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Nuclear membrane blebbing in amoeboid cells. Time-lapse analysis of DU145 DIAPH3-depleted cells treated with EGF and IL6 (exacerbates amoeboid features (13)) and stained with lipid fluorescent dye CellMask Orange.

Abnormalities in nuclear shape are a well-known feature of cancer, but their contribution to malignant progression remains poorly understood. Here, we show that depletion of the cytoskeletal regulator, Diaphanous-related formin 3 (DIAPH3), or the nuclear membrane–associated proteins, lamin A/C, in prostate and breast cancer cells, induces nuclear shape instability, with a corresponding gain in malignant properties, including secretion of extracellular vesicles that contain genomic material. This transformation is characterized by a reduction and/or mislocalization of the inner nuclear membrane protein, emerin. Consistent with this, depletion of emerin evokes nuclear shape instability and promotes metastasis. By visualizing emerin localization, evidence for nuclear shape instability was observed in cultured tumor cells, in experimental models of prostate cancer, in human prostate cancer tissues, and in circulating tumor cells from patients with metastatic disease. Quantitation of emerin mislocalization discriminated cancer from benign tissue and correlated with disease progression in a prostate cancer cohort. Taken together, these results identify emerin as a mediator of nuclear shape stability in cancer and show that destabilization of emerin can promote metastasis.Significance: This study identifies a novel mechanism integrating the control of nuclear structure with the metastatic phenotype, and our inclusion of two types of human specimens (cancer tissues and circulating tumor cells) demonstrates direct relevance to human cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6086/F1.large.jpg. Cancer Res; 78(21); 6086–97. ©2018 AACR.

Analysis all available cancer types in TCGA data sets. Correlation analysis of METABRIC data between stages or aggressiveness of cancer vs. ECM proteins expression.

Emerin deregulation links nuclear shape instability to metastatic potential

Emerin deregulation links nuclear shape instability to metastatic potential

Patients with COVID-19 demonstrate higher rates of cardiovascular complications, including thromboses and thromboembolism. One may suppose that the action of SARS-CoV-2 transforms stable atherosclerotic plaques into unstable status. Cardiovascular complications in COVID-19 may be caused by progressive viral alteration the blood vessels, including vasa vasorum. A lethal case of ischemic brain disease caused by cerebral atherosclerosis and exacerbated with a stroke during COVID-19 infection is briefly described. The results of autopsy showed perivascular lymphocytic infiltration and signs of vasa vasorum vasculitis with thrombi of adventitial microvasculature. The data discussed in the article are interpreted in context of the concept giving the important role in atherogenesis to vasa vasorum.

We show that CD39 is a critical modulator of TGF-β1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-β1-induced CD39 expression generates a negative feedback loop that attenuates cardiac fibroblast activation. In the absence of CD39 activity, collagen deposition is increased, elastin expression is decreased, and diastolic dysfunction is worsened. Treatment with ecto-apyrase attenuates the TGF-β1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis.

Lung adenocarcinoma (ADC) is a heterogeneous group of tumors associated with different survival rates, even when detected at an early stage. Here, we aim to investigate whether CyTOF identifies cellular and molecular predictors of tumor behavior. We developed and validated a CyTOF panel of 34 antibodies in four ADC cell lines and PBMC. We tested our panel in a set of 10 ADCs, classified into long- (LPS) (n = 4) and short-predicted survival (SPS) (n = 6) based on radiomics features. We identified cellular subpopulations of epithelial cancer cells (ECC) and their microenvironment and validated our results by multiplex immunofluorescence (mIF) applied to a tissue microarray (TMA) of LPS and SPS ADCs. The antibody panel captured the phenotypical differences in ADC cell lines and PBMC. LPS ADCs had a higher proportion of immune cells. ECC clusters (ECCc) were identified and uncovered two ADC groups. ECCc with high HLA-DR expression were correlated with CD4+ and CD8+ T cells, with LPS samples being enriched for those clusters. We confirmed a positive correlation between HLA-DR expression on ECC and T cell number by mIF staining on TMA slides. Spatial analysis demonstrated shorter distances from T cells to the nearest ECC in LPS. Our results demonstrate a distinctive cellular profile of ECC and their microenvironment in ADC. We showed that HLA-DR expression in ECC is correlated with T cell infiltration, and that a set of ADCs with high abundance of HLA-DR+ ECCc and T cells is enriched in LPS samples. This suggests new insights into the role of antigen presenting tumor cells in tumorigenesis.

Surgeons are not trained to decipher the pathogenesis of diseases, which they operate on. They are used to repair, remove, or replace defective tissues and organs. Yet, we often see typical pathomorphological or pathophysiological phenomena, characteristic of a specific disorder that can only be observed during surgery. Such patterns would not be recognized easily by current imaging techniques, and their visibility would require a living organism. In modern terminology, one could call them “surgical biomarkers”. Many disease entities, today, are still not completely deciphered regarding initial links of their pathogenesis, despite decades of experimental and clinical research. In such disorders, characteristically named “idiopathic”, surgical observations may be helpful to clarify disease mechanisms, two of which we offer here for one of these disease entities, namely pulmonary hypertension.

Modern technologies designed for tissue structure visualization like brightfield microscopy, fluorescent microscopy, mass cytometry imaging (MCI) and mass spectrometry imaging (MSI) provide large amounts of quantitative and spatial information about cells and tissue structures like vessels, bronchioles etc. Many published reports have demonstrated that the structural features of cells and extracellular matrix (ECM) and their interactions strongly predict disease development and progression. Computational image analysis methods in combination with spatial analysis and machine learning can reveal novel structural patterns in normal and diseased tissue. Here, we have developed a Python package designed for integrated analysis of cells and ECM in a spatially dependent manner. The package performs segmentation, labeling and feature analysis of ECM fibers, combines this information with pre-generated single-cell based datasets and realizes cell-cell and cell-fiber spatial analysis. To demonstrate performance and compatibility of our computational tool, we integrated it with a pipeline designed for cell segmentation, classification, and feature analysis in the KNIME analytical platform. For validation, we used a set of mouse mammary gland tumors and human lung adenocarcinoma tissue samples stained for multiple cellular markers and collagen as the main ECM protein. The developed package provides sufficient performance and precision to be used as a novel method to investigate cell-ECM relationships in the tissue, as well as detect structural patterns correlated with specific disease outcomes.

Cancer associated fibroblasts (CAF) play a key role in cancer progression and metastasis. Diminished TGFβ response on CAF correlates with poor outcome and recurrence in cancer patients. Mechanisms behind lost TGFβ signaling on CAF are poorly understood, but, utilizing MMTV-PyMT mouse model, we have previously demonstrated that in tumor microenvironment myeloid cells, producing adenosine, contribute to downregulated TGFβ signaling on CAFs. In the current work, we performed serial in vitro studies to investigate the role of adenosine/TGFβ axis in mouse mammary fibroblast functions, i.e., proliferation, protein expression, migration, and contractility. We found that adenosine analog NECA diminished TGFβ-induced CCL5 and MMP9 expression. Additionally, we discovered that NECA completely inhibited effect of TGFβ to upregulate αSMA, key protein of cytoskeletal rearrangements, necessary for migration and contractility of fibroblasts. Our results show that TGFβ increases contractility of mouse mammary fibroblasts and human fibroblast cell lines, and NECA attenuates theses effects. Using pharmacological approach and genetically modified animals, we determined that NECA effects on TGFβ pathway occur via A2A/A2B adenosine receptor—AC—PKA dependent manner. Using isolated CD11b+ cells from tumor tissue of CD73-KO and CD39-KO animals in co-culture experiments with ATP and AMP, we confirmed that myeloid cells can affect functions of mammary fibroblasts through adenosine signaling. Our data suggest a novel mechanism of interaction between adenosine and TGFβ signaling pathways that can impact phenotype of fibroblasts in a tumor microenvironment.

The search for various biomarkers for the diagnosis and treatment effectiveness assessment of the various pathological conditions does not lose relevance. Aim: To compare levels of markers of an extracellular matrix destruction (ECM) in blood with morphological characteristics of activity (MCA) and to consider a possibility of using them for choosing of tuberkuloma (TUB) treatment tactic. Materials and Methods: The blood concentration matrix metalloproteinases (MMPs) of 87 patient with verified diagnosis of TUB were investigated: MMP-1,-3,-8,-9, tissue inhibitor (TIMP-1), alpha 2-macroglobulin (MG), neutrophil elastase (NE) and proteinase inhibitor (PI) were measured by ELISA. MCA was carried out according to the classification, based on the ratio of the state of caseous masses, capsules and surrounding lung tissue. We used Mann–Whitney U-test, Spearman correlation and the method of projective classification (Alexeeva N.2014). Results: It is established that, TUB was characterized by disturbance of balance of MMP and NE with inhibitors: by increase in the MMP-1,-8,-9, NE levels, by decrease in MG and at the absence of changes levels of MMP-3, TIMP-1 and PI. The levels of markers correspond to MCA: the levels of MMP-1 and MG associated with caseous necrosis in the TUB center. Changes in capsule - with MMP-8 and MG variation. Various combinations of markers of destruction of ECM predicted morphological outcomes with an accuracy of 86% - 92%. Conclusion: Various combinations of ECM destruction markers may be useful as predictors of morphological outcomes. The biochemical data should be added into tactic of TUB treatment.

TGFβ plays a crucial role in the tumor microenvironment by regulating cell–cell and cell–stroma interactions. We previously demonstrated that TGFβ signaling on myeloid cells regulates expression of CD73, a key enzyme for production of adenosine, a protumorigenic metabolite implicated in regulation of tumor cell behaviors, immune response, and angiogenesis. Here, using an MMTV-PyMT mouse mammary tumor model, we discovered that deletion of TGFβ signaling on myeloid cells (PyMT/TGFβRIILysM) affects extracellular matrix (ECM) formation in tumor tissue, specifically increasing collagen and decreasing fibronectin deposition. These changes were associated with mitigated tumor growth and reduced metastases. Reduced TGFβ signaling on fibroblasts was associated with their proximity to CD73+ myeloid cells in tumor tissue. Consistent with these findings, adenosine significantly downregulated TGFβ signaling on fibroblasts, an effect regulated by A2A and A2B adenosine receptors. METABRIC dataset analysis revealed that patients with triple-negative breast cancer and basal type harbored a similar signature of adenosine and ECM profiles; high expression of A2B adenosine receptors correlated with decreased expression of Col1 and was associated with poor outcome. Taken together, our studies reveal a new role for TGFβ signaling on myeloid cells in tumorigenesis. This discovered cross-talk between TGFβ/CD73 on myeloid cells and TGFβ signaling on fibroblasts can contribute to ECM remodeling and protumorigenic actions of cancer-associated fibroblasts. Significance: TGFβ signaling on fibroblasts is decreased in breast cancer, correlates with poor prognosis, and appears to be driven by adenosine that accelerates tumor progression and metastasis via ECM remodeling.

Tumor microenvironment (TME) represents an integrated system that affects cancer cell behavior and contributes directly to disease outcome. Systemic approach to analysis of TME should uncover its complexity and facilitate discovery of mechanisms orchestrating tumor development and metastasis. Multiplex fluorescence tissue staining followed by spatial analysis of tumor tissue architecture can provide insights to pivotal interactions of cellular and acellular components of TME. Extracellular matrix (ECM is represented mainly by collagen deposition. Number of reports indicates that ECM contribution to TME state not only depends upon amount of accumulated collagen but its geometrical features and spatial orientation of fibers. These characteristics of collagen fibers contribute directly to physical and mechanical properties of tissue and can change tumor growth and metastasis. Current methods of computational image analysis of tissue implement assessment of cellular or acellular components separately. The goal of current work was to develop a new computational tool to perform integrated analysis of fibrous and cellular components of tumor tissue in spatial dependent manner to achieve detailed tumor tissue mapping and structural patterns recognition. To pursue this goal, we generated images of human lung adenocarcinoma tissue characterized by indolent and aggressive behavior. We performed multiplex immunofluorescence staining for following markers: CD3 - marker of T-lymphocytes, PanCytokeratin - marker of epithelial/tumor cells, collagen hybridizing peptide (3Helix) - marker of collagen, DAPI - nuclear counterstain. To develop image analysis pipeline, we utilized an open source graphical interface analytical platform KNIME, where we generated modular workflow. For ECM analysis, we integrated Python written code into KNIME node. Segmentation of collagen fibers was performed using skeletonization with subsequent calculation of geometrical properties (length, alignment, widths) and orientation of each fiber. Data, collected from single cell analysis and ECM architecture assessment, were combined and forwarded to downstream spatial analysis, where distances from cell to cell or cell to ECM were computed and neighborhood analysis was performed. We demonstrated that tumor cells in aggressive adenocarcinoma samples were co-localized with a smaller number of collagen fibers. In addition, length of that fibers was less in comparison to indolent group. Correlation analysis revealed positive correlation between length of collagen fibers and number of tumor cells in indolent group, but we did not observe this phenomenon in indolent group. Developed computational method provides additional dimensionality to tissue image analysis and can reveal underrecognized structural patterns of the tumor microenvironment. Citation Format: Georgii Vasiukov, Tatiana Novitskaya, Maria-Fernanda Senosain, Anna Menshikh, Andries Zijlstra, Sergey Novitskiy, Pierre Massion. Integrated computational image analysis of cellular and acellular tissue components as a method for detailed tumor tissue mapping and structural patterns recognition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 239.

Following myocardial infarction, purinergic nucleotides and nucleosides are released via non-specific and specific mechanisms in response to cellular activation, stress, or injury. These extracellular nucleotides are potent mediators of physiologic and pathologic responses, contributing to the inflammatory and fibrotic milieu within the injured myocardium. Via autocrine or paracrine signaling, cell-specific effects occur through differentially expressed purinergic receptors of the P2X, P2Y, and P1 families. Nucleotide activation of the ionotropic (ligand-gated) purine receptors (P2X) and several of the metabotropic (G-protein-coupled) purine receptors (P2Y) or adenosine activation of the P1 receptors can have profound effects on inflammatory cell function, fibroblast function, and cardiomyocyte function. Extracellular nucleotidases that hydrolyze released nucleotides regulate the magnitude and duration of purinergic signaling. While there are numerous studies on the role of the purinergic signaling pathway in cardiovascular disease, the extent to which the purinergic signaling pathway modulates cardiac fibrosis is incompletely understood. Here we provide an overview of the current understanding of how the purinergic signaling pathway modulates cardiac fibroblast function and myocardial fibrosis.

Lung adenocarcinoma (ADC) is a heterogeneous group of tumors associated with dramatically different survival rates, even when detected at an early stage. We hypothesized that a single cell proteomic approach would allow the dissection of cellular determinants of early lung ADC behavior. We developed a mass cytometry panel of 34 antibodies and validated their performance in four ADC cell lines (A459, H23, PC9 and H3211) and immune cells. We tested our panel in a set of 10 early stage lung ADCs, classified into long- (LPS) (n=4) and short-predicted survival (SPS) (n=6) based on radiomics features. Tumors were disaggregated and cryopreserved until mass cytometry analysis. We identified cellular subpopulations by clustering and analyzed differences in their distribution both within the tumor microenvironment and the epithelial compartment. To validate our results a tissue micro array was generated from lung tissue blocks from patients with LPS and SPS lung adenocarcinoma. Fluorescent staining was performed for PanCK, CD45, CD3, HLA-DR, DAPI. Cell nuclei were segmented using deep learning algorithm and were further processed in KNIME analytical platform where cell segmentation, feature extraction and cell classification were performed. The antibody panel captured the phenotypical differences in ADCs cell lines and PBMCs. When tumors were analyzed long-predicted survival tumors had a higher proportion of immune cells, whereas some short-predicted survival tumors had a higher proportion of fibroblasts/mesenchymal cells. Additionally, tumors show high degree of heterogeneity with distinct protein expression profiles among epithelial subpopulations, and some subsets with high HLA-DR expression were positively correlated with CD4+ and CD8+ T cells with LPS samples being enriched for such subsets. These results were further validated by Fluorescent staining on TMA slides. We found a positive correlation between HLA-DR expression on tumor cells and T cell number (r = 0.25, p = 2.2e-05). For this, in neighborhoods of 100 um diameter for each tumor cell, HLA-DR median signal intensity on neighboring tumor cells and number of T cells were calculated in Python and used as inputs for correlative analysis. Spatial analysis was performed in KNIME by calculation of distances from each T cell to nearest 1st and 2nd tumor cell, for which LPS tumors showed smaller distances for both 1st and 2nd tumor cell compared to SPS tumors (p = 0.039, p = 0.21). Our results demonstrate a distinct cellular profile of epithelial and stromal cells among indolent vs aggressive ADCs with higher HLA-DR expression in indolent tumors, which is associated with greater T cell infiltration. Our results illustrate the heterogeneity of T cell responses and HLA DR expression in lung adenocarcinoma and should further our understanding of mechanisms related to tumorigenesis. This work deserves further validation at the cellular and molecular level to gain further insights into tumor behavior. The work was supported by CA196405 to PPM. Citation Format: Maria-Fernanda Senosain, Tatiana Novitskaya, Georgii Vasiukov, Yong Zou, Aneri Balar, Deon B. Doxie, Jonathan M. Lehman, Rosana Eisenberg, Fabien Maldonado, Andries Zijlstra, Sergey V. Novitskiy, Jonathan M. Irish, Pierre P. Massion. Single cell proteomic analysis of lung adenocarcinoma identifies high HLA-DR expression to be associated with indolent tumor behavior [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-045.

TGF-beta plays a crucial role in tumor microenvironment by regulating cell-cell and cell-stroma interactions. We previously demonstrated that TGF-beta signaling on myeloid cells regulates expression of CD73, a key enzyme for production of adenosine, a pro-tumorigenic metabolite implicated in regulation of tumor cell behaviors, immune response and angiogenesis. Using MMTV-PyMT mouse mammary tumor model, we discovered that deletion of TGF-beta signaling on myeloid cells (PyMT/TGFbRII-LysM) affects ECM formation in tumor tissue, specifically increases collagen and decreases fibronectin deposition, and these changes associate with mitigated tumor growth and reduced metastases. Using multiplexed five-color immunofluorescent staining and spatial analysis on a single-cell level, we discovered that reduced TGF-beta signaling on fibroblasts associates with their proximity to CD73 positive myeloid cells in tumor tissue. Consistent with these findings, in vitro gel contraction assay and Western blotting for Collagen I and pSMAD proteins confirmed that adenosine significantly downregulates TGF-beta signaling on fibroblasts. Using in vitro pharmacological approach, we found that this effect is regulated by A2a and A2b adenosine receptors. TCGA data base analysis revealed that patients with triple negative breast cancer and basal type have similar signature of adenosine and ECM profiles: high expression of A2b adenosine receptors correlates with decreased expression of Col1 and is associates with poor outcome. Taken together, our studies reveal a new role for TGF-beta signaling on myeloid cells in tumorigenesis. Discovered crosstalk between TGF-beta/CD73 on myeloid cells and TGF-beta signaling on fibroblasts can contribute to ECM remodeling and pro-tumorigenic actions of CAFs. Citation Format: Georgii Vasiukov, Tatiana Novitskaya, Samantha Schwager, Harold Moses, Timothy Blackwell, Igor Feoktistov, Sergey Novitskiy. TGF-beta signaling on myeloid cells regulates ECM deposition in mammary carcinoma via adenosine dependent mechanisms [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 1714A.

Tumor microenvironment (TME) represents a dynamic niche that regulates cancer cell behavior contributing directly to disease outcome. Systemic approach to analysis of TME should uncover its complexity and facilitate discovery of mechanisms orchestrating tumor development and metastasis. Multiplexed fluorescent tissue stain followed by spatial analysis of tumor tissue architecture can provide insights to pivotal interactions of cellular and acellular components of TME. Extracellular matrix (ECM), one of constitutive of TME, is represented mainly by collagen deposition. Recently it is became increasingly recognized that ECM contribution to TME dynamics not only depend upon amount of accumulated collagen but its geometrical features and spatial orientation of fibers. These characteristics of collagen contribute directly to physical and mechanical properties of tissue and can contribute to tumor growth and metastasis. Current algorithms of tissue stain-based methods include estimation of ECM deposition by measuring percent of positive area and separate to it cell classification and cell count. The goal of current work was to develop a new computational tool to perform spatial distribution analysis based on geometrical features and orientation of collagen fibers in combination with cell class assessment aimed to achieve detailed tumor tissue mapping. To pursue this goal, we generated fluorescent images of human breast tumor tissue, stained for following markers: CD3 - marker of T-lymphocytes, PanCytokeratin - marker of epithelial/tumor cells, collagen hybridizing peptide (3Helix) - marker of collagen, DAPI - nuclear counterstain. To develop image analysis pipeline, we utilized open source graphical interface analytical platform KNIME, where we generated modular workflow. For ECM analysis, we integrated Python written code into KNIME node. Segmentation of collagen fibers was performed using skeletonization with subsequent calculation of geometrical properties (length, alignment, widths) and orientation of each fiber. Data, collected from single cell analysis and ECM architecture assessment, were combined and forwarded to downstream spatial analysis, where distances from cell to cell or cell to ECM were computed and neighborhood analysis was performed. We demonstrate different patterns in tumor microenvironment organization that correlate with cancer outcome. Developed image analysis algorithm provides additional dimensionality to fluorescent tissue stains and can reveal underrecognized patterns of tumor microenvironment that can contribute to better understanding of tumorigenesis and metastasis. Citation Format: GEORGII VASIUKOV, Tatiana Novitskaya, Sergey Gutor, Anna Menshikh, Andries Zijlstra, Sergey Novitskiy. Extracellular matrix segmentation combined with cell classification as a novel method for detailed tumor tissue mapping [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 4425.

Purpose: Machine-learning assisted histopathology using markers of basal and luminal differentiation was employed to profile the intratumoral heterogeneity of bladder cancer from cystectomy patients and predict disease-free survival in this high-risk patient population. Methods: Urothelial carcinomas are biologically heterogeneous and vary greatly in clinical progression as well as treatment response. Delineation of molecular subtypes by gene expression analysis of luminal and basal markers has indicated differential outcomes associated with basal and luminal subtypes. However, histologic validation of this classification using protein markers (basal = KRT5/6, P63; luminal = KRT20/GATA3) has been challenging. While using multiplex-immunofluorescence to subtype a retrospective cystectomy cohort (a TMA of 380 patients), we determined that nearly 50% of tumors did not exhibit cytokeratin markers. Subtyping was further confounded by frequent loss of basal-to-luminal stratification and the emergence of intratumoral spatial heterogeneity with the basal and luminal subtypes being completely intermixed throughout the tumor. These observations caused us to hypothesize that previously undefined but clinically relevant subtypes might exist. To address this challenge we developed a single-cell image analysis pipeline that leveraged machine learning to classify molecular subtype and spatial heterogeneity within each tumor. Using the informatics software KNIME we achieved single-cell segmentation and extracted 285 features for 5 protein markers (P63, GATA3, collagen, nuclear stain, and pan-cytokeratin) from each ~20,000 cells contained in 2 cores of tumor and adjacent benign for each patient. Under guidance from a pathologist, definitive urothelial cells (luminal, intermediate, and basal cells) as well as stromal cells were selected from 25 cores normal urothelium to form the ground truth for XGboost-based machine-learning. Summary Findings: Single-cell profiling with machine learning on transcription factors could classify basal and luminal subtypes with greater than 97% accuracy according to validation in normal urothelium using keratin markers. While we were able to recapitulate differential survival associated with a pure basal subtype, it was the intratumoral heterogeneity of basal and luminal cells that was the predominant driver of disease-free survival. Conclusion: A newly identified bladder cancer subtype defined by intratumoral heterogeneity is a clinically relevant driver of disease-free survival. This abstract is also being presented as Poster B22. Citation Format: Adel Eskaros, Tatiana Novitskaya, Andries Zijlstra. Profiling intratumoral heterogeneity of bladder cancer subtypes at the single-cell level using machine-learning assisted histopathology [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr PR02.

For decades, histopathology with routine hematoxylin and eosin staining has been and remains the gold standard for reaching a morphologic diagnosis in tissue samples from humans and veterinary species. However, within the past decade, there has been exponential growth in advanced techniques for in situ tissue biomarker imaging that bridge the divide between anatomic and molecular pathology. It is now possible to simultaneously observe localization and expression magnitude of multiple protein, nucleic acid, and molecular targets in tissue sections and apply machine learning to synthesize vast, image-derived datasets. As these technologies become more sophisticated and widely available, a team-science approach involving subspecialists with medical, engineering, and physics backgrounds is critical to upholding quality and validity in studies generating these data. The purpose of this manuscript is to detail the scientific premise, tools and training, quality control, and data collection and analysis considerations needed for the most prominent advanced imaging technologies currently applied in tissue sections: immunofluorescence, in situ hybridization, laser capture microdissection, matrix-assisted laser desorption ionization imaging mass spectrometry, and spectroscopic/optical methods. We conclude with a brief overview of future directions for ex vivo and in vivo imaging techniques.

Abnormalities in nuclear shape are a well-known feature of cancer, but their contribution to malignant progression remains poorly understood. Here, we show that depletion of the cytoskeletal regulator, Diaphanous-related formin 3 (DIAPH3), or the nuclear membrane–associated proteins, lamin A/C, in prostate and breast cancer cells, induces nuclear shape instability, with a corresponding gain in malignant properties, including secretion of extracellular vesicles that contain genomic material. This transformation is characterized by a reduction and/or mislocalization of the inner nuclear membrane protein, emerin. Consistent with this, depletion of emerin evokes nuclear shape instability and promotes metastasis. By visualizing emerin localization, evidence for nuclear shape instability was observed in cultured tumor cells, in experimental models of prostate cancer, in human prostate cancer tissues, and in circulating tumor cells from patients with metastatic disease. Quantitation of emerin mislocalization discriminated cancer from benign tissue and correlated with disease progression in a prostate cancer cohort. Taken together, these results identify emerin as a mediator of nuclear shape stability in cancer and show that destabilization of emerin can promote metastasis. Significance: This study identifies a novel mechanism integrating the control of nuclear structure with the metastatic phenotype, and our inclusion of two types of human specimens (cancer tissues and circulating tumor cells) demonstrates direct relevance to human cancer. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6086/F1.large.jpg. Cancer Res; 78(21); 6086–97. ©2018 AACR.

Background— Despite increased secondary cardiovascular events in patients with ischemic cardiomyopathy (ICM), the expression of innate cardiac protective molecules in the hearts of patients with ICM is incompletely characterized. Therefore, we used a nonbiased RNAseq approach to determine whether differences in cardiac protective molecules occur with ICM. Methods and Results— RNAseq analysis of human control and ICM left ventricular samples demonstrated a significant decrease in KCNJ11 expression with ICM. KCNJ11 encodes the Kir6.2 subunit of the cardioprotective K ATP channel. Using wild-type mice and kcnj11 -deficient ( kcnj11 -null) mice, we examined the effect of kcnj11 expression on cardiac function during ischemia-reperfusion injury. Reactive oxygen species generation increased in kcnj11 -null hearts above that found in wild-type mice hearts after ischemia-reperfusion injury. Continuous left ventricular pressure measurement during ischemia and reperfusion demonstrated a more compromised diastolic function in kcnj11 -null compared with wild-type mice during reperfusion. Analysis of key calcium-regulating proteins revealed significant differences in kcnj11 -null mice. Despite impaired relaxation, kcnj11 -null hearts increased phospholamban Ser16 phosphorylation, a modification that results in the dissociation of phospholamban from sarcoendoplasmic reticulum Ca 2+ , thereby increasing sarcoendoplasmic reticulum Ca 2+ –mediated calcium reuptake. However, kcnj11 -null mice also had increased 3-nitrotyrosine modification of the sarcoendoplasmic reticulum Ca 2+ -ATPase, a modification that irreversibly impairs sarcoendoplasmic reticulum Ca 2+ function, thereby contributing to diastolic dysfunction. Conclusions— KCNJ11 expression is decreased in human ICM. Lack of kcnj11 expression increases peroxynitrite-mediated modification of the key calcium-handling protein sarcoendoplasmic reticulum Ca 2+ -ATPase after myocardial ischemia-reperfusion injury, contributing to impaired diastolic function. These data suggest a mechanism for ischemia-induced diastolic dysfunction in patients with ICM.

Phenylthiobutanoic acids (PTBAs) are a new class of histone deacetylase (HDAC) inhibitors that accelerate recovery and reduce postinjury fibrosis after ischemia-reperfusion-induced acute kidney injury. However, unlike the more common scenario in which patients present with protracted and less clearly defined onset of renal injury, this model of acute kidney injury gives rise to a clearly defined injury that begins to resolve over a short period of time. In these studies, we show for the first time that treatment with the PTBA analog methyl-4-(phenylthio)butanoate (M4PTB) accelerates recovery and reduces postinjury fibrosis in a progressive model of acute kidney injury and renal fibrosis that occurs after aristolochic acid injection in mice. These effects are apparent when M4PTB treatment is delayed 4 days after the initiating injury and are associated with increased proliferation and decreased G2/M arrest of regenerating renal tubular epithelial cells. In addition, there is reduced peritubular macrophage infiltration and decreased expression of the macrophage chemokines CX3Cl1 and CCL2. Since macrophage infiltration plays a role in promoting kidney injury, and since renal tubular epithelial cells show defective repair and a marked increase in maladaptive G2/M arrest after aristolochic acid injury, these findings suggest M4PTB may be particularly beneficial in reducing injury and enhancing intrinsic cellular repair even when administered days after aristolochic acid ingestion.

Introduction: The manifestation of cancer malignancy occurs at the cellular level where individual cells escape tissue confinements and disseminate. Identifying this malignant behavior at a cellular level is necessary in order to deconvolve the tumor heterogeneity, identify disease progression and predict cancer-related morbidity and mortality. To achieve that goal, we established a unique computer-assisted single-cell histopathology analysis of prostate tissue that evaluates nuclear-membrane instability and the associated extracellular vesicle (EV) biogenesis to identify malignant potential and assess future disease progression. In recent years cancer EVs have been identified as important mediators of intercellular communication. The potential for using EVs as a liquid biopsy has promoted research on profiling EVs in biofluids. However, the direct analysis of EV biogenesis in tumor tissue has been largely omitted, even thought identifying such cells is likely to be a specific and sensitive measure of disease malignancy. We have previously demonstrated that highly migratory and metastatic cancer cells shed atypically large EVs, known as large oncosomes (LO, Di Vizio et. al., 2012). LO play distinct functions and contain a specific repertoire of molecules that can be used for detection of tumor-derived cargo in plasma (Minciacchi et. al., 2015). The recent discovery that the biogenesis of large oncosomes (LO) in prostate cancer is associated with nuclear membrane instability (Reis-Sobreiro et. al., 2018) offered an opportunity to investigate such biogenesis in patient specimens. We have developed a machine-learning assisted histopathology (HistoMAP) that quantitatively identifies nuclear-membrane instability in prostate cancer tissue and demonstrate a direct correlation between this molecular biogenesis of vesicles and biochemical recurrence after prostatectomy. Experimental procedures: We developed a multiplex immunofluorescent technique to detect and quantify nuclear-derived LO in formalin fixed paraffin embedded prostatectomy tissues. Using computer-assisted image segmentation and machine-learning we distinguished these LO from all other intracellular compartments and assess malignancy in a cohort of Vanderbilt prostate cancer patients. Results: LO production was significantly elevated in prostate cancer in comparison to benign tissue and particularly evident in lymph node metastases. Moreover, LO production was associated with the risk of future metastatic disease which re-enforced its relevance in cancer progression. Conclusions: Our findings demonstrate that, given knowledge of the EV machinery, EV biogenesis can be detected in patient tissues and provide critical information related to patient disease status and assist with the prediction of future clinical performance. Citation Format: Andries Zijlstra, Tatiana Novitskaya, Dolores Di Vizio, Mariana Reis- Sobreiro, Michael Freeman. Machine-learning assisted histopathology (HistoMAP) links nuclear membrane instability to disease progression in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4448.

At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio)butanoate, which we subsequently administered to zebrafish larvae and mice 24-48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.

Aims Prior work suggests that ischemic preconditioning increases the level of CD39 in the heart and contributes to cardiac protection. Therefore, we examined if targeted cardiac expression of CD39 protects against myocardial injury. Main methods Mice with cardiac-specific expression of human CD39 (αMHC/hCD39-Tg) were generated, characterized and subjected to left coronary artery ischemia-reperfusion injury and infarct size at 24 h following injury quantified. Key findings αMHC/hCD39-Tg mice have increased in cardiac ATPase and ADPase activity compared to WT littermates. The increased activity in αMHC/hCD39-mice was inhibited by the CD39 antagonist sodium polyoxotungstate (POM-1). Measurement of basal cardiac function by echocardiography revealed that αMHC/hCD39-Tg mice have a lower resting heart rate and increased stroke volume. In response to myocardial ischemia, systolic and diastolic function was better preserved in αMHC/hCD39-Tg compared to WT mice. Comparison of Tau also revealed preserved cardiac relaxation during ischemia in αMHC/hCD39-Tg hearts. Assessment of myocardial infarct size in response to 60 min of ischemia and 24 h of reperfusion demonstrated a significant reduction in infarct size in αMHC/hCD39-Tg hearts. Analysis of isolated cardiomyocytes revealed no basal difference in calcium transients between WT and αMHC/hCD39-Tg cardiomyocytes. However, in response to isoproterenol stimulation, there was a trend toward lower calcium transients in αMHC/hCD39 cardiomyocytes suggesting less calcium accumulation in response to metabolic stress. Significance Cardiac-specific expression of CD39 reduces myocardial dysfunction and infarct size following ischemia-reperfusion injury. Increasing nucleotidase expression in the heart may be a novel approach to protect the heart from ischemic injury.

Objective— Circulating blood cells and endothelial cells express ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73). CD39 hydrolyzes extracellular ATP or ADP to AMP. CD73 hydrolyzes AMP to adenosine. The goal of this study was to examine the interplay between CD39 and CD73 cascade in arterial thrombosis. Approach and Results— To determine how CD73 activity influences in vivo thrombosis, the time to ferric chloride–induced arterial thrombosis was measured in CD73-null mice. In response to 5% FeCl 3 , but not to 10% FeCl 3 , there was a significant decrease in the time to thrombosis in CD73-null mice compared with wild-type mice. In mice overexpressing CD39, ablation of CD73 did not inhibit the prolongation in the time to thrombosis conveyed by CD39 overexpression. However, the CD73 inhibitor α-β-methylene-ADP nullified the prolongation in the time to thrombosis in human CD39 transgenic (hC39-Tg)/CD73-null mice. To determine whether hematopoietic-derived cells or endothelial cell CD39 activity regulates in vivo arterial thrombus, bone marrow transplant studies were conducted. FeCl 3 -induced arterial thrombosis in chimeric mice revealed a significant prolongation in the time to thrombosis in hCD39-Tg reconstituted wild-type mice, but not on wild-type reconstituted hCD39-Tg mice. Monocyte depletion with clodronate-loaded liposomes normalized the time to thrombosis in hCD39-Tg mice compared with hCD39-Tg mice treated with control liposomes, demonstrating that increased CD39 expression on monocytes protects against thrombosis. Conclusions— These data demonstrate that ablation of CD73 minimally effects in vivo thrombosis, but increased CD39 expression on hematopoietic-derived cells, especially monocytes, attenuates in vivo arterial thrombosis.

Objective: To elucidate the molecular mechanisms leading to accelerated atherosclerosis after a myocardial infarction (MI). Approach and Results: Human studies suggest that atherosclerosis is accelerated following MI. Although, this has been recapitulated in apolipoprotein E deficient (apoE -/- ) mice, the mechanisms by which MI-accelerated atherosclerosis have not been elucidated. Here, we demonstrate that, similar to apoE -/- mice, MI accelerates atherosclerosis in high-fat diet-fed LDLr -/- mice. We fed high-fat diet (21% saturated fat, 0.15% cholesterol) to LDLr -/- mice for 1 week prior to coronary artery ligation (CAL) or sham surgery. After surgery, LDLr -/- mice were maintained on high-fat diet and sacrificed after 3 weeks. Total serum cholesterol (Sham: 1349.0 ± 50.09 and CAL: 1355 ± 20.09, p = 0.19) and triglycerides (Sham: 394.6 ± 32.4 and MI 421.1 ± 6.27, p =0.48) were comparable between sham and CAL mice. Examination of aortic sinus by oil red O-stained cross-sections revealed an increase in atherosclerotic lesion area in LDLr -/- that received an MI (CAL: 78,465 ± 15,239 microm 2 /section and sham 42,697 ± 4757 microm 2 /section, p= 0.03). Additionally, we found a statistically significant increase in the number (sham: 17,943 ± 7762, n=8 and CAL: 58,129 ± 13,444, n=6, p = 0.017) and percentages (sham: 2.2 ± 0.41, n=8, CAL: 5.32 ± 1.105, n=6, p = 0.01) of Ly6G + neutrophil infiltrate in para-aortic lymph nodes in LDLr -/- mice that received MI versus sham surgery. Analysis of cellular composition in atherosclerotic lesions is ongoing. Conclusion: Atherosclerosis is accelerated following an MI in high-fat fed LDLr -/- mice.

Objective: To determine how leukocyte nucleotidase affects arterial thrombosis. Approach and Results: Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) is expressed on circulating cells, endothelium and smooth muscle cells where it hydrolyzes extracellular ATP or ADP to AMP. We have demonstrated that transgenic mice with a global overexpression of human CD39 (hCD39-Tg) are protected against ferric chloride-induced carotid artery thrombosis. Furthermore, transplant of hCD39-Tg bone marrow into WT recipient mice increases the time to thrombosis when compared to recipient mice (wild-type or hCD39-Tg) receiving wild-type bone marrow. Based upon these data and previously published work, we hypothesized that CD39 expression on leukocytes is responsible for the prolongation of the time to thrombosis measured in hCD39-Tg mice. To test this hypothesis, we first performed ex vivo mixing experiments. Addition of hCD39-Tg monocytes to WT blood inhibits the expression of activated glycoprotein IIb/IIIa on platelets in response to ADP as measured by FACS analysis (Baseline: 1224 ± 94.9 MFI vs hCD39-Tg monocytes: 663.5 ± 61.5 activated glycoprotein IIb/IIIa MFI: n=4; p< 0.001). Subsequently, in vivo we demonstrated that monocytes with increased CD39 contribute to extending the time to thrombosis. Clodronate liposome depletion of monocytes (WT: 69% decrease; hCD39-Tg: 63% decrease) resulted in a normalization of the time to thrombosis in hCD39-Tg mice (8.0 ± 1.07 minutes, n = 10) when compared to control loaded liposomes (120.0 ± 0.0, n = 14). No changes in the time to thrombosis were detected in wild-type mice treated with clodronate (8.6 ± 1.35 minutes, n = 8) or control liposomes (7.8 ± 0.80 minutes, n=8). Conclusion: Increased expression of CD39 on monocytes can inhibit platelet activation and extend the time to thrombosis following ferric chloride-induced carotid artery injury.

Late gestational placental insufficiency resulting in asymmetric intrauterine organ growth restriction (IUGR) is associated with an increased incidence of diabetes, cardiovascular and renal disease in adults. The molecular mechanisms mediating these defects are poorly understood. To explore this, we investigated the mechanisms leading to IUGR in Cited1 knockout mice, a genetic model of late gestational placental insufficiency. We show that loss of placental Cited1 leads to asymmetric IUGR with decreased liver, lung, and kidney sizes and preservation of fetal brain weight. IGF and insulin signaling regulate embryonic organ growth. IGF-I and IGF-II protein and mRNA expression are reduced in livers, lungs, and kidneys of embryonic d 18.5 embryos with IUGR. Decreased IGF-I is associated with reduced activating phosphorylation of the type 1 IGF receptor (pIGF-IR) in the kidney, whereas reduced IGF-II is associated with decreased phosphorylation of the insulin receptor (pIR) in the lung. In contrast, decreased pIR is associated with reduced IGF-I but not IGF-II in the liver. However, pancreatic β-cell mass and serum insulin levels are also decreased in mice with IUGR, suggesting that hepatic IR signaling may be regulated by alterations in fetal insulin production. These findings contrast with observations in IUGR fetal brains in which there is no change in IGF-IR/IR phosphorylation, and IGF-I and IGF-II expression is actually increased. In conclusion, IUGR disrupts normal fetal IGF and insulin production and is associated with organ-specific defects in IGF-IR and IR signaling that may regulate asymmetric IUGR in late gestational placental insufficiency.

The bone morphogenetic protein (BMP) type 2 receptor ligand, Bmp2, is upregulated in the peripheral pulmonary vasculature during hypoxia-induced pulmonary hypertension (PH). This contrasts with the expression of Bmp4, which is expressed in respiratory epithelia throughout the lung. Unlike heterozygous null Bmp4 mice ( Bmp4LacZ/+), which are protected from the development of hypoxic PH, mice that are heterozygous null for Bmp2 ( Bmp2+/−) develop more severe hypoxic PH than their wild-type littermates. This is associated with reduced endothelial nitric oxide synthase (eNOS) expression and activity in the pulmonary vasculature of hypoxic Bmp2+/−but not Bmp4LacZ/+mutant mice. Furthermore, exogenous BMP2 upregulates eNOS expression and activity in intrapulmonary artery and pulmonary endothelial cell preparations, indicating that eNOS is a target of Bmp2 signaling in the pulmonary vasculature. Together, these data demonstrate that Bmp2 and Bmp4 exert opposing roles in hypoxic PH and suggest that the protective effects of Bmp2 are mediated by increasing eNOS expression and activity in the hypoxic pulmonary vasculature.

No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury.

Background: CD39 (ectonucleoside triphosphate diphosphohydrolase) is a nucleotidase expressed on endothelial cells, vascular smooth muscles cells, and leukocytes. CD39 plays a key role in vascular homeostasis, hydrolyzing extracellular ATP and ADP. CD39 has been shown to be important in models of ischemic preconditioning and cardiac ischemia reperfusion. However, the effect of CD39 activity on functional recovery of heart after myocardial infarction (MI) has not been evaluated. Hypothesis: Genetic ablation of CD39 expression exacerbates post-myocardial infarction cardiac function and fibrosis. Methods: Wild-type (WT) and CD39-null mice were subjected to coronary artery ligation. Cardiac function and protein evaluation of fibrotic markers was performed at day 28 post-MI. Results: Evaluation at Day 28 post-MI revealed that while mice of both genotypes had similarly reduced ejection fraction and equally compromised contractile function (dP/dtmax), there was a more pronounced negative effect on lusitropy (dP/dtmin) and increased left ventricular end-diastolic pressure in CD39-null mice. Therefore, cd39 gene ablation associates with the development of worsening cardiac performance. Histological analysis revealed increased collagen deposition and abundance of alpha-smooth muscle actin (αSMA) positive interstitial cells in the CD39-null hearts compared to WT hearts. To quantify these findings immunoblot analysis for collagen and αSMA was performed. We found that collagen and αSMA were increased at Day 28 post-MI, in CD39-null hearts compared to WT hearts. Conclusion: CD39 ablation has detrimental effects on post-MI recovery, resulting in diminished cardiac performance and increased fibrosis.

Introduction: CD39 is an ATP and ADP hydrolyzing ectonucleotidase found on the endothelium, leukocytes and platelets. Prior work has shown that the genotype at the single nucleotide polymorphism rs10748643 correlates with CD39 expression (GG:high, AG:intermediate, AA: low) in lymphocytes. However the influence on CD39 expression in platelets is unresolved. Objective: The aim of this study was to determine whether the rs10748643 genotype associates with low-dose ADP-induced platelet aggregation ex vivo . Methods: Whole blood was obtained from healthy volunteers not on aspirin (baseline) and then treated with aspirin for 14 days (81 mg). Serum thromboxane levels were also measured to demonstrate ASA adherence. Platelet rich plasma was generated and platelet aggregation in response to 2.5 uM ADP was measured. DNA from whole blood was isolated and used for genotyping at rs10748643 by the Vanderbilt DNA Resources Core. Results: The study population included 65 subjects.Genotyping revealed 23 GG, 28 AG and 14 AA at rs10748643 subjects. There was no difference in age (Years: AA: 32.4±11.7, AG: 34.2±9.5, GG: 33.5±9.4; P =0.72) or sex (Male: AA: 57%, AG: 76%, GG: 60%; P =0.34). Stimulation of platelets from untreated subjects with 2.5 uM ADP showed no difference in the percent aggregation (AA: 77.8±7.05%; AG: 75.9±4.85%, GG: 78.7±4.31%; P =0.233). Following aspirin treatment there was a significant decrease in thromboxane levels ( P P P =0.148). Conclusions: These data suggests that the rs10748643 genotype does not associate with differences in platelet aggregation at baseline or following aspirin therapy. However, the small size of the cohort examined is a clear limitation of the study. The trend for lower aggregation in AA individuals on aspirin is intriguing and studies in a larger cohort will determine if there is significance of these findings.

Introduction: CD39 (ectonucleoside triphosphate diphosphohydrolase) is a nucleotidase expressed on endothelial cells, vascular smooth muscles cells, leukocytes, and platelets. CD39 plays a key role in vascular homeostasis, hydrolyzing extracellular ATP or ADP to AMP. Prior work has demonstrated that the genotype at the single nucleotide polymorphism (SNP) rs10748643 correlates with CD39 expression on HapMap lymphocytes (GG: high, GA: intermediate, AA: baseline). Hypothesis: The aim of this study was to determine if an association exists between the rs10748643 genotype and secondary cardiovascular events. Methods: Subjects treated with placement of an intracoronary stent and on clopidogrel therapy for coronary artery disease were identified in the BioVU DNA repository that links DNA to Synthetic Derivative, a de-identified electronic health record. Selection of the study population was based on a combination of ICD 9th edition, Current Procedural Terminology codes, laboratory values, and natural language processing in physician notes. “Case” or “control” status was assigned depending on recurrence of a secondary cardiovascular event (myocardial infarction, revascularization and/or death) within two years. Genotyping for rs10748643 was performed using TaqMan. Results: The study population included 189 cases and 379 controls, with comparable demographics. There were 47 MIs, 14 deaths, and 166 revascularizations. We compared the composite outcomes of MI and/or death or MI, death and/or revascularization. We found no difference in the composite end-point of MI, death and revascularization, however the GG-genotype at rs10748443 was associated with an increase in the composite end-point of MI and/or death (HR GG:AA: 3.82 95% CI 1.10 - 13.29; P=0.039). Conclusions: The GG genotype, was associated with an increased incidence of the composite end-point of myocardial infarction and death in patients who received intracoronary stents for coronary artery disease and were treated with clopidogrel. This is the first study to demonstrate an association of a CD39-associated SNP with human cardiac pathology. Confirmatory studies are underway in a larger cohort.

Introduction: CD39 (ectonucleoside triphosphate diphosphohydrolase) is a nucleotidase expressed on endothelial cells, vascular smooth muscles cells, and leukocytes. CD39 plays a key role in vascular homeostasis, hydrolyzing extracellular ATP or ADP to AMP. CD73 (ecto-5’-nculeotidase) hydrolyzes AMP to adenosine. Both CD39 and CD73 are key modulators of purinergic signaling and have been shown to be important in ischemic preconditioning and ischemia reperfusion in animal models. Hypothesis: We hypothesized that the expression of the purine nucleotide-metabolizing pathway consisting of CD39 and CD73 in human hearts with ischemic cardiomyopathy is unregulated in response to ischemia. Methods: We compared the expression of CD39 and CD73 in left ventricular samples from patients with end-stage ischemic cardiomyopathy and normal hearts. Left ventricular tissue obtained from 7 end-stage ischemic cardiomyopathy hearts and 7 normal hearts not used for transplantation were homogenized and the levels of CD39 and CD73 mRNA were measured by qRT-PCR. Using the same tissue, immunoblot analysis for the protein level of CD39 and CD73 was conducted. Furthermore, immunohistochemical analysis of CD39 and CD73 expression was conducted on left ventricular tissue from the same subjects. Results: The total mRNA levels of CD39 and CD73 were increased in patients with end-stage ischemic cardiomyopathy. However, while there was a reduction in the total CD73 protein levels that did not reach significance, there was a marked reduction in the the total protein level of CD39 in ICM hearts (Figure). Immunohistochemical analysis demonstrated that the expression pattern of CD39 was significantly different in ICM hearts compared to normal hearts. Conclusions: These data provide important evidence that the extracellular nucleotide metabolizing pathway is altered in ICM. CD39 protein levels are significantly reduced in human end-stage ischemic cardiomyopathy.

Objective: To determine the effect of increased CD39 expression on circulating blood components on in vivo arterial thrombosis in mice. Approach and Results: We have previously demonstrated that increased global expression of hCD39 conveys an anti-thrombotic phenotype in mice suggesting a pivotal role for CD39 in modulating the balance between a pro-thrombotic and anti-thrombotic milieu. We hypothesized the expression of CD39 on circulating blood components can regulate occlusive thrombus formation. Bone marrow transplantations were performed (WT to WT; WT to hCD39-Tg; hCD39-Tg to hCD39-Tg; hCD39-Tg to WT) to determine the contribution of CD39 expressed on circulating blood cells and platelets on occlusive arterial thrombus formation in mice. Complete blood counts and fluorescence-activated cell sorting (FACS) analyses were performed on whole blood obtained from mice 60 days after transplantation of bone marrow. No significant differences in the circulating levels of white blood cells, red blood cells, and platelets were observed between the groups. FACS analysis confirmed that the circulating blood components were reconstituted from the transplanted bone marrow. Carotid artery thrombosis was induced by ferric chloride (FeCl 3 ) application and the time to thrombotic occlusion (blood flow of 0 ml/min) was measured. The operator was blinded to the animal genotype and treatment during all experiments. Transplantation of CD39-Tg bone marrow into WT mice resulted in a marked resistance to thrombosis (CD39-Tg to WT: 315.7.00 ± 54.6 min; WT to WT: 7.58 ± 0.89 min; P P Conclusion: Increased expression of CD39 on circulating bone-marrow-derived blood components markedly prolongs the time to carotid artery occlusion following ferric-chloride-induced injury in mice.

Purpose Pax3cre-mediated deletion of fibroblast growth factor receptor 2 (Fgfr2) broadly in renal and urinary tract mesenchyme led to ureteric bud (UB) induction defects and vesicoureteral reflux (VUR), although the mechanisms were unclear. Here, we investigated whether Fgfr2 acts specifically in peri-Wolffian duct stroma (ST) to regulate UB induction and development of VUR and the mechanisms of Fgfr2 activity. Methods We conditionally deleted Fgfr2 in ST (Fgfr2ST−/−) using Tbx18cre mice. To look for ureteric bud induction defects in young embryos, we assessed length and apoptosis of common nephric ducts (CNDs). We performed 3D reconstructions and histological analyses of urinary tracts of embryos and postnatal mice and cystograms in postnatal mice to test for VUR. We performed in situ hybridization and real-time PCR in young embryos to determine mechanisms underlying UB induction defects. Results We confirmed that Fgfr2 is expressed in ST and that Fgfr2 was efficiently deleted in this tissue in Fgfr2ST−/− mice at embryonic day (E) 10.5. E11.5 Fgfr2ST−/− mice had randomized UB induction sites with approximately 1/3 arising too high and 1/3 too low from the Wolffian duct; however, apoptosis was unaltered in E12.5 mutant CNDs. While ureters were histologically normal, E15.5 Fgfr2ST−/− mice exhibit improper ureteral insertion sites into the bladder, consistent with the ureteric induction defects. While ureter and bladder histology appeared normal, postnatal day (P) 1 mutants had high rates of VUR versus controls (75% versus 3%, p = 0.001) and occasionally other defects including renal hypoplasia and duplex systems. P1 mutant mice also had improper ureteral bladder insertion sites and shortened intravesicular tunnel lengths that correlated with VUR. E10.5 Fgfr2ST−/− mice had decreases in Bmp4 mRNA in stromal tissues, suggesting a mechanism underlying the ureteric induction and VUR phenotypes. Conclusion Mutations in FGFR2 could possibly cause VUR in humans.

Obesity is commonly associated with development of insulin resistance and systemic evidence of inflammation. Macrophages contribute to inflammatory amplification in obesity and may contribute directly to insulin resistance and the development of nonalcoholic fatty liver disease through the production of inflammatory cytokines, including tumor necrosis factor (TNF)-α. To test this hypothesis, we transplanted male wild-type (WT) and TNF-α deficient (KO) mice with either TNF-α-sufficient (TNF-α+/+) or TNF-α-deficient (TNF-α−/−) bone marrow. After consuming a high-fat diet for 26 wk, metabolic and morphometric characteristics of the animals were analyzed. While there were no differences in terms of relative weight gain, body composition analysis yielded a lower relative adipose and higher relative lean mass in mice lacking TNF-α, which was partially explained by reduced epididymal fat pad and liver weight. TNF-α−/−→KO mice exhibited enhanced insulin sensitivity compared with that observed in TNF-α+/+→KO mice; remarkably, no protection against insulin resistance was provided by transplanting TNF-α−/−bone marrow in WT mice compared with TNF-α+/+→WT. The preserved insulin sensitivity seen in TNF-α−/−→KO mice provided protection against the development of hepatic steatosis. Taken together, these data indicate that macrophage-derived TNF-α contributes to the pattern and extent of fat accumulation and insulin resistance in diet-induced obesity; however, this contribution is negligible in the presence of host-derived TNF-α.

Plasma levels of plasminogen activator inhibitor-1 (PAI-1) are elevated in obesity and correlate with body mass index. The increase in PAI-1 associated with obesity likely contributes to increased cardiovascular risk and may predict the development of type 2 diabetes mellitus. Although adipocytes are capable of synthesizing PAI-1, the bulk of evidence indicates that cells residing in the stromal fraction of visceral fat are the primary source of PAI-1. We hypothesized that bone marrow-derived PAI-1, e.g. derived from macrophages located in visceral fat, contributes to the development of diet-induced obesity. To test this hypothesis, male C57BL/6 wild-type mice and C57BL/6 PAI-1 deficient mice were transplanted with either PAI-1(-/-), PAI-1(+/-), or PAI-1(+/+) bone marrow. The transplanted animals were subsequently fed a high fat diet for 24 weeks. Our findings show that only the complete absence of PAI-1 protects from the development of diet-induced obesity, whereas the absence of bone marrow-derived PAI-1 protects against expansion of the visceral fat mass. Remarkably, there is a link between the PAI-1 levels, the degree of inflammation in adipose tissue, and the development of obesity. Based on these findings we suggest that bone marrow-derived PAI-1 has an effect on the development of obesity through its effect on inflammation.