Molecular Profiling of 50 734 Bethesda III-VI Thyroid Nodules by ThyroSeq v3: Implications for Personalized Management.

CONTEXT: Comprehensive genomic analysis of thyroid nodules for multiple classes of molecular alterations detected in a large series of fine needle aspiration (FNA) samples has not been reported. OBJECTIVE: To determine the prevalence of clinically relevant molecular alterations in Bethesda categories III-VI (BCIII-VI) thyroid nodules. METHODS: This retrospective analysis of FNA samples, tested by ThyroSeq v3 using Genomic Classifier and Cancer Risk Classifier at UPMC Molecular and Genomic Pathology laboratory, analyzed the prevalence of diagnostic, prognostic, and targetable genetic alterations in a total of 50 734 BCIII-VI nodules from 48 225 patients. RESULTS: Among 50 734 informative FNA samples, 65.3% were test-negative, 33.9% positive, 0.2% positive for medullary carcinoma, and 0.6% positive for parathyroid. The benign call rate in BCIII-IV nodules was 68%. Among test-positive samples, 73.3% had mutations, 11.3% gene fusions, and 10.8% isolated copy number alterations. Comparing BCIII-IV nodules with BCV-VI nodules revealed a shift from predominantly RAS-like alterations to BRAF V600E-like alterations and fusions involving receptor tyrosine kinases (RTK). Using ThyroSeq Cancer Risk Classifier, a high-risk profile, which typically included TERT or TP53 mutations, was found in 6% of samples, more frequently BCV-VI. RNA-Seq confirmed ThyroSeq detection of novel RTK fusions in 98.9% of cases. CONCLUSION: In this series, 68% of BCIII-IV nodules were classified as negative by ThyroSeq, potentially preventing diagnostic surgery in this subset of patients. Specific genetic alterations were detected in most BCV-VI nodules, with a higher prevalence of BRAF and TERT mutations and targetable gene fusions compared to BCIII-IV nodules, offering prognostic and therapeutic information for patient management.

Validation of Linear Range HER2/Estrogen Receptor/Progesterone Receptor IHControls for Daily Quality Assurance.

OBJECTIVES: To evaluate a new US Food and Drug Administration (FDA)-cleared immunohistochemistry (IHC) control (IHControls [Boston Cell Standards]) comprising peptide epitopes for HER2, estrogen receptor (ER), and progesterone receptor (PR) attached to cell-sized microspheres and to compare its performance against conventional tissue controls. METHODS: IHControls and tissue/cell line controls for HER2, ER, and PR were compared side by side daily at 5 clinical IHC laboratories for 1 to 2 months. Separately, the sensitivity of the 2 types of controls was evaluated in simulated IHC assay failure experiments by diluting the primary antibody. Additional evaluations included lot-to-lot manufacturing reproducibility of 3 independent lots and specificity against 26 antigenically irrelevant IHC stains. RESULTS: Side-by-side testing revealed a 99.6% concordance between IHControls and tissue controls across 5 IHC laboratories and 766 individual evaluations. Three discordant quality control events were the result of operator error. Simulated assay failure data showed that both IHControls and tissue controls are similarly capable of detecting IHC staining errors. Manufacturing reproducibility of IHControls showed less than 10% variability (coefficient of variation). No cross-reactions were detected from 26 antigenically irrelevant IHC stains. CONCLUSIONS: IHControls, the first FDA-cleared IHC controls, can sensitively and accurately detect IHC assay problems, similar to tissue controls.

Optimizing molecular testing of lung cancer needle biopsy specimens: potential solutions from an interdisciplinary qualitative study.

BACKGROUND: Molecular testing can detect actionable genomic alterations and tumor cell surface proteins in patients with non-small cell lung cancer (NSCLC). However, utilization remains suboptimal, representing missed treatment opportunities. This study aimed to identify challenges and potential solutions to obtaining percutaneous lung needle biopsy specimens for successful molecular testing in patients with advanced NSCLC. METHODS: This interdisciplinary qualitative study included ten radiologists and four pathologists from academic and community settings across the United States who routinely perform and analyze percutaneous lung needle biopsies. Participants underwent semi-structured one-on-one interviews (Phase 1). Interview questionnaires were constructed based on a literature review of key lines of inquiry and conducted by professional market researchers using the theoretical domains framework. Primary barriers to molecular testing were identified using thematic analysis. Subsequently, multidisciplinary focus groups were convened to identify potential solutions (Phase 2). RESULTS: Four themes emerged as barriers to molecular testing and were matched to the clinical workflow: (1) biopsy request, (2) biopsy procedure, (3) specimen analysis, and (4) communication. The nineteen potential solutions included adding a "checkbox" to indicate molecular testing in the biopsy request, leveraging pre-procedural imaging to guide biopsies, conserving tissue through appropriate allocation strategies and next generation sequencing panels instead of sequential single-gene assays, instituting reflex-molecular testing upon NSCLC diagnosis, tracking and communicating biopsy outcomes at multidisciplinary tumor boards, and improving integration of radiologists and pathologists into oncology care teams. CONCLUSIONS: Potential solutions exist to increase successful molecular testing of lung needle biopsy specimens in patients with advanced NSCLC.

Fluorescence Polarization Imaging of Methylene Blue Facilitates Quantitative Detection of Thyroid Cancer in Single Cells.

BACKGROUND: Diagnostic accuracy of the standard of care fine-needle aspiration cytology (FNAC) remains a significant problem in thyroid oncology. Therefore, a robust and accurate method for reducing uncertainty of cytopathological evaluation would be invaluable. METHODS: In this double-blind study, we employed fluorescence emission and quantitative fluorescence polarization (Fpol) confocal imaging for sorting thyroid cells into benign/malignant categories. Samples were collected from malignant tumors, benign nodules, and normal thyroid epithelial tissues. RESULTS: A total of 32 samples, including 12 from cytologically indeterminate categories, were stained using aqueous methylene blue (MB) solution, imaged, and analyzed. Fluorescence emission images yielded diagnostically relevant information on cytomorphology. Significantly higher MB Fpol was measured in thyroid cancer as compared to benign and normal cells. The results obtained from 12 indeterminate samples revealed that MB Fpol accurately differentiated benign and malignant thyroid nodules. CONCLUSIONS: The developed imaging approach holds the potential to provide an accurate and objective biomarker for thyroid cancer, improve diagnostic accuracy of cytopathology, and decrease the number of lobectomy and near-total thyroidectomy procedures.

Multisite Quality Improvement Study of a Patient-Pathologist Consultation Program.

OBJECTIVES: The aim of this multisite quality improvement study was to evaluate patients' experiences with the patient-centered pathology (PCP) consultation program and to determine whether PCP enhanced their care experience. METHODS: Patients were invited to attend PCP consultations to review their pathology report and slides and have their questions answered by the pathologist privately, with the option to attend the appointment with family members or friends for support. A patient experience questionnaire (PEQ) was administered to patients, who participated voluntarily in the PCP, and survey data were collected and stored in REDCap. Statistical analysis was performed using SAS 9.4 (SAS Institute). RESULTS: Sixty-seven patients (95.5% female) aged 18 to 84 years across 4 institutions completed the PEQ. Overall, 58% and 15.8% of patients had breast and brain tumors, respectively, and 59.7% of tumors were newly diagnosed. Most patients thought it was important for them to learn as much as they could about their health condition. However, the majority of patients reported some degree of difficulty learning about their health condition based on written information, despite 97% having completed high school and/or further education. The majority of patients rated their pathologist as "excellent" across communication metrics. Ultimately, 100% of respondents were satisfied, found their visits to be useful, and would recommend the PCP to other patients. CONCLUSIONS: Patients found that personalized clinical encounters with pathologists improved their understanding of their health condition and their satisfaction with their care experience. Patients thought pathologists communicated respectfully, effectively, and empathetically.

In Vivo and In Vitro Effects of Vasopressin V2 Receptor Antagonism on Myocardial Fibrosis in Rats.

BACKGROUND: Myocardial fibrosis is a major pathophysiologic substrate of heart failure with preserved ejection fraction. Vasopressin is an important therapeutic target in heart failure with preserved ejection fraction since it can modulate fluid balance, and based on a few studies, myocardial matrix deposition. Hence we examined the role of vasopressin antagonism in modulating myocardial matrix metabolism in vivo and in vitro. MATERIALS AND METHODS: In vivo studies utilized an established model of hyperhomocysteinemia-induced myocardial fibrosis in Sprague-Dawley rats combined with high salt diet; in vivo studies also utilized the same profibrotic stimuli of homocysteine and NaCl in cultured rat cardiac fibroblasts. RESULTS: Hyperhomocysteinemia combined with high-salt diet promoted myocardial fibrosis, profibrotic and matrix gene expression and tolvaptan attenuated all these in vivo effects. In cultured cardiac fibroblasts, combined treatment with homocysteine and NaCl increased profibrotic and matrix gene expression and activation of PI3/Akt pathway; all these effects were attenuated by tolvaptan Vasopressin levels, gene expression and V2 receptor expression were increased in vivo and in vitro on exposure to profibrotic stimuli, and tolvaptan attenuated these in vivo and in vitro effects. CONCLUSIONS: Antagonism of vasopressin V2 receptor, via direct actions on cardiac fibroblast, attenuates myocardial matrix deposition.

"Please Help Me See the Dragon I Am Slaying": Implementation of a Novel Patient-Pathologist Consultation Program and Survey of Patient Experience.

CONTEXT.­: Pathologists evaluate human disease and teach medical students, residents, and clinicians. Historically recognized as the "doctor's doctor," pathologists are well suited to be a direct patient resource of individualized, accurate information. OBJECTIVE.­: To develop and implement a pathology consultation service whereby patients review their tissue slides directly with pathologists. DESIGN.­: A pathologist conducted patient consultations, reviewing biopsy or surgery findings on a multiheaded microscope or computer screen. The pathologist evaluated patients' understanding of their disease and invited patients to ask specific questions. We recorded patient demographic data and assessed utilization with a short patient satisfaction survey using 6 questions with a 5-point Likert scale and 2 questions for open response. RESULTS.­: A total of 31 patients came for consultation; 39% (12 of 31) were accompanied by a friend or family member. Patients' median age was 59 years, with a strong female predominance (90%; 28 of 31). The majority of patients had breast cancer (58%; 18 of 31) or hematologic malignancy (19%; 6 of 31). Of the 31 patients, the survey response rate was 58% (18 of 31). Top-box scoring demonstrated program support, with 89% (16 of 18) of respondents strongly recommending the experience to another patient. Additionally, 78% (14 of 18) strongly agreed that they felt more empowered after seeing their disease. Mean scores for Likert-based questions all were higher than 4.0. CONCLUSIONS.­: To our knowledge, this study is the first report of direct patient-pathologist consultation. Early data suggest that the program may provide effective patient-specific education. The high response rate and favorable assessment of the program suggest that it may be a valuable resource for some patients.

Integration of basic science and clinical medicine: the innovative approach of the cadaver biopsy project at the Boston University School of Medicine.

PROBLEM: Curricular integration has emerged as a consistent theme in medical education reform. Vertical integration of topics such as pathology offers the potential to bring basic science content into the clinical arena, but faculty/student acceptance and curricular design pose challenges for such integration. APPROACH: The authors describe the Cadaver Biopsy Project (CBP) at Boston University School of Medicine as a sustainable model of vertical integration. Faculty and select senior medical students obtained biopsies of cadavers during the first-year gross anatomy course (fall 2009) and used these to develop clinical cases for courses in histology (spring 2010), pathology (fall 2010-spring 2011), and radiology (fall 2011 or spring 2012), thereby linking students' first experiences in basic sciences with other basic science courses and later clinical courses. Project goals included engaging medical stu dents in applying basic science princi ples in all aspects of patient care as they acquire skills. The educational intervention used a patient (cadaver)-centered approach and small-group, collaborative, case-based learning. OUTCOMES: Through this project, the authors involved clinical and basic science faculty-plus senior medical students-in a collaborative project to design and implement an integrated curriculum through which students revisited, at several different points, the microscopic structure and pathophysiology of common diseases. NEXT STEPS: Developing appropriate, measurable out comes for medical education initiatives, including the CBP, is challenging. Accumu lation of qualitative feedback from surveys will guide continuous improvement of the CBP. Documenting longer-term impact of the curricular innovation on test scores and other competency-based outcomes is an ultimate goal.

Devising the optimal preclinical oncology curriculum for undergraduate medical students in the United States.

A third of women and a near majority of men in the United States will be diagnosed with cancer in their lifetimes. To prepare future physicians for this reality, we have developed a preclinical oncology curriculum that introduces second-year medical students to essential concepts and practices in oncology to improve their abilities to appropriately care for these patients. We surveyed the oncology and education literature and compiled subjects important to students' education including basic science and clinical aspects of oncology and addressing patients' psychosocial needs. Along with the proposed curriculum content, scheduling, independent learning exercises, and case studies, we discuss practical considerations for curriculum implementation based on experience at our institution. Given the changing oncology healthcare landscape, all (new) physicians must competently address their cancer patients' needs, regardless of chosen specialty. A thorough and logically organized cancer curriculum for preclinical medical students should help achieve these aims. This new model curriculum, with accompanying strategies to evaluate its efforts, is essential to update how medical students are educated about cancer.

B lymphocytes in human subcutaneous adipose crown-like structures.

Accumulation of macrophages and T cells within crown-like structures (CLS) in subcutaneous adipose tissue predicts disease severity in obesity-related insulin resistance (OIR). Although rodent data suggest the B cell is an important feature of these lesions, B cells have not been described within the human CLS. In order to identify B cells in the human subcutaneous CLS (sCLS) in obese subjects and determine whether the presence of B cells predict insulin resistance, we examined archived samples of subcutaneous and omental fat from 32 obese men and women and related findings to clinical parameters. Using immunohistochemistry, we identified B (CD19(+)) and T cells (CD3 (+)) within the sCLS and perivascular space. The presence and density of B cells (B cells per high-power field (pHPF), T cells pHPF, and B cell:T cell (B:T) ratio) were compared with measures of insulin resistance (homeostasis model assessment (HOMA)) and other variables. In 16 of 32 subjects (50%) CD19(+) B cells were localized within sCLS and were relatively more numerous than T cells. HOMA was not different between subjects with CD19(+) vs. CD19(-) sCLS (5.5 vs. 5.3, P = 0.88). After controlling for diabetes and glycemia (hemoglobin A(1c) (HbA(1c))), the B:T ratio correlated with current metformin treatment (r = 0.89, P = 0.001). These results indicate that in human OIR, B cells are an integral component of organized inflammation in subcutaneous fat, and defining their role will lead to a better understanding of OIR pathogenesis and potentially impact treatment.

Reduced adipose tissue inflammation represents an intermediate cardiometabolic phenotype in obesity.

OBJECTIVES: The purpose of this study was to determine whether obese individuals with reduced adipose tissue inflammation exhibit a more favorable cardiovascular risk profile. BACKGROUND: Obesity is associated with a low-grade state of chronic inflammation that might be causally related to cardiometabolic disease. METHODS: With immunohistochemistry, we categorized obese individuals dichotomously as having inflamed fat (n = 78) or noninflamed fat (n = 31) on the basis of the presence (+) or absence (-) of macrophage crown-like structures (CLS) in subcutaneous abdominal fat biopsy samples. We compared their metabolic, vascular, and adipose tissue characteristics with lean subjects (n = 17). RESULTS: Inflamed CLS+ obese individuals displayed higher plasma insulin, homeostasis model assessment, triglycerides, glucose, blood pressure, high-sensitivity C-reactive protein, low-density lipoprotein cholesterol, lower high-density lipoprotein cholesterol, and brachial artery flow-mediated dilation compared with lean subjects (p < 0.05). Adipose messenger ribonucleic acid expression of inflammatory genes including CD68, leptin, matrix metalloproteinase-9, CD163, and CD8A were significantly greater and vascular endothelial growth factor was lower in the CLS+ group (p < 0.05). In contrast, obese subjects with noninflamed fat exhibited a mixed clinical phenotype with lower insulin resistance, reduced proatherogenic gene expression, and preserved vascular function as in lean subjects. In multiple linear regression adjusting for age and sex, CLS status (beta = -0.28, p = 0.008) and waist circumference (beta = -0.25, p = 0.03) were independent predictors of flow-mediated dilation. CONCLUSIONS: These findings lend support to the novel concept that factors in addition to absolute weight burden, such as qualitative features of adipose tissue, might be important determinants of cardiovascular disease. Therapeutic modulation of the adipose phenotype might represent a target for treatment in obesity.

Altered mitochondrial dynamics contributes to endothelial dysfunction in diabetes mellitus.

BACKGROUND: Endothelial dysfunction contributes to the development of atherosclerosis in patients with diabetes mellitus, but the mechanisms of endothelial dysfunction in this setting are incompletely understood. Recent studies have shown altered mitochondrial dynamics in diabetes mellitus with increased mitochondrial fission and production of reactive oxygen species. We investigated the contribution of altered dynamics to endothelial dysfunction in diabetes mellitus. METHODS AND RESULTS: We observed mitochondrial fragmentation (P=0.002) and increased expression of fission-1 protein (Fis1; P<0.0001) in venous endothelial cells freshly isolated from patients with diabetes mellitus (n=10) compared with healthy control subjects (n=9). In cultured human aortic endothelial cells exposed to 30 mmol/L glucose, we observed a similar loss of mitochondrial networks and increased expression of Fis1 and dynamin-related protein-1 (Drp1), proteins required for mitochondrial fission. Altered mitochondrial dynamics was associated with increased mitochondrial reactive oxygen species production and a marked impairment of agonist-stimulated activation of endothelial nitric oxide synthase and cGMP production. Silencing Fis1 or Drp1 expression with siRNA blunted high glucose-induced alterations in mitochondrial networks, reactive oxygen species production, endothelial nitric oxide synthase activation, and cGMP production. An intracellular reactive oxygen species scavenger provided no additional benefit, suggesting that increased mitochondrial fission may impair endothelial function via increased reactive oxygen species. CONCLUSION: These findings implicate increased mitochondrial fission as a contributing mechanism for endothelial dysfunction in diabetic states.

Expression of inducible nitric oxide synthase in conduits used in patients with diabetes mellitus undergoing coronary revascularization.

BACKGROUND: Expression of inducible nitric oxide synthase (iNOS) is a marker of vascular inflammation which can result in thrombosis and atherosclerosis. This study was undertaken to examine the difference in iNOS expression in the internal mammary artery (IMA) and saphenous veins (SVs) of patients with diabetes mellitus undergoing coronary artery bypass graft (CABG) surgery using both qualitative and quantitative methodology. METHODS: Segments of IMA and SV harvested in 100 diabetic patients with diabetes mellitus undergoing CABG surgery were fixed in formalin and immunostained to detect the presence of iNOS. Sections were graded using a qualitative score (0 = absence of iNOS expression to 3 = extensive expression of iNOS) and a quantitative computer-aided image analysis (area of staining/area of endothelium). Linear regression analyses were performed to assess the association of the degree of iNOS expression in both the IMA and SV with the type of diabetes control (insulin, oral, diet), and the serum levels of HbAlc, glucose, free fatty acids (ffa), C-reactive protein (CRP), and low-density liproprotein (LDL) at the time of conduit harvest. RESULTS: The degree of iNOS expression was significantly lower in the IMA compared to the SV by both qualitative (0.88 +/- 0.74 SD IMA vs. 1.38 +/- 0.68 SV; p < 0.0001) and quantitative (11.76 +/- 3.34% IMA vs. 17.10 +/- 2.54% SV; p = 0.01) methods. The Spearman rank correlation analysis showed a highly statistically significant association between the two methodologies (p < 0.0001). There was no correlation between iNOS expression in either the IMA or SV and the type of diabetes control, or levels of HA1c, glucose, ffa, and CRP. However, there was a significant (p = 0.04) correlation between LDL and iNOS expression in the SV graft, but not the IMA. CONCLUSIONS: iNOS expression is significantly decreased in the IMA compared to the SV in patients with diabetes mellitus undergoing CABG surgery. The degree of iNOS expression is unrelated to the level of glycemic control at the time of conduit harvest, but is associated with serum LDL levels in the SV, but not in the IMA grafts.

Nonmyocardial production of ST2 protein in human hypertrophy and failure is related to diastolic load.

OBJECTIVES: This study was designed to investigate: 1) relationships between serum ST2 levels and hemodynamic/neurohormonal variables; 2) myocardial ST2 production; and the 3) expression of ST2, membrane-anchored ST2L, and its ligand, interleukin (IL)-33, in myocardium, endothelium, and leukocytes from patients with left ventricular (LV) pressure overload and congestive cardiomyopathy. BACKGROUND: Serum levels of ST2 are elevated in heart failure. The relationship of ST2 to hemodynamic variables, source of ST2, and expression of ST2L and IL-33 in the cardiovascular system are unknown. METHODS: Serum ST2 (pg/ml; median [25th, 75th percentile]) was measured in patients with LV hypertrophy (aortic stenosis) (n = 45), congestive cardiomyopathy (n = 53), and controls (n = 23). ST2 was correlated to N-terminal pro-brain natriuretic peptide, C-reactive protein, and hemodynamic variables. Coronary sinus and arterial blood sampling determined myocardial gradient (production) of ST2. The levels of ST2, ST2L, and IL-33 were measured (reverse transcriptase-polymerase chain reaction) in myocardial biopsies and leukocytes. The ST2 protein production was evaluated in human endothelial cells. The IL-33 protein expression was determined (immunohistochemistry) in coronary artery endothelium. RESULTS: The ST2 protein was elevated in aortic stenosis (103 [65, 165] pg/ml, p < 0.05) and congestive cardiomyopathy (194 [69, 551] pg/ml, p < 0.01) versus controls (49 [4, 89] pg/ml) and correlated with B-type natriuretic peptide (r = 0.5, p < 0.05), C-reactive protein (r = 0.6, p < 0.01), and LV end-diastolic pressure (r = 0.38, p < 0.03). The LV ST2 messenger ribonucleic acid was similar in aortic stenosis and congestive cardiomyopathy versus control (p = NS). No myocardial ST2 protein gradient was observed. Endothelial cells secreted ST2. The IL-33 protein was expressed in coronary artery endothelium. Leukocyte ST2L and IL-33 levels were highly correlated (r = 0.97, p < 0.001). CONCLUSIONS: In human hypertrophy and failure, serum ST2 correlates with the diastolic load. Though the heart, endothelium, and leukocytes express components of ST2/ST2L/IL-33 pathway, the source of circulating serum ST2 is extra-myocardial.

Effect of anti-oxidant treatment on hyperhomocysteinemia-induced myocardial fibrosis and diastolic dysfunction.

BACKGROUND: Recent studies demonstrate that hyperhomocysteinemia is a risk factor for heart failure. Oxidant stress is a major mediator of the pathogenic effects of hyperhomocysteinemia. METHODS: We utilized a rat model of diet-induced hyperhomocysteinemia to examine whether treatment with an anti-oxidant vitamin (C&E) combination will prevent hyperhomocysteinemia-induced myocardial fibrosis. RESULTS: Dietary anti-oxidant therapy attenuated hyperhomocysteinemia-induced increases in myocardial oxidant stress and myocardial fibrosis, and diastolic dysfunction. CONCLUSIONS: Hyperhomocysteinemia acts via oxidant stress to promote myocardial fibrosis and dysfunction. Dietary anti-oxidant therapy could be an important preventive and therapeutic strategy in diastolic heart failure.

Flow-induced arterial remodeling relates to endothelial function in the human forearm.

BACKGROUND: Chronic changes in blood flow stimulate arterial remodeling, which contributes to the maintenance of vascular homeostasis. Experimental studies suggest that remodeling represents a response to local changes in endothelial shear stress and is nitric oxide-dependent. METHODS AND RESULTS: To investigate determinants of outward arterial remodeling in humans, we measured ulnar artery flow, diameter, and flow-mediated dilation before and after removal of the adjacent radial artery in 53 patients who were undergoing coronary bypass surgery (age 60+/-11 years; 13% female). Removal of the radial artery increased ulnar artery blood flow by 35% (P=0.009) and increased ulnar artery diameter by 9% (P<0.001) 4 to 8 weeks after surgery. At 1 week, ulnar artery shear stress was increased by 58% (P<0.001), but it was no longer different from baseline at longer-term follow-up. The contralateral ulnar artery was unaffected, which suggests that these findings were not attributable to the systemic effects of medications or the postoperative state. Extent of outward remodeling correlated with the increase in blood flow (r=0.50, P=0.001) and with flow-mediated dilation at baseline (r=0.50, P=0.001). Remodeling correlated inversely with baseline endothelial expression of P-selectin in the radial artery (r=-0.76, P=0.004, n=14). CONCLUSIONS: A sustained increase in blood flow in the ulnar artery induced outward arterial remodeling despite the presence of risk factors and coronary artery disease. The remodeling response was related to endothelial phenotype, as reflected by flow-mediated dilation and expression of P-selectin. These findings provide evidence that the endothelium plays an important role in the regulation of vascular structure in humans.

Adipose macrophage infiltration is associated with insulin resistance and vascular endothelial dysfunction in obese subjects.

OBJECTIVE: Experimental studies suggest that adipose inflammation is etiologically linked to obesity-induced systemic disease. Our goal was to characterize the state of inflammation in human fat in relation to vascular function and metabolic parameters in obese individuals. METHODS AND RESULTS: We collected subcutaneous abdominal fat in 77 obese subjects (BMI >or=30 kg/m(2)) and quantified adipose macrophage population using targeted immunohistochemistry. Brachial artery vasodilator function was examined using high-resolution vascular ultrasound. In 50 subjects, an inflamed adipose phenotype characterized by tissue macrophage accumulation in crown-like structures was associated with systemic hyperinsulinemia and insulin resistance (HOMA-IR 5.5+/-4.5 versus 2.6+/-1.9, P=0.002) and impaired endothelium-dependent flow-mediated vasodilation (8.5+/-4.4% versus 10.8+/-3.8%, P<0.05), as compared to subjects with quiescent noninflamed adipose architecture (n=27). Macrophage retention in fat was linked to upregulated tissue CD68 and tumor necrosis factor (TNF)-alpha mRNA expression in addition to increased plasma hs-CRP. CONCLUSIONS: In a cohort of obese subjects, we demonstrate that proinflammatory changes in adipose tissue are associated with systemic arterial dysfunction and insulin resistance. These findings suggest that adipose inflammation may be linked to vascular injury and increased cardiovascular risk in obese subjects.

Dietary homocysteine promotes atherosclerosis in apoE-deficient mice by inducing scavenger receptors expression.

Elevated plasma homocysteine (Hcy) levels have been recognized as an independent risk factor for atherosclerosis leading to cardiovascular diseases. However, the mechanisms contributing to atherosclerosis have not been delineated. Since, scavenger receptors mediated uptake of oxidized-LDL (oxLDL) by macrophages resulting in foam cell formation is an early event in atherosclerosis, we hypothesized that atherogenic effects of Hcy may be mediated via regulating expression of scavenger receptor(s). We have tested this hypothesis using apoE-/- female mice fed normal rodent chow (NC) diet or NC supplemented with Hcy in drinking water (9 g/L). Hcy-fed mice showed increased fatty streak lesions in aortic sinus/root compared to NC group without alterations in plasma lipid profiles. Similar findings were observed in the enface analysis of the descending aorta. To determine the molecular mechanisms underlying Hcy-mediated progression of fatty streak lesions, expression of scavenger receptors such as CD36 and lectin-like oxidized LDL binding protein-1 (LOX-1) in the aortic lesions were analyzed. Interestingly, Hcy-fed mice had increased immuno-positive staining for CD36 and LOX-1 in the atherosclerotic lesions compared to NC-fed mice. In vitro analyses showed neither Hcy nor HcyLDL directly affect the expression of CD36 and LOX-1 on mouse macrophages. However, Hcy supplementation in apoE-/- mice resulted in elevated oxLDL levels in plasma. Since oxLDL has been shown to upregulate the expression of CD36 and LOX-1, these findings suggest that Hcy may exert its atherogenic effect in part by elevating the levels of oxLDL. Interestingly, interaction of monocytes with Hcy-activated endothelial cells resulted in upregulation of CD36 expression on monocytes, suggesting a possible mechanism by which Hcy may upregulate CD36 expression at the lesion site. Further, these findings suggest a novel mechanism by which Hcy may promote atherogenesis.