Notch signaling regulates CXCR4 expression and the migration of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have been used to repair injured tissues through immune-suppression and/or cell replace mechanisms. However, a significant barrier to MSC therapy is insufficient MSC engraftment in injured tissues after systemic administration. Here, we report that cell surface, total protein, and mRNA levels of CXCR4 were significantly increased in MSCs when Notch signaling was interrupted by γ-secretase inhibitor (GSI) or knockout of the transcription factor RBP-J, which mediates signaling from all four mammalian Notch receptors. The GSI-treated or RBP-J deficient MSCs showed stronger migration toward stromal cell-derived factor-1α (SDF-1α) than that of the control. In a mouse hepatic ischemia/reperfusion model, RBP-J deficient MSCs migrated into the injured liver tissues at a significantly higher efficiency than that of the control MSCs. Mice transfused with RBP-J deficient MSCs showed reduced liver damage. Therefore, Notch signaling regulates MSC migration and function, at least partially via the modulation of CXCR4 expression.

Disruption of the transcription factor RBP-J results in osteopenia attributable to attenuated osteoclast differentiation.

The transcription factor recombination signal binding protein-Jκ (RBP-J) is the critical transcription factor downstream to all four mammalian Notch receptors. Although it has been reported that Notch signaling pathway is involved in bone remodeling, the importance of RBP-J in osteoclastogenesis has not been fully explored. To investigate the role of RBP-J in osteoclastogenesis, we conditionally deleted RBP-J systemically in bone marrow (BM) or specifically in macrophages. We found that disruption of RBP-J in BM resulted in an obvious decrease in trabecular bone mass associated with an increase in osteoclasts, leading to osteopenia. Disruption of RBP-J in macrophages phenocopied the phenotypes of RBP-J deletion in BM with respect to osteoclastogenesis, suggesting that the osteopenia in RBP-J deficient mice is essentially resulted from increased osteoclastogenesis. Furthermore, we found that RBP-J deletion in osteoclasts resulted in a dramatic increase in tartrate-resistant acid phosphatase expression. These findings demonstrate a negatively role of RBP-J in the differentiation of osteoclasts and suggest that Notch pathway may be a new therapeutic target for bone diseases related to increased osteoclastogenesis.

Soluble extracellular domains of human SIRPα and CD47 expressed in Escherichia coli enhances the phagocytosis of leukemia cells by macrophages in vitro.

Signal regulatory protein (SIRP) α, a transmembrane protein belonging to the immunoglobulin superfamily, is a receptor for CD47. The interaction between SIRPα and CD47 plays an important role in regulating the phagocytosis of leukemia cells and leukemia stem cells (LSCs) by macrophages. Blocking antibodies against CD47 have been shown to promote phagocytosis of LSCs by macrophages. Here, we consider an alternative way to interrupt the interaction between CD47 and SIRPα. We expressed the extracellular domains of the human SIRPα (hSIRP(ext)) and the human CD47 (hCD47(ext)) in Escherichia coli as Trx fusion proteins, and purified them by using affinity chromatography. We show that the purified fusion protein Trx-SIRP(ext) could interact in vitro with Trx-hCD47(ext). Moreover, Trx-SIRP(ext) could effectively bind to Jurkat T-ALL cells, which expressed CD47 at a high level. CD47(ext), on the other hand, bound to human macrophages. In vitro phagocytosis assay showed that these fusion proteins could enhance the phagocytosis of Jurkat cells by macrophage, with Trx-hSIRP(ext) showed a higher efficiency than Trx-CD47(ext). These results indicated that the soluble Trx-hSIRP(ext) and Trx-CD47(ext) polypeptides could be alternative molecules to interrupt CD47-SIRPα interaction between leukemia cells and macrophages, and might be potentially useful for the targeted therapy of leukemia.

Simulated microgravity hampers Notch signaling in the fight against myocardial ischemia­reperfusion injury.

The gravitational field is an important determinant of cardiovascular function. Exposure to microgravity during spaceflight may lead to a series of maladaptive alterations in the cardiovascular system. The authors have previously demonstrated that microgravity can increase the susceptibility to myocardial ischemia­reperfusion (IR) injury under simulated microgravity. Although Notch1 signaling protects against myocardial IR injury, whether Notch1 protects against myocardial IR injury under simulated weightlessness remains unknown. The present study is designed to investigate the role of the Notch1 receptor in myocardial IR injury under simulated weightlessness. The differences in Notch signaling expression and myocardial infarct size following myocardial IR were compared between normal rats and tail­suspended rats that were kept in 30˚ head­down tilt and hindlimb unloading position. The data revealed low expression levels of Notch1 receptor and its endogenous ligand Jagged1 in normal adult rat hearts. However, significantly higher expression of Notch1 was observed in the border zone compared with the infarcted area and the remote zone following myocardial IR. Notch1 expression was notably reduced in the infarcted hearts of tail­suspended rats compared with the control group. Conversely, the myocardial infarct size was significantly increased in tail­suspended rats compared with the control rats. In conclusion, these data suggested that the proper function of Notch signaling may be hampered under simulated microgravity.

Scorpion inhibits epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most malignant diseases worldwide. The unfavorable clinical outcome and poor prognosis are due to high rates of recurrence and metastasis after treatments. Some scholars of traditional Chinese medicine suggested that endogenous wind-evil had played an important role in metastasis of malignant tumor. Therefore, the drug of dispelling wind-evil could be used to prevent cancer metastasis and improve the poor prognosis. So we wondered whether Scorpion, one of the most important wind calming drugs, has antitumor effect especially in epithelial-mesenchymal transition (EMT) and metastasis of HCC in this research. We found that Scorpion-medicated serum could inhibit proliferation, induce apoptosis, and decrease migration and invasion capacity of Hepa1-6 cells in vitro. Meanwhile, we observed that water decoction of Scorpion restrained tumor growth and metastasis in nude mouse of HCC metastasis models. Further experiments showed that Scorpion could suppress EMT, which is characterized by increased epithelial marker E-cadherin expression and decreased mesenchymal markers N-cadherin and Snail expression following Scorpion treatment both in vitro and in vivo. These results suggested that the Scorpion could inhibit Hepa1-6 cells' invasion and metastasis in part by reversing EMT and providing a possible potential approach for preventing HCC metastasis. Impact statement The unfavorable clinical outcome and poor prognosis of hepatocellular carcinoma (HCC) are due to high rates of recurrence and metastasis after treatments. Here we found Scorpion, one of the most important wind calming drugs, has antitumor effect. Scorpion-medicated serum inhibited the proliferation, induced apoptosis, and decreased migration and invasion capacity of Hepa1-6 cells in vitro. Water decoction of Scorpion restrained tumor growth and metastasis in nude mouse of HCC metastasis models. Further experiments showed that Scorpion could suppress EMT of HCC both in vitro and in vivo. Our results suggested that the Scorpion could inhibit Hepa1-6 cells' invasion and metastasis in part by reversing EMT and providing a possible potential approach for preventing HCC metastasis.

Identification of differential gene expression profile from peripheral blood cells of military pilots with hypertension by RNA sequencing analysis.

BACKGROUND: Elevated blood pressure is an important risk factor for cardiovascular disease and is also an important factor in global mortality. Military pilots are at high risk of cardiovascular disease because they undergo persistent noise, high mental tension, high altitude hypoxia, high acceleration and high calorie diet. Hypertension is the leading cause of cardiovascular disease in military pilots. In this study, we want to identify key genes from peripheral blood cells of military pilots with hypertension. Identification of these genes may help diagnose and control hypertension and extend flight career for military pilots. METHODS: We use RNA sequencing technology, bioinformatics analysis and Western blotting to identify key genes from peripheral blood cells of military pilots with hypertension. RESULTS: Our study detected 121 up-regulated genes and 623 down-regulated genes in the peripheral blood mononuclear cells (PBMCs) from hypertensive military pilots. We have also identified 8 important genes (NME4, PNPLA7, GGT5, PTGS2, IGF1R, NT5C2, ENTPD1 and PTEN), a number of gene ontology categories and biological pathways that may be associated with military pilot hypertension. CONCLUSIONS: Our study may provide effective means for the prevention, diagnosis and treatment of hypertension for military pilot and extend their flight career.

Inhibition of tumor angiogenesis and tumor growth by the DSL domain of human Delta-like 1 targeted to vascular endothelial cells.

The growth of solid tumors depends on neovascularization. Several therapies targeting tumor angiogenesis have been developed. However, poor response in some tumors and emerging resistance necessitate further investigations of new drug targets. Notch signal pathway plays a pivotal role in vascular development and tumor angiogenesis. Either blockade or forced activation of this pathway can inhibit angiogenesis. As blocking Notch pathway results in the formation of vascular neoplasm, activation of Notch pathway to prevent tumor angiogenesis might be an alternative choice. However, an in vivo deliverable reagent with highly efficient Notch-activating capacity has not been developed. Here, we generated a polypeptide, hD1R, which consists of the Delta-Serrate-Lag-2 fragment of the human Notch ligand Delta-like 1 and an arginine-glycine-aspartate (RGD) motif targeting endothelial cells (ECs). We showed that hD1R could bind to ECs specifically through its RGD motif and effectively triggered Notch signaling in ECs. We demonstrated both in vitro and in vivo that hD1R inhibited angiogenic sprouting and EC proliferation. In tumor-bearing mice, the injection of hD1R effectively repressed tumor growth, most likely through increasing tumor hypoxia and tissue necrosis. The amount and width of vessels reduced remarkably in tumors of mice treated with hD1R. Moreover, vessels in tumors of mice treated with hD1R recruited more NG2(+) perivascular cells and were better perfused. Combined application of hD1R and chemotherapy with cisplatin and teniposide revealed that these two treatments had additive antitumor effects. Our study provided a new strategy for antiangiogenic tumor therapy.

Endothelium-targeted Delta-like 1 promotes hematopoietic stem cell expansion ex vivo and engraftment in hematopoietic tissues in vivo.

BACKGROUND: Notch ligands enhance ex vivo expansion of hematopoietic stem cells (HSCs). But to use Notch ligands in HSC therapies of human diseases, efforts are required to improve ex vivo expansion efficiency and in vivo transplant engraftment. DESIGN AND METHODS: We designed and produced an endothelium-targeted soluble Notch ligand, the DSL domain of Delta-like 1 fused with a RGD motif (D1R), and examined the effects of this protein on HSCs ex vivo and in vivo. RESULTS: D1R efficiently promoted ex vivo expansion of both mouse bone marrow (BM) and human umbilical cord blood HSCs. HSCs expanded with D1R up-regulated many of the stemness-related genes, and showed high BM engraftment efficacy with long-term repopulation capacity after transplantation. Moreover, in vivo administration of D1R increased the number of BM HSCs in mice, and facilitated BM recovery of mice after irradiation. Injection of D1R significantly improved HSC engraftment and myeloid recovery after BM transplantation in irradiated mice. D1R enhanced HSC engraftment not only in BM, but also in the liver and spleen after BM transplantation in mice. D1R induced the formation of compact cell clusters containing the transplanted HSCs in close contact with endothelial cells, reminiscent of HSC niches, in the liver and spleen. CONCLUSIONS: D1R might be applied in improving both HSC expansion ex vivo and HSC engraftment in vivo in transplantation.

Overexpression of Notch ligand Dll1 in B16 melanoma cells leads to reduced tumor growth due to attenuated vascularization.

Notch signaling plays an important role in vascular development and tumor angiogenesis. It has been shown that disruption of Dll4-triggered Notch signal activation effectively inhibits tumor growth, but this treatment also results in the formation of vascular neoplasms. In this study, we investigate the effects of over-expressing Notch ligand Dll1 in B16 melanoma cells on tumor cell proliferation and tumor growth in vitro and in vivo. Our results showed that over-expression of Dll1 could activate Notch signaling in tumor cells, and promote tumor cell proliferation in vitro. In contrast, growth of Dll1-over-expressing tumors in vivo was reduced, due to abnormal tumor vessel formation. Impaired tumor vasculature enhanced hypoxia and necrosis in tumor tissues, leading to retarded tumor growth. These results suggest that activation of Notch signaling may serve as an anti-angiogenesis strategy in the treatment of malignant tumors.