Oscar M. Pello

Role of platelets as mediators that link inflammation and thrombosis in atherosclerosis

Platelets, crucial mediators of the acute complications of atherosclerosis that cause life-threatening ischemic events at late stages of the disease, are also key effectors of inflammation throughout plaque development through their interaction with endothelial and immune cells in the injured vessel wall. During the first steps of atherosclerosis, blood inflammatory leukocytes interact with the damaged endothelium in areas rich in platelet aggregates. In late stages of the disease, platelets secrete several inflammatory molecules, even without forming aggregates. These molecules exacerbate the inflammation and induce the transition from chronic to acute disease, featuring increased instability of the atherosclerotic lesion that results in plaque rupture and thrombosis. Moreover, platelets play an important role in vascular wall remodeling induced by chronic inflammation by controlling vascular cell differentiation and proliferation. In this review, we discuss the role of platelets as cell mediators that link inflammation and thrombosis in atherosclerotic disease and their potential in the development of new therapeutic tools to fight cardiovascular disease.

A glimpse on the phenomenon of macrophage polarization during atherosclerosis

Atherosclerosis and associated cardiovascular disease are the leading causes of mortality in developed countries and the World Health Organization has estimated that by 2020 these disorders will be the main sanitary and socio-economic problem world-wide due in part to the progressive aging of our societies. Atherosclerosis is a complex chronic inflammatory process triggered and perpetuated by cardiovascular risk factors which cause endothelial dysfunction and leukocyte infiltration within the subendothelial space in the artery wall. In this review, we summarize the mechanisms that govern the recruitment of circulating monocytes into the incipient atherosclerotic lesion and their differentiation into macrophages. Moreover, we discuss current knowledge on macrophage polarization, a phenomenon of increasing interest given recent work suggesting that different stages in the progression of atherosclerosis are associated with the presence of distinct macrophage subtypes. Understanding the molecular mechanisms that orchestrate macrophage polarization and the precise role of distinct macrophage subsets should provide a basis for novel treatment strategies to limit the progression of atherosclerosis.

In Vivo Inhibition of c-MYC in Myeloid Cells Impairs Tumor-Associated Macrophage Maturation and Pro-Tumoral Activities

Although tumor-associated macrophages (TAMs) are involved in tumor growth and metastasis, the mechanisms controlling their pro-tumoral activities remain largely unknown. The transcription factor c-MYC has been recently shown to regulate in vitro human macrophage polarization and be expressed in macrophages infiltrating human tumors. In this study, we exploited the predominant expression of LysM in myeloid cells to generate c-Mycfl/fl LysMcre/+ mice, which lack c-Myc in macrophages, to investigate the role of macrophage c-MYC expression in cancer. Under steady-state conditions, immune system parameters in c-Mycfl/fl LysMcre/+ mice appeared normal, including the abundance of different subsets of bone marrow hematopoietic stem cells, precursors and circulating cells, macrophage density, and immune organ structure. In a model of melanoma, however, TAMs lacking c-Myc displayed a delay in maturation and showed an attenuation of pro-tumoral functions (e.g., reduced expression of VEGF, MMP9, and HIF1α) that was associated with impaired tissue remodeling and angiogenesis and limited tumor growth in c-Mycfl/fl LysMcre/+ mice. Macrophage c-Myc deletion also diminished fibrosarcoma growth. These data identify c-Myc as a positive regulator of the pro-tumoral program of TAMs and suggest c-Myc inactivation as an attractive target for anti-cancer therapy.

Nitric Oxide Prevents Aortic Neointimal Hyperplasia by Controlling Macrophage Polarization

Objective— Nitric oxide synthase 3 (NOS3) prevents neointima hyperplasia by still unknown mechanisms. To demonstrate the significance of endothelial nitric oxide in the polarization of infiltrated macrophages through the expression of matrix metalloproteinase (MMP)-13 in neointima formation. Approach and Results— After aortic endothelial denudation, NOS3 null mice show elevated neointima formation, detecting increased mobilization of LSK (lineage-negative [Lin]-stem-cell antigen 1 [SCA1]+KIT+) progenitor cells, and high ratios of M1 (proinflammatory) to M2 (resolving) macrophages, accompanied by high expression of interleukin-5, interleukin-6, MCP-1 (monocyte chemoattractant protein), VEGF (vascular endothelial growth factor), GM-CSF (granulocyte-macrophage colony stimulating factor), interleukin-1&bgr;, and interferon-&ggr;. In conditional c-Myc knockout mice, in which M2 polarization is defective, denuded aortas showed extensive wall thickening as well. Conditioned medium from NOS3-deficient endothelium induced extensive repolarization of M2 macrophages to an M1 phenotype, and vascular smooth muscle cells proliferated and migrated faster in conditioned medium from M1 macrophages. Among the different proteins participating in cell migration, MMP-13 was preferentially expressed by M1 macrophages. M1-mediated vascular smooth muscle cell migration was inhibited when macrophages were isolated from MMP-13–deficient mice, whereas exogenous administration of MMP-13 to vascular smooth muscle cell fully restored migration. Excess vessel wall thickening in mice lacking NOS3 was partially reversed by simultaneous deletion of MMP-13, indicating that NOS3 prevents neointimal hyperplasia by preventing MMP-13 activity. An excess of M1-polarized macrophages that coexpress MMP-13 was also detected in human carotid samples from endarterectomized patients. Conclusions— These findings indicate that at least M1 macrophage-mediated expression of MMP-13 in NOS3 null mice induces neointima formation after vascular injury, suggesting that MMP-13 may represent a new promising target in vascular disease.

Aryl hydrocarbon receptor contributes to the MEK/ERK-dependent maintenance of the immature state of human dendritic cells

Dendritic cells (DCs) promote tolerance or immunity depending on their maturation state, which is enhanced or accelerated upon MEK-ERK signaling pathway inhibition. We have determined the contribution of MEK-ERK activation to the profile of gene expression of human immature monocyte-derived dendritic cells (MDDCs) and peripheral blood myeloid DCs. ERK inhibition altered the expression of genes that mediate Chemokine (C-C motif) ligand 19 (CCL19)-directed migration (CCR7) and low-density lipoprotein (LDL) binding (CD36, SCARB1, OLR1, CXCL16) by immature DCs. In addition, ERK upregulated CCL2 expression while impairing the expression of DC maturation markers (RUNX3, ITGB7, IDO1). MEK-ERK-regulated genes exhibited an overrepresentation of cognate sequences for the aryl hydrocarbon receptor (AhR) transcription factor, whose transcriptional and DNA-binding activities increased in MDDCs upon exposure to the MEK1/2 inhibitor U0126. Therefore, the MEK-ERK signaling pathway regulates antigen capture, lymph node homing, and acquisition of maturation-associated genes, and its contribution to the maintenance of the immature state of MDDCs and myeloid DCs is partly dependent on the activity of AhR. Since pharmacologic modulation of the MEK-ERK signaling pathway has been proposed as a potential therapeutic strategy for cancer, our findings indicate that ERK inhibitors might influence antitumor responses through regulation of critical DC effector functions.

Role of c-MYC in tumor-associated macrophages and cancer progression

Transcription factors of the MYC family regulate several homeostatic cell functions, and their role as proto-oncogenes has been the focus of interest for decades. We have recently demonstrated that c-MYC is expressed by tumor-associated macrophages (TAMs) and regulates their phenotype and pro-tumor activities in vivo.

Inactivation of Nuclear Factor-Y Inhibits Vascular Smooth Muscle Cell Proliferation and Neointima Formation

Objective—Atherosclerosis and restenosis are multifactorial diseases associated with abnormal vascular smooth muscle cell (VSMC) proliferation. Nuclear factor-Y (NF-Y) plays a major role in transcriptional activation of the CYCLIN B1 gene (CCNB1), a key positive regulator of cell proliferation and neointimal thickening. Here, we investigated the role of NF-Y in occlusive vascular disease. Approach and Results—We performed molecular and expression studies in cultured cells, animal models, and human tissues. We find upregulation of NF-Y and cyclin B1 expression in proliferative regions of murine atherosclerotic plaques and mechanically induced lesions, which correlates with higher binding of NF-Y to target sequences in the CCNB1 promoter. NF-YA expression in neointimal lesions is detected in VSMCs, macrophages, and endothelial cells. Platelet-derived growth factor-BB, a main inductor of VSMC growth and neointima development, induces the recruitment of NF-Y to the CCNB1 promoter and augments both CCNB1 mRNA expression and cell proliferation through extracellular signal–regulated kinase 1/2 and Akt activation in rat and human VSMCs. Moreover, adenovirus-mediated overexpression of a NF-YA-dominant negative mutant inhibits platelet-derived growth factor-BB–induced CCNB1 expression and VSMC proliferation in vitro and neointimal lesion formation in a mouse model of femoral artery injury. We also detect NF-Y expression and DNA-binding activity in human neointimal lesions. Conclusions—Our results identify NF-Y as a key downstream effector of the platelet-derived growth factor-BB–dependent mitogenic pathway that is activated in experimental and human vasculoproliferative diseases. They also identify NF-Y inhibition as a novel and attractive strategy for the local treatment of neointimal formation induced by vessel denudation.

Abstract 2937: Editing central memory T lymphocyte specificity for safe and effective adoptive immunotherapy of leukemia

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The transfer of high-avidity T cell receptors (TCR) isolated from tumor-specific lymphocytes into polyclonal T cells is an attractive strategy for the adoptive immunotherapy of tumors. However, three major hurdles hinder the effectiveness of this strategy: i) inefficient gene transfer and unstable transgene expression, ii) exhaustion of gene-modified cells, and iii) mispairing of endogenous and exogenous TCR chains. To solve these issues, we used a bi-directional lentiviral vector (LV) expressing a codon-optimized, cysteine-modified TCR for the Wilms tumor antigen 1 (WT1) to genetically modified human central memory T lymphocytes (TCM). TCM have been reported superior to effectors in mediating long-lasting anti-tumor responses in vivo. Transduced cells showed high (∼16%) and stable (70 days in culture) levels of WT1-pentamer binding and cytotoxic activity towards WT1+ primary AML leukemic blasts. To further enhance the safety profile and potency of modified TCM, we developed a platform to genome edit TCM based on Zinc Finger Nucleases (ZFN). In order to ensure stable and consistent transgene expression, we demonstrated the targeted insertion of the transgene into a predefined genomic locus (CCR5) by homologous recombination in up to 6% of treated TCM, with concurrent inactivation of 18% of the targeted CCR5 gene by non-homologous end joining (NHEJ). These findings prompted us to address the major technological hurdle of TCR gene transfer: the co-expression of endogenous and tumor specific TCRs in the same cell. Indeed, this result in competition for the CD3 co-receptor molecules, leading to TCR dilution, and in TCR mispairing, giving rise to unpredictable specificities that are potentially autoreactive. To solve these issues, we developed ZFN specific for the endogenous TCR β chain gene to promote the disruption of TCR β by NHEJ. We obtained functional inactivation of this gene in up to 7% of TCM. Targeted lymphocytes failed in expressing the CD3/TCR complex on cell surface, and could be sorted as CD3neg. CD3neg proved stable in culture, maintained a TCM phenotype and were resistant to further TCR-mediated triggering. For a complete editing of T cell specificity, the WT1-specific TCR was transferred with high efficiency (>20%) by LV on CD3neg cells. WT1-TCR rescued CD3 surface expression and was expressed in edited lymphocytes at higher levels to that observed upon conventional TCR gene transfer. Accordingly, TCR-edited cells proved superior to cells undergoing conventional TCR gene transfer in promoting killing of relevant targets, and in recognizing low (10 nM) peptide concentrations. Overall, these data show the feasibility of rapid and efficient editing of TCM to generate safe and functional tumor-specific T cells and may open new perspectives to cancer adoptive immunotherapy. (PG and EP: equal contribution) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2937.