We have previously shown that CX3CR1 knockout mice display a 58% decrease in intimal hyperplasia in a vascular injury model relative to wildtype animals [8
]. In order to determine any relationship between the involvement of CX3CR1 and CCR2 in the inflammatory response in this model, we tested the effect of a specific CCR2 antagonist (MRL-677) in CX3CR1 knockout mice.
MRL-677 has a murine whole blood potency (IC50) of 3.2 nM as determined by the ability to block 125I labeled-mCCL2 binding to peripheral blood mononuclear cells (PBMC) after in vitro whole blood treatment (Figure ). This is consistent with the potency of the compound to block mCCL2 binding to cells expressing recombinant mCCR2 (IC50 = 1.8 ± 0.4 nM), with a slight shift noted in whole blood, likely due to protein binding. MRL-677 was at least 100-fold more effective in inhibiting CCR2 compared to other chemokine receptors in standard competition binding experiments: murine CCR5 (selectivity >200 fold), human CCR1 (selectivity > 5000 fold), CCR3 (selectivity >500 fold), CCR8 (selectivity >500 fold), CXCR1 (selectivity >5000 fold), CXCR2 (selectivity >5000 fold), and CXCR3 (selectivity >500 fold). Table summarizes the findings. All assays were performed as competition binding assays using radioactively labeled chemokine receptor ligands.
Figure 1 A) MRL-677 added to murine whole blood at the concentrations indicated inhibited the binding of mCCR2 on PBMCs . Each point represents the average of three measurements and the error bars represent the standard error of the mean. The potency (IC50) of (more ...)
CCR2 Antagonist MRL-677 Competition
To confirm the activity of MRL-677 in vivo, we evaluated its effect of CCR2 blockade on monocyte recruitment in response to inflammation in C57Bl6 mice. The intraperitoneal administration of thioglycollate evoked a robust monocyte infiltration to the murine peritoneal cavity (26 ± 6 × 106 cells) compared to mice without application of thioglycollate (1 × 106 cells). Administration of MRL-677 at doses of 3, 10, or 30 mg/kg/day inhibited monocyte recruitment in the peritoneal cavity upon thioglycollate stimulation by approximately 49%, 75%, and 89% respectively (Figure ). Drug levels of MRL-677 assessed at trough for these experiments were below the limit of detection (<1 nM) 24 hours after 3 and 10 mg/kg/day doses and was 5 nM 24 hours after the highest dose tested at 30 mg/kg/day. Therefore, MRL-677 was administered by oral gavage at 30 mg/kg for the first two days and thereafter in feed at a dose of ~15 mg/kg/day in order to evaluate the effect of inhibiting CCR2 on vascular inflammation in response to injury. In addition to reducing animal handling and the stress that accompanies it, the administration of the drug in feed was anticipated to lead to higher trough drug levels to ensure complete blockade.
In the vascular injury experiments, we first measured the blood concentration of MRL-677 everyday for the complete experimental course of 14 days in both wildtype and CX3CR1 KO mice. We achieved an average trough level of 0.43 ± 0.11 μM for wildtype mice and 0.68 ± 0.41 μM for CX3CR1 KO mice. This indicates that sustained therapeutic drug concentrations considerably greater than those anticipated to give >95% coverage of CCR2 based on the mouse whole blood potency (whole blood IC95 = 0.06 μM, corresponding to 19 fold the IC50) were achieved during the 14 day treatment.
Then we examined the intimal hyperplasia by measuring the ratio of the area of the intima over the media (I/M ratio) 14 days after the femoral artery injury procedure. Treatment of wildtype mice with the CCR2 antagonist resulted in 56% reduction in intimal hyperplasia compared to wildtype mice without antagonist treatment (0.482 ± 0.097 vs. 0.213 ± 0.05, p < 0.05) (Figure ). In addition, the degree of protection from intimal hyperplasia observed in the normal mice fed the drug is similar to that seen in CCR2 deficient mice (0.213 ± 0.05 vs. 0.133 ± 0.084, p = NS). Furthermore, there was a dramatic reduction in vascular injury observed in the CX3CR1 KO animals given the CCR2 antagonist (I/M ratio of 0.056 ± 0.017) in which there was an 88% decrease in the development of intimal hyperplasia relative to untreated wildtype animals. Thus, knocking out the CX3CR1 pathway together with blocking CCR2 function via MRL-677 significantly decreased the intimal hyperplasia and this decrease is greater than inhibiting CCR2 alone (p = 0.009, Figure ). In order to determine whether or not monocyte trafficking played a role in the change in intimal hyperplasia observed, we performed immunohistochemistry on sections. Figure shows that while monocytes are seen in injured femoral arteries from wildtype mice, there is a significant decrease in monocyte numbers in MRL-677 treated animals. These data suggest that although CX3CR1 and CCR2 can both influence vascular inflammation and remodeling, their roles are non-redundant and additive.
Figure 2 CME stained cross section of mouse femoral artery (a) with no injury and (b)14 days after endoluminal injury in wildtype mouse, (c) wildtype mouse fed CCR2 antagonist, and (d) CX3CR1 KO mouse fed CCR2 antagonist . IEL is marked by arrows. Note the lack (more ...)
Figure 3 Shown are the average intimal/media ratios for each experimental arm . Graph depicts the mean ± standard error in each group. Statistical significance was achieved for differences between WT and WT+MRL-677, as well as between WT+MRL-677 and CX3CR1 (more ...)
Figure 4 Monocytes within the femoral artery were detected by immunohistochemistry using the F4/80 antibody . A representative section from wildtype animals and those treated with MRL-677 is shown. The top pair of pictures was counterstained in Gills solution. (more ...)