Developmental Project 1 - Nanoagents for chemokine receptor imaging in atherosclerosis & acute vascular injury.
Project Leader - Michael J. Welch, Ph.D.
The goal of this Developmental Project is to test the hypothesis that novel NPs engineered to target chemokine receptors will provide an innovative means to both detect and characterize the acuity and severity of in vivo atherosclerotic inflammation. Accordingly, the following Specific Aims are proposed:
- Validate comb-XXXX as a novel nanoagent to specifically target chemokine receptors. Cell culture and flow cytometry studies will be used to investigate the binding specificity of the comb-XXXX NP. Immunohistochemistry with FITC-labeled comb-XXXX will be used to investigate its binding to chemokine receptors in tissue at varying points after acute vascular injury.
- Utilize comb-XXXX as a novel NP to image vascular inflammation related to de novo atherosclerosis and in response to acute vascular injury. The temporal and spatial expression pattern of this NP and its chemokine receptor targets will be correlated to the intensity of the inflammatory response as assessed through biochemical and histological evaluation.
Chemokines are a family of small molecule, secreted chemotatic cytokines characterized by the number and arrangement of conserved cysteine residues (i.e. CC, CXC, CX3C, XC). Chemokines are produced by a variety of cells, including endothelial and smooth muscle cells in response to injury and inflammation. Chemokines bind to their cognate receptors on circulating leukocytes to direct these cells to areas of injury, contributing to the inflammatory burden. While the chemokine system has been well characterized in inflammatory disease states, including rheumatoid arthritis and asthma, several lines of evidence suggest that chemokines and their receptors play important roles in atherosclerosis as well. Immunohistochemical analyses of human atherosclerotic tissue have demonstrated increased expression of various chemokines and chemokine receptors, including CCR3 and CX3CR1. Additionally, studies utilizing hyperlipidemic ApoE-null mice undergoing wire-induced arterial injury reveal increased expression of multiple chemokines in the injured areas. The contribution of the chemokine system in the progression of atherosclerotic lesions was shown in studies utilizing wire injury in ApoE-null or wildtype mice rendered functionally deficient for either chemokine receptor CCR2 or CCR5 by genetic modification or specific inhibitors and resulted in significant decreases in neointimal lesion area. This concept has been corroborated in models of de novo atherosclerosis lacking any element of injury as well. Specifically, hyperlipidemic ApoE-null/CX3CR1-null mice at fifteen weeks showed significantly decreased spontaneous atherosclerosis as assessed by lesion area and macrophage infiltration, compared to identically treated ApoE-null mice. Taken together, chemokines and their receptors play important roles in the development and progression of atherosclerosis, largely through cell recruitment mechanisms.
While the expression of chemokines in response to acute vascular injury is well described, much less is known about chemokine receptor expression after such injury. While conventionally thought that chemokine receptors are expressed primarily on activated monocytes and macrophages, in vitro studies have demonstrated that both endothelial and smooth muscle cells express these receptors. Recently Jabs et al., using balloon overstretch injury in porcine coronary arteries, delineated the early temporal expression of the chemokine receptors CCR2, CCR5, and CXCR4 over a 14 day period. These chemokine receptors were detected in the neointima, media, and adventia by immunostaining 7 days after injury; by 14 days CXCR4 was not detected and CCR2 and CCR5 expression was markedly decreased. RT-PCR data confirmed this temporal expression pattern, suggesting that the expression of different chemokine receptors may provide a composite profile of the localized inflammatory response. Thus, at varying time points after injury, NPs targeting chemokine receptors may possibly represent a novel, innovative means to detect and also characterize the severity and acuity of vascular inflammation associated with atherosclerosis and acute vascular injury. Importantly, the antagonism of these receptors by NPs may have important modulatory effects on macrophage function in vivo and may suggest that such novel nanoagents could be uniquely endowed with diagnostic as well as therapeutic utility.
atherosclerotic inflammation has only recently been described. Hartung et al.
demonstrated that intravenous 99m
Tc-labeled CCL2 could be used to image areas of arterial injury in hyperlipidemic New Zealand rabbits. Immunohistochemical analyses demonstrated that 99m
Tc-labeled CCL2 co-localized with activated macrophages in the neointima of the developing lesions. This was consistent with previous studies that demonstrated chemokine receptor CCR2 on activated macrophages mediated CCL2 uptake. Thus, using specific imaging agents that target chemokine receptors on circulating macrophages is a viable means to deliver imaging payloads to areas of vascular injury to characterize the acuity and severity of the inflammatory response. We will exploit the inherent advantages of NPs of both multivalent target ligand binding as well as signal amplification to test the hypothesis that novel synthetic NPs engineered to target chemokine receptors will provide an innovative means to both detect and characterize the acuity and severity of in vivo atherosclerotic inflammation.