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 δ-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.

Interaction between platelets and endothelial progenitor cells via LPA-Edg-2 axis is augmented by PPAR-δ activation.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-δ is a nuclear receptor regulating cell metabolism. The role of PPAR-δ in late endothelial progenitor cells (EPCs) has not been fully elucidated. We aim to understand the effects of PPAR-δ activation on late EPC and to reveal the underlying mechanism. METHODS AND RESULTS: Treatment with a highly selective PPAR-δ agonist (GW501516) induced proliferation of late EPCs and enhanced their vasculogenic potential. Search for the target molecule of PPAR-δ activation revealed endothelial differentiation gene (Edg)-2. Chromatin immunoprecipitation and promoter assays demonstrated that Edg-2 gene was specifically induced by PPAR-δ through direct transcriptional activation. Lysophosphatidic acid (LPA), an Edg ligand, mimicked the pro-vasculogenic effects of GW501516 in late EPCs whereas Edg antagonist (Ki16425) blocked these effects. Edg-2 is a membrane receptor for LPA which is a major growth factor from activated platelets. Thus, the interaction between platelets and late EPCs via the LPA-Edg-2 axis was assessed. Platelet supernatant boosted the pro-vasculogenic effects of GW501516, which was reversed by antagonist to PPAR-δ (GSK0660) or Edg (Ki16425). Both of in vivo Matrigel plug model and mouse skin punch-wound model demonstrated that the combination of platelets and PPAR-δ-activated late EPCs synergistically enhanced vascular regeneration. CONCLUSIONS: There exists a synergistic interaction between human platelets and late EPCs leading to vascular regeneration. This interaction consists of LPA from platelets and its receptor Edg-2 on the surface of EPCs and can be potentiated by PPAR-δ activation in EPCs. A PPAR-δ agonist is a good candidate to achieve vasculogenesis for ischemic vascular disease.

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.

Secondary sphere formation enhances the functionality of cardiac progenitor cells.

Loss of cardiomyocytes impairs cardiac function after myocardial infarction (MI). Recent studies suggest that cardiac stem/progenitor cells could repair the damaged heart. However, cardiac progenitor cells are difficult to maintain in terms of purity and multipotency when propagated in two-dimensional culture systems. Here, we investigated a new strategy that enhances potency and enriches progenitor cells. We applied the repeated sphere formation strategy (cardiac explant → primary cardiosphere (CS) formation → sphere-derived cells (SDCs) in adherent culture condition → secondary CS formation by three-dimensional culture). Cells in secondary CS showed higher differentiation potentials than SDCs. When transplanted into the infarcted myocardium, secondary CSs engrafted robustly, improved left ventricular (LV) dysfunction, and reduced infarct sizes more than SDCs did. In addition to the cardiovascular differentiation of transplanted secondary CSs, robust vascular endothelial growth factor (VEGF) synthesis and secretion enhanced neovascularization in the infarcted myocardium. Microarray pathway analysis and blocking experiments using E-selectin knock-out hearts, specific chemicals, and small interfering RNAs (siRNAs) for each pathway revealed that E-selectin was indispensable to sphere initiation and ERK/Sp1/VEGF autoparacrine loop was responsible for sphere maturation. These results provide a simple strategy for enhancing cellular potency for cardiac repair. Furthermore, this strategy may be implemented to other types of stem/progenitor cell-based therapy.

Gata6 in pluripotent stem cells enhance the potential to differentiate into cardiomyocytes.

Pluripotent stem cell (PSC) variations can cause significant differences in the efficiency of cardiac differentiation. This process is unpredictable, as there is not an adequate indicator at the undifferentiated stage of the PSCs. We compared global gene expression profiles of two PSCs showing significant differences in cardiac differentiation potential. We identified 12 up-regulated genes related to heart development, and we found that 4 genes interacted with multiple genes. Among these genes, Gata6 is the only gene that was significantly induced at the early stage of differentiation of PSCs to cardiomyocytes. Gata6 knock-down in PSCs decreased the efficiency of cardiomyocyte production. In addition, we analyzed 6 mESC lines and 3 iPSC lines and confirmed that a positive correlation exists between Gata6 levels and efficiency of differentiation into cardiomyocytes. In conclusion, Gata6 could be utilized as a biomarker to select the best PSC lines to produce PSC-derived cardiomyocytes for therapeutic purposes. [BMB Reports 2018; 51(2): 85-91].