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There is an increasing need for more advanced, composite biomarkers that can model the complex systems biology driving response and resistance to cancer therapy. However, many cancer diagnostic platforms to date, with their focus on mutational changes in a relatively small panel of genes, provide limited data to support integrative, multidimensional biomarkers that can better predict immunotherapy response. To enable the identification of composite biomarkers that combine tumor- and immune-related information from both DNA and RNA, we have developed ImmunoID NeXT, an enhanced exome/transcriptome-based diagnostic platform that can simultaneously profile the tumor and immune system from a single FFPE sample, across all of the approximately 20,000 genes. By co-optimizing assay and analytics design, we enable sensitive evaluation of clinically-relevant cancer biomarkers from >=25ng of co-extracted DNA/RNA, while also providing a broader evaluation of neoantigens, HLA typing and LOH, antigen processing machinery (APM), TCR/BCR repertoire, immune expression signatures, tumor-infiltrating lymphocytes (TILs), oncoviruses, and germline variants. Leveraging this expansive feature set, we developed methods that combine individual analytes to construct composite biomarker scores that correlate with immunotherapy response. Validation of ImmunoID NeXT demonstrated high sensitivity and specificity to somatic and structural variants across ~20,000 genes at allelic fractions as low as 5%, with clinical diagnostic reporting on actionable mutations (SNVs, indels, CNAs, fusions) in 248 cancer-driver genes that have been boosted further for higher sensitivity, as well as reporting on TMB and MSI status. For neoantigen prediction, immuno-peptidomic data from monoallelic HLA-transfected cell lines were used to train neural networks to predict pMHC binding with higher precision than public tools. For TCRα/β analysis in FFPE tumor samples, strong correlation with targeted TCR kit results was shown (R^2>0.9 and >0.94). For TILs, we developed signatures for eight immune cell types, demonstrating concordance with orthogonal immunofluorescence methods. We achieved genotyping accuracy of 99.1% for HLA Class I, and 95% for HLA Class II, and have developed and verified the performance of a tool for HLA LOH detection. In a cohort of 55 late-stage melanoma patients, the integration of neoantigen burden, HLA LOH, and APM mutational data formed a composite neoantigen score that more accurately predicted response to checkpoint blockade than other markers such as TMB. With ImmunoID NeXT, we have developed a broad diagnostic platform that can be leveraged for the development of advanced composite biomarkers (and novel resistance mechanisms) that combine both tumor and immune features from DNA and RNA; enabling more accurate stratification of patient response to immunotherapy. The platform has been validated and optimized for use with limited FFPE tissue samples, making it ideal for both research and clinical applications. Citation Format: Robert Peter Power, Gabor Bartha, Jason Harris, Sean M. Boyle, Eric Levy, Pamela Milani, Prateek Tandon, Paul McNitt, Mandy Lee, Massimo Morra, Sejal Desai, Sebastian Salvidar, Michael J. Clark, Christian Haudenschild, Sekwon Jang, John West, Richard Chen. A diagnostic platform for precision cancer therapy enabling composite biomarkers by combining tumor and immune features from an enhanced exome and transcriptome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1334.

e21715 Background: Important questions have been raised about whether real world data (RWD) can be relied upon to support clinical and regulatory decision-making. The aims of this study were to assess overall survival (OS), time-to-treatment (TTT), follow-up time, and treatment pathways in metastatic non-small cell lung cancer (mNSCLC) patients treated with programmed cell death protein 1 inhibitors (PD-1i). Methods: Two mNSCLC cohorts were identified in a US based EMR network. To explore treatment pathways, patients had to have an advanced (stage 3/4) diagnosis of NSCLC confirmed by a tumor registry record. A line of treatment (LOT) was defined as a PD-1i or chemotherapy taken within 30 days. OS, time to treatment, and follow-up time were also calculated. In another cohort patients had to have at least one PD-1i (pembrolizumab, nivolumab, durvalumab, or atezolizmab) and an oncology treatment within 3 months of an advanced stage diagnosis. OS was calculated using Kaplan-Meier analysis and stratified by time after nivolumab approval, quarter of PD-1i initiation, and line of therapy of first PD-1i. Patient characteristics were defined by ICD, CPT, LOINC, and RxNorm terminology. Results: Median overall survival was found to be 213 days (IQR 109, 425.25). In the treatment pathways analysis, 58.3% of patients started on a non-PD-1i chemotherapy as an initial line of treatment for mNSCLC. PD-1i was the most common second line treatment and 41.4% of patients who started on a non-PD-1i therapy switched to a PD-1i as their second line therapy. Median time from advanced diagnosis to PD-1i inhibitor initiation (9.6 months (IQR 3.45, 21.45)) and median structured follow-up times from advanced diagnosis (21.87 months (IQR 11.94, 38.97)) and from inhibitor initiation (8.71 months (3.06, 17.26)) were comparable to results found in other RWD. Conclusions: Overall survival, time to treatment, and other outcomes were consistent with comparable RWD sources, (Stewart M, 2019; Sean K 2018) regardless of treatment timeframe. We demonstrated that our real world evidence based approach provides a robust method for analyzing clinical outcomes, supporting the validity of real world data to complement clinical trials and inform clinical decisions.

With the advent of next-generation sequencing techniques, tumor mutations that guide therapeutic decisions can be identified on a large scale. The ability to accurately detect these mutations is often impaired by obstacles such as sequencing coverage gaps, small sample amount, low cellularity, and tumor heterogeneity. Two methods commonly used to interrogate the genomic profile of a tumor are: i) cancer gene panels, where a small number of genes (in the range of ~5-500, depending on the specific panel design) is assessed, and ii) cancer exome sequencing, where most human genes are evaluated. Additionally, tumor RNA can be studied to elucidate gene fusions and expression features that may impact treatment. Depending on the goals of the investigator, both methods have certain advantages and disadvantages. Differences between these assays must be clearly understood in order for cancer researchers to make the best decision to achieve their objective. We developed a set of unique sequencing assays to augment and improve detection of major cancer mutations by enhancing coverage over known sequencing gaps and GC-rich regions. We utilize these protocols to create an accuracy and content enhanced whole-exome (ACE Exome) as well as an extended, augmented cancer gene panel (ACE Extended Cancer Panel), targeting over 1,300 cancer genes and 200 miRNA genes at very high depth. We compared these ACE assays to standard exome and gene panels currently available. We then compared the ACE panel to the ACE exome using a set of well-characterized cancer cell lines, cell line mixtures with known genomic perturbations, and primary tumor and matched normal pairs involved in drug resistance. We also performed whole-transcriptome and targeted RNA sequencing as companion assays to the DNA exome and panel, respectively. All data were analyzed using a cancer bioinformatics pipeline optimized for detection of low-representation small variants and indels, as well as somatic copy-number aberrations, gene expression and fusions. The ACE assays were able to detect somatic variants with higher accuracy than standard assays across regions targeted by both. For example, a low-representation BRAF mutation detected by standard exome was confirmed as a false positive using the ACE exome due to 90 times more coverage over that region. Between the ACE assays, we found that 88% of somatic variants identified through ACE exome were also identified with our comprehensive panel within regions targeted by both. Conversely, more than half of the somatic variants discovered in the ACE panel were missed in the ACE exome due to insufficient read depth. For instance, the panel detected two EGFR variants known to be present at 1% that were missed in the ACE exome. About 20 times more somatic variants across some ~18,000 genes were detected in whole-exome that are not targeted on the augmented gene panel. These genes have not yet been associated with cancer in current literature, but may be of potential interest for discovery research. Copy number profiles, although more comprehensive in the exome, were recapitulated using the panel, despite large distances between panel targets. Finally, the integration of RNA analysis to the DNA panel was able to detect expression and fusion patterns. In particular, we detected a DNA mutation associated with resistance in a post-treatment specimen, but accompanying RNA data proved this mutant unexpressed and thereby unlikely to be the driver of resistance. For NGS-based cancer analysis to effectively guide clinical decisions and research, accuracy across all relevant cancer genes is of paramount importance. An enhanced exome may offer a solution for researchers interested in novel cancer associations who desire broad, even coverage. A comprehensive, high-depth panel targeting a large set of genes associated with cancer, accompanied by RNA analysis, may accomplish research goals across a wide range of translational cancer research. Citation Format: Elena Helman, Michael James Clark, Sean Boyle, Richard Chen, Shujun Luo, Christian Haudenschild, Jason Harris, Gabor Bartha, Deanna Church, John West. Augmented targeted NGS in cancer diagnostics: Comparing gene panels and whole exome sequencing for accurate detection of driver mutations. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-39.

Immunogenomic profiling of the tumor and the tumor microenvironment (TME) is critical for identifying new biomarkers of immunotherapy response, understanding resistance, and enabling the development of personalized immunotherapies. However, running a comprehensive array of biomarker assays for each patient sample is often impractical given limited sample quantity, processing complexity, and prohibitive cost. To address these challenges, we developed a novel, augmented exome and transcriptome-based platform that simultaneously characterizes the tumor and TME from a single FFPE sample. We co-optimized the design of our sequencing assays and analytics to increase performance for the detection of somatic SNVs, indels, CNAs, and fusions across ~20,000 genes, as well as the evaluation of neoantigens, expression signatures, HLA typing and LOH, TCR/BCR repertoires, oncoviruses, tumor-infiltrating lymphocytes (TILs), clinically-actionable mutations, tumor mutational burden (TMB), and MSI status. We developed novel methods to sequence difficult regions of the exome and to extend coverage to key immunogenomic biomarkers. Analytic pipelines were designed to utilize assay optimizations to achieve higher accuracy than with other platforms. We then validated the platform for diagnostic and therapeutic use. With as little as 50ng of DNA per FFPE sample and co-extracted RNA, this platform completely covers between 17% to 40% more genes compared to a non-augmented exome, thus increasing sensitivity to somatic mutations and putative neoantigens. For neoantigen performance, we generated immune-peptidomic data from mono-allelic HLA transfected cell-lines and trained neural networks to predict neoepitope binding to MHC, demonstrating a higher precision (0.88) across alleles than publicly available tools (0.9 and >0.94, respectively). For TILs, we developed signatures for CD4, CD8 T-cells, and other immune cells, demonstrating concordance with synthetic and CyTOF-derived validation sets. For HLA typing, we achieve an accuracy of 99.1% for HLA Class I, and 95% for HLA Class II typing calls, and have developed a novel tool for HLA LOH detection. We demonstrate sensitive detection of HPV, EBV, HCV, HTLV, and KSHV in known samples, and accurate MSI and TMB assessment. Finally, for diagnostic reporting, we achieve high sensitivity and specificity for clinically-reportable mutations comparable with diagnostic cancer panels. With this platform, we have developed a novel immunogenomics platform that can characterize both the tumor and TME from a single sample. By co-optimizing our assay and analytics for immuno-oncology, we enhance biomarker sensitivity compared to non-optimized genomics assays. Validation of the platform extends its use to diagnostics and personalized immunotherapy development. Citation Format: Robert Power, Gabor Bartha, Jason Harris, Sean Michael Boyle, Eric Levy, Pamela Milani, Prateek Tandon, Robin Li, Manjula Chinnappa, Paul McNitt, Rena McClory, Massimo Morra, Sebastian Saldivar, Michael Clark, Christian Haudenschild, Erin Newburn, Christelle Johnson, John West. A comprehensive, highly accurate genomics platform for precision immunotherapy: Simultaneously characterize tumors and the TME from a single FFPE sample [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A051. doi:10.1158/1535-7163.TARG-19-A051

Cardiovascular disease presents significant variations in human populations with respect to the atherosclerotic plaque progression, inflammation, thrombosis, and rupture. To gain a more comprehensive picture of the pathogenic mechanism of atherosclerosis and the variations seen in patients, efficient methods to identify proteins from the normal and diseased arteries need to be developed. To accomplish this goal, we tested the feasibility and efficiency of protein identification by a recently developed method, termed direct tissue proteomics (DTP). We analyzed frozen and paraformaldehyde-fixed archival coronary arteries with the DTP method. We also validated the distinct expression of four proteins by immunohistochemistry. In addition, we demonstrated the compatibility of the DTP method with laser capture microdissection and the possibility of monitoring specific cytokines and growth factors by the absolute quantification of abundance method. Major findings from this feasibility study are that 1) DTP can be used to efficiently identify proteins from paraformaldehyde-fixed, paraffin-embedded, and frozen coronary arteries; 2) approximately twice the number of proteins were identified from the frozen sections when compared with the paraformaldehyde-fixed sections; 3) laser capture microdissection is compatible with DTP; and 4) detection of low abundance cytokines and growth factors in the coronary arteries required selective reaction monitoring experiments coupled to absolute quantification of abundance. The analysis of 35 human coronary atherosclerotic samples allowed identification of a total of 806 proteins. The present study provides the first large scale proteomics map of human coronary atherosclerotic plaques.

Background Enumeration of circulating tumor cells (CTCs) isolated from the peripheral blood of breast cancer patients holds promise as a clinically relevant, minimally invasive diagnostic test. However, CTC utility has been limited as a prognostic indicator of survival by the inability to stratify patients beyond general enumeration. In comparison, histological biopsy examinations remain the standard method for confirming malignancy and grading malignant cells, allowing for cancer identification and then assessing patient cohorts for prognostic and predictive value. Typically, CTC identification relies on immunofluorescent staining assessed as absent/present, which is somewhat subjective and limited in its ability to characterize these cells. In contrast, the physical features used in histological cytology comprise the gold standard method used to identify and preliminarily characterize the cancer cells. Here, we superimpose the methods, cytologically subtyping CTCs labeled with immunohistochemical fluorescence stains to improve their prognostic value in relation to survival. Methods In this single-blind prospective pilot study, we tracked 36 patients with late-stage breast cancer over 24 months to compare overall survival between simple CTC enumeration and subtyping mitotic CTCs. A power analysis (1-β = 0. 9, α = 0.05) determined that a pilot size of 30 patients was sufficient to stratify this patient cohort; 36 in total were enrolled. Results Our results confirmed that CTC number is a prognostic indicator of patient survival, with a hazard ratio 5.2, p = 0.005 (95 % CI 1.6–16.5). However, by simply subtyping the same population based on CTCs in cytological mitosis, the hazard ratio increased dramatically to 11.1, p

Recent studies have suggested the IL-4Rα expressed on lung epithelium is necessary for TH2-mediated goblet cell differentiation and mucus hypersecretion in a murine model of allergic lung disease. However, the IL-4Rα is expressed on numerous cell types that could contribute to the overall pathology and severity of asthma. The relative role of the receptor on these cells has not yet been conclusively delineated. To dissect the contribution of IL-4Rα in the development of pulmonary allergic responses, we generated murine radiation bone marrow (BM) chimeras. BM from IL-4Rα+ or IL-4Rα− mice was transferred into recipient mice that expressed or lacked IL-4Rα. In the absence of IL-4Rα in recipient mice, there was no goblet cell metaplasia or mucus hypersecretion in response to OVA, even in the presence of TH2 cells and substantial eosinophilic infiltration. More importantly, we found that expression of the IL-4Rα on a nonlymphoid, MHC class II+, BM-derived cell type contributes to the severity of inflammation and mucus production. These results suggest that IL-4 and IL-13 contribute to the development of allergic inflammation by stimulating a complex interaction between IL-4Rα+ cell types of both bone marrow and non-bone marrow origin.

Treatment of HIV with AZT (zidovudine) may have toxic side effects as a result of multiple mechanisms. It is known that patients with AIDS may suffer from magnesium deficiency (MgD). We studied selected biochemical and histopathologic consequences of AZT administration (0.7 mg/mL in drinking water) with concurrent Mg-deficient (20% of normal) diet in male C57Bl/6N mice for 3 wk. Significant decreases in red blood cell glutathione (GSH) were evident in the Mg-deficient mice with or without AZT treatment, suggesting compromised antioxidant capacity in the blood. Although MgD alone led to a 1.9-fold increase in plasma thromboxane B(2) (TXB(2), derived from the highly vasoconstrictive TXA(2)), AZT + MgD increased the TXB(2) level 3.5-fold. AZT (+/-MgD) provoked prominent hepatic damage expressed by distortion of lobular architecture, nuclear and cellular swelling, and inflammatory lesions and loss of hepatocytes. AZT alone caused mild cardiac lesions, resulting in partial cardiac fibrosis, especially in the atrium. AZT + MgD caused only scattered small-size cardiac lesions consisting of microscopic foci of inflammatory infiltrates in the ventricles but led to more prominent lesions, fibrosis, and scars in the atrium. MgD or AZT alone caused varying degrees of skeletal muscle degeneration; in combination, more intense degeneration and regeneration of muscle cells were evident. In conclusion, it is suggested that both the decreased blood GSH and elevated plasma TXA(2) might contribute, at least in part, to the aggravated pathological damages observed in the atrium and skeletal muscle of the AZT-treated Mg-deficient mice.

Dietary flaxseed meal reduces proteinuria and ameliorates nephropathy in an animal model of type II diabetes mellitus.BackgroundEvidence is emerging that varying the type or source of dietary protein intake can have beneficial effects on chronic renal disease. Consumption of soybean and soy-based food products, as the source of plant protein, can retard the development and progression of chronic renal disease. We studied the obese spontaneously hypertensive/NIH-corpulent (SHR/N-cp) rat, a model of obesity and type II diabetes mellitus that consistently develops nephropathy resembling diabetic nephropathy. We specifically sought to determine whether changing the source of protein intake from animal protein, casein, to plant protein in the form of either soy protein concentrate or flaxseed protein in the diet has a different impact on renal function and nephropathy in this model.MethodsMale obese SHR/N-cp rats were randomly assigned to one of three diets containing either 20% casein, 20% soy protein concentrate, or 20% flaxseed meal. Except for the protein source, all three diets were identical and contained similar amounts of protein, fat, carbohydrates, minerals, and vitamins. All animals were maintained on these diets for 6months. At the end of the study, blood sampling and 24-hour urine collections were performed for renal functional measurements, and the kidneys were harvested and examined for histologic evaluation.ResultsAll three groups had similar amounts of food intake and body weight gain and exhibited fasting hyperglycemia and hyperinsulinemia. Plasma glucose levels did not differ among the three groups, but plasma insulin concentration was significantly lower in rats fed flaxseed meal than those fed either casein or soy protein concentrate. Mean plasma creatinine, creatinine clearance, and urinary urea excretion also did not differ significantly between the three groups. By contrast, urinary protein excretion was significantly lower (P < 0.01) in rats fed flaxseed than in rats fed either casein or soy protein concentrate. Morphologic analysis of renal structural lesions showed that the percentage of abnormal glomeruli with mesangial expansion and the tubulointerstitial score (an index of severity of tubulointerstitial damage) were significantly reduced in rats fed flaxmeal compared to those fed casein or soy protein concentrate.ConclusionWe conclude that dietary protein substitution with flaxseed meal reduces proteinuria and glomerular and tubulointerstitial lesions in obese SHR/N-cp rats and that flaxseed meal is more effective than soy protein in reducing proteinuria and renal histologic abnormalities in this model. The reduction in proteinuria and renal injury was independent of the amount of protein intake and glycemic control. Which dietary component(s) present in flaxseed meal is (are) responsible for the renal protective effect remains to be determined.

Objective— Oxidized LDL (oxLDL) was shown to trigger the release of acidic fibroblast growth factor (FGF-1). Because these components are likely to be present simultaneously in the atherosclerotic milieu, we investigated whether oxLDL interacts with FGF-1 and whether this interaction affects FGF-1 functioning. Methods and Results— Using molecular sieve and electrophoretic mobility shift assays, we found that FGF-1 forms a complex with oxLDL in vitro, in contrast to its low affinity for nonatherogenic, native LDL. The FGF-1/oxLDL complex had a dramatically decreased ability to bind heparin and was nonmitogenic on cultured smooth muscle cells. In human atherosclerotic lesions, the highest FGF-1 immunoreactivity was found in macrophages. With respect to oxLDL accumulation, 2 patterns were distinguished: (1) moderate, intracellular in matrix-rich regions containing viable cells and (2) massive, both cell-associated and extracellular oxLDL deposits in foam cell–rich regions with necrotic areas. The proliferating cell nuclear antigen readings for proliferating cells reflected that the mitogenic activity of FGF-1 was confined to the regions where oxLDL was strictly intracellular and was inhibited in the regions with extracellular oxLDL deposition. Conclusions— oxLDL, besides being a bulky component of the atherosclerotic lesion, possibly manifests its pathogenicity by complexing FGF-1 and inhibiting its growth-promoting function during atherogenesis.

SHP-2, a ubiquitously expressed Src hmology 2 (SH2) domain-containing tyrosine phosphatase, plays a critical role in the regulation of growth factor and cytokine signal transduction. Here we report a novel function of this phosphatase in DNA damage-induced cellular responses. Mutant embryonic fibroblast cells lacking functional SHP-2 showed significantly decreased apoptosis in response to DNA damage. Following cisplatin treatment, induction of p73 and its downstream effector p21(Cip1) was essentially blocked in SHP-2 mutant cells. Further investigation revealed that activation of the nuclear tyrosine kinase c-Abl, an essential mediator in DNA damage induction of p73, was impaired in the mutant cells, suggesting a functional requirement of SHP-2 in c-Abl activation. Consistent with this observation, the effect of overexpression of c-Abl kinase in SHP-2 mutant cells on sensitizing the cells to DNA damage-induced death was abolished. Additionally, we found that in embryonic fibroblast cells 30-40% of SHP-2 was localized in the nuclei, and that a fraction of nuclear SHP-2 was constitutively associated with c-Abl via its SH3 domain. Phosphatase activity of nuclear but not cytoplasmic SHP-2 was significantly enhanced in response to DNA damage. These results together suggest a novel nuclear function for SHP-2 phosphatase in the regulation of DNA damage-induced apoptotic responses.

Matriptase/MT-SP1 is a novel tumor-associated type II transmembrane serine protease that is highly expressed in the epidermis, thymic stroma, and other epithelia. A null mutation was introduced into the Matriptase/MT-SP1 gene of mice to determine the role of Matriptase/MT-SP1 in epidermal development and neoplasia. Matriptase/MT-SP1-deficient mice developed to term but uniformly died within 48 h of birth. All epidermal surfaces of newborn mice were grossly abnormal with a dry, red, shiny, and wrinkled appearance. Matriptase/MT-SP1-deficiency caused striking malformations of the stratum corneum, characterized by dysmorphic and pleomorphic corneocytes and the absence of vesicular bodies in transitional layer cells. This aberrant skin development seriously compromised both inward and outward epidermal barrier function, leading to the rapid and fatal dehydration of Matriptase/MT-SP1-deficient pups. Loss of Matriptase/MT-SP1 also seriously affected hair follicle development resulting in generalized follicular hypoplasia, absence of erupted vibrissae, lack of vibrissal hair canal formation, ingrown vibrissae, and wholesale abortion of vibrissal follicles. Furthermore, Matriptase/MT-SP1-deficiency resulted in dramatically increased thymocyte apoptosis, and depletion of thymocytes. This study demonstrates that Matriptase/MT-SP1 has pleiotropic functions in the development of the epidermis, hair follicles, and cellular immune system.

ObjectivesWe evaluated the long-term influence of intramural delivery of advanced c-myc neutrally charged antisense oligonucleotides (Resten-NG) on neointimal hyperplasia after stenting in a pig model.BackgroundNeointimal hyperplasia after percutaneous coronary interventions is one of the key components of the restenotic process. The c-myc is a critical cell division cycle protein involved in the formation of neointima.MethodsIn short-term experiments, different doses (from 500 μg to 5 mg) of Resten-NG or saline were delivered to the stent implantation site with an infiltrator delivery system (Interventional Technologies, San Diego, California). Animals were euthanized at 2, 6 and 18 h after interventions, and excised vessels were analyzed for c-myc expression by Western blot. In long-term experiments, either saline or a dose of 1, 5 or 10 mg of Resten-NG was delivered in the same fashion, and animals were euthanized at 28 days after the intervention.ResultsWestern blot analysis demonstrated inhibition of c-myc expression and was dose dependent. Morphometry showed that the intimal area was 3.88 ± 1.04 mm2in the control. There was statistically significant reduction of intimal areas in the 5 and 10 mg groups (2.01 ± 0.66 and 1.95 ± 0.91, respectively, p < 0.001) but no significant reduction in the 1 mg group (2.81 ± 0.56, p > 0.5) in comparison with control.ConclusionsThis study demonstrated that intramural delivery of advanced c-myc neutrally charged antisense morpholino compound completely inhibits c-myc expression and dramatically reduces neointimal formation in a dose dependent fashion in a porcine coronary stent restenosis model, while allowing for complete vascular healing.

Recent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch(®) CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45-/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45- cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45- cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45-/DAPI+ cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using non-EpCAM dependent isolation. © 2014 The Authors. Published by Wiley Periodicals, Inc.

Myointimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA) is a key component of the process of restenosis. The c-myc is a critical cell-cycle division protein involved in the formation of neointima. We evaluated the long-term impact of local delivery of c-myc neutrally charged antisense oligonucleotides (Resten-NG) on myointimal hyperplasia after PTCA in a rabbit model. PTCA was performed in the iliac arteries of 25 New Zealand white rabbits, using a Transport catheter at 8 atm for 30 sec, three times; 500 microg Resten-NG (n = 11) or saline (n = 14) was delivered to the PTCA site at 2 atm with the outer balloon for 2 min. The diet was supplemented with 0.25% cholesterol for 10 days before and 60 days after PTCA. Angiography was performed at harvest, and vessels were fixed in formalin, processed, and stained with hematoxylin and eosin (H&E) and Movat. Quantitative angiography showed that local delivery of antisense c-myc at PTCA reduced late luminal loss from 1.8 +/- 0.30 mm in control animals to 0.90 +/- 0.30 mm in the treatment group (P = 0.001). Histological analysis by planimetry showed that intimal areas were 1.67 +/- 0.44 mm(2) and 0.82 +/- 0.32 mm(2) in the control and antisense delivery groups, respectively (P < 0.05). We conclude that local delivery of Resten-NG inhibited myointimal hyperplasia after PTCA in cholesterol-fed rabbits for up to 60 days.

OBJECTIVES The objectives of this study were 1) to improve the attachment of reimplanted endothelial cells (EC) using a fibrin glue, and 2) to assess the impact of endothelial reseeding on restenosis eight weeks after balloon angioplasty. BACKGROUND A possible mechanism contributing to restenosis after balloon angioplasty is the loss of the EC lining. Previous attempts to reseed EC had little effect due to rapid loss of the seeded cells. METHODS Twelve atherosclerotic rabbits were subjected to angioplasty of iliac arteries and reseeding procedure. One iliac artery was subjected to EC/glue reconstruction and a contralateral site to EC seeding without glue. The animals were sacrificed after 4 h. In another series 12 rabbits were treated in the same fashion and were restudied at eight weeks. Additionally, in 10 animals one iliac was subjected to glue treatment, and another served as control. RESULTS Histological examination demonstrated the ability of this method to reattach the EC/glue matrix circumferentially to 68.0 ± 6.7% of the arterial wall in comparison with 13.5 ± 3.9% reattachment after EC seeding. Morphometry at eight weeks showed that the lumen area was significantly greater in the EC/glue group (1.23 ± 0.35 mm2) than in the EC seeding alone (0.65 ± 0.02 mm2) and 0.72 ± 0.41 mm2 in the glue group. This was principally accounted for by the statistically significant differences in the intimal area (0.76 ± 0.18 mm2 vs. 1.25 ± 0.26 mm2 and 1.01 ± 0.53 mm2, respectively). CONCLUSIONS The attachment of EC after angioplasty can be greatly improved with fibrin glue matrix. The near 70% endothelial coverage achieved by this method resulted in a significant reduction of restenosis in atherosclerotic rabbit.

Abstract —In the C57BL/6J mice model, we investigated whether obesity affects the function or expression of components of the tissue renin-angiotensin system and whether endothelin (ET)-1 contributes to these changes. ACE activity (nmol · L His-Leu · mg protein −1 ) was measured in lung, kidney, and liver in control (receiving standard chow) and obese animals treated for 30 weeks with a high-fat, low cholesterol diet alone or in combination with LU135252 , an orally active ET A receptor antagonist. ACE mRNA expression was measured in the kidney, and the effects of LU135252 on purified human ACE were determined. Aortic and renal tissue ET-1 protein content was measured, and the vascular contractility to angiotensin II was assessed. Obesity was associated with a tissue-specific increase in ACE activity in the kidney (55±4 versus 33±3 nmol/L) but not in the lung (34±2 versus 32±2 nmol/L). Long-term LU135252 treatment completely prevented this activation (13.3±0.3 versus 55±4 nmol/L, P LU135252 treatment (6±0.4% versus 33±5% KCl). No effect of LU135252 on purified ACE was observed. Thus, obesity is associated with the activation of renal ACE in vivo independent of its mRNA expression and enhanced vascular contractility to angiotensin II. These effects are regulated by ET in an organ-specific manner, providing novel mechanisms by which ET antagonists may exert organ protection.

Abstract —Myxomatous tissue is a characteristic component of human coronary artery lesions, found more often in restenotic lesions. It represents a bulky accumulation of stellate-shaped cells of unknown histogenesis that are embedded in a loose stroma. We analyzed 64 atherectomy specimens containing substantial amounts of myxomatous tissue by using immunohistochemistry, in situ hybridization, and electron microscopy techniques. Stellate cells represented a heterogeneous population, sharing features of smooth muscle cells (SMCs), macrophages, as well as antigen-presenting dendritic cells. Like quiescent medial SMCs, the stellate cells in all specimens expressed high levels of SM α-actin message and protein and showed heterogeneity with respect to heavy-chain myosin, SM22, desmin, and vimentin. Ultrastructurally, stellate cells resembled SMCs, with some peculiarities that distinguish them from both differentiated and dedifferentiated SMCs. In contrast to quiescent SMCs, the stellate cells expressed high levels of acidic fibroblast growth factor mRNA and protein similar to cells of monocyte/macrophage lineage. However, stellate cells did not express the marker of mature macrophages, HAM56, and were heterogeneous with respect to CD68. Moreover, unlike SMCs, the stellate cells bore some of the major phenotypic markers of dendritic cells: they were S100-positive and showed various reactivity with respect to CD1a and human leukocyte antigen (HLA)-DR. Invasion of myxomatous tissue with CD45RO-positive T lymphocytes was correlated with strong expression of CD1a in these specimens. Stellate cells also expressed a pericyte marker, high-molecular-weight melanoma-associated antigen. We conclude that stellate cells of myxomatous tissue represent a specific phenotype of mesenchymal cells (possibly pericytes), which is activated to express some markers of antigen-presenting cells. These findings suggest involvement of the stellate cells in a local immune response.

Objective. To relate local arterial geometry with markers that are thought to be related to plaque rupture. Background. Plaque rupture often occurs at sites with minor luminal stenosis and has retrospectively been characterized by colocalization of inflammatory cells. Recent studies have demonstrated that luminal narrowing is related with the mode of atherosclerotic arterial remodeling. Methods. We obtained 1,521 cross section slices at regular intervals from 50 atherosclerotic femoral arteries. Per artery, the slices with the largest and smallest lumen area, vessel area and plaque area were selected for staining on the presence of macrophages (CD68), T-lymphocytes (CD45RO), smooth muscle cells (alpha-actin) and collagen. Results. Inflammation of the cap or shoulder of the plaque was observed in 33% of all cross sections. Significantly more CD68 and CD45RO positive cells, more atheroma, less collagen and less alpha-actin positive staining was observed in cross sections with the largest plaque area and largest vessel area vs. cross sections with the smallest plaque area and smallest vessel area, respectively. No difference in the number of inflammatory cells was observed between cross sections with the largest and smallest lumen area. Conclusion. Intraindividually, pathohistologic markers previously reported to be related to plaque vulnerability were associated with a larger plaque area and vessel area. In addition, inflammation of the cap and shoulder of the plaque was a common finding in the atherosclerotic femoral artery.

The ninth type III module of murine fibronectin was expressed in E. coli and folded into a compact homogeneous monomer whose unfolding and refolding were then investigated by fluorescence, circular dichroism, calorimetry and electron microscopy. The isolated module is unusually labile under physiological conditions. When heated at 1 deg. C/minute it exhibits an irreversible endothermic transition between 35 and 42 degrees C depending on the protein concentration. The transition is accompanied by changes in secondary and tertiary structure with partial exposure of the single tryptophan and increased binding of the hydrophobic probe, 1,8-anilinonaphthalene-sulfonate. The partially unfolded intermediate undergoes rapid self-association leading to the formation of large stable multimers that, like the original monomer, contain substantial amounts of beta sheet structure. The multimers melt and dissociate reversibly in a second endothermic transition between 60 and 90 degrees C also depending on the protein concentration. This second transition destroys the remaining secondary structure and further exposes the tryptophan. Visualization of negatively stained specimens in the electron microscope reveals that partially unfolded rmIII-9 slowly forms amyloid-like fibrils of approximately 10 nm width and indeterminate length. A subdomain swapping mechanism is proposed in which beta strands from one partially unfolded molecule interact with complementary regions of another to form oligomers and polymers. The possibility that similar interactions could play a role in the formation of fibrils by fibronectin in vivo is discussed.

Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FGF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.

A cDNA clone for the serine proteinase inhibitor (serpin), neuroserpin, was isolated from a human whole brain cDNA library, and recombinant protein was expressed in insect cells. The purified protein is an efficient inhibitor of tissue type plasminogen activator (tPA), having an apparent second-order rate constant of 6. 2 x 10(5) M-1 s-1 for the two-chain form. However, unlike other known plasminogen activator inhibitors, neuroserpin is a more effective inactivator of tPA than of urokinase-type plasminogen activator. Neuroserpin also effectively inhibited trypsin and nerve growth factor-gamma but reacted only slowly with plasmin and thrombin. Northern blot analysis showed a 1.8 kilobase messenger RNA expressed predominantly in adult human brain and spinal cord, and immunohistochemical studies of normal mouse tissue detected strong staining primarily in neuronal cells with occasionally positive microglial cells. Staining was most prominent in the ependymal cells of the choroid plexus, Purkinje cells of the cerebellum, select neurons of the hypothalamus and hippocampus, and in the myelinated axons of the commissura. Expression of tPA within these regions is reported to be high and has previously been correlated with both motor learning and neuronal survival. Taken together, these data suggest that neuroserpin is likely to be a critical regulator of tPA activity in the central nervous system, and as such may play an important role in neuronal plasticity and/or maintenance.

Abstract Luminal stenosis can be based on large atherosclerotic plaques in compensatory enlarged segments or on relatively little plaques in shrunken segments. In the present study, the contribution of plaque formation and remodeling to luminal narrowing was compared among six types of arteries prone to symptomatic atherosclerosis. Cross-sections (n=5195) were obtained at regular intervals from 329 arteries. For each artery, the cross-section that contained the least amount of plaque was considered to be the reference. For each cross-section, the percentage of lumen area decrease was expressed as a percentage of the lumen area at the reference site (luminal stenosis). Similarly, the area encompassed by the internal elastic lamina (IEL area) was expressed as a percentage of the IEL area at the reference site (relative IEL area). All cross-sections were categorized in three groups: relative IEL area >105% (enlargement), 95% to 105% (no remodeling), and P

The cyclooxygenase (Cox) enzyme catalyzes the rate-limiting oxidative and peroxidative enzymatic steps in the biosynthesis of prostanoids. Both Cox-1 and -2 genes encode the two isoenzymes that carry out similar enzymatic steps. Enhanced Cox activity is associated with proliferative diseases such as colon cancer. To determine if a cause and effect relationship exists between Cox isoenzyme overexpression and tumorigenesis, the human Cox-1 and Cox-2 isoenzymes were transfected into ECV immortalized endothelial cells. Although numerous clones of Cox-1 expressing cells were obtained, Cox-2 overexpression resulted in growth disadvantage and increased cell death. In contrast, Cox-1 overexpressing cells expressed high levels of the functional Cox-1 polypeptide in the endoplasmic reticulum and the nucleus. In vitro proliferation of these cells was reduced compared with vector-transfected ECV cells. Cox-1 overexpression also enhanced the tumor necrosis factor-α-induced apoptosis of ECV cells 2-fold. In contrast to the in vitrobehavior, ECV-Cox-1 cells proliferated aggressively and formed tumors in athymic “nude” mice, whereas the vector-transfected counterparts did not. The growth of Cox-1-induced tumors was not inhibited by indomethacin, suggesting a nonprostanoid function of Cox-1. ECV-Cox-1-derived tumors were angiosarcoma-like and contained numerous host-derived neovessels. These data suggest that Cox-1 overexpression in immortalized ECV endothelial cells results in nuclear localization of the polypeptide and tumorigenesis.

Cortactin, a prominent substrate for pp60(c-src), is a filamentous actin (F-actin) binding protein. We show here that cortactin can promote sedimentation of F-actin at centrifugation forces under which F-actin is otherwise not able to be precipitated. Electron microscopic analysis after negative staining further revealed that actin filaments in the presence of cortactin are cross-linked into bundles of various degrees of thickness. Hence, cortactin is also an F-actin cross-linking protein. We also demonstrate that the optimal F-actin cross-linking activity of cortactin requires a physiological pH in a range of 7.3-7.5. Furthermore, pp60(c-src) phosphorylates cortactin in vitro, resulting in a dramatic reduction of its F-actin cross-linking activity in a manner depending on levels of tyrosine phosphorylation. In addition, pp60(c-src) moderately inhibits the F-actin binding activity of cortactin. This study presents the first evidence that pp60(c-src) can directly regulate the activity of its substrate toward the cytoskeleton and implies a role of cortactin as an F-actin modulator in tyrosine kinase-regulated cytoskeleton reorganization.

Abstract Fibroblast growth factor-1 (FGF-1) and lipoproteins play an important role in atherogenesis. In the present study, we explored a possible mechanism by which abnormal lipid metabolism could be linked to the proliferative aspects of the disease. We tested oxidized LDL (oxLDL) as a possible pathophysiological mediator of the release of FGF-1, using FGF-1–transfected mouse NIH 3T3 cells and FGF-1–transfected rabbit smooth muscle cells, and compared it with the release caused by elevated temperature. Immunoblot analysis showed that oxLDL induced the release of FGF-1 in a concentration-dependent manner from 10 to 100 μg/mL. The effect correlated with the extent of oxidative modification of LDL and was maximal within 4 hours of exposure of cells to oxLDL. In contrast to the temperature stress–induced FGF-1 secretion pathway, FGF-1 released in response to oxLDL (1) appeared in the conditioned medium as a monomer, (2) appeared independently of the presence of either actinomycin D or cycloheximide, and (3) was neither enhanced nor inhibited by brefeldin A. We did not detect cell loss, significant morphological changes, changes in growth characteristics, or other indications of lethal toxicity in oxLDL-treated cells. Although the level of lactate dehydrogenase activity was elevated after oxLDL exposure, the calculations showed that >90% of the FGF-1 was released by viable cells. We propose that oxLDL-induced FGF-1 release is mediated by sublethal and apparently transient changes in cell membrane permeability. In the environment of an atherosclerotic lesion, oxLDL-induced FGF-1 release may be among the mediators of endothelial and smooth muscle cell proliferation.

Cortactin is a potent filamentous actin-binding protein acting as a prominent substrate of Src tyrosine kinases. We have evaluated cortactin expression in a series of murine tissues and shown an abundant expression of cortactin in megakaryocytes and platelets. Cortactin, but not its related protein HS1, is upregulated during the phorbol 12-myristate 13-acetate (PMA)-mediated maturation of a human megakaryoblastic cell line CMK. Although the expression of Src-related kinases is also upregulated more rapidly than cortactin in PMA-treated CMK cells, tyrosine phosphorylation of cortactin appears to be only transiently elevated 4 days after PMA stimulation. In addition, cortactin expression is induced by thrombopoietin and interleukin-3 in megakaryocytes derived from murine bone marrow cells. Thus, cortactin represents a megakaryocyte-specific gene in bone marrow and the interaction of Src kinases with cortactin may be involved in the maturation of megakaryocytes.

Geometric remodeling in atherosclerosis and restenosis comprises a change of the total arterial circumference that, together with plaque growth or neointima formation, determine the lumen of the artery. The change in total arterial circumference relative to a reference cross section ranges from excessive enlargement with an actual increase in lumen to a shrinkage contributing to lumen narrowing. The forces that determine whether an artery shrinks or is able to enlarge as a consequence of atherosclerotic lesions or of post-interventional intimal hyperplasia are unknown but are likely to affect matrix and specifically collagen redistribution. Identifying these forces is of importance because they might be used to promote favorable remodeling. The cellular and humoral triggers of collagen degradation and synthesis, as well as conformation changes of collagen matrices after their deposition, need to be investigated. As with injury-induced intimal hyperplasia and plaque growth in de novo atherosclerosis, injury models may serve as a paradigm for geometric remodeling in atherosclerosis.

The fibulins are an emerging family of extracellular matrix and blood proteins presently having two members designated fibulin-1 and −2. Fibulin-1 is the predominant fibulin in blood, present at a concentration of 30-40 μg/ml (~1000-fold higher than fibulin-2). During the course of isolating fibulin-1 from plasma by immunoaffinity chromatography, a 340-kDa polypeptide was consistently found to co-purify. This protein was identified as fibrinogen (Fg) based on its electrophoretic behavior and reactivity with Fg monoclonal antibodies. Radioiodinated fibulin-1 was shown to bind to Fg transferred onto nitrocellulose filters after SDS-polyacrylamide gel electrophoresis. In enzyme-linked immunosorbent assay, fibulin-1 bound to Fg (and fibrin) adsorbed onto microtiter well plastic, and conversely, Fg bound to fibulin-1-coated wells. The binding of Fg to fibulin-1 was also observed in surface plasmon resonance assays, and a dissociation constant (Kd) of 2.9 ± 1.6 μM was derived. In addition, fluorescence anisotropy experiments demonstrated that the interaction was also able to occur in fluid phase, which suggests that complexes of fibulin-1 and Fg could exist in the blood. To localize the portion of Fg that is responsible for interacting with fibulin-1, proteolytic fragments of Fg were evaluated for their ability to promote fibulin-1 binding. Fragments containing the carboxyl-terminal region of the Bβ chain (residues 216-468) were able to bind to fibulin-1. In addition, it was found that fibulin-1 was able to incorporate into fibrin clots formed in vitro and was immunologically detected within newly formed fibrin-containing thrombi associated with human atherectomy specimens. The interaction between fibulin-1 and Fg highlights potential new roles for fibulin-1 in hemostasis as well as thrombosis.

Targeted sequencing assays are increasingly used to identify tumor mutations that guide therapeutic decisions. Interpretation of a cancer variant's origin and therapeutic impact poses analytical challenges. Recent studies have indicated that jointly analyzing a tumor with its matched normal can accurately discriminate between tumor-specific (somatic) and inherited (germline) mutations. Moreover, a NHGRI/NCI Clinical Sequencing Exploratory Research Consortium Tumor Working Group just released a set of guidelines recommending that laboratories performing cancer sequencing tests should include germline variants. However, procurement of a matched sample is often logistically impractical. In the absence of a matched normal, large databases and analytical techniques are currently used to identify cancer variants in tumor sequencing data. Whether the benefits outweigh the additional burden of sequencing the matched normal for accurate detection of cancer-relevant mutations remains an open question. To compare tumor-only and tumor/normal analysis of cancer samples, we collected a set of >100 formalin-fixed (FFPE) and fresh frozen cancer samples of various tumor types, where matched normal blood or adjacent tissue was available. We performed augmented target enrichment sequencing (exome and large cancer gene panel) of both DNA and RNA. The data was analyzed using cancer bioinformatics pipelines that detect base substitutions, small insertions/deletions, copy number alterations, and gene fusions in both tumor-only and tumor/normal modes. Variants were annotated using described clinical actionability filtering strategies. Analysis of germline variants for secondary findings was performed. We find that 67% of mutations detected in tumor-only mode are reclassified as germline variants when analyzed together with the matched normal sample. These include mutations in hereditary cancer predisposition genes, such as BRCA1, VHL, and other genes with ACMG guidelines that warrant germline variant classification and appropriate management. Clinically actionable mutations may be miscalled as somatic when a matched normal is not available; however, we find the definition of ‘actionable’ can greatly impact the results of this analysis. Finally, the use of newly available large datasets, such as ExAC, substantially decreases the number of miscalled somatic variants in the absence of a matched normal. The effects of administering targeted therapies to patients with germline mutations in the relevant gene are largely unknown. Mutations of putative germline origin may be important for hereditary cancer knowledge and tumor treatment, and should be reported as such. For NGS-based cancer interpretation to guide clinical decisions in a practical and cost-effective manner, highly optimized tumor-only and tumor/normal analyses must be available with proper attention to germline consent, classification and education. Citation Format: Elena Helman, Michael J. Clark, Ravi Alla, Sean M. Boyle, Shujun Luo, Selene Virk, Deanna Church, Parin Sripakdeevong, Jason Harris, Mirian karbelashvili, Christian Haudenschild, John West, Richard Chen. The benefits and burdens of assaying matched normal tissue when sequencing cancer genomes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3169.

The objective of this study was to determine whether basic fibroblast growth factor (bFGF), a known angiogenic factor, can promote new vessel growth when infused within the pericardial space in a model of chronic myocardial ischemia. Intravenous angiotensin II (AII) was infused to induce left ventricular hypertrophy and concomitant ischemia in New Zealand white rabbits. Basic FGF was infused into the intrapericardial space with an osmotic pump. Animals were assigned to one of four groups: group 1 received intrapericardial bFGF and intravenous AII, group 2 received intrapericardial bFGF and intravenous saline solution, group 3 received intrapericardial albumin and intravenous AII, and group 4 received intravenous AII only. Epicardial angiogenesis was graded histologically on a scale of 0 to 2. Animals receiving intravenous administration of AII displayed left ventricular hypertrophy that disproportionately affected the interventricular septum with a wall thickness of 5.62 +/- 1.00 mm versus 3.98 +/- 0.61 mm in the AII group and the saline solution control group, respectively (p0.005). A highly localized angiogenic effect of bFGF was observed. The mean angiogenesis scores were 1.9, 1.4, 1.3, and 0.2 (p0.001) with an angiogenesis score of 2 (marked increase in vascularity) noted in 86%, 40%, 43%, and 0% of hearts in groups 1 through 4, respectively. We conclude that intrapericardial bFGF enhances new epicardial small-vessel growth in a rabbit model; furthermore this effect is enhanced in the presence of left ventricular hypertrophy.

Summary Reocclusion after fibrinolysis is a thrombotic event most likely due to the persistence of the thrombogenic stimuli such as plaque fissures that caused the occlusion in the first place. After angioplasty, more severe vascular wall disruption and elastic recoil are additional mechanisms of early reocclusion. Either spontaneous fissures or angioplastic modifications of the vascular wall initiate a remodeling process that primarily restores and possibly stabilizes and reinforces the wall tissue. The mechanisms which turn this desirable healing process into restenosis include failure of coordination and down-regulation of initiating stimuli and responses, poorly adapted tissue fragments, exposure of additional thrombogenic surfaces, excessive cell growth and synthesis of matrix, and contracture of the newly formed scar tissue, leading to a chronic compromise of the effective lumen.

BACKGROUND In de novo human atherosclerosis, compensatory vessel enlargement limits the effect of intimal plaque formation on lumen narrowing. We hypothesized that arterial remodeling may also play an important role in determining the chronic lumen size after angioplasty and tested this hypothesis using the hypercholesterolemic rabbit iliac artery angioplasty model. METHODS AND RESULTS Morphometric analysis of histological cross-sectional areas of vessels from animals killed immediately after angioplasty (acute group, n = 11) were compared with the same areas from animals killed 4 weeks after the procedure (chronic group, n = 37), when restenosis occurs in this model. The area circumscribed by the internal elastic lamina (IEL) increased by 20% from acute to 4 week follow-up after angioplasty (acute group, 2.36 +/- 0.45 mm2, chronic group, 2.84 +/- 0.89 mm2). Over the same time period, intimal area increased by 0.82 mm2. Despite this increase in intimal area, lumen area decreased by only 0.34 mm2 because of the compensatory enlargement of the IEL area. In the chronic group, polynomial regression analysis revealed a quadratic relation between intimal area and lumen area (R2 = .35, P < .001). A lumen area of 0.45 mm2 (the nadir of the quadratic relation) was used to divide the chronic group into two subgroups: restenotic (n = 21; lumen area, < 0.45 mm2) and nonrestenotic (n = 16; lumen area, > 0.45 mm2). By definition, there was a significant difference in lumen area between the two subgroups (0.15 +/- 0.15 mm2 for restenotic; 0.73 +/- 0.18 mm2 for nonrestenotic). Surprisingly, the intimal areas in the two subgroups were virtually identical (2.41 +/- 0.92 mm2 for restenotic, 2.49 +/- 0.69 mm2 for nonrestenotic, P = NS). The difference in the lumen area between restenotic and nonrestenotic vessels was a result of the significantly greater IEL area in the nonrestenotic subgroup (3.22 +/- 0.83 mm2 for nonrestenotic, 2.56 +/- 0.84 mm2 for restenotic, P < .05). In both restenotic and nonrestenotic vessels, the IEL area increased with increases in intimal area. In the restenotic arteries, the slope of this correlation was < 1, showing inadequate compensatory enlargement for the intimal plaque. In the nonrestenotic vessels, the slope was > 1, limiting the effect of intimal plaque on luminal narrowing. CONCLUSIONS These data indicate that the iliac artery in an atherosclerotic rabbit model compensates for intimal formation after angioplasty by vessel enlargement. Furthermore, the degree of vessel enlargement is more important than intimal area in determining the chronic lumen size.

Plasminogen activator inhibitor-1 (PAI-1), as the name implies, is the primary in vivo inhibitor of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). PAI-1 also binds to other nonproteinase ligands, including the matrix protein vitronectin, glycosaminoglycans such as heparin, and the endocytic clearance receptor, the low-density-lipoprotein-receptor-related protein (LRP). PAI-1 belongs to the superfamily of serine proteinase inhibitors (serpins), and, like other serpins, it acts as "suicide inhibitor" that reacts only once with a target proteinase. The suicide mechanism results in irreversible modification of the serpin and an extensive change in its conformation. In the case of PAI-1, this conformational change is important not only for inhibition of the proteinase, but it also causes changes in affinity for vitronectin and LRP. These changes have important consequences for cell migration.

Oxidized low-density lipoproteins (LDL), which may play an important role in atherogenesis, inhibit endothelium-dependent relaxations of normal arteries in vitro. The effects of probucol, an inhibitor of LDL oxidation, on endothelium-dependent relaxations in thoracic aorta of rabbits that received a cholesterol-rich (0.5%) or standard diet for 10 weeks were determined. In some rabbits in each group, the diet was supplemented with probucol (1%) for the last 6 weeks. The cholesterol-rich diet markedly increased plasma cholesterol and resulted in increased plasma lipid peroxides. Probucol prevented the increase in lipid peroxides, but had no effect on plasma cholesterol. Rings of aorta were mounted in organ chambers for measurement of isometric tension and contracted with phenylephrine. Endothelium-dependent relaxations to acetylcholine and A23187 were significantly impaired in aortic rings from cholesterol-fed rabbits. Aortic rings from rabbits fed cholesterol and treated with probucol relaxed normally to both vasodilators. Relaxations to acetylcholine and A23187 were not significantly changed in rings from rabbits that received probucol-supplemented standard diet. Endothelium-independent relaxations to sodium nitroprusside (SNP) were not influenced by the cholesterol diet or probucol. Thus, probucol preserves endothelium-dependent relaxations of hypercholesterolemic rabbit aorta without reducing plasma cholesterol. As demonstrated by the reduction in plasma lipid peroxides, the effect of probucol may be related to its antioxidant properties and may imply that oxidized lipids have a role in endothelial cell dysfunction of atherosclerotic arteries in vivo.

Atherosclerosis, a chronic systemic disease of the vasculature with an inflammatory component, is the primary cause of cardiovascular morbidity and mortality in industrialized countries. Impairment of vascular endothelial cell function in atherosclerosis and in conditions associated with increased cardiovascular risk are important determinants of disease progression. Reduced endothelium-dependent relaxation in the coronary and systemic circulation due to decreased bioavailability of nitric oxide (NO) and increased release of oxygen-derived free radicals promotes the adhesion of leukocytes, thrombosis, inflammation, cell proliferation, and increases in vascular tone. In addition to decreases in bioactive NO, enhanced production of the 21-amino acid peptide endothelin-1 contributes to the progression of atherosclerosis. This paper discusses mechanisms and therapeutic approaches to improving endothelial pathways in atherosclerosis. Restoration of endothelium-derived NO bioactivity through inhibition of the renin-angiotensin system, the endothelin system, or statin therapy improves vascular function in experimental hypercholesterolemia, hypertension and heart failure. These treatments may also have therapeutic benefit for patients at risk or with overt atherosclerosis, and are likely to reduce vascular and myocardial complications of this disease.

Internal mammary artery specimens from 17 patients were each divided into three separate rings. One ring (control) remained in Krebs solution and the other two were clamped for 30 minutes with either a soft or hard jaw clamp. Isometric tensions were measured in an organ chamber by contracting the rings twice with a thromboxane A2 mimetic, U46619, and relaxing the rings first with the endothelium-dependent agent acetylcholine followed by the endothelium-independent agent sodium nitroprusside. Endothelium-dependent maximal relaxation of the rings was impaired from control after both soft (20% versus 91%; p horac C ardiovasc S urg 1992;104:966-71)

This study was undertaken to study the effects of hyperlipidemia and hypertension on the coronary circulation and on the myocardium of Watanabe heritable hyperlipidemic (WHHL) rabbits. Surgery to induce hypertension by the one-kidney, one-clip technique was performed on the WHHL rabbits at 3 months of age. At 3 and 6 months after surgery, the right and left coronary arteries and the left ventricle and posterior papillary muscle from normotensive and hypertensive animals were assessed. Atherosclerotic involvement was found at the coronary origin in 94% of the arteries evaluated. Lesions were usually confined to the proximal 1-2 mm of the coronary artery. The prevalence of coronary atherosclerosis in the WHHL rabbit was found to be higher than previously reported in rabbits of the same age. Hypertension-induced muscular and vascular changes such as left ventricular hypertrophy, medial thickening of the arteries, and hyaline arteriolosclerosis were found in most of the hypertensive animals. These changes were rarely seen in the normotensive rabbits. Characteristics of ischemia and cell injury such as eosinophilic fibers, fiber vacuolization, and contraction band necrosis were found more often in hypertensive than in normotensive WHHL rabbits. Confluent areas of severe necrosis indicative of myocardial infarction were not found; myocardial damage was diffuse and involved individual cells and small microscopic areas. This model may be valuable in further studies of coronary artery disease and myocardial injury that result from the combination of hypercholesterolemia and hypertension.

This is an investigation of the relationship between graft preparation techniques and the subsequent fate of vein grafts. Vein grafts intentionally injured by warm saline storage demonstrated endothelial and smooth muscle cell damage. In the acute postimplantation period, platelet adhesion/activation and white cell infiltration were present. By 7 days the endothelium had “healed,” but the underlying smooth muscle cells had modulated and were of the synthetic phenotype. This persisted at 30 days, but by 60 days the graft wall remodeled with smooth muscle cells that were of the contractile phenotype, with an organized extracellular matrix. None of these injurious responses were noted in optimally prepared papaverine-treated vein grafts. Optimal preparation of vein grafts is effective in minimizing endothelial and smooth muscle cell injury before and after arterial reconstruction. Prevention of vein graft injury during harvesting prevents the morphologic changes characteristic of the “arterialization response.” (J T horac C ardiovasc S urg 1992;103:671-7)

Fibulin-1 is a fibrinogen-binding blood protein and a component of many extracellular matrices (ECM) including those of blood vessels. In this study, the deposition patterns of fibulin-1 and fibrinogen were examined in human coronary artery atherosclerotic lesions excised by atherectomy from 20 patients. Fibulin-1 deposition was found to be closely overlapping with fibrinogen located within the atherosclerotic lesions and in regions containing fresh thrombi. Pronounced intracellular fibulin-1 immunostaining was apparent in lesion areas rich in macrophages and foam cells, although THP-1 macrophages and foam cells were found not to express fibulin-1. Strong ECM deposition of fibulin-1 was observed in acellular atheromatous and myxomatous regions. By contrast, fibulin-1 was present at relatively low levels in the ECM associated with smooth muscle cells within and outside of lesions and was not detected in sclerotic regions. These results reveal the pattern of fibulin-1 within human atherosclerotic lesions and highlight the potential for fibulin-1, perhaps derived from the blood and acting in conjunction with fibrinogen, to play a role in the etiology and cardiovascular disease progression, particularly with respect to thrombotic aspects of atherosclerosis.

Laser thermal recanalization has been used clinically as an adjunct to balloon angioplasty in the treatment of peripheral arteriosclerotic disease, with improved initial success rates in total peripheral occlusions and greater 1 yr vessel patency suggested, as compared to balloon angioplasty alone. However, the morphological effects of laser-assisted balloon angioplasty are unknown. Therefore the goals of the present study were to evaluate 1) the angiographic and nistologic effects of laser thermal recanalization followed by balloon angioplasty and 2) the hypothesis that balloon-catheter-induced neointimal fracture could be sealed by subsequent laser thermal angioplasty in an experimental rabbit iliac artery atherosclerotic model. In Group 1(7 vessels), a 1.5 mm metal capped argon laser fiberoptic was introduced via femoral arteriotomy and 10 W of thermal power was applied to the iliac artery stenosis for 5 sec while maintaining constant back-and-forth motion. Thereafter, balloon angioplasty was performed in the same vessel segment with a 2.5 mm balloon catheter inflated 3 times at 5 atm for 30 sec each. Mean angiographic luminal diameter increased from 1.1 mm to 2.0 mm after both procedures, and mean final post balloon dissection grade was 0.6 on a scale of 0,1 +, and 2 +. Perforation occurred once with the laser probe and once with the balloon catheter. Histologic examination of these vessels was characterized by irregular thermal erosions with minimal reactive thrombosis. In Group 2 (10 vessels), the sequence was reversed, with laser thermal angioplasty following balloon dilation. Mean angiographic luminal diameter improved from 1.2 mm to 1.8 mm after both procedures. The mean post laser thermal angioplasty dissection grade was 1.3, which was twice that of Group 1. Perforation occurred once with the balloon catheter and twice with the laser probe. In only 1 of the 10 vessels was there histologic evidence suggestive of neointimal sealing. In conclusion, laser thermal recanalization prior to balloon angioplasty appears to decrease the degree of angiographic dissection. Histologically, the thermal effect may be eccentric, and thrombosis appears to be limited. However, laser thermal sealing was not found to be feasible with the current 1.5 mm metal-capped fiberoptic after dilation with a 2.5 mm balloon catheter. This is possibly due to inadequate contact of the probe with the vessel wall. There may also be anincreased tendency towards laser probe perforation following the fracture and dissection which results from balloon dilation.

Gap junctions between vessel wall cells provide a pathway for the intercellular exchange of ions and small molecules. Pure cultures of microvascular and macrovascular endothelial and smooth muscle cells, vascular pericytes, and several nonvascular cell lines were tested for junctional communication by fluorescent dye transfer. All of the vascular wall cells were capable of dye transfer. Since gap junctions are formed by a family of related proteins (connexins) whose unique domains may confer physiological regulatory properties, we tested total RNA from these cultures by Northern blot analysis for expression of the currently available, characterized, and cloned mammalian gap junction proteins: connexin26, connexin32, and connexin43. All of the vascular wall cells expressed connexin43 messenger RNA. Connexin43 was expressed in vascular cells from bovine, porcine, rat, and human sources. Several nonvascular cell lines of mesenchymal origin also expressed connexin43 messenger RNA. When high stringency Northern blots were used, messenger RNAs for connexin32 or connexin26 were not detected in any of the vascular wall cells but were expressed in several cell lines of epithelial origin. Freshly isolated and purified aortic endothelial and smooth muscle RNA preparations similarly contained only connexin43 messenger RNA, excluding the possibility of culture-induced alterations in gene expression. The expression of connexin43 by all vascular wall cells may provide a mechanism for the functional integration of the vessel wall by gap junctions.

Choosing a genomic assay for cancer research is complicated by trade-offs. Cancer gene panels are a common choice, but they target mutational hotspots in a relatively small number of genes, often for cancers that are most commonly tested and that have common genetic etiologies. A common alternative is exome sequencing, which includes all the coding genes but, due to its larger genomic footprint, cannot reach the same depths as panels and therefore is less able to deal with low tumor purity and heterogeneity. Whole genome sequencing trades off very shallow depth and coverage over vast regions of uninterpretable genomic sequence in exchange for the identification of intergenic variants and structural variant breakpoints. All of these assays can be supplemented with RNA sequencing in order to capture gene fusions, allelic expression, splice isoforms, and gene expression. RNAseq comes with its own costs: the need to extract RNA from the same tissue, the need to perform a second assay, and the need to analyze a very different type of data from DNA sequencing. The trade-offs generally come down to three major issues: depth of sequencing, specific genes targeted, and cost. To solve these, we designed an extended, optimized cancer gene panel facilitating high depth sequencing at low cost. We started by identifying a comprehensive list of over 1,300 cancer genes. These genes were chosen through exhaustive cancer gene database and literature curation, and include genes from all major cancer pathways and from the Cancer Gene Census. We then took this gene list and applied an augmented targeting design strategy that we have previously used to create an augmented exome enrichment platform which fills in gaps that standard technical exomes miss. To validate the panel and analysis, we identified test samples including well-described cancer cell lines, cell line mixtures with engineered cancer variations, and formalin-fixed neoplastic tissues. We then performed a series of tests with these samples to measure the panel's small variant sensitivity and specificity, gauge its limits of detection, validate the detection of gene fusions, and demonstrate its ability to identify copy number alterations and loss of heterozygosity. In engineered cell lines, we detected 100% of small variants down to 5% allele frequency. We also mixed the cancer cell lines in various ratios and found similarly high sensitivity as well as very high specificity for small variant detection. We further compared our structural variation calls in the DNA and our fusion calls in the RNA with known data and found that we had very high concordance with known variations. These studies demonstrate that an extended, augmented cancer gene panel strategy solves many genomic assay trade-offs and leads to high accuracy and variant yield for cancer research applications. Citation Format: Michael J. Clark, Sean M. Boyle, Elena Helman, Shujun Luo, Gabor Bartha, Massimo Morra, Anil Patwardhan, Christian Haudenschild, Mirian Karbelashvili, Parin Sripakdeevong, Jason Harris, Deanna Church, Stephen Chervitz, John West, Richard Chen. Solving genomic assay trade-offs with an optimized, extended cancer gene panel for research and clinical applications. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4744. doi:10.1158/1538-7445.AM2015-4744

11029 Background: It has been well documented that enumeration of Circulating Tumor Cells (CTCs) isolated from the peripheral blood of breast cancer patients can be used as a prognostic indicator o...

Background Chimeric proteins may be used to direct cell-specific activity. Heparin-binding growth-associated molecule (HBGAM) binds to cell receptors that are relatively more robust on endothelial cells, and it may confer endothelial cell selectivity to potent angiogens such as fibroblast growth factor-1 (FGF-1). Methods By ligating fibroblast growth factor or its potent mutant, S130K, to HBGAM, we tested their effect on re-endothelialization after angioplasty injury by using a canine model. Results Both HBGAM/S130K- and HBGAM/FGF-1–treated arteries had increased neointimal mitotic index and re-endothelialization rates at 30 days compared with control arteries without inducing a significant increase in the neointimal thickness or the ratio of neointimal to medial thickness between treatment and control groups. Conclusion HBGAM/S130K and HBGAM/FGF-1 facilitates endothelial healing without myointimal thickening after canine carotid artery balloon angioplasty injury. Application of these growth factors in fibrin glue may improve endothelialization clinically after angioplasty or endarterectomy.

Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH2-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1–deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH2-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation.

Atherosclerotic vascular disease Atherosclerosis: a chronic inflammatory condition Atherosclerosis, a chronic systemic disease of the vasculature with an inflammatory component, remains the major cause of disease and death in industrialized countries [1–3]. Early atheromatous vascular changes are already present during fetal life and maternal plasma cholesterol levels determine the severity of fatty streak formation in the aorta of the fetus [4,5]. Intimal accumulation of low-density lipoproteins (LDLs) precedes monocyte recruitment [4], thus a direct inflammatory effect of native, non-oxidized LDLs is likely to be involved in vascular lesion formation [6]. Fatty streaks persist in adolescents [1] and form the basis for a chronic process developing over decades with progressive atheroma formation. Loss of organ function due to infarction is a frequent consequence of advanced disease, which in most cases is due to rupture of the fibrous cap covering the atheromatous plaque [1,3]. The inflammatory mechanisms underlying plaque development and rupture are regulated by a number of conditions as indicated by the multiple risk factors involved in atherogenesis [1]. As discussed in the next paragraph, inflammatory changes in the endothelium play a crucial role for the onset and continuation of the disease.