application) The overall goal of this proposal is to determine the role of chemokine receptors in vascular disease. Chemokines (chemotatic cytokines) specifically attract leukocytes by activating G-protein-coupled receptors. The investigators have focused their efforts on two of these receptors, CCR2 and CCR5, which were cloned in this laboratory and which are the major chemokine receptors on monocytes. During the first funding period, the investigators examined the structure/activity relationships of these two receptors with regard to ligand binding and signaling. The work proposed in the current application will extend these in vitro studies to in vivo models, taking advantage of our recent creation of a CCR2 knockout mouse. The first goal of the proposal is to determine the role of CCR2 in two forms of vascular disease: atherosclerosis and the accelerated arteriosclerosis of allogeneic cardiac transplants. The investigators will cross the CCR2-/- mice with apoE-/- mice and perform detailed atherosclerosis studies. The investigators will also perform heterotopic transplants of allogeneic hearts into the CCR2-/- mice and determine whether CCR2 plays a role in chronic myocardial rejection and transplant arteriosclerosis. The second goal is creating CCR2/CCR5 double-knockout mice. The investigators will selectively delete these receptors and simultaneously """"""""knock in"""""""" lacZ under the control of the CCR2 promoter and green fluorescent protein (GFP) under the control of the CCR5 promoter. This approach will generate chemokine receptor knockout animals, as well as provide a sensitive means for detecting the endogenous in vivo expression of CCR2 and CCR5. Differential regulation of chemokine receptors clustered on chromosome 3 (3p21.3-24) may provide specificity in inflammation and immunity. The third goal is to determine the tissue-specific expression and transcriptional regulation if selected CC receptors. The investigators will use the hemizygous CCR2/CCR5 double-knockout mice (CCR2+/LacZ, CCR5=/GfP), as well as transgenic mice created with large fragments of human genomic DNA, to follow the expression of these two receptors in models of human disease. the experiments proposed in this grant will utilize novel and complementary approaches to provide the first detailed information on the role of chemokines and chemokine receptors in vascular disease.
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