Jae-Il Choi

CD82/KAI1 Maintains the Dormancy of Long-Term Hematopoietic Stem Cells through Interaction with DARC-Expressing Macrophages

Hematopoiesis is regulated by crosstalk between long-term repopulating hematopoietic stem cells (LT-HSCs) and supporting niche cells in the bone marrow (BM). Here, we examine the role of CD82/KAI1 in niche-mediated LT-HSC maintenance. We found that CD82/KAI1 is expressed predominantly on LT-HSCs and rarely on other hematopoietic stem-progenitor cells (HSPCs). In Cd82(-/-) mice, LT-HSCs were selectively lost as they exited from quiescence and differentiated. Mechanistically, CD82-based TGF-β1/Smad3 signaling leads to induction of CDK inhibitors and cell-cycle inhibition. The CD82 binding partner DARC/CD234 is expressed on macrophages and stabilizes CD82 on LT-HSCs, promoting their quiescence. When DARC(+) BM macrophages were ablated, the level of surface CD82 on LT-HSCs decreased, leading to cell-cycle entry, proliferation, and differentiation. A similar interaction appears to be relevant for human HSPCs. Thus, CD82 is a functional surface marker of LT-HSCs that maintains quiescence through interaction with DARC-expressing macrophages in the BM stem cell niche.

High glucose-induced jagged 1 in endothelial cells disturbs notch signaling for angiogenesis: A novel mechanism of diabetic vasculopathy

Angiogenesis is a multistep process which is orchestrated by intercellular signaling. We developed an in vitro model of human angiogenesis to identify a pathologic angiogenesis and intercellular signaling in high glucose condition. We co-cultivated human endothelial cells (ECs) and smooth muscle cells (SMCs) in a spheroid on an SMC monolayer for 7 days either in high glucose or in control condition. We analyzed vascular growth and expression of notch or its ligands with confocal microscopy. Abnormal angiogenesis by high glucose condition was characterized by (1) increased sprouting and branching (high glucose vs. normal: number of sprouts 20.3±1.5 vs. 13.7±2.9, p=0.024; number of branching points 7.6±2.5 vs. 2.3±2.1, p=0.047), (2) decreased vascular diameter (diameter of the tubes 13.4±6. 1μm vs. 19.1±8.8 μm, p=0.012) and (3) destabilization of the tubes. We identified that high glucose induced jagged 1 and suppressed notch1 in ECs whereas it did not affect Dll4. Constitutive jagged 1 overexpression or inhibition of notch1 in ECs induced abnormal angiogenesis as the high glucose condition did. Endothelial-specific shRNA targeting jagged 1 rescued the aberrant angiogenesis in high glucose condition. High glucose condition induced an abnormal endothelial intercellular signaling leading to aberrant angiogenesis. It is a novel mechanism of diabetic microvasculopathy which can be a therapeutic target beyond glucose control.

Highly angiogenic CXCR4+CD31+ monocyte subset derived from 3D culture of human peripheral blood

Ex vivo expansion of human circulating angiogenic cells is a major challenge in autologous cell therapy for ischemic diseases. Here, we demonstrate that hematosphere-derived CXCR4(+)CD31(+) myeloid cells using peripheral blood possess robust proangiogenic capacity such as formation of vessel-like structures and tip cell-like morphology in Matrigel. We also found that CD31 positive myeloid cells are principal cellular component of hematospheres by magnetic cell sorting. Flow cytometry analysis showed that fresh peripheral blood contained 40.3 ± 15.2% of CXCR4(+)CD31(+) myeloid cells, but at day 5 of hematosphere culture, most of myeloid cells were CXCR4(+)CD31(+) by 86.9 ± 5.4%. Hematosphere culture significantly increased the production of angiogenic niche-supporting cytokines. Moreover, CD31-homophilic interaction and VEGF-VEGF receptor loop signaling were essential for sphere formation and acquisition of angiogenic capacity in hematospheres. Matrigel plug and ischemic hindlimb model provide in vivo evidence that hematosphere-derived myeloid cells have highly vasculogenic capacities, participate in new and mature vessel formation, and exert therapeutic effects on ischemic hindlimb. In conclusion, our strategy for ex vivo expansion of human CXCR4(+)CD31(+) angiogenic cells using hematospheres provides an autologous therapeutic cell source for ischemic diseases and a new model for investigating the microenvironment of angiogenesis.

Diabetes-Induced Jagged1 Overexpression in Endothelial Cells Causes Retinal Capillary Regression in a Murine Model of Diabetes Mellitus: Insights Into Diabetic Retinopathy.

Background: Several mechanisms have been proposed to account for diabetes-induced microvasculopathy (DMV). Although Notch signaling was reported to be affected by glucose metabolism in endothelial cells during developmental angiogenesis, it has not been investigated in vascular remodeling of adult capillaries in relation to diabetes mellitus. Methods: We induced diabetes mellitus in 8-week-old adult mice by intravenously administering streptozotocin. After 6 weeks, we harvested organs, including retina, heart, and skeletal muscle, and evaluated the capillaries with immunofluorescence and confocal microscopy. We modulated endothelial Notch signaling using chemical inhibitors in wild-type mice or transgenic mice, inducing conditional knockout of Jagged1 or Mib1. Results: DMV was characterized by capillary remodeling, regression, and decreased density. Notch ligand Jagged1, but not &dgr;-like ligand 4, was markedly increased in endothelial cells of diabetic mice. Using endothelium-specific Jagged1 knockdown mice, we found that blocking Jagged1 prevented DMV even under diabetic conditions. Furthermore, in the inducible endothelium-specific Jagged1 knockdown mice, blocking Jagged1 even at 4 weeks after the establishment of DMV could reverse it, leading to normalization of retinal vasculature. A search for downstream signals revealed that diabetes mellitus decreased the nuclear localization of Notch1 intracellular domain and reduced the expression of VE-cadherin and N-cadherin in endothelial cells. Chemical Notch inhibition phenocopied DMV in normal mice. Conclusions: Our findings indicate that diabetes mellitus induces Jagged1 overexpression and suppresses Notch signaling in endothelial cells, leading to DMV in adult mice. We conclude that dysregulated intercellular Notch signaling may be a novel mechanism of DMV.

Erythropoietin priming improves the vasculogenic potential of G-CSF mobilized human peripheral blood mononuclear cells

AIMS From our previous clinical trials, intracoronary infusion of granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells ((mob)PBMCs) proved to be effective in improving myocardial contractility and reducing infarct volume in acute myocardial infarction. We tested the effect of priming (mob)PBMCs with erythropoietin (EPO) to augment its therapeutic efficacy. METHODS AND RESULTS (mob)PBMCs were obtained from healthy volunteers after a 3-day subcutaneous injection of G-CSF (10 μg/kg). About 40% of (mob)PBMCs were EPO receptor (EPOR) (+) and responded to 6 h EPO-priming (10 IU/mL) by increasing the expression of vasculogenic factors (i.e. IL8, IL10, bFGF, PDGF, MMP9) and adhesion molecules (i.e. integrin αV, β1, β2, β8) through the JAK2 and Akt pathway. These responses were also observed in PBMCs from elderly patients with coronary disease. The conditioned media from EPO-primed (mob)PBMCs contained various cytokines such as IL8, IL10, TNFα, and PDGF, which enhanced the migration and tube formation capability of endothelial cells. EPO-primed (mob)PBMCs also showed increased adhesion on endothelial cells or fibronectin. Augmented vasculogenic potential of EPO-primed (mob)PBMCs was confirmed in a Matrigel plug assay, ischaemic hindlimb, and myocardial infarction models of athymic nude mice. There were two action mechanisms: (i) cellular effects confirmed by direct incorporation of human (mob)PBSCs into mouse vasculature and (ii) indirect humoral effects confirmed by the therapeutic effect of the supernatant of EPO-primed (mob)PBMCs. CONCLUSION Brief ex vivo EPO-priming is a novel method to augment the vasculogenic potential of human (mob)PBMCs, which would help to achieve better results after intracoronary infusion in myocardial infarction patients.

Human Podoplanin-positive Monocytes and Platelets Enhance Lymphangiogenesis Through the Activation of the Podoplanin/CLEC-2 Axis

Emerging studies suggested that murine podoplanin-positive monocytes (PPMs) are involved in lymphangiogenesis. The goal of this study was to demonstrate the therapeutic lymphangiogenesis of human PPMs by the interaction with platelets. Aggregation culture of human peripheral blood mononuclear cells (PBMCs) resulted in cellular aggregates termed hematospheres. During 5-day culture, PPMs expanded exponentially and expressed several lymphatic endothelial cell-specific markers including vascular endothelial growth factor receptor (VEGFR)-3 and well-established lymphangiogenic transcription factors. Next, we investigated the potential interaction of PPMs with platelets that had C-type lectin-like receptor-2 (CLEC-2), a receptor of podoplanin. In vitro coculture of PPMs and platelets stimulated PPMs to strongly express lymphatic endothelial markers and upregulate lymphangiogenic cytokines. Recombinant human CLEC-2 also stimulated PPMs through Akt and Erk signaling. Likewise, platelets in coculture with PPMs augmented secretion of a lymphangiogenic cytokine, interleukin (IL)-1-β, via podoplanin/CLEC-2 axis. The supernatant obtained from coculture was able to enhance the migration, viability, and proliferation of lymphatic endothelial cell. Local injection of hematospheres with platelets significantly increased lymphatic neovascularization and facilitated wound healing in the full-thickness skin wounds of nude mice. Cotreatment with PPMs and platelets augments lymphangiogenesis through podoplanin/CLEC-2 axis, which thus would be a promising novel strategy of cell therapy to treat human lymphatic vessel disease.

New method to differentiate human peripheral blood monocytes into insulin producing cells: Human hematosphere culture

Strategy to differentiate stem cells into insulin producing cells (IPCs) in vitro has been a promising one to get cell source of β-cell replacement therapy for diabetes. It has been suggested that islets and neurons share features and nestin-positive cells could differentiate into IPCs. We have recently developed a three-dimensional culture system using human peripheral blood cells named as blood-born hematosphere (BBHS). Here we showed that most of BBHS were composed of nestin-positive cells. Under the four-stage differentiation protocol for IPCs, we plated nestin-positive BBHS onto fibronectin-coated dish. These cells form islet-like clusters and most of them expressed insulin. Pancreatic specific genes were turned on, such as transcription factors (Pdx-1, Ngn3 and Nkx6.1), genes related to endocrine function (Glut-2 and PC2) or β cell function (Kir6.2, SUR1). Furthermore islet differentiation was confirmed by dithizone (DTZ) staining to detect zinc ion which binds insulin protein within the cells. Finally, IPCs derived from BBHS showed capability to secrete insulin in response to glucose stimulation. Taken together, our novel protocol successfully induced islet-like human insulin producing cells out of BBHS. This strategy of ex vivo expansion of IPCs using BBHS provides an autologous therapeutic cell source for the treatment of diabetes.

Shedding light on the DARC knight as a guardian of hematopoietic stem cell quiescence

In-depth understanding of the crosstalk between hematopoietic stem cells (HSCs) and their niche, and identification of ‘functional’ surface markers of HSCs are vital in the advancement of therapies for hematological disorders (e.g., ex vivo expansion or transplantation of HSCs). MacNamara (1) and Perez-Fernandez et al . (2) respectively provided constructive comments on our results and made very interesting suggestions regarding clinical application of the CD82/DARC axis. In this Correspondence, we aim to add to the discussion (1,2) on our recent article (3).

Abstract 917: CD82/KAI1 maintains the dormancy of long-term hematopoietic stem cells through interaction with DARC-expressing macrophages

Hematopoiesis is regulated by crosstalk between long-term repopulating hematopoietic stem cells (LT-HSCs) and supporting niche cells in the bone marrow (BM). Here, we examine the role of CD82/KAI1 (CD82 hereafter) in niche-mediated LT-HSC maintenance. We found that CD82 is expressed predominantly on LT-HSCs and rarely on other hematopoietic stem-progenitor cells (HSPCs). In Cd82 -/- mice, LT-HSCs were selectively lost as they exited from quiescence and differentiated. Mechanistically, CD82-based TGF-b1/Smad3 signaling leads to induction of CDK inhibitors and cell-cycle inhibition. The CD82 binding partner DARC/CD234 is expressed on macrophages and stabilizes CD82 on LT-HSCs, promoting their quiescence. When DARC + BM macrophages were ablated, the level of surface CD82 on LT-HSCs decreased, leading to cell-cycle entry, proliferation, and differentiation. A similar interaction appears to be relevant for human HSPCs. Thus, CD82 is a functional surface marker of LT-HSCs that maintains quiescence through interaction with DARC-expressing macrophages in the BM stem cell niche. Finally, we hope that further development of our study will allow us to improve leukemia treatment by awakening not only “good” stem cells to keep beneficial stem cell functions, but also “bad” stem cells (e.g. cancer stem cells) in order to increase the sensitivity to treatment and avoid relapse. Citation Format: Jae-Il Choi, Jin Hur, Hwan Lee, Pniel Nham, Cheong-Whan Chae, Young-Eun Choi, Taewan Kim, Ga-Young Lee, Sung Hee Baek, Hyo-Soo Kim. CD82/KAI1 maintains the dormancy of long-term hematopoietic stem cells through interaction with DARC-expressing macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 917. doi:10.1158/1538-7445.AM2017-917

Diabetes-Induced Jagged1 Overexpression in Endothelial Cells Causes Retinal Capillary Regression in a Murine Model of Diabetes MellitusClinical Perspective: Insights Into Diabetic Retinopathy

Background: Several mechanisms have been proposed to account for diabetes-induced microvasculopathy (DMV). Although Notch signaling was reported to be affected by glucose metabolism in endothelial cells during developmental angiogenesis, it has not been investigated in vascular remodeling of adult capillaries in relation to diabetes mellitus. Methods: We induced diabetes mellitus in 8-week-old adult mice by intravenously administering streptozotocin. After 6 weeks, we harvested organs, including retina, heart, and skeletal muscle, and evaluated the capillaries with immunofluorescence and confocal microscopy. We modulated endothelial Notch signaling using chemical inhibitors in wild-type mice or transgenic mice, inducing conditional knockout of Jagged1 or Mib1. Results: DMV was characterized by capillary remodeling, regression, and decreased density. Notch ligand Jagged1, but not &dgr;-like ligand 4, was markedly increased in endothelial cells of diabetic mice. Using endothelium-specific Jagged1 knockdown mice, we found that blocking Jagged1 prevented DMV even under diabetic conditions. Furthermore, in the inducible endothelium-specific Jagged1 knockdown mice, blocking Jagged1 even at 4 weeks after the establishment of DMV could reverse it, leading to normalization of retinal vasculature. A search for downstream signals revealed that diabetes mellitus decreased the nuclear localization of Notch1 intracellular domain and reduced the expression of VE-cadherin and N-cadherin in endothelial cells. Chemical Notch inhibition phenocopied DMV in normal mice. Conclusions: Our findings indicate that diabetes mellitus induces Jagged1 overexpression and suppresses Notch signaling in endothelial cells, leading to DMV in adult mice. We conclude that dysregulated intercellular Notch signaling may be a novel mechanism of DMV.