Atherosclerosis is a leading cause of death worldwide. Macrophages are key components of vascular inflammation, which contributes to the development and complications of atherosclerosis. Ferritin, an iron storage and transport protein, has been found to accumulate in macrophages in human atherosclerotic plaques. We hypothesized that ferritin could serve as an intrinsic nanoplatform to target delivery of imaging agents to vascular macrophages to detect high-risk atherosclerotic plaques. Here we show that engineered human ferritin protein cages, either conjugated to the fluorescent Cy5.5 molecule or encapsulating a magnetite nanoparticle, are taken up in vivo by macrophages in murine atherosclerotic carotid arteries and can be imaged by fluorescence and magnetic resonance imaging. These results indicate that human ferritin can serve as a nanoparticle platform to image vascular inflammation in vivo.

Cage-like protein nano particles are promising platforms for cell and tissue specific targeted delivery of imaging and therapeutic agents. Here, we have successfully modified the 12 nm small heat shock protein from Methanococcus jannaschii (MjHsp) to detect atherosclerotic plaque lesions in a mouse model system. As macrophages are centrally involved in the initiation and progression of atherosclerosis, targeted imaging of macrophages is valuable to assess the biologic status of the blood vessel wall. LyP-1, a nine residue peptide, has been shown to target tumor-associated macrophages. Thus, LyP-1 was genetically incorporated onto the exterior surface of MjHsp, while a fluorescent molecule (Cy5.5) was conjugated on the interior cavity. This bioengineered protein cage, LyP-Hsp, exhibited enhanced affinity to macrophage in vitro. Furthermore, in vivo injection of LyP-Hsp allowed visualization of macrophage-rich murine carotid lesions by in situ and ex vivo fluorescence imaging. These results demonstrate the potential of LyP-1-conjugated protein cages as nano-scale platforms for delivery of imaging agents for the diagnosis of atherosclerosis.

Abstract-Hepatocyte growth factor (HGF) plays a role in cell protection, antiapoptosis, antifibrosis, and angiogenesis.However, the role of HGF in the immune system is not well defined. We examined the influence of HGF on T cells and the effects of HGF therapy in acute myocarditis. Lewis rats were immunized on day 0 with cardiac myosin to establish experimental autoimmune myocarditis (EAM). Human HGF gene with hemagglutinating virus of the Japan-envelope vector was injected directly into the myocardium on day 0 or on day 14 (two groups of treated rats). Rats were killed on day 21. Expression of c-Met/HGF receptor in splenocytes and myocardial infiltrating cells was confirmed by immunohistochemical staining or FACS analysis. Myocarditis-affected areas were smaller in the treated rats than in control rats. Cardiac function in the treated rats was markedly improved. An antigen-specific T cell proliferation assay was done with CD4-positive T cells isolated from control rats stimulated with cardiac myosin. HGF suppressed T cell proliferation and production of IFN-␥ and increased production of IL-4 and IL-10 secreted from CD4-positive T cells in vitro.

Background-Programmed death-1 (PD-1), a member of the CD28 family, has been identified. PD-1 is involved in the negative regulation of some immune responses. We evaluated the role of PD-ligand 1 (PD-L1) in graft arterial disease (GAD) of cardiac allografts and in smooth muscle cells (SMCs). Methods and Results-C57BL/6 murine hearts were transplanted into B6.C-H2Ͻbm12ϾKhEg mice for examination of GAD. PD-L1 was expressed in SMCs of the thickened intima in the graft coronary arteries, and administration of anti-PD-L1 monoclonal antibody (mAb) enhanced the progression of GAD (luminal occlusion: 55Ϯ5.0% versus 9.8Ϯ4.3%, PϽ0.05). The expressions of interferon ␥ (IFN-␥) and tumor necrosis factor ␣ of cardiac allografts were upregulated in response to anti-PD-L1 mAb treatment. In vitro, PD-L1 expression was induced in SMCs in response to IFN-␥ stimulation. Sensitized splenocytes increased SMC proliferation, and anti-PD-L1 mAb in combination with IFN-␥ stimulation increased this proliferation. Key Words: transplantation Ⅲ graft arterial disease Ⅲ smooth muscle cell Ⅲ immune system Ⅲ B7 family C ardiac allograft transplantation developed as a therapy for end-stage congestive heart failure. The survival rate at 1 year after transplantation has increased to Ͼ80% by introduction of immunosuppressive drugs. 1 However, the use of the drugs also results in opportunistic infection, and these drugs do not prevent graft arterial disease (GAD), which occurs in chronic rejection. Conclusions-TheProgrammed death-1 (PD-1) is a member of the CD28 family that was identified in a T cell line undergoing programmed cell death, 2 but subsequent studies have shown that its expression is associated with lymphocyte activation rather than cell death. 3,4 PD-1 contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic tail. 5 C57BL/6 mice lacking PD-1 develop lupus-like arthritis and glomerulonephritis, 6 and Balb/c mice lacking PD-1 develop fatal dilated cardiomyopathy. 7 Okazaki et al purified the 30-kDa protein from heart extract and identified it as cardiac troponin I in these dilated cardiomyopathy mice, and administration of anti-cardiac troponin I (anti-cTnI) antibody induced heart dilation and dysfunction in wild-type mice. 8 The data suggest that PD-1 receptor engagement leads to downregulation of immune responses. Furthermore, PDligand 1 (PD-L1) was recently identified. 9 PD-1 ligand shows a tissue distribution profile distinct from that of the other B7 family members. Expression of PD-L1 is upregulated on activation of antigen presenting cells, including dendritic cells, monocytes, and B cells. In addition, PD-L1 has been detected in lymphoid as well as in nonlymphoid organs. 10,11 Although Ozkaynak et al reported the expressions and functional roles of PD-1 and PD-L1 in the pathogenesis of cardiac transplants, 12 the relation between the PD-1/PD-L1 pathway and the proliferation of smooth muscle cells (SMCs) has not been investigated. In the present study, we examined the expression of PD-1 and PD-L1 in relation to...