CRISPR/Cas9 Edited sRAGE-MSCs Protect Neuronal Death in Parkinson's Disease Model

BACKGROUND AND OBJECTIVES: Parkinson’s disease (PD) is a fatal and progressive degenerative disease of the nervous system. Until recently, its promising treatment and underlying mechanisms for neuronal death are poorly understood. This study was investigated to identify the molecular mechanism of neuronal death in the substantia nigra and corpus striatum of PD. METHODS: The soluble RAGE (sRAGE) secreting Umbilical Cord Blood—derived Mesenchymal Stem Cell (UCB-MSC) was generated by gene editing method using clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9). These cells were transplanted into Corpus Striatum of rotenone-induced PD animal models then behavioral test, morphological analysis, and immunohistochemical experiments were performed to determine the neuronal cell death and recovery of movement. RESULTS: The neuronal cell death in Corpus Striatum and Substantia Nigra was dramatically reduced and the movement was improved after sRAGE secreting UCB-MSC treatment in PD mice by inhibition of RAGE in neuronal cells. CONCLUSIONS: We suggest that sRAGE secreting UCB-MSC based therapeutic approach could be a potential treatment strategy for neurodegenerative disease including PD.

Thrombospondin-1 and Inhibition of Tumor Growth / 체질인류학회지

Thrmobospondin-1 is the multifunctional protein that modulates endothelial cell and tumor cell behavior via several cell surface receptors and inhibits angiogenesis. In vitro, thrombospondin-1 alters adhesion, proliferation, motility, and survival of endothelial and cancer cells. Studies have confirmed that increased TSP-1 expression suppresses growth or metastasis of some tumors and inhibits angiogenesis. In the past three decades, inhibitors of angiogenesis have been developed as regulators target the vascular endothelial growth factor (VEGF) signaling pathway and small molecule tyrosine kinase inhibitors have been clinically approved. TSP-1 has several functional domain structures and inhibits tumor angiogenesis by engaging receptors CD36 and CD47. TSP-1 binding to CD47 dissociates it from VEGFR2, inhibiting downstream AKT activation and functional responses of endothelial cells to VEGF. Recently, macrophage phagocytosis and cytotoxic T-cell induction of tumor cells mediated by CD47-specific blocking antibodies have been proposed. These findings provide a new therapeutic paradigm for elinination of cancer cells and inhibition of angiogenesis of tumor by TSP-1.

Inhibition of Angiogenesis by the First Type I Repeat Peptides of Thrombospondin-1 / 체질인류학회지

Angiogenesis is the fundamental biological phenomenon in the development of vertebrates and various pathophysiological process such as cancer, inflammation and wound healing. Thrombospondin-1 is a well-known anti-angiogenic molecule which is distributed in the extracellular matrix of various tissues. The second and third type I repeats of human TSP-1 have inhibitory effects on endothelial cell migration and induce angiogenesis inhibition. However the role of the first type I repeat was not elucidated. In addition, the first type I repeat of bovine TSP-1 has CSVTCG amino acid sequence which is known to have anti-angiogenic activity. In the present study, we compared the inhibition of angiogenesis to investigate the role of the first type I repeat of the human and bovine TSP-1. Matrigel was mixed with or without TSR-1 peptides and then injected into C57BL/6J mice. We compared angiogenesis inhibition activity by hemoglobin assay, microvessel density and optical density value after 7 days. Furthermore, inhibition of angiogenesis was confirmed on CAM assay by TSR-1 peptides. For in vitro angiogenesis assay, TSR-1 peptides were treated on the proliferation, migration, and tube formation assay of HUVEC. Apoptosis effect of TSR-1 peptides was confirmed by apoptosis assay kit and flow cytometry. Bovine and human TSR-1 peptides blocked neovascularization in in vivo Matrigel plug assay and CAM assay at 10 microM. Bovine TSR-1 peptides have shown stronger angiogenesis inhibition in bFGF-induced angiogenesis than human TSR-1 and CSVTCG peptides. However, all of TSR-1 peptides inhibit migration and tube formation of HUVEC in in vitro. Furthermore, these peptides also induced apoptosis of HUVEC. These results suggest that TSR-1 peptides of bovine and human TSP-1 have angiogenesis inhibition activity.

Caffeine-induced endothelial cell death and the inhibition of angiogenesis / 대한해부학회지

Numerous studies have shown that adenosine or adenosine agonists can stimulate angiogenesis. However, the effect of caffeine (a known adenosine receptor antagonist) on angiogenesis has not been previously studied. Accordingly, this study was undertaken to examine the effect of caffeine on angiogenesis and to clarify the mechanism involved. Chick chorioallantoic membrane assays were used to investigate the effect of caffeine on angiogenesis and proliferation assays using human umbilical vein endothelial cells (HUVECs), were used to study its effects on specific aspects of angiogenesis. The expressions of caspase-3 and Bcl-2 were examined by western blotting, immunofluorescence staining was used to identify HUVEC morphological changes, and fluorescence activated cell sorting (FACS) and DAPI staining were used to detect HUVEC apoptosis. Caffeine was found to inhibit blood vessel formation dose-dependently and to inhibit the proliferation of HUVECs time- and dose-dependently. FACS analysis and DAPI staining showed that inhibitory effect of caffeine on HUVEC proliferation was the result of apoptosis and the up-regulation of thrombospondin-1 (TSP-1). Furthermore, TSP-1 levels were down-regulated by NECA but were unaffected by CGS21680, indicating that caffeine regulated TSP-1 expression via adenosine A2B receptor. In addition, caffeine up-regulated caspase-3 and down-regulated Bcl-2 at the protein level. These results suggest that the inhibitory effect of caffeine on angiogenesis is associated, at least in part, with its induction of endothelial cell apoptosis, probably mediated by a caspase-3 dependent mechanism.

Activated microglial cells synthesize and secrete AGE-albumin / 대한해부학회지

A holy grail of curing neurodegenerative diseases is to identify the main causes and mechanisms underlying neuronal death. Many studies have sought to identify these targets in a wide variety of ways, but a more important task is to identify critical molecular targets and their origins. Potential molecular targets include advanced glycation end products (AGEs) that can promote neuronal cell death, thereby contributing to neurodegenerative disorders such as Alzheimer disease or Parkinson disease. In this study, we showed that AGE-albumin (glycated albumin) is synthesized in microglial cells and secreted in the human brain. Our results provide new insight into which microglial cells can promote the receptor for AGE-mediated neuronal cell death, eventually leading to neurodegenerative diseases.

Vascular Endothelial Growth Factor - Its Relation to Neovascular ization and Their Significance as Prognostic Factors in Renal Cell Carcinoma

Angiogenesis is a series of processes that include endothelial proliferation, migration and tube formation. Vascular endothelial growth factor (VEGF) is regarded as a potent mediator of angiogenesis, vascular permeability and tumor cell growth in renal cell carcinoma. This study was designed to evaluate the expression of VEGF and the microvessel count (MVC) and to determine their prediction efficacies for prognosis in renal cell carcinoma. The relationship between the expression of VEGF and MVC were evaluated immunohistochemically in 50 patients with renal cell carcinoma who received a radical nephrectomy at Wonju Christian Hospital between 1989 and 1997. Microvessels were identified by immunostaining endothelial cells for CD-31 antigen. The mean follow-up was 96 months (3 - 133 months). Overall 5-year survival rate was 71.5%. VEGF was expressed in the tumor cell cytoplasm. Of the 50 tumors, 23 (46%) were weak to strongly positive for VEGF but 27 (54%) were unreactive. The respective 5-year survival rates for patients with positive and negative expressions of VEGF were 70% and 73% (p > 0.05). The overall mean MVC was 13.4 in a 400x field. Mean MVCs were significantly higher in VEGF-positive tumors (17.6 +/- 12.1) than in VEGF-negative tumors (9.9 +/- 5.4), and the MVCs of the high vascular density group and the low ascular density groups were significantly different. The 5-year survival rates of patients with high vascular density and low vascular density were 59% and 86%. The median survival period for patients with MVCs higher than or equal to 10 vessels/field was 85 months, whereas for those with MVCs lower than 10 vessels/field the median survival time was 102 months. These results suggest that MVC may be a better prognostic factor in renal cell carcinoma than the expression of VEGF.

Expression of alpha3beta1 Integrin in ECV304 Endothelial Cells and Angiogenesis / 대한해부학회지

Angiogenesis is a fundamental biological process including endothelial cell adhesion, migration, invasion and tube formation. Integrin receptors of endothelial cells play important roles in angiogenesis. They mediate cell-cell contact and cell adhesion to extracellular matrix. Roles of integrins have been described for a number of cell types. ECV304 endothelial cells were known to overexpress alpha3beta1 integrin and to form tube like structure in 3-D Matrigel culture. However the function of alpha3beta1 integrin in endothelial cells remains to be determined. Therefore, we have investigated morphological characteristics of ECV304 cells and roles of alpha3beta1 integrin in angiogenesis. To elucidate several characteristics, ECV304 endothelial cells were compared with HUVEC in the aspect of morphology, localization of integrins, angiogenesis pattern. In addition, role of alpha3beta1 integrin were analyzed in the aspect of endothelial cell binding, migration, invasion and tube formation on Matrigel. The result showed that alpha3beta1 integrin overexpressed ECV304 endothelial cells showed strong adhesiveness to extracellular matrix proteins, and high migration and invasion activities. Furthermore, expression of alpha3beta1 integrin was increased according to time course during in vitro culture and was continuously strong in ECV304 cells on 3-D Matrigel culture. These results indicate that alpha3beta1 integrin is able to be a critical component in control of angiogenesis by regulation of cell adhesion, migration, invasion and tube formation of ECV304 endothelial cells.

Observation of Endothelial Cell Differentiation by 3D Cell culture system: Angiogenesis inhibition of Thrombospondin-1 / 대한해부학회지

Angiogenesis plays a fundamental role in development of circulation system, reorganization of reproductive system, wound healing. Pathological angiogenesis is deeply involved in a variety of diseases, particularly solid tumor growth and metastasis. However, it is not easy to study the mechanism of angiogenesis because endothelial cells proceed complex differentiation by interaction with extracellular matrix proteins and growth factors. However, human umbilical vein endothelial cells (HUVEC) form polygonal networks of capillary-like tubes in 3D Matrigel cultures. Differentiation of endothelial cells will be observed accurately by application of videomicroscopy. Thrombospondin-1 is secreted by a wide variety of cells including endothelial cells and is incorporated into their matrix. Thrombospondin-1 can modulate differentiation of endothelial cells by increasing cell-cell interactions as well as cell-substrate interactions. The current study was undertaken to determine which mechanism is involved in inhibition of angiogenesis by Thrombospondin-1. They was secreted from HUVEC during the process of angiogenesis in 3D Matrigel culture. When applied to endothelial cells attachment to the surface of Matrigel was not decreased, but spreading was decreased. In addition, bigger clusters was formed by enhancement of cell to cell binding by Thrombospondin-1. They inhibit cord and tube formation of HUVEC by inhibition of migration. These results suggest that Thrombospondin-1 inhibits angiogenesis by blocking differentiation of endothelial cells to motile phenotype in 3D Matrigel culture.

Light and scanning electron microscopic observation of the mouse oocytes ovulated before metaphase II stage / 대한불임학회지

No abstract available.