PEDF promotes the repair of bone marrow endothelial cell injury and accelerates hematopoietic reconstruction after bone marrow transplantation

Ju, Wen; Lu, Wenjing; Ding, Lan; Bao, Yurong; Fu, Hong; Chen, Yuting; Gao, Hui; Xu, Xiao‐Qi; Wang, Guozhang; Wang, Weiwei; Zhang, Xi; Fu, Chunling; Qi, Kunming; Li, Zhenyu; Xu, Kailin; Qiao, Jianlin; Zeng, Lingyu

doi:10.1186/s12929-020-00685-4

Cited by 17 publications

(14 citation statements)

References 46 publications

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“…Like our previous study on another anti-angiogenic molecule, PEDF [ 71 ], Robo4 in ECs ameliorates the IR-induced inhibitory effect on HSC functions in our coculture model. HSC has various biological characteristics, the most important of which is the ability to self-renew and infinitely proliferate [ 72 ].…”

Section: Discussionsupporting

confidence: 80%

Endothelial Robo4 suppresses endothelial-to-mesenchymal transition induced by irradiation and improves hematopoietic reconstitution

Adzraku,

Cao,

Zhou

et al. 2024

Cell Death Dis

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Bone marrow ablation is routinely performed before hematopoietic stem cell transplantation (HSCT). Hematopoietic stem and progenitor cells (HSPCs) require a stable bone marrow microenvironment to expand and refill the peripheral blood cell pool after ablation. Roundabout guidance receptor 4 (Robo4) is a transmembrane protein exclusive to endothelial cells and is vital in preserving vascular integrity. Hence, the hypothesis is that Robo4 maintains the integrity of bone marrow endothelial cells following radiotherapy. We created an endothelial cell injury model with γ-radiation before Robo4 gene manipulation using lentiviral-mediated RNAi and gene overexpression techniques. We demonstrate that Robo4 and specific mesenchymal proteins (Fibronectin, Vimentin, αSma, and S100A4) are upregulated in endothelial cells exposed to irradiation (IR). We found that Robo4 depletion increases the expression of endoglin (CD105), an auxiliary receptor for the transforming growth factor (TGF-β) family of proteins, and promotes endothelial-to-mesenchymal transition (End-MT) through activation of both the canonical (Smad) and non-canonical (AKT/NF-κB) signaling pathways to facilitate Snail1 activation and its nuclear translocation. Endothelial Robo4 overexpression stimulates the expression of immunoglobulin-like adhesion molecules (ICAM-1 and VCAM-1) and alleviates irradiation-induced End-MT. Our coculture model showed that transcriptional downregulation of endothelial Robo4 reduces HSPC proliferation and increases HSC quiescence and apoptosis. However, Robo4 overexpression mitigated the damaged endothelium’s suppressive effects on HSC proliferation and differentiation. These findings indicate that by controlling End-MT, Robo4 preserves microvascular integrity after radiation preconditioning, protects endothelial function, and lessens the inhibitory effect of damaged endothelium on hematopoietic reconstitution.

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Section: Discussionsupporting

confidence: 80%

Endothelial Robo4 suppresses endothelial-to-mesenchymal transition induced by irradiation and improves hematopoietic reconstitution

Adzraku,

Cao,

Zhou

et al. 2024

Cell Death Dis

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“…Even though there were no considerable changes in claudin-5 and ZO-1 distribution after Robo4 knockdown, we observed irregular patterning and disrupted organization of these TJs following irradiation. Several reports suggest that IR disrupts claudin-5, occludin, PECAM-1, and ZO-1 [ 55 , 56 ], but what is unknown is the role Robo4 plays in regulating these TJ molecules against the damaging effects of irradiation. Apart from occludin, the expression of claudin-5, PECAM-1, and ZO-1 were upregulated after lentiviral-mediated Robo4 overexpression.…”

Section: Discussionmentioning

confidence: 99%

Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions

et al. 2023

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Background Hematopoietic stem cell transplantation involves irradiation preconditioning which causes bone marrow endothelial cell dysfunction. While much emphasis is on the reconstitution of hematopoietic stem cells in the bone marrow microenvironment, endothelial cell preservation is indispensable to overcome the preconditioning damages. This study aims to ascertain the role of Roundabout 4 (Robo4) in regulating irradiation-induced damage to the endothelium. Methods Microvascular endothelial cells were treated with γ-radiation to establish an endothelial cell injury model. Robo4 expression in the endothelial cells was manipulated employing lentiviral-mediated RNAi and gene overexpression technology before irradiation treatment. The permeability of endothelial cells was measured using qPCR, immunocytochemistry, and immunoblotting to analyze the effect on the expression and distribution of junctional molecules, adherens junctions, tight junctions, and gap junctions. Using Transwell endothelial monolayer staining, FITC-Dextran permeability, and gap junction-mediated intercellular communication (GJIC) assays, we determined the changes in endothelial functions after Robo4 gene manipulation and irradiation. Moreover, we measured the proportion of CD31 expression in endothelial cells by flow cytometry. We analyzed variations between two or multiple groups using Student’s t-tests and ANOVA. Results Ionizing radiation upregulates Robo4 expression but disrupts endothelial junctional molecules. Robo4 deletion causes further degradation of endothelial junctions hence increasing the permeability of the endothelial cell monolayer. Robo4 knockdown in microvascular endothelial cells increases the degradation and delocalization of ZO-1, PECAM-1, occludin, and claudin-5 molecules after irradiation. Conversely, connexin 43 expression increases after silencing Robo4 in endothelial cells to induce permeability but are readily destroyed when exposed to 10 Gy of gamma radiation. Also, Robo4 knockdown enhances Y731-VE-cadherin phosphorylation leading to the depletion and destabilization of VE-cadherin at the endothelial junctions following irradiation. However, Robo4 overexpression mitigates irradiation-induced degradation of tight junctional proteins and stabilizes claudin-5 and ZO-1 distribution. Finally, the enhanced expression of Robo4 ameliorates the irradiation-induced depletion of VE-cadherin and connexin 43, improves the integrity of microvascular endothelial cell junctions, and decreases permeability. Conclusion This study reveals that Robo4 maintains microvascular integrity after radiation preconditioning treatment by regulating endothelial permeability and protecting endothelial functions. Our results also provided a potential mechanism to repair the bone marrow vascular niche after irradiation by modulating Robo4 expression.

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“…Several markers of mouse HSCs have been reported, including lineage-negative (Lin − ) c-kit + CD34 + Sca-1 + in mouse fetuses or neonates, and Lin − c-kit + CD34 − Sca-1 + in mouse adults. 35,36 CD150 + CD48 − CD41 − is also reported to be a common HSC marker profile in adult bone marrow and fetal liver, 7,31,37,38 making this combination of markers worth investigating. Here, high purification using multiple markers enhances HSC purity, but also reduces cell numbers in the HSC fraction.…”

Section: Discussionmentioning

confidence: 99%

Effects of mouse fetal liver cell culture density on hematopoietic cell expansion in three-dimensional cocultures with stromal cells

et al. 2021

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Objective: An effective ex vivo expansion system of primitive hematopoietic cells (HCs) is required for wider application of hematopoietic stem cell transplantation. In this study, we examined effects of culture density on mouse fetal liver cells (FLCs) used as an HC source for the expansion of primitive HCs in three-dimensional (3D) cocultures with two kinds of mouse stromal cell lines (OP9 or C3H10T1/2). Materials and methods: FLCs were seeded at different densities (1, 2, and 10 × 107 cells/cm3) into porous polymer scaffolds with or without stromal cell layers and HCs were expanded in the cultures for 2 weeks without exogenous cytokines. Results: Differential effects of culture density on HC expansion were observed between cocultures and solitary FLC controls. In stromal cell cocultures, high expansion of HCs was achieved when FLCs were seeded at low densities. In contrast, the expansion in the controls was enhanced with increasing culture densities. With respect to expansion of primitive HCs existing in the FLCs, cocultures with C3H10T1/2 cells were superior to those with OP9 cells with a 29.3-fold expansion for c-kit+ hematopoietic progenitor cells and 8.3-fold expansion for CD34+ hematopoietic stem cells. In the controls, HC expansion was lower than in any cocultures, demonstrating the advantages of coculturing for HC expansion. Conclusion: Stromal cell lines are useful in expanding primitive HCs derived from FLCs in 3D cocultures. Culture density is a pivotal factor for the effective expansion of primitive HCs and this effect differs by culture condition.

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PEDF promotes the repair of bone marrow endothelial cell injury and accelerates hematopoietic reconstruction after bone marrow transplantation

Cited by 17 publications

References 46 publications

Endothelial Robo4 suppresses endothelial-to-mesenchymal transition induced by irradiation and improves hematopoietic reconstitution

Endothelial Robo4 suppresses endothelial-to-mesenchymal transition induced by irradiation and improves hematopoietic reconstitution

Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions

Effects of mouse fetal liver cell culture density on hematopoietic cell expansion in three-dimensional cocultures with stromal cells

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