Endothelial Cell-selective Adhesion Molecule Research Articles

Role of endothelial cell-selective adhesion molecule in the regulation of pulmonary vascular resistance

Endothelial cell-selective adhesion molecule (ESAM) is a member of the endothelial cell junction adhesion molecules family, which plays an important role in the maintenance of endothelial barrier function in the pulmonary circulation. The role of ESAM in affecting pulmonary vascular resistance remains unknown. To examine the role of ESAM in pulmonary vascular resistance, whole lungs were isolated from 16-18 weeks old ESAM knock out (KO) mice and wild type (WT) littermates, ventilated and perfused. We found that pulmonary vascular resistance is significantly increased in the lung of ESAM KO mice compared to WT mice. Bradykinin-induced, endothelium-dependent reduction in the pulmonary vascular resistance was impaired in ESAM KO mice, whereas the direct NO donor, sodium nitroprusside-induced endothelium independent responses remained similar in WT and ESAM KO mice. U46619 mediated increases in pulmonary vascular resistance was significantly higher in ESAM KO mice. Inhibition of the NO synthesis with the L-NAME (0.2 mM for 10 minutes) augmented the U46619-mediated increase in pulmonary vascular resistance in WT mice, but it did not affect the U46619 response in the ESAM KO mice. The mRNA expression of pro-inflammatory cytokines, including interleukin-6 and tumor necrosis factor, was increased in the lungs of ESAM KO mice, whereas expression of collagen isoforms (type I and type III) and fibronectin were similar in WT and ESAM KO mice. Collectively, we propose that ESAM plays an important role in the maintenance of pulmonary vascular resistance, likely via preserving endothelial nitric oxide production in pulmonary arteries. AHA predoctoral award ID 917057. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Integrated causal inference modeling uncovers novel causal factors and potential therapeutic targets of Qingjin Yiqi granules for chronic fatigue syndrome

Objective: Chronic fatigue syndrome (CFS) is a prevalent symptom of post-coronavirus disease 2019 (COVID-19) and is associated with unclear disease mechanisms. The herbal medicine Qingjin Yiqi granules (QJYQ) constitute a clinically approved formula for treating post-COVID-19; however, its potential as a drug target for treating CFS remains largely unknown. This study aimed to identify novel causal factors for CFS and elucidate the potential targets and pharmacological mechanisms of action of QJYQ in treating CFS. Methods: This prospective cohort analysis included 4,212 adults aged ≥65 years who were followed up for 7 years with 435 incident CFS cases. Causal modeling and multivariate logistic regression analysis were performed to identify the potential causal determinants of CFS. A proteome-wide, two-sample Mendelian randomization (MR) analysis was employed to explore the proteins associated with the identified causal factors of CFS, which may serve as potential drug targets. Furthermore, we performed a virtual screening analysis to assess the binding affinity between the bioactive compounds in QJYQ and CFS-associated proteins. Results: Among 4,212 participants (47.5% men) with a median age of 69 years (interquartile range: 69–70 years) enrolled in 2004, 435 developed CFS by 2011. Causal graph analysis with multivariate logistic regression identified frequent cough (odds ratio: 1.74, 95% confidence interval [CI]: 1.15–2.63) and insomnia (odds ratio: 2.59, 95% CI: 1.77–3.79) as novel causal factors of CFS. Proteome-wide MR analysis revealed that the upregulation of endothelial cell-selective adhesion molecule (ESAM) was causally linked to both chronic cough (odds ratio: 1.019, 95% CI: 1.012–1.026, P = 2.75 e−05) and insomnia (odds ratio: 1.015, 95% CI: 1.008–1.022, P = 4.40 e−08) in CFS. The major bioactive compounds of QJYQ, ginsenoside Rb2 (docking score: −6.03) and RG4 (docking score: −6.15), bound to ESAM with high affinity based on virtual screening. Conclusions: Our integrated analytical framework combining epidemiological, genetic, and in silico data provides a novel strategy for elucidating complex disease mechanisms, such as CFS, and informing models of action of traditional Chinese medicines, such as QJYQ. Further validation in animal models is warranted to confirm the potential pharmacological effects of QJYQ on ESAM and as a treatment for CFS.

Transcriptomic analysis of glucosidase II beta subunit (GluIIß) knockout A549 cells reveals its roles in regulation of cell adhesion molecules (CAMs) and anti-tumor immunity

Glucosidase II beta subunit (GluIIß), encoded from PRKCSH, is a subunit of the glucosidase II enzyme responsible for quality control of N-linked glycoprotein folding and suppression of GluIIß led to inhibitory effect of the receptor tyrosine kinase (RTKs) activities known to be critical for survival and development of cancer. In this study, we investigated the effect of GluIIß knockout on the global gene expression of cancer cells and its impact on functions of immune cells. GluIIß knockout lung adenocarcinoma A549 cell line was generated using CRISPR/Cas9-based genome editing system and subjected to transcriptomic analysis. Among 23,502 expressed transcripts, 1068 genes were significantly up-regulated and 807 genes greatly down-regulated. The KEGG enrichment analysis showed significant down-regulation of genes related extracellular matrix (ECM), ECM-receptor interaction, cytokine-cytokine receptor interaction and cell adhesion molecules (CAMs) in GluIIß knockout cells. Of 9 CAMs encoded DEG identified by KEGG enrichment analysis, real time RT-PCR confirmed 8 genes to be significantly down-regulated in all 3 different GluIIß knockout clones, which includes cadherin 4 (CDH4), cadherin 2 (CDH2), versican (VCAN), integrin subunit alpha 4 (ITGA4), endothelial cell-selective adhesion molecule (ESAM), CD274 (program death ligand-1 (PD-L1)), Cell Adhesion Molecule 1 (CADM1), and Nectin Cell Adhesion Molecule 3 (NECTIN3). Whereas PTPRF (Protein Tyrosine Phosphatase Receptor Type F) was significantly decreased only in 1 out of 3 knockout clones. Microscopic analysis revealed distinctively different cell morphology of GluIIβ knockout cells with lesser cytoplasmic and cell surface area compared to parental A549 cells and non-targeted transfected cells.Further investigations revealed that Jurkat E6.1 T cells or human peripheral blood mononuclear cells (PBMCs) co-cultured with GluIIß knockout A549 exhibited significantly increased viability and tumor cell killing activity compared to those co-cultured with non-target transfected cells. Analysis of cytokine released from Jurkat E6.1 T cells co-cultured with GluIIß knockout A549 cells showed significant increased level of angiogenin and significant decreased level of ENA-78. In conclusion, knockout of GluIIß from cancer cells induced altered gene expression profile that improved anti-tumor activities of co-cultured T lymphocytes and PBMCs thus suppression of GluIIß may represent a novel approach of boosting anti-tumor immunity.

Abstract 635: ESAM Deficiency-related Coronary Microvascular Dysfunction Contributes To Left Ventricle Diastolic Dysfunction In HFpEF

Coronary microvascular dysfunction and vascular inflammation have been linked to heart failure with preserved ejection fraction (HFpEF), but the underlying molecular mechanisms remain incompletely understood. Endothelial cell-selective adhesion molecule (ESAM) has been recently implicated as one of the biomarkers for heart failure. The biological function of ESAM is to regulate inflammatory cell adhesion, permeability and angiogenesis. Here, we examined the mechanistic role of ESAM in contributing left ventricle (LV) diastolic dysfunction in a mouse model of human HFpEF. We employed wild type (WT) mice and mice with homozygous genetic deficiency of ESAM (ESAM -/- ), with or without uninephrectomy and aldosterone infusion (UNX-Aldo, for 4-week). Using echocardiography, we found that WT mice with UNX-Aldo develop LV diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time (IVRT) and E wave deceleration time. UNX-Aldo significantly increased the serum soluble ESAM levels, along with elevated MMP2/9 levels, as potential mediators of ESAM ectodomain shedding. When compared to WT, ESAM -/- mice with UNX-Aldo displayed significant worsening of LV diastolic dysfunction, as indicated by increased IVRT, and the development of pulmonary edema and fibrosis. An unbiased automated tracing and 3D reconstruction of coronary microvasculature revealed that ESAM -/- mice had a significantly reduced coronary microvascular density. Arteries of ESAM -/- mice exhibited impaired endothelial sprouting and in cultured endothelial cells siRNA-mediated ESAM knockdown reduced tube formation. Thus, we propose that ESAM plays a role in the proper myocardial vascularization, hence the maintenance of myocardial perfusion. Deficiency, or augmented ectodomain shedding of ESAM impairs myocardial vascularization, whereby it contributes to the development of LV diastolic dysfunction, and could have mechanistic and diagnostic implications in human HFpEF.

Endothelial Cell-Selective Adhesion Molecule Enhances Tumor Growth in a HFpEF Mouse Model

Accumulating epidemiological evidence shows that the incidence of multiple types of cancer, such as melanoma and GI cancers, is significantly higher in patients with heart failure with preserved ejection fraction (HFpEF). However, the mechanistic relationships between HFpEF and cancer development are poorly understood. Impaired angiogenesis, upregulation of endothelial adhesion molecules and chronic low-grade inflammation are common in HFpEF. Here we examined the role of endothelial cell-selective adhesion molecule (ESAM) in the progression of malignant melanoma in a HFpEF mouse model. Orthotopic melanoma xenografts (B16F10-Luc2) were used and implanted subcutaneously in mice underwent uninephrectomy and aldosterone infusion (UNX-Aldo with 1% high salt drinking water for 4 weeks) and tumor growth was monitored with bioluminescent imaging. We found that ESAM expression and serum levels were elevated in the UNX-Aldo mice, which was accompanied by enhanced melanoma growth and metastasis rate. ESAM promoted tube formation and increased phospho-Akt, phospho-PKCα and phospho-ERK1/2 in cultured endothelial cells. Flow cytometry analysis showed elevated number of tumor-associated neutrophils (CD11b+Ly6G+Ly6C-) and myeloid-derived suppressor cells (MDSCs, CD11b+F4/80-Gr-1+) in the UNX-Aldo mice. UNX-Aldo mice with genetic deletion of ESAM (ESAM KO) showed a reduced tumor growth and reduced number of tumor-associated neutrophils and MDSCs. Collectively, our results indicate that in HFpEF, a proinflammatory ESAM activation may act to promote tumor angiogenesis, neutrophil and MDSCs tumor infiltration, thereby mitigating a proper immune response and facilitate tumor growth. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antigen loaded monocytes prime CD4+ T cell responses through Notch2 dependent cDC2

Abstract Cellular vaccines, primarily monocyte-derived dendritic cells (MoDC), have recently been realized as therapeutic cancer vaccines with modest clinical efficacy. Understanding the mechanism by which these vaccines lead to antigen presentation is key to improving their potency. A significant advancement was the discovery that MoDC vaccines do not directly present antigen, but instead rely on endogenous dendritic cells for their antigen presenting cell (APC) function. Subsequent research has also demonstrated that primary monocytes can be loaded with antigen and used as cellular vaccines, outperforming MoDC vaccines in mouse models of melanoma and glioblastoma. In this study, we characterized how monocyte vaccines generate CD4+ T cell responses. We utilized the CD11c-Cre RBPJfl/flmouse model, which lack a subset of Notch2 dependent cDC2 characterized by expression of Endothelial cell-selective adhesion molecule (ESAM hi). We demonstrate that the ESAM hicDC2 subset is required for effective CD4+ OT-II T cell priming after vaccination with ovalbumin (OVA) loaded monocytes. Interestingly, depletion of the ESAM hiDC population does not affect monocyte vaccine-induced CD8+ T cell priming, a process previously shown to be dependent on cDC1. This indicates that monocyte vaccines generate T cell responses by independently transferring antigen to both ESAM hiand CD8+ DCs, leveraging their APC function to prime CD4+ and CD8+ T cell responses, respectively. This work furthers the mechanistic understanding of monocyte vaccines, and will inform the design of future monocyte vaccines as they enter clinical trials as therapeutic cancer vaccines. Supported by NCI P50 CA 190991 and funding from the Stead Scholarship Program of the Duke University Department of Medicine.

Mice with endothelial cell-selective adhesion molecule deficiency develop coronary microvascular rarefaction and left ventricle diastolic dysfunction.

Endothelial cell-selective adhesion molecule (ESAM) regulates inflammatory cell adhesion and transmigration and promotes angiogenesis. Here, we examined the role of ESAM in cardiac vascularization, inflammatory cell infiltration, and left ventricle (LV) diastolic function under basal and hemodynamic stress conditions. We employed mice with homozygous genetic deletion of ESAM (ESAM-/- ) and also performed uninephrectomy and aldosterone infusion (UNX-Aldo) to induce volume and pressure overload. Using echocardiography, we found that ESAM-/- mice display no change in systolic function. However, they develop LV diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e' ratio, isovolumic relaxation time (IVRT), and E wave deceleration time. An unbiased automated tracing and 3D reconstruction of coronary vasculature revealed that ESAM-/- mice had reduced coronary vascular density. Arteries of ESAM-/- mice exhibited impaired endothelial sprouting and in cultured endothelial cells siRNA-mediated ESAM knockdown reduced tube formation. Changes in ESAM-/- mice were accompanied by elevated myocardial inflammatory cytokine and myeloperoxidase-positive neutrophil levels. Furthermore, UNX-Aldo procedure in wild type mice induced LV diastolic dysfunction, which was accompanied by significantly increased serum ESAM levels. When compared to wild types, ESAM-/- mice with UNX-Aldo displayed worsening of LV diastolic function, as indicated by increased IVRT and pulmonary edema. Thus, we propose that ESAM plays a mechanistic role in proper myocardial vascularization and the maintenance of LV diastolic function under basal and hemodynamic stress conditions.

Propionate regulates tight junction barrier by increasing endothelial-cell selective adhesion molecule in human intestinal Caco-2 cells

Regulation of the intestinal barrier is closely associated with intestinal microbial metabolism. This study investigated the role of propionate, a major short-chain fatty acid produced by intestinal microorganisms, in the regulation of the tight junction (TJ) barrier in human intestinal Caco-2 cells. Propionate strengthened TJ barrier integrity, as indicated by decreased permeability to macromolecules and increased transepithelial electrical resistance in Caco-2 cells. DNA microarray analysis revealed that propionate upregulated endothelial cell-selective adhesion molecule (ESAM), a TJ-associated protein, without any increase in other TJ proteins. The upregulation of ESAM was confirmed using quantitative reverse transcription-PCR, immunoblotting, and immunofluorescence analyses. Luciferase promoter analysis demonstrated that propionate induced the transcriptional activation of ESAM. The effects of propionate were sensitive to nilotinib inhibition of NR2C2. Overexpression of human ESAM (hESAM) in canine kidney epithelial MDCK-II cells lowered the permeability to macromolecules in a manner similar to that of propionate-treated Caco-2 cells. hESAM overexpression facilitated calcium-induced assembly of the TJ complex in MDCK-II cells. Taken together, propionate strengthened the intestinal TJ barrier by increasing ESAM levels in Caco-2 cells.

Abstract 15421: Mice Deficient in Endothelial Cell-Selective Adhesion Molecule Display Coronary Microvascular Rarefaction and Left Ventricle Diastolic Dysfunction

Coronary microvascular dysfunction and chronic inflammation play a role in the development of left ventricular diastolic dysfunction (LVDD) in heart failure with preserved ejection fraction (HFpEF). Endothelial cell-selective adhesion molecule (ESAM) is part of the endothelial cell junction complex, which plays a role in the maintenance of cell barrier function, inflammatory cell transmigration and angiogenesis. Here, we examined the role of ESAM in affecting coronary microvascular density and left ventricle diastolic function by employing mice with genetic deletion of ESAM (ESAM -/- ). Using echocardiography, we found that ESAM -/- mice display LVDD, as indicated by a significantly (p<0.05) reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time and E wave deceleration time, with no change in the ejection fraction. Using an unbiased, automated tracing and 3D reconstruction of microvascular network revealed that ESAM -/- mice have a reduced coronary microvascular density, which was accompanied by myocardial accumulation of myeloperoxidase-positive neutrophils and elevated myocardial levels of inflammatory cytokines and chemokines. ESAM -/- mice exhibited a reduced endothelial sprout formation in the carotid artery, whereas in cultured endothelial cells siRNA-mediated ESAM knockdown impaired tube formation. Thus, ESAM deficiency in mice leads to coronary microvascular rarefaction, which likely via a myocardial hypoperfusion-related neutrophil accumulation and inflammatory mediator production impairs LV diastolic function.

Mice Deficient in Endothelial Cell‐Selective Adhesion Molecule Develop Vascular Endothelial and Left Ventricle Diastolic Dysfunction

The Endothelial Cell‐Selective Adhesion Molecule (ESAM) mediates inflammatory cell transmigration and also involved in pathological angiogenesis. Here we investigated the role of ESAM in affecting vasodilator and cardiac contractile function. To that end ESAM knockout and wild type (WT) mice were employed. Using transthoracic echocardiography it was found that ESAM knockout mice displayed left ventricle (LV) diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time (IVRT) and E wave deceleration time, with no change in the ejection fraction. The systolic blood pressure, as measured by tail‐cuff plethysmography, was not different between ESAM knockout and WT mice, whereas the heart to body weight ratio is increased in ESAM knockout mice. Using an unbiased automated tracing of the microvasculature we found a decreased vascularization (total vascular length) of the myocardium in ESAM knockout mice. In addition, vasodilator function of the pulmonary artery and aorta was assessed using wire myography. We found that the endothelium‐dependent, acetylcholine‐induced relaxation of the pulmonary artery was significantly reduced in ESAM knockout mice, whereas endothelium‐independent, sodium nitroprusside‐induced relaxation was similar in two groups. Thus, we conclude that ESAM deficiency causes vasodilator dysfunction, impaired angiogenesis underlying the development LV diastolic dysfunction.

Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.

Abstract Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. To dissect early lymphoid specification events, we generated two transgenic mouse models reporting and tracing the expression of terminal deoxynucleotidyl transferase (TdT). Surprisingly, transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset which lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction by MPPs reflected a transcriptionally dynamic but uncommitted stage characterized by low expression of lineage-associated genes. Further, analysis using single-cell CITE-Seq indicated that multipotency is associated with expression of Endothelial Cell-Selective Adhesion Molecule (ESAM). Developmental progression along all lineages is defined by downregulation of ESAM and by stable TdT expression within the lymphoid trajectory. Collectively, we propose a new developmental hierarchy of early hematopoietic progenitors for which we provide their transcriptional profile as well as their phenotypic and functional properties at single cell resolution. Supported by NIH intramural funding

3D Visualization of Human Blood Vascular Networks Using Single-Domain Antibodies Directed against Endothelial Cell-Selective Adhesion Molecule (ESAM).

High-quality three-dimensional (3D) microscopy allows detailed, unrestricted and non-destructive imaging of entire volumetric tissue specimens and can therefore increase the diagnostic accuracy of histopathological tissue analysis. However, commonly used IgG antibodies are oftentimes not applicable to 3D imaging, due to their relatively large size and consequently inadequate tissue penetration and penetration speed. The lack of suitable reagents for 3D histopathology can be overcome by an emerging class of single-domain antibodies, referred to as nanobodies (Nbs), which can facilitate rapid and superior 2D and 3D histological stainings. Here, we report the generation and experimental validation of Nbs directed against the human endothelial cell-selective adhesion molecule (hESAM), which enables spatial visualization of blood vascular networks in whole-mount 3D imaging. After analysis of Nb binding properties and quality, selected Nb clones were validated in 2D and 3D imaging approaches, demonstrating comparable staining qualities to commercially available hESAM antibodies in 2D, as well as rapid and complete staining of entire specimens in 3D. We propose that the presented hESAM-Nbs can serve as novel blood vessel markers in academic research and can potentially improve 3D histopathological diagnostics of entire human tissue specimens, leading to improved treatment and superior patient outcomes.

Exploring new molecules that regulate hematopoietic stem cells and early stages of lymphoid hematopoiesis: the functional significance of ESAM and SATB1

Hematopoietic stem cells (HSCs) possess multilineage differentiation capability, which sustains the production of blood and immune cells throughout life. However, the precise mechanisms by which HSCs initiate differentiation toward a particular lineage and the factors that attenuate their lymphopoietic potential with aging are yet to be elucidated. Our group has investigated this issue for over two decades. We initially developed a method for segregating early lymphoid progenitors from HSCs and identified two molecules: endothelial cell-selective adhesion molecule (ESAM), highly expressed in HSCs, and special AT-rich sequence binding protein 1 (SATB1), expressed in early lymphoid progenitors. ESAM marks HSCs across species, including humans. In addition to its significance in stress-induced hematopoiesis, ESAM is also useful in identifying features of human acute myeloid leukemia stem cells. Further, we determined the role of SATB1 in the early HSC differentiation processes toward the lymphoid lineage. Remarkably, SATB1 expression in HSCs significantly decreased with aging, whereas its exogenous induction in aged HSCs rejuvenated their lymphopoietic potential. Furthermore, SATB1-expressing HSCs demonstrated robust lymphopoietic and long-term reconstituting capability, whereas HSCs without SATB1 skewed toward the myeloid lineage. Thus, our continuing research has revealed the significance of ESAM and SATB1 in the fundamental biology of HSCs.

Functions of two distinct Kupffer cells in the liver

Tissue-resident macrophages play critically important roles in host homeostasis and pathogenesis of diseases, with the functions of phagocytosis, metabolism, and immune modulation. Recently, two research studies accomplished by a collaborated group of researchers showed that there are two populations of liver resident Kupffer cells (KCs), including a major cluster of differentiation 206 low expression (CD206low)endothelial cell-selective adhesion molecule negative (ESAM-) population (KC1) and a minor CD206highESAM+ population (KC2). Both KC1 and KC2 express KC markers, such as C-type lectin domain family 4 member F (CLEC4F) and T-cell membrane protein 4 (Tim4). In fatty liver, the frequency of KC2 was increased, and those KC2 expressed some markers like liver sinusoidal endothelial cells (LSECs), such as CD31 and ESAM. In addition, KC2 population had a relatively higher expression of CD36, as fatty acid transporter, which was implicated in the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, this collaborated group also showed that KC2 can cross-present hepatocellular antigens to prime antiviral function of CD8+ T cells by sensing interleukin-2 (IL-2) in hepatitis B virus (HBV) replication-competent transgenic mice. Increasing evidence shows that targeting hepatic macrophages can prevent and reverse non-alcoholic fatty liver disease (NAFLD), with a new suggested name metabolic dysfunction-associated fatty liver disease (MAFLD) to include metabolic dysfunction-associated fatty liver diseases, such as viruses and alcohol. In summary, differentiating specific populations of hepatic macrophages is critically important for the treatment of MAFLD or NAFLD, and their overlaps. Markers specifically expressed on sub-types of hepatic macrophages may be applied for liver disease diagnosis.

Abstract 14412: Endothelial Cell-Selective Adhesion Molecule Deficiency in Mice Causes Vascular Endothelial Dysfunction and Impairs Left Ventricle Diastolic Function

Dysfunction of vascular endothelium plays a key role in the development of left ventricular diastolic dysfunction (LVDD) in patients with heart failure with preserved left ventricular ejection fraction (HFpEF). Recently, Endothelial Cell-Selective Adhesion Molecule (ESAM) was proposed as a novel biomarker in HFpEF. We hypothesized that ESAM plays a mechanistic role in the development of both microvascular dysfunction and LVDD. ESAM knockout and wild type (WT) mice were employed. Using transthoracic echocardiography it was found that ESAM knockout mice displayed LV diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time (IVRT) and increased E wave deceleration time, with no change in the ejection fraction. The systolic blood pressure, as measured by tail-cuff plethysmography, was not different between ESAM knockout and WT mice; and no cardiac hypertrophy was detected in the ESAM knockout mice. Vasodilator function of the pulmonary artery and aorta was assessed using wire myography. We found that the endothelium-dependent, acetylcholine-induced relaxation of the aorta and pulmonary artery was significantly reduced in ESAM knockout mice, whereas endothelium-independent, nitric oxide donor, sodium nitroprusside-induced relaxation was similar in the two groups. Thus, we concluded that ESAM deficiency in mice display a HFpEF phenotype, in that ESAM knockout mice develop endothelial dysfunction and LVDD.

Endothelial Cell-Selective Adhesion Molecule (ESAM) Plays Important Roles in the Adult-Type Hemoglobin Synthesis during Fetal Erythropoiesis

Endothelial cell-selective adhesion molecule (ESAM) is a lifelong marker for HSCs and is highly expressed on the first definitive HSCs. In addition, ESAM expression is functionally essential for adult HSCs because ESAM deficiency causes life-threatening myelo-suppression, especially severe anemia, after administration of 5-FU. In the last ASH annual meeting, we reported that ESAM deletion perturbed the development of definitive hematopoiesis, especially definitive erythropoiesis in the fetal liver (FL). In this study, we precisely analyzed the mechanism via which ESAM deletion affects erythroid lineage development.Although the number and the size of ESAM homozygous (homo) knockout (KO) fetuses were comparable to that of wild type (WT) or heterozygous KO littermates until embryonic day (E) 14.5, some ESAM homo KO fetuses exhibited severe anemia after E15.5 and approximately half of the ESAM homo KO fetuses died before E17.5. Quantitative real-time PCR analyses for assessing the expression of erythropoiesis-related genes in E16.5 ESAM-null FLs revealed significant reduction in mRNA levels for adult globins (Hba and Hbb-b1) and the erythroid-specific isoenzyme of 5-aminolevulinic acid synthase 2 (Alas2). In contrast, transcript levels of erythropoietin (Epo), Epo-receptor, and several transcription factors essential for erythropoiesis, such as Gata1, Klf1, and c-myb, were not reduced by ESAM deletion.Although no apparent malformation was evident in the E14.5 ESAM-null fetuses, the developmental abnormality in erythropoiesis already existed, because the proportion of enucleated erythrocytes was smaller and the number of mature Ter119+ erythroid cells was significantly decreased.Next, we examined the quality of ESAM deficient HSCs in culture and in vivo transplantation. In methylcellulose cultures, although E14.5 LSK CD48- FL cells of ESAM-null mice produced sufficient numbers of BFU-E colonies, the mRNA expression levels of adult globins and Alas2 in the BFU-E colonies produced by ESAM-null HSCs were significantly reduced. In competitive repopulation assays, 4 × 102 LSK CD48- HSCs sorted from CD45.2+ E14.5 ESAM-null or WT FLs were transplanted into lethally irradiated CD45.1+ congenic WT mice with 2 × 105 CD45.1+ bone marrow cells and analyzed after 15 weeks. Although donor chimerism and the number of CD45.2+ HSCs did not differ between the two groups, peripheral blood hemoglobin level in ESAM-null HSC-transplanted recipient mice was significantly reduced.Next, to understand the molecular mechanisms involved in the developmental failure of definitive erythropoiesis in the ESAM-null FL, we conducted RNA-sequencing (RNA-seq) and compared the gene expression patterns of WT and ESAM-null HSCs. We found that the expression of many erythropoiesis-related genes was reduced in ESAM-null HSCs. Among them, the reduction of ALAS2 expression was most remarkable. In addition, transcripts for both adult globins (Hba-a1, Hba-a2 and Hbb-b1) and embryonic globins (Hbb-y and Hbb-bh1) were dramatically decreased. These results corroborated our hypothesis that hemoglobin synthesis is impaired by ESAM deficiency, and further suggested that the cause of developmental failure in the ESAM-null fetuses can be attributed to aberrant gene expression in ESAM-null “definitive” HSCs.We exploited an antibody cross-linking method to address whether ESAM directly affected gene expression profiles in HSCs. WT E14.5 LSK CD48- cells were either untreated or incubated with a rat mAb against mouse ESAM. Then, both cells were incubated with goat anti-rat IgG Abs-conjugated microbeads for 4 h, and were subsequently applied to RNA-seq. The IPA network analysis indicated that “Hematological System Development and Function, Increased Levels of Hematocrit, Molecular Transport” network, which were associated with hemoglobin synthesis including Hbb-bh1, were the most affected. We also found up-regulation of GDF15, which promotes hemoglobin synthesis by suppressing hepcidin secretion.In conclusion, we showed that approximately half of ESAM-null fetuses died between E15.5 and E17.5, mainly due to the disturbance of adult-type hemoglobin synthesis. Although indirect mechanisms could be involved in the anemic phenotype of ESAM-null fetuses, we propose that ESAM-mediated interactions directly modulate the expression pattern of erythropoiesis-related genes in the developing HSCs. DisclosuresYokota:MSD K.K.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Celgene: Research Funding. Ezoe:Taiho Pharmaceutical Co., LTD.: Research Funding. Shibayama:Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; MOCHIDA PHARMACEUTICAL: Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Kanakura:Astellas: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Bristol Myers: Research Funding; Nippon Shinyaku: Research Funding; Kyowa Hakko Kirin: Research Funding; Eisai: Research Funding; Chugai Pharmaceutical: Research Funding; Fujimotoseiyaku: Research Funding; Pfizer: Research Funding; Shionogi: Research Funding; Toyama Chemical: Research Funding.

Variable Expression Levels of the Hematopoietic Stem Cell Surface Antigen ESAM Depict Heterogeneity and Fluctuations in Leukemic Stem Cells

Traditionally, hematopoietic stem cells (HSCs) have been regarded as a homogeneous cell population. However, many studies have shown that populations of HSCs are essentially heterogenous. Moreover, several recent studies have proposed that HSCs exhibit fluctuating biological characteristics. Although the physiological significance of this heterogeneity and fluctuation is unclear, we presume that these qualities enable HSCs to flexibly demonstrate their self-renewal or differentiation abilities dependent on physiological demands. It is, therefore, unsurprising that such heterogeneity and fluctuation is also seen in leukemic stem cells (LSCs), and that they are involved in the development of resistance to treatment.We have previously demonstrated that the endothelial-cell selective adhesion molecule (ESAM) is an important marker of HSCs in humans and mice (Blood 2009, Exp Hematol 2013), and that the expression level of this molecule indicates the activation status of HSCs (J Immunol 2012). Furthermore, we found that a subset of leukemic cells exhibit elevated levels of ESAM expression on their surfaces (Exp Hematol 2013). Approximately two-thirds of acute myelogenous leukemia cases in humans were found to be positive for ESAM expression. Since ESAM was found to be useful in monitoring the fluctuating status of HSCs after 5-FU treatment, we posited that the marker could be used to characterize LSCs.In this study, we used ESAM expression levels as an index to analyze the heterogeneity and fluctuation in LSC populations. Flow cytometric analysis of the human leukemia cell lines KG1a and CMK revealed wide-ranging heterogeneity in ESAM expression levels, with up to a 1000-fold difference in variation, even among individual cells derived from the same clone. When these cell populations were roughly fractionated into ESAM-high or ESAM-low expression groups, the ESAM-high expression group showed a higher proliferative capacity in culture. Interestingly, however, the expression levels of ESAM were not maintained indefinitely at the same level in these groups. In four weeks, both ESAM-high and ESAM-low cell fractions developed heterogeneous ESAM expression distributions similar to that in the parent cell lines. Furthermore, when single cells were sorted and cultured, each cell showed a self-renewal ability and the heterogenous nature of ESAM expression levels was reconstructed in each population derived from these single cells. These results suggest that populations of LSCs develop heterogeneity and have a fluctuating nature.When the transcriptomes of ESAM-low and ESAM-high KG1a cells were compared using RT-PCR, the expression of the transforming growth factor beta receptor type 2 (TGFBR2) gene was found to be higher in the ESAM-low cells. Reportedly, the transforming growth factor beta (TGFβ) signaling pathway is involved in various biological processes such as cell proliferation, differentiation, and migration. In tumor cells, this pathway is involved in reducing proliferation and accelerating invasion. Based on this information, it was considered likely that the TGFβ signaling pathway is involved in the heterogeneity of ESAM expression. When TGFβ1 (5 ng/ml) was added to a culture of KG1a cells (grown in RPMI 1640 medium with 10% fetal bovine serum (FBS)), the heterogeneity of ESAM expression in the population became more pronounced, mainly due to a decrease in ESAM expression in some cells.A transcriptomic analysis of cells exhibiting decreased ESAM expression after TGFβ1 stimulation showed that these cells had a higher expression of TGFBR2 than cells not exhibiting a decrease in ESAM expression. Since the presence of 10% FBS in the culture medium provides natural TGFβ1 at a concentration of ~1 ng/ml, we conducted culture experiments without FBS. When KG1a cells were cultured in serum-free medium, the ESAM-low fraction disappeared and the heterogeneity of ESAM expression could not be replicated. However, the heterogeneity of ESAM expression in KG1a cultures could be replicated in serum-free medium by the addition of 1 ng/ml TGFβ1. These results suggest that the TGFβ signaling pathway is involved in inducing heterogeneity in ESAM expression in cell populations. The fluctuation of ESAM expression levels could be caused by differences in TGF sensitivity, which in turn, may confer certain benefits to LSCs, such as drug resistance and promoting invasiveness. DisclosuresYokota:Celgene: Research Funding; MSD K.K.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding. Ezoe:Taiho Pharmaceutical Co., LTD.: Research Funding. Shibayama:Novartis Pharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:MOCHIDA PHARMACEUTICAL: Speakers Bureau; Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Kanakura:Kyowa Hakko Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Fujimotoseiyaku: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Nippon Shinyaku: Research Funding; Toyama Chemical: Research Funding; Shionogi: Research Funding.

Autonomous TGFβ signaling induces phenotypic variation in human acute myeloid leukemia

Heterogeneity of leukemia stem cells (LSCs) is involved in their collective chemoresistance. To eradicate LSCs, it is necessary to understand the mechanisms underlying their heterogeneity. Here, we aimed to identify signals responsible for heterogeneity and variation of LSCs in human acute myeloid leukemia (AML). Monitoring expression levels of endothelial cell‐selective adhesion molecule (ESAM), a hematopoietic stem cell‐related marker, was useful to detect the plasticity of AML cells. While healthy human hematopoietic stem/progenitor cells robustly expressed ESAM, AML cells exhibited heterogeneous ESAM expression. Interestingly, ESAM− and ESAM+ leukemia cells obtained from AML patients were mutually interconvertible in culture. KG1a and CMK, human AML clones, also represented the heterogeneity in terms of ESAM expression. Single cell culture with ESAM− or ESAM+ AML clones recapitulated the phenotypic interconversion. The phenotypic alteration was regulated at the gene expression level, and RNA sequencing revealed activation of TGFβ signaling in these cells. AML cells secreted TGFβ1, which autonomously activated TGFβ pathway and induced their phenotypic variation. Surprisingly, TGFβ signaling blockade inhibited not only the variation but also the proliferation of AML cells. Therefore, autonomous activation of TGFβ signaling underlies the LSC heterogeneity, which may be a promising therapeutic target for AML.

Sox17-mediated expression of adherent molecules is required for the maintenance of undifferentiated hematopoietic cluster formation in midgestation mouse embryos

Hematopoietic stem cell-containing intra-aortic hematopoietic cell clusters (IAHCs) emerge in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation mouse embryos. We previously showed that transduction of Sox17 in CD45lowc-Kithigh cells, which are one component of IAHCs, maintained the cluster formation and the undifferentiated state, but the mechanism of the cluster formation by Sox17 has not been clarified. By microarray gene expression analysis, we found that genes for vascular endothelial-cadherin (VE-cad) and endothelial cell-selective adhesion molecule (ESAM) were expressed at high levels in Sox17-transduced c-Kit+ cells. Here we show the functional role of these adhesion molecules in the formation of IAHCs and the maintenance of the undifferentiated state by in vitro experiments. We detected VE-cad and ESAM expression in endothelial cells of dorsal aorta and IAHCs in E10.5 embryos by whole mount immunohistochemistry. Cells with the middle expression level of VE-cad and the low expression level of ESAM had the highest colony-forming ability. Tamoxifen-dependent nuclear translocation of Sox17-ERT fusion protein induced the formation of cell clusters and the expression of Cdh5 (VE-cad) and ESAM genes. We showed the induction of the Cdh5 (VE-cad) and ESAM expression and the direct interaction of Sox17 with their promoter by luciferase assay and chromatin immunoprecipitation assay, respectively. Moreover, shRNA-mediated knockdown of either Cdh5 (VE-cad) or ESAM gene in Sox17-transduced cells decreased the multilineage-colony forming potential. These findings suggest that VE-cad and ESAM play an important role in the high hematopoietic activity of IAHCs and cluster formation.

MiR-7 Reduces Breast Cancer Stem Cell Metastasis via Inhibiting RELA to Decrease ESAM Expression

This study aimed to present evidence that miR-7 inhibited the metastasis of breast cancer stem cells (BCSCs) and elucidated the mechanisms that have remained unknown. The samples collected from miR-7 agomir-treated, BCSC-driven tumors were subjected to a protein array to analyze the protein expression profiles. A dual-luciferase reporter and chromatin immunoprecipitation-PCR were used to validate and evaluate the molecular expressions of interest in the collected breast cancer tissues and cell lines. miR-7 overexpression affecting metastasis of BCSCs was further evaluated in mice. The endothelial cell-selective adhesion molecule (ESAM) was highly expressed in breast cancer tissues and in BCSC-driven xenografts. Results of the dual-luciferase reporter and chromatin immunoprecipitation-PCR indicated that the miR-7 mimic reduced RELA expression by directly targeting the 3′ UTR of RELA to inhibit ESAM expression in MDA-MB-231 cells. Moreover, the expression levels of RELA, CD44, and ESAM were significantly decreased in lentivirus (Lenti)-miR-7-BCSC-driven xenografts compared with the control xenografts, accompanied with an increase in E-cadherin and a decrease in vimentin expression, as well as reduction in tumor growth and metastasis to lungs. Our data demonstrated that miR-7 overexpression reduced the metastasis of BCSCs via inhibiting ESAM, suggesting that ESAM could be a potential target for breast cancer therapy.