Yuri Sheikine

CXCL16/SR-PSOX is an interferon-gamma-regulated chemokine and scavenger receptor expressed in atherosclerotic lesions

Objective—Atherosclerosis is an inflammatory disease. Several chemokines are important for monocyte/macrophage and T-cell recruitment to the lesion. CXCL16 is a recently discovered chemokine that is expressed in soluble and transmembrane forms, ligates CXCR6 chemokine receptor, and guides migration of activated Th1 and Tc1 cells. It is identical to scavenger receptor SR-PSOX, which mediates uptake of oxidized low-density lipoprotein. We investigated whether CXCL16 expression is controlled by interferon-&ggr; (IFN-&ggr;)-cytokine abundant in atherosclerotic lesions. Methods and Results—CXCL16 and CXCR6 expression was identified by polymerase chain reaction and histochemistry in atherosclerotic lesions from humans and apolipoprotein-E–deficient mice. In vitro IFN-&ggr; induced CXCL16 in human monocytic THP-1 cells and primary human monocytes, which led to increased uptake of oxidized low-density lipoprotein in THP-1 cells, which could be blocked by peptide antibodies against CXCL16. In vivo IFN-&ggr; induced CXCL16 expression in murine atherosclerotic lesions. Conclusions—We demonstrate a novel role of IFN-&ggr; in foam cell formation through upregulation of CXCL16/SR-PSOX. CXCR6 expression in the plaque confirms the presence of cells able to respond to CXCL16. Therefore, this chemokine/scavenger receptor could serve as a molecular link between lipid metabolism and immune activity in the atherosclerotic lesion.

CD137 Is Expressed in Human Atherosclerosis and Promotes Development of Plaque Inflammation in Hypercholesterolemic Mice

Background— Atherosclerosis is a multifactorial disease in which inflammatory processes play an important role. Inflammation underlies lesion evolution at all stages, from establishment to plaque rupture and thrombosis. Costimulatory molecules of the tumor necrosis factor superfamily such as CD40/CD40L and OX40/OX40L have been implicated in atherosclerosis. Methods and Results— This study shows that the tumor necrosis factor superfamily members CD137 and CD137 ligand (CD137L), which play a major role in several autoimmune diseases, may constitute a pathogenic pair in atherogenesis. We detected CD137 protein in human atherosclerotic lesions not only on T cells but also on endothelial cells and showed that CD137 in cultured endothelial cells and smooth muscle cells was induced by proinflammatory cytokines implicated in atherosclerosis. Activation of CD137 by CD137L induced adhesion molecule expression on endothelial cells and reduced smooth muscle cell proliferation. In addition, treatment of atherosclerosis-prone apolipoprotein E–deficient mice with a CD137 agonist caused increased inflammation. T-cell infiltration, mainly of CD8+ cells, and expression of the murine major histocompatibility complex class II molecule I-Ab increased significantly in atherosclerotic lesions, as did the aortic expression of proinflammatory cytokines. Conclusions— Taken together, these observations suggest that CD137-CD137L interactions in the vasculature may contribute to the progression of atherosclerosis via augmented leukocyte recruitment, increased inflammation, and development of a more disease-prone phenotype.

Increased Glucose Uptake in Visceral Versus Subcutaneous Adipose Tissue Revealed by PET Imaging

OBJECTIVES The current study tested the hypothesis that glucose utilization differs between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), and investigated potential mechanisms for such a finding. BACKGROUND VAT burden correlates better with cardiovascular risk than does SAT burden. Beyond volumetric measurement, glucose uptake in adipose tissue (AT) might reflect metabolic activity and provide pathophysiologic insight and aid risk stratification. METHODS We retrospectively studied tissue-specific glucose uptake in vivo in clinically obtained whole-body fluorodeoxyglucose positron emission tomography (FDG-PET) scans in humans. We also assessed glucose uptake in vitro, using stromal vascular cells isolated from SAT and VAT of diet-induced obese C57BL/6 mice. Quantitative polymerase chain reaction (PCR) evaluated the expression of multiple genes involved in cellular glucose metabolism, including glucose transporters (GLUT-1, -3, and -4) and hexokinases (HK-1 and -2) in SAT and VAT of obese C57BL/6 mice. RESULTS We analyzed whole-body FDG-PET scans from 31 obese and 26 lean patients. VAT exhibited higher FDG uptake compared with SAT (p < 0.0001) independent of age, sex, body mass index, comorbidities, and medications. To investigate mechanisms underlying this observation, we studied glucose uptake in the stromal vascular cell fraction of AT, which is rich in inflammatory cells. Stromal vascular cells from VAT of diet-induced obese C57BL/6 mice exhibited higher glucose uptake than those from SAT (p = 0.01). Evaluation of expression of glucose transporters (GLUT-1, -3, and -4) and hexokinases (HK-1 and -2), revealed increased expression of HK-1 in VAT-derived compared with SAT-derived stromal vascular cells, and also in visceral versus subcutaneous unfractionated AT. CONCLUSIONS In humans in vivo, VAT has increased glucose uptake compared with SAT, as determined noninvasively with FDG PET imaging. Differential stromal metabolic activity may be 1 mechanism underlying differences in metabolic activity of visceral and subcutaneous AT.

CXCL16/SR-PSOX--a friend or a foe in atherosclerosis?

Chemokines, scavenger receptors and adhesion molecules have long been known as important players in the pathogenesis of atherosclerosis. A series of studies conducted in the past few years described CXCL16/SR-PSOX--a new molecule combining those three functions, and suggested that CXCL16/SR-PSOX can be a potential player in atherogenesis. Initial ex vivo studies showed that CXCL16/SR-PSOX is abundant in human and murine atherosclerotic lesions. Following in vitro studies suggested that as an adhesion molecule CXCL16/SR-PSOX might mediate T-cell adhesion to the endothelium, as a chemokine - drive T-cell migration, stimulate cell proliferation and elicit inflammatory phenotype in smooth muscle cells (SMC) and, finally, as a scavenger receptor - mediate uptake of atherogenic lipoproteins by macrophages and SMC. All these effects are known to be pro-atherogenic. Surprisingly, in vivo studies performed in murine models of atherosclerosis suggested that CXCL16/SR-PSOX is atheroprotective, while its receptor CXCR6 is harmful. In addition, studies investigating the association of circulating CXCL16/SR-PSOX plasma concentrations with the presence and extent of coronary artery disease (CAD) in humans are controversial suggesting both positive, negative and no association. To finally answer the question whether CXCL16/SR-PSOX can serve as a causative factor, biomarker or even a therapeutic target in atherosclerosis, we are currently in need of carefully designed animal and human studies investigating the effects of CXCL16/SR-PSOX and CXCR6 deficiency, inhibition and over-expression on the progression of atherosclerosis. Such complex approach will help us unravel the mystery of CXCL16/SR-PSOX in atherosclerosis and hopefully develop better ways of treating atherosclerosis by targeting this interesting molecule.

FDG–PET imaging of atherosclerosis: Do we know what we see?

Imaging atherosclerosis may help to identify subjects harboring rupture-prone atherosclerotic plaques who may benefit from preventive interventions. Potential of plaques to rupture depends on their structural changes and metabolic activation, which are difficult to assess using anatomic imaging modalities. Recent studies suggested that functional imaging with positron emission tomography (PET) utilizing fluorine-18-labeled 2-deoxy-d-glucose (FDG) has the potential to assess plaque metabolism and add to prediction of vascular risk. Aortic, iliac, and carotid plaques can be detected with FDG-PET, even though not all plaques exhibit high FDG uptake. Detection of coronary artery plaques is more cumbersome due to technical limitations of PET and fast movement of these vessels during cardiac and respiratory cycles. Studies on substrate accumulating FDG in plaques are contradictory and mostly do not extend beyond correlation analyses. Vascular FDG uptake has an excellent short-term stability, but larger fluctuations of uptake long-term, which may complicate interpretation of such changes in therapeutic trials. FDG uptake in major arteries correlates with some cardiovascular risk factors and atherosclerosis markers, but clinical utility of such correlations is unclear. What is more important is that recently reported studies in cancer patients showed correlation between higher baseline FDG uptake and subsequent cardiovascular mortality. Anti-atherogenic therapy and therapeutic lifestyle changes seem to decrease vascular FDG uptake but it is not clear whether the latter predicts subsequent lower morbidity and mortality. These initial findings suggest that vascular FDG-PET may in the future find some utility in management of patients with atherosclerosis, but a number of important issues need to be addressed first. We need to: (1) determine optimal standard ways of performing imaging and quantifying vascular FDG uptake; (2) understand molecular mechanisms governing FDG accumulation in plaques; (3) perform studies prospectively linking vascular FDG uptake to cardiovascular events in non-cancer patients. As of today, vascular FDG-PET is not ready for its prime time in clinical practice.

Assessment of breast pathologies using nonlinear microscopy

Significance Development of methods for rapid intraoperative assessment of breast pathologies is important for decreasing the rate of surgical reexcisions during breast-conserving therapy. Frozen-section analysis is the most widely used method for intraoperative margin assessment but is time consuming, has limited diagnostic accuracy, and produces freezing artifacts. This manuscript demonstrates nonlinear microscopy for rapid assessment of surgical breast specimens without need for fixation, embedding, and sectioning required for conventional histology. Blinded reading of nonlinear microscopy images by three pathologists achieved 95.4% sensitivity, 93.3% specificity, and 94.1% overall accuracy compared with histology for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. The results suggest that nonlinear microscopy is a promising method for intraoperative assessment of breast surgical excision specimens. Rapid intraoperative assessment of breast excision specimens is clinically important because up to 40% of patients undergoing breast-conserving cancer surgery require reexcision for positive or close margins. We demonstrate nonlinear microscopy (NLM) for the assessment of benign and malignant breast pathologies in fresh surgical specimens. A total of 179 specimens from 50 patients was imaged with NLM using rapid extrinsic nuclear staining with acridine orange and intrinsic second harmonic contrast generation from collagen. Imaging was performed on fresh, intact specimens without the need for fixation, embedding, and sectioning required for conventional histopathology. A visualization method to aid pathological interpretation is presented that maps NLM contrast from two-photon fluorescence and second harmonic signals to features closely resembling histopathology using hematoxylin and eosin staining. Mosaicking is used to overcome trade-offs between resolution and field of view, enabling imaging of subcellular features over square-centimeter specimens. After NLM examination, specimens were processed for standard paraffin-embedded histology using a protocol that coregistered histological sections to NLM images for paired assessment. Blinded NLM reading by three pathologists achieved 95.4% sensitivity and 93.3% specificity, compared with paraffin-embedded histology, for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. Interobserver agreement was κ = 0.88 for NLM and κ = 0.89 for histology. These results show that NLM achieves high diagnostic accuracy, can be rapidly performed on unfixed specimens, and is a promising method for intraoperative margin assessment.

Chemokines as Potential Therapeutic Targets in Atherosclerosis

Atherosclerosis is a chronic disease with high morbidity and mortality around the globe. It is characterized by chronic inflammation of the vessel wall, which is perpetuated by the continuous migration of cells to and within the atherosclerotic lesion. Chemokines (CK) and chemokine receptors (CKR) together with other chemoattractants and adhesion molecules are major mediators facilitating this process. Many CK/CKR (CC, CX3C and CXC) and other chemoattractants (e.g. leukotrienes) have been implicated in atherogenesis, but only a few have been validated as pathogenic by in vitro assays, in vivo experiments using gene-targeted animal models and genetic studies. Promising attempts are currently made to inhibit CK-dependent cell recruitment to lesion by using neutralizing antibodies, mutant proteins, viral and synthetic inhibitors or receptor antagonists. Some of the therapeutics have already entered clinical trials for other conditions and are about to be tested in human atherosclerosis. However, our limited understanding of the complex CK system and the functional specialization of individual CK/CKR, translatability of animal research into human population, limitations of current imaging techniques and surrogate markers for evaluation of the benefits of potential anti-CK compounds are still hampering therapeutic exploitation of the CK system in atherosclerosis. Hopefully we will be able to solve many of these issues in the near future and use this approach to control atherosclerotic disease in man.

Swept source optical coherence microscopy using a 1310 nm VCSEL light source

We demonstrate high speed, swept source optical coherence microscopy (OCM) using a MEMS tunable vertical cavity surface-emitting laser (VCSEL) light source. The light source had a sweep rate of 280 kHz, providing a bidirectional axial scan rate of 560 kHz. The sweep bandwidth was 117 nm centered at 1310 nm, corresponding to an axial resolution of 13.1 µm in air, corresponding to 8.1 µm (9.6 µm spectrally shaped) in tissue. Dispersion mismatch from different objectives was compensated numerically, enabling magnification and field of view to be easily changed. OCM images were acquired with transverse resolutions between 0.86 µm - 3.42 µm using interchangeable 40X, 20X and 10X objectives with ~600 µm x 600 µm, ~1 mm x 1 mm and ~2 mm x 2 mm field-of-view (FOV), respectively. Parasitic variations in path length with beam scanning were corrected numerically. These features enable swept source OCM to be integrated with a wide range of existing scanning microscopes. Large FOV mosaics were generated by serially acquiring adjacent overlapping microscopic fields and combining them in post-processing. Fresh human colon, thyroid and kidney specimens were imaged ex vivo and compared to matching histology sections, demonstrating the ability of OCM to image tissue specimens.

Ultrahigh speed spectral-domain optical coherence microscopy.

We demonstrate a compact, ultrahigh speed spectral-domain optical coherence microscopy (SD-OCM) system for multiscale imaging of specimens at 840 nm. Using a high speed 512-pixel line scan camera, an imaging speed of 210,000 A-scans per second was demonstrated. Interchangeable water immersion objectives with magnifications of 10×, 20×, and 40× provided co-registered en face cellular-resolution imaging over several size scales. Volumetric OCM data sets and en face OCM images were demonstrated on both normal and pathological human colon and kidney specimens ex vivo with an axial resolution of ~4.2 µm, and transverse resolutions of ~2.9 µm (10×), ~1.7 µm (20×), and ~1.1 µm (40×) in tissue. In addition, en face OCM images acquired with high numerical aperture over an extended field-of-view (FOV) were demonstrated using image mosaicking. Comparison between en face OCM images among different transverse and axial resolutions was demonstrated, which promises to help the design and evaluation of imaging performance of Fourier domain OCM systems at different resolution regimes.

CXCR3 Controls T-Cell Accumulation in Fat Inflammation

Objective—Obesity associates with increased numbers of inflammatory cells in adipose tissue (AT), including T cells, but the mechanism of T-cell recruitment remains unknown. This study tested the hypothesis that the chemokine (C-X-C motif) receptor 3 (CXCR3) participates in T-cell accumulation in AT of obese mice and thus in the regulation of local inflammation and systemic metabolism. Approach and Results—Obese wild-type mice exhibited higher mRNA expression of CXCR3 in periepididymal AT-derived stromal vascular cells compared with lean mice. We evaluated the function of CXCR3 in AT inflammation in vivo using CXCR3-deficient and wild-type control mice that consumed a high-fat diet. Periepididymal AT from obese CXCR3-deficient mice contained fewer T cells than obese controls after 8 and 16 weeks on high-fat diet, as assessed by flow cytometry. Obese CXCR3-deficient mice had greater glucose tolerance than obese controls after 8 weeks, but not after 16 weeks. CXCR3-deficient mice fed high-fat diet had reduced mRNA expression of proinflammatory mediators, such as monocyte chemoattractant protein-1 and regulated on activation, normal T cell expressed and secreted, and anti-inflammatory genes, such as Foxp3, IL-10, and arginase-1 in periepididymal AT, compared with obese controls. Conclusions—These results demonstrate that CXCR3 contributes to T-cell accumulation in periepididymal AT of obese mice. Our results also suggest that CXCR3 regulates the accumulation of distinct subsets of T cells and that the ratio between these functional subsets across time likely modulates local inflammation and systemic metabolism.