents. Methods and Results—Rat abdominal aortas were perfused with saline (control) or porcine pancreatic elastase and studied on postperfusion days 1, 2, 4, 7, and 14 (n=5 per treatment group per day). Neutrophil (polymorphonucleur leukocyte, PMN) and macrophage counts per high-powered field (HPF) were performed on fixed sections. L-selectin expression and protein levels in aortic tissue were determined by polymerase chain reaction and Western blot, respectively. Elastase-perfused aortic diameters were significantly increased compared with control aortas at all time points except day 1 (P<0.05). PMN counts significantly increased in elastase-perfused aortas compared with control aortas at days 1, 2, and 4, reaching maximum levels at day 7 (40.8 versus 0.3 PMNs/HPF, P=0.001). L-selectin mRNA expression in elastase-perfused aortas was 18 (P=0.018), 17 (P<0.001), and 8 times (P=0.02) times greater than control aortas at days 1, 2, and 4, respectively. Western blot demonstrated a significant 69% increase in L-selectin protein at day 7 in elastase- as compared with saline-perfused aortas (P=0.005). Subsequent experiments involved similar studies on postperfusion days 4, 7, and 14 of aortas from C57BL/6 wild-type (WT) mice (n=21) and L-selectin–knockout (LKO) mice (n=19). LKO mice had significantly smaller aortic diameters at day 14 as compared with WT mice (88% versus 123%, P=0.02). PMN counts were significantly greater in elastase-perfused WT mouse aortas as compared with LKO mouse aortas at day 4 after perfusion (12.8 versus 4.8 PMNs/HPF, P=0.02). Macrophage counts were significantly greater at all time points after perfusion in elastase-perfused WT mouse aortas compared with elastase-perfused LKO mouse aortas, with a maximum difference at day 7 after perfusion (13.3 versus 0.5 macrophages/HPF, P<0.001). Conclusion—L-selectin–mediated neutrophil recruitment may be a critical early step in AAA formation.

Neutrophils modulate post-thrombotic vein wall remodeling but not thrombus neovascularization

Early deep venous thrombosis (DVT) resolution is associated with neutrophil (PMN) influx. This study examined the role of PMNs in thrombus neovascularization and vein wall injury after DVT. A rat model of DVT by inferior vena cava (IVC) ligation was performed with control serum or rabbit anti-rat PMN serum administered perioperatively with sacrifice at 2 and 7 days. At 2 days, neutropenic rats had 1.6-fold larger thrombi (P = .04) and 1.4-fold higher femoral venous pressures by water manometry (P = .008) but no difference in thrombus neovascularization was observed. By 7 days, DVT sizes were similar, but vein wall injury persisted in the neutropenic rats with a 2.0-fold increase in vein wall stiffness by microtensiometry (P < .05), as well as a 1.2-fold increased thickness (P = .04). Collagen and profibrotic growth factors were significantly increased in neutropenic IVC at 7 days (all P < .05). Vein wall and intrathrombus uPA byWestern immunoblotting, and intrathrombus MMP-9 gelatinase activity were significantly less in neutropenic rats than controls (P < .001). Conversely, MMP-2 was significantly elevated in neutropenic IVC at 2 days after DVT. However, neutropenia induced 24 hours after DVT formation resulted in no significant increase in vein wall stiffness or collagen levels at 7 days, despite 1.4-fold larger thrombi (P < .05). These data suggest a critical early role for PMN in post DVT vein wall remodeling.