Noninvasive assessment of the lung inflammation-fibrosis axis by targeted imaging of CMKLR1.

Precision management of fibrotic lung diseases is challenging due to their diverse clinical trajectories and lack of reliable biomarkers for risk stratification and therapeutic monitoring. Here, we validated the accuracy of CMKLR1 as an imaging biomarker of the lung inflammation-fibrosis axis. By analyzing single-cell RNA sequencing datasets, we demonstrated CMKLR1 expression as a transient signature of monocyte-derived macrophages (MDMφ) enriched in patients with idiopathic pulmonary fibrosis (IPF). Consistently, we identified MDMφ as the major driver of the uptake of CMKLR1-targeting peptides in a murine model of bleomycin-induced lung fibrosis. Furthermore, CMKLR1-targeted positron emission tomography in the murine model enabled quantification and spatial mapping of inflamed lung regions infiltrated by CMKLR1-expressing macrophages and emerged as a robust predictor of subsequent lung fibrosis. Last, high CMKLR1 expression by bronchoalveolar lavage cells identified an inflammatory endotype of IPF with poor survival. Our investigation supports the potential of CMKLR1 as an imaging biomarker for endotyping and risk stratification of fibrotic lung diseases.

Glutaredoxin 1 controls monocyte reprogramming during nutrient stress and protects mice against obesity and atherosclerosis in a sex-specific manner.

High-calorie diet-induced nutrient stress promotes thiol oxidative stress and the reprogramming of blood monocytes, giving rise to dysregulated, obesogenic, proatherogenic monocyte-derived macrophages. We report that in chow-fed, reproductively senescent female mice but not in age-matched male mice, deficiency in the thiol transferase glutaredoxin 1 (Grx1) promotes dysregulated macrophage phenotypes as well as rapid weight gain and atherogenesis. Grx1 deficiency derepresses distinct expression patterns of reactive oxygen species and reactive nitrogen species generators in male versus female macrophages, poising female but not male macrophages for increased peroxynitrate production. Hematopoietic Grx1 deficiency recapitulates this sexual dimorphism in high-calorie diet-fed LDLR-/- mice, whereas macrophage-restricted overexpression of Grx1 eliminates the sex differences unmasked by high-calorie diet-feeding and protects both males and females against atherogenesis. We conclude that loss of monocytic Grx1 activity disrupts the immunometabolic balance in mice and derepresses sexually dimorphic oxidative stress responses in macrophages. This mechanism may contribute to the sex differences reported in cardiovascular disease and obesity in humans.

Post-GWAS functional analysis identifies CUX1 as a regulator of p16INK4a and cellular senescence.

Accumulation of senescent cells with age is an important driver of aging and age-related diseases. However, the mechanisms and signaling pathways that regulate senescence remain elusive. In this report, we performed post-genome-wide association studies (GWAS) functional studies on the CDKN2A/B locus, a locus known to be associated with multiple age-related diseases and overall human lifespan. We demonstrate that transcription factor CUX1 (Cut-Like Homeobox 1) specifically binds to an atherosclerosis-associated functional single-nucleotide polymorphism (fSNP) (rs1537371) within the locus and regulates the CDKN2A/B-encoded proteins p14ARF, p15INK4b and p16INK4a and the antisense noncoding RNA in the CDK4 (INK4) locus (ANRIL) in endothelial cells (ECs). Endothelial CUX1 expression correlates with telomeric length and is induced by both DNA-damaging agents and oxidative stress. Moreover, induction of CUX1 expression triggers both replicative and stress-induced senescence via activation of p16INK4a expression. Thus, our studies identify CUX1 as a regulator of p16INK4a-dependent endothelial senescence and a potential therapeutic target for atherosclerosis and other age-related diseases.

Divergence of acetate uptake in proinflammatory and inflammation-resolving macrophages: implications for imaging atherosclerosis.

BACKGROUND: Metabolic divergence of macrophages polarized into different phenotypes represents a mechanistically relevant target for non-invasive characterization of atherosclerotic plaques using positron emission tomography (PET). Carbon-11 (11C)-labeled acetate is a clinically available tracer which accumulates in atherosclerotic plaques, but its biological and clinical correlates in atherosclerosis are undefined. METHODS AND RESULTS: Histological correlates of 14C-acetate uptake were determined in brachiocephalic arteries of western diet-fed apoE-/- mice. The effect of polarizing stimuli on 14C-acetate uptake was determined by proinflammatory (interferon-γ + lipopolysaccharide) vs inflammation-resolving (interleukin-4) stimulation of murine macrophages and human carotid endarterectomy specimens over 2 days. 14C-acetate accumulated in atherosclerotic regions of arteries. CD68-positive monocytes/macrophages vs smooth muscle actin-positive smooth muscle cells were the dominant cells in regions with high vs low 14C-acetate uptake. 14C-acetate uptake progressively decreased in proinflammatory macrophages to 25.9 ± 4.5% of baseline (P < .001). A delayed increase in 14C-acetate uptake was induced in inflammation-resolving macrophages, reaching to 164.1 ± 21.4% (P < .01) of baseline. Consistently, stimulation of endarterectomy specimens with interferon-γ + lipopolysaccharide decreased 14C-acetate uptake to 66.5 ± 14.5%, while interleukin-4 increased 14C-acetate uptake to 151.5 ± 25.8% compared to non-stimulated plaques (P < .05). CONCLUSIONS: Acetate uptake by macrophages diverges upon proinflammatory and inflammation-resolving stimulation, which may be exploited for immunometabolic characterization of atherosclerosis.

Molecular Imaging of Very Late Antigen-4 in Acute Lung Injury.

Inflammation plays a central role in the pathogenesis of acute lung injury (ALI) during both the acute pneumonitis stage and progression into the chronic fibroproliferative phase, leading to pulmonary fibrosis. Currently, there is an unmet clinical and research need for noninvasive ways to monitor lung inflammation through targeting of immunoregulatory pathways contributing to ALI pathogenesis. In this study, we evaluated the role of targeted imaging of very late antigen-4 (VLA-4), as a key integrin mediating the adhesion and recruitment of immune cells to inflamed tissues, in quantifying lung inflammation in a mouse model of lipopolysaccharide-induced ALI. Methods: ALI was induced by a single intratracheal administration of lipopolysaccharide (10, 20, or 40 µg per mouse) in C57BL/6J mice. Control mice were intratracheally instilled with sterile phosphate-buffered saline. VLA-4-targeted PET/CT was performed 24 h after intravenous injection of a 64Cu-labeled high-affinity peptidomimetic ligand referred to as 64Cu-LLP2A, which is conjugated with the chelator (1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) and a polyethylene glycol 4 linker, at day 2 after the induction of ALI. Ex vivo biodistribution of 64Cu-LLP2A was determined by γ-counting of harvested organs. The severity of lung inflammation was assessed histologically and by measuring the expression of inflammatory markers in the lung tissue lysates using reverse transcription quantitative polymerase chain reaction. Results: Intratracheal lipopolysaccharide instillation led to an acute inflammatory response in the lungs, characterized by increased expression of multiple inflammatory markers and infiltration of myeloid cells, along with a significant and specific increase in 64Cu-LLP2A uptake, predominantly in a peribronchial distribution. There was a strong correlation between the lipopolysaccharide dose and 64Cu-LLP2A uptake, as quantified by in vivo PET (R = 0.69, P < 0.01). Expression levels of both subunits of VLA-4, that is, integrins α4 and ß1, significantly correlated with the expression of multiple inflammatory markers, including tumor necrosis factor-α, interleukin-1ß, and nitric oxide synthase-2, highlighting the potential of VLA-4 as a surrogate marker of acute lung inflammation. Notably, in vivo 64Cu-LLP2A uptake significantly correlated with the expression of multiple inflammatory markers and VLA-4. Conclusion: Our study demonstrates the feasibility of molecular imaging of VLA-4, as a mechanistically relevant target in ALI, and the accuracy of VLA-4-targeted PET in quantification of ongoing lung inflammation in a murine model.

Characterization of Macrophage Polarization States Using Combined Measurement of 2-Deoxyglucose and Glutamine Accumulation: Implications for Imaging of Atherosclerosis.

OBJECTIVE: Despite the early promising results of 18F-fluorodeoxyglucose positron emission tomography for assessment of vessel wall inflammation, its accuracy in prospective identification of vulnerable plaques has remained limited. Additionally, previous studies have indicated that 18F-fluorodeoxyglucose uptake alone may not allow for accurate identification of specific macrophage activation states. We aimed to determine whether combined measurement of glucose and glutamine accumulation-the 2 most important bioenergetic substrates for macrophages-improves the distinction of macrophage inflammatory states and can be utilized to image atherosclerosis. APPROACH AND RESULTS: Murine peritoneal macrophages (MΦ) were activated ex vivo into proinflammatory states with either lipopolysaccharide (MΦLPS) or interferon-γ+tumor necrosis factor-α (MΦIFN-γ+TNF-α). An alternative polarization phenotype was induced with interleukin-4 (MΦIL-4). The pronounced increase in 2-deoxyglucose uptake distinguishes MΦLPS from MΦIFN-γ+TNF-α, MΦIL-4, and unstimulated macrophages (MΦ0). Despite having comparable levels of 2-deoxyglucose accumulation, MΦIL-4 can be distinguished from both MΦIFN-γ+TNF-α and MΦ0 based on the enhanced glutamine accumulation, which was associated with increased expression of a glutamine transporter, Slc1a5. Ex vivo autoradiography experiments demonstrated distinct and heterogenous patterns of 18F-fluorodeoxyglucose and 14C-glutamine accumulation in atherosclerotic lesions of low-density lipoprotein receptor-null mice fed a high-fat diet. CONCLUSIONS: Combined assessment of glutamine and 2-deoxyglucose accumulation improves the ex vivo identification of macrophage activation states. Combined ex vivo metabolic imaging demonstrates heterogenous and distinct patterns of substrate accumulation in atherosclerotic lesions. Further studies are required to define the in vivo significance of glutamine uptake in atherosclerosis and its potential application in identification of vulnerable plaques.

Differential Regulation of Macrophage Glucose Metabolism by Macrophage Colony-stimulating Factor and Granulocyte-Macrophage Colony-stimulating Factor: Implications for 18F FDG PET Imaging of Vessel Wall Inflammation.

Purpose To determine the divergence of immunometabolic phenotypes of macrophages stimulated with macrophage colony-stimulating factor (M-CSF) and granulocyte-M-CSF (GM-CSF) and its implications for fluorine 18 (18F) fluorodeoxyglucose (FDG) imaging of atherosclerosis. Materials and Methods This study was approved by the animal care committee. Uptake of 2-deoxyglucose and various indexes of oxidative and glycolytic metabolism were evaluated in nonactivated murine peritoneal macrophages (MΦ0) and macrophages stimulated with M-CSF (MΦM-CSF) or GM-CSF (MΦGM-CSF). Intracellular glucose flux was measured by using stable isotope tracing of glycolytic and tricyclic acid intermediary metabolites. 18F-FDG uptake was evaluated in murine atherosclerotic aortas after stimulation with M-CSF or GM-CSF by using quantitative autoradiography. Results Despite inducing distinct activation states, GM-CSF and M-CSF stimulated progressive but similar levels of increased 2-deoxyglucose uptake in macrophages that reached up to sixfold compared with MΦ0. The expression of glucose transporters, oxidative metabolism, and mitochondrial biogenesis were induced to similar levels in MΦM-CSF and MΦGM-CSF. Unexpectedly, there was a 1.7-fold increase in extracellular acidification rate, a 1.4-fold increase in lactate production, and overexpression of several critical glycolytic enzymes in MΦM-CSF compared with MΦGM-CSF with associated increased glucose flux through glycolytic pathway. Quantitative autoradiography demonstrated a 1.6-fold induction of 18F-FDG uptake in murine atherosclerotic plaques by both M-CSF and GM-CSF. Conclusion The proinflammatory and inflammation-resolving activation states of macrophages induced by GM-CSF and M-CSF in either cell culture or atherosclerotic plaques may not be distinguishable by the assessment of glucose uptake. © RSNA, 2016 Online supplemental material is available for this article.

Monocytic MKP-1 is a Sensor of the Metabolic Environment and Regulates Function and Phenotypic Fate of Monocyte-Derived Macrophages in Atherosclerosis.

Diabetes promotes the S-glutathionylation, inactivation and subsequent degradation of mitogen-activated protein kinase phosphatase 1 (MKP-1) in blood monocytes, and hematopoietic MKP-1-deficiency in atherosclerosis-prone mice accelerates atherosclerotic lesion formation, but the underlying mechanisms were not known. Our aim was to determine the mechanisms through which MKP-1 deficiency in monocytes and macrophages promotes atherogenesis. Transplantation of MKP-1-deficient bone marrow into LDL-R-/- (MKP-1LeuKO) mice accelerated high-fat diet (HFD)-induced atherosclerotic lesion formation. After 12 weeks of HFD feeding, MKP-1LeuKO mice showed increased lesion size in both the aortic root (1.2-fold) and the aorta (1.6-fold), despite reduced plasma cholesterol levels. Macrophage content was increased in lesions of MKP-1LeuKO mice compared to mice that received wildtype bone marrow. After only 6 weeks on a HFD, in vivo chemotactic activity of monocytes was already significantly increased in MKP-1LeuKO mice. MKP-1 deficiency in monocytes and macrophages promotes and accelerates atherosclerotic lesion formation by hyper-sensitizing monocytes to chemokine-induced recruitment, predisposing macrophages to M1 polarization, decreased autophagy and oxysterol-induced cell death whereas overexpression of MKP-1 protects macrophages against metabolic stress-induced dysfunction. MKP-1 serves as a master-regulator of macrophage phenotype and function and its dysregulation by metabolic stress may be a major contributor to atherogenesis and the progression of atherosclerotic plaques.

Protein Thiol Redox Signaling in Monocytes and Macrophages.

SIGNIFICANCE: Monocyte and macrophage dysfunction plays a critical role in a wide range of inflammatory disease processes, including obesity, impaired wound healing diabetic complications, and atherosclerosis. Emerging evidence suggests that the earliest events in monocyte or macrophage dysregulation include elevated reactive oxygen species production, thiol modifications, and disruption of redox-sensitive signaling pathways. This review focuses on the current state of research in thiol redox signaling in monocytes and macrophages, including (i) the molecular mechanisms by which reversible protein-S-glutathionylation occurs, (ii) the identification of bona fide S-glutathionylated proteins that occur under physiological conditions, and (iii) how disruptions of thiol redox signaling affect monocyte and macrophage functions and contribute to atherosclerosis. Recent Advances: Recent advances in redox biochemistry and biology as well as redox proteomic techniques have led to the identification of many new thiol redox-regulated proteins and pathways. In addition, major advances have been made in expanding the list of S-glutathionylated proteins and assessing the role that protein-S-glutathionylation and S-glutathionylation-regulating enzymes play in monocyte and macrophage functions, including monocyte transmigration, macrophage polarization, foam cell formation, and macrophage cell death. CRITICAL ISSUES: Protein-S-glutathionylation/deglutathionylation in monocytes and macrophages has emerged as a new and important signaling paradigm, which provides a molecular basis for the well-established relationship between metabolic disorders, oxidative stress, and cardiovascular diseases. FUTURE DIRECTIONS: The identification of specific S-glutathionylated proteins as well as the mechanisms that control this post-translational protein modification in monocytes and macrophages will facilitate the development of new preventive and therapeutic strategies to combat atherosclerosis and other metabolic diseases. Antioxid. Redox Signal. 25, 816-835.

Protein S-Glutathionylation Mediates Macrophage Responses to Metabolic Cues from the Extracellular Environment.

AIMS: Protein S-glutathionylation, the formation of a mixed disulfide between glutathione and protein thiols, is an oxidative modification that has emerged as a new signaling paradigm, potentially linking oxidative stress to chronic inflammation associated with heart disease, diabetes, cancer, lung disease, and aging. Using a novel, highly sensitive, and selective proteomic approach to identify S-glutathionylated proteins, we tested the hypothesis that monocytes and macrophages sense changes in their microenvironment and respond to metabolic stress by altering their protein thiol S-glutathionylation status. RESULTS: We identified over 130 S-glutathionylated proteins, which were associated with a variety of cellular functions, including metabolism, transcription and translation, protein folding, free radical scavenging, cell motility, and cell death. Over 90% of S-glutathionylated proteins identified in metabolically stressed THP-1 monocytes were also found in hydrogen peroxide (H2O2)-treated cells, suggesting that H2O2 mediates metabolic stress-induced protein S-glutathionylation in monocytes and macrophages. We validated our findings in mouse peritoneal macrophages isolated from both healthy and dyslipidemic atherosclerotic mice and found that 52% of the S-glutathionylated proteins found in THP-1 monocytes were also identified in vivo. Changes in macrophage protein S-glutathionylation induced by dyslipidemia were sexually dimorphic. INNOVATION: We provide a novel mechanistic link between metabolic (and thiol oxidative) stress, macrophage dysfunction, and chronic inflammatory diseases associated with metabolic disorders. CONCLUSION: Our data support the concept that changes in the extracellular metabolic microenvironment induce S-glutathionylation of proteins central to macrophage metabolism and a wide array of cellular signaling pathways and functions, which in turn initiate and promote functional and phenotypic changes in macrophages. Antioxid. Redox Signal. 25, 836-851.

Ursolic acid protects monocytes against metabolic stress-induced priming and dysfunction by preventing the induction of Nox4.

AIMS: Dietary supplementation with ursolic acid (UA) prevents monocyte dysfunction in diabetic mice and protects mice against atherosclerosis and loss of renal function. The goal of this study was to determine the molecular mechanism by which UA prevents monocyte dysfunction induced by metabolic stress. METHODS AND RESULTS: Metabolic stress sensitizes or "primes" human THP-1 monocytes and murine peritoneal macrophages to the chemoattractant MCP-1, converting these cells into a hyper-chemotactic phenotype. UA protected THP-1 monocytes and peritoneal macrophages against metabolic priming and prevented their hyper-reactivity to MCP-1. UA blocked the metabolic stress-induced increase in global protein-S-glutathionylation, a measure of cellular thiol oxidative stress, and normalized actin-S-glutathionylation. UA also restored MAPK phosphatase-1 (MKP1) protein expression and phosphatase activity, decreased by metabolic priming, and normalized p38 MAPK activation. Neither metabolic stress nor UA supplementation altered mRNA or protein levels of glutaredoxin-1, the principal enzyme responsible for the reduction of mixed disulfides between glutathione and protein thiols in these cells. However, the induction of Nox4 by metabolic stress, required for metabolic priming, was inhibited by UA in both THP-1 monocytes and peritoneal macrophages. CONCLUSION: UA protects THP-1 monocytes against dysfunction by suppressing metabolic stress-induced Nox4 expression, thereby preventing the Nox4-dependent dysregulation of redox-sensitive processes, including actin turnover and MAPK-signaling, two key processes that control monocyte migration and adhesion. This study provides a novel mechanism for the anti-inflammatory and athero- and renoprotective properties of UA and suggests that dysfunctional blood monocytes may be primary targets of UA and related compounds.

Bioenergetic profiles diverge during macrophage polarization: implications for the interpretation of 18F-FDG PET imaging of atherosclerosis.

UNLABELLED: Conventional cardiovascular imaging is invaluable for the assessment of late sequelae of atherosclerosis, such as diminished perfusion reserve and luminal stenosis. Molecular imaging provides complementary information about plaque composition and ongoing biologic processes in the vessel wall, allowing the early diagnosis and risk stratification of patients. Detection of enhanced glucose uptake, using (18)F-FDG PET, has been proposed as a noninvasive approach to track macrophage activation as a critical event in the development and progression of atherosclerosis. In this study, we determined the impact of macrophage polarization on glucose metabolism and oxidative phosphorylation. METHODS: Murine peritoneal macrophages were incubated in the presence of interferon-γ (IFN-γ) plus tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), or interleukin-4 (IL-4) to induce classic (M1 and M(LPS)) or alternative (M2) polarization, respectively. Glucose uptake was measured using (3)H-deoxyglucose. Oxidative phosphorylation was evaluated using an extracellular flux analyzer. Mitochondrial DNA copy numbers were quantified by polymerase chain reaction. The expression of glucose transporter-1 (Glut-1), hexokinase-1 and -2 (Hk-1 and Hk-2, respectively), mitochondrial transcription factor-1 (Tfam), and cytochrome c oxidase subunit I (Cox-1) was determined by quantitative reverse transcription polymerase chain reaction. RESULTS: Stimulation of macrophages by LPS, but not polarization with either IFN-γ plus TNF-α (M1) or IL-4 (M2), resulted in a 2.5-fold increase in (3)H-deoxyglucose uptake. Enhanced glucose uptake by M(LPS) macrophages paralleled the overexpression of rate-limiting proteins involved in transmembrane transport and intracellular trapping of glucose--that is, Glut-1, Hk-1, and Hk-2. Alternatively polarized M2 macrophages developed a markedly higher spare respiratory capacity than both nonpolarized and classically polarized M1 macrophages. M2 polarization was associated with a 4.6-fold increase in mitochondrial content of the cells, compared with nonpolarized macrophages. The expression of Tfam, a major regulator of mitochondrial biogenesis, and Cox-1, a critical component of respiratory chain, was significantly increased in M2 polarized macrophages. CONCLUSION: Polarization of macrophages induces distinct metabolic profiles with respect to glycolysis versus oxidative phosphorylation, with alternatively polarized macrophages shifting to mitochondria as their main source of adenosine triphosphate. Only M(LPS), but not M1 or M2 polarized macrophages, showed increased glucose uptake, suggesting that glucose metabolism is regulated independent of the polarization state and macrophage polarization may not be detectable by (18)F-FDG PET.

Contrast-enhanced cardiac magnetic resonance imaging.

Cardiac magnetic resonance (CMR) imaging has significantly evolved in the past decade and is well established in the evaluation of coronary artery disease (CAD). The evaluation of cardiac anatomy and contractility by high-resolution CMR can be improved by using intravenous administration of gadolinium-based contrast agents. Delayed enhancement CMR imaging has become the gold standard for quantification of myocardial viability in CAD. Contrast-enhanced CMR imaging may circumvent the need for endomyocardial biopsy or localize the involved regions, thereby improving the diagnostic yield of this invasive procedure. The application of contrast-enhanced CMR as an advanced imaging technique for ischemic and nonischemic diseases is reviewed.

Reactive oxygen species and thiol redox signaling in the macrophage biology of atherosclerosis.

SIGNIFICANCE: Despite the recent decline in the prevalence of cardiovascular diseases, atherosclerosis remains the leading cause of death in industrialized countries. Monocyte recruitment into the vessel wall is a rate-limiting step in atherogenesis. Death of macrophage-derived foam cells promotes lesion progression and the majority of acute complications of atherosclerotic disease (e.g., myocardial infarction) occur in lesions that are intensely infiltrated with monocyte-derived macrophages, underlining the critical roles monocytes and macrophages play in this complex chronic inflammatory disease. RECENT ADVANCES: A rapidly growing body of literature supports a critical role for reactive oxygen species (ROS) in the regulation of monocyte and macrophage (dys)function associated with atherogenesis and macrophage death in atherosclerotic plaque. CRITICAL ISSUES: In this review we highlight the important roles of NADHP oxidase 4 recently identified in monocytes and macrophages and the role of ROS and (thiol) redox signaling in different aspects of monocytes and macrophage biology associated with atherosclerosis. FUTURE DIRECTIONS: Studies aimed at identifying the intracellular targets of ROS involved in redox signaling in macrophages and at elucidating the redox signaling mechanisms that control differentiation, activation, polarization, and death of monocytes and macrophages may ultimately lead to the development of novel preventive and therapeutic strategies for atherosclerosis.

Integrin-targeted imaging of inflammation in vascular remodeling.

OBJECTIVE: Inflammation plays a key role in the development of vascular diseases. Monocytes and macrophages express α(v)ß(3) integrin. We used an α(v) integrin-specific tracer, (99m)Tc-NC100692, to investigate integrin-targeted imaging for detection vessel wall inflammation. METHODS AND RESULTS: The binding of a fluorescent homologue of NC100692 to α(v)ß(3) on human monocytes and macrophages was shown by flow cytometry. Vessel wall inflammation and remodeling was induced in murine carotid arteries through adventitial exposure to CaCl(2). NC100692 micro single photon computed tomography/CT imaging was performed after 2 and 4 weeks and showed significantly higher uptake of the tracer in CaCl(2)-exposed left carotids compared with sham-operated contralateral arteries. Histological analysis at 4 weeks demonstrated significant remodeling of left carotid arteries and considerable macrophage infiltration, which was confirmed by real-time polymerase chain reaction. There was no significant difference in normalized α(v), ß(3), or ß(5) mRNA expression between right and left carotid arteries. Finally, NC100692 uptake strongly correlated with macrophage marker expression in carotid arteries. CONCLUSIONS: NC100692 imaging can detect vessel wall inflammation in vivo. If further validated, α(v)-targeted imaging may provide a noninvasive approach for identifying patients who are at high risk for vascular events and tracking the effect of antiinflammatory treatments.

Matrix metalloproteinase activation predicts amelioration of remodeling after dietary modification in injured arteries.

OBJECTIVE: To establish and validate early noninvasive imaging of matrix metalloproteinase (MMP) activation for monitoring the progression of vascular remodeling and response to dietary modification. METHODS AND RESULTS: Apolipoprotein E(-/-) mice that were fed a high-fat diet underwent left common carotid artery wire injury. One week after surgery, a group of animals were withdrawn from the high-fat diet. The other group of animals continued that diet throughout the study. Micro single-photon emission computed tomographic (microSPECT)/CT imaging with RP805 (a (99m)Tc-labeled tracer targeting activated MMPs) was repeatedly performed at 2 and 4 weeks after surgery. Histological analysis at 4 weeks showed significant left carotid neointima formation, monocyte/macrophage infiltration, and upregulation of several MMPs, which were ameliorated by withdrawal from the high-fat diet. In vivo microSPECT/CT images visualized significant RP805 uptake, reflecting MMP activation, in the injured carotid arteries. MMP activation was reduced as early as 1 week after withdrawal from the high-fat diet and significantly correlated with neointimal area at 4 weeks after surgery. CONCLUSIONS: MMP activation predicts the progression of vascular remodeling and can track the effect of dietary modification after vascular injury.

VEGF blockade inhibits lymphocyte recruitment and ameliorates immune-mediated vascular remodeling.

RATIONALE: There are conflicting data on the effects of vascular endothelial growth factor (VEGF) in vascular remodeling. Furthermore, there are species-specific differences in leukocyte and vascular cell biology and little is known about the role of VEGF in remodeling of human arteries. OBJECTIVE: We sought to address the role of VEGF blockade on remodeling of human arteries in vivo. METHODS AND RESULTS: We used an anti-VEGF antibody, bevacizumab, to study the effect of VEGF blockade on remodeling of human coronary artery transplants in severe combined immunodeficient mice. Bevacizumab ameliorated peripheral blood mononuclear cell-induced but not interferon-gamma-induced neointimal formation. This inhibitory effect was associated with a reduction in graft T-cell accumulation without affecting T-cell activation. VEGF enhanced T-cell capture by activated endothelium under flow conditions. The VEGF effect could be recapitulated when a combination of recombinant intercellular adhesion molecule 1 and vascular cell adhesion molecule-1 rather than endothelial cells was used to capture T cells. A subpopulation of CD3+ T cells expressed VEGF receptor (VEGFR)-1 by immunostaining and FACS analysis. VEGFR-1 mRNA was also detectable in purified CD4+ T cells and Jurkat and HSB-2 T-cell lines. Stimulation of HSB-2 and T cells with VEGF triggered downstream ERK phosphorylation, demonstrating the functionality of VEGFR-1 in human T cells. CONCLUSIONS: VEGF contributes to vascular remodeling in human arteries through a direct effect on human T cells that enhances their recruitment to the vessel. These findings raise the possibility of novel therapeutic approaches to vascular remodeling based on inhibition of VEGF signaling.