Therapeutic Effect of Extracellular Vesicles Derived from HIF Prolyl Hydroxylase Domain Enzyme Inhibitor-Treated Cells on Renal Ischemia/Reperfusion Injury.

Introduction: Acute kidney injury (AKI) is a major public health problem worldwide. However, there is no definitive therapies to treat established AKI. In this study, we used FG-4592 to induce hypoxia inducible factor (HIF) expression in cells and then explored whether the extracellular vesicles (EVs) secreted by HIF-upregulated cells could alleviate ischemia/reperfusion injury (IRI)-induced AKI. Methods: FG-4592/HK2-EVs and FG-4592/HEK293-EVs were prepared by treating HK2 or HEK293 cells with FG-4592 for 24 h, respectively. HK2 cells under hypoxia were treated with FG-4592/HK2-EVs or FG-4592/HEK293-EVs to observe the therapeutic effect of EVs on H/R-induced apoptosis and inflammation. Mice were treated with FG-4592/HEK293-EVs after IRI to observe whether FG-4592/HEK293-EVs treatment could alleviate ischemic AKI. Results: The expression of HIF was induced by FG-4592 in a dose-dependent manner in HK2 and HEK293 cells under normoxia. In vitro, FG-4592/HK2-EVs and FG-4592/HEK293-EVs inhibited apoptosis and inflammation induced by H/R. In vivo, treatment with FG-4592/HEK293-EVs significantly ameliorated renal tubular injury and inflammation caused by IRI. In addition, the expression of HIF-1α in cells and kidneys was significantly downregulated by FG-4592/HK2-EVs and FG-4592/HEK293-EVs treatment. Conclusion: This study demonstrated that EVs derived from HK2 or HEK293 cells after FG-4592 treatment could alleviate renal tubular injury and inflammation, suggesting a novel therapeutic role of FG-4592/EVs in the treatment of AKI.

A Programmable Multifunctional 3D Cancer Cell Invasion Micro Platform.

In the research of cancer cell invasion and metastasis, recreation of physiologically relevant and faithful three-dimensional (3D) tumor models that recapitulate spatial architecture, spatiotemporal control of cell communication and signaling pathways, and integration of extracellular cues remains an open challenge. Here, a programmable multifunctional 3D cancer cell invasion microbuckets-hydrogel (Mb-H) platform is developed by integrating various function-variable microbuckets and extracellular matrix (ECM)-like hydrogels. Based on this Mb-H micro platform, the aggregation of multi-cancer cells is well controlled to form cancer cell spheroids, and the guiding relationship of single-cell migration and collective cell migration during the epithelial-mesenchymal transition (EMT) of cancer cell invasion are demonstrated. By programming and precisely assembling multiple functions in one system, the Mb-H platform with spatial-temporal controlled release of cytokine transforming growth factor beta (TGF-ß) and various functionalized Mb-H platforms with intelligent adjustment of cell-matrix interactions are engineered to coordinate the 3D invasive migration of cancer cell spheroids. This programmable and adaptable 3D cancer cell invasion micro platform takes a new step toward mimicking the dynamically changing (localized) tumor microenvironment and exhibits wide potential applications in cancer research, bio-fabrication, cell signaling, and drug screening.

Kim-1 Targeted Extracellular Vesicles: A New Therapeutic Platform for RNAi to Treat AKI.

BACKGROUND: AKI is a significant public health problem with high morbidity and mortality. Unfortunately, no definitive treatment is available for AKI. RNA interference (RNAi) provides a new and potent method for gene therapy to tackle this issue. METHODS: We engineered red blood cell-derived extracellular vesicles (REVs) with targeting peptides and therapeutic siRNAs to treat experimental AKI in a mouse model after renal ischemia/reperfusion (I/R) injury and unilateral ureteral obstruction (UUO). Phage display identified peptides that bind to the kidney injury molecule-1 (Kim-1). RNA-sequencing (RNA-seq) characterized the transcriptome of ischemic kidney to explore potential therapeutic targets. RESULTS: REVs targeted with Kim-1-binding LTH peptide (REVLTH) efficiently homed to and accumulated at the injured tubules in kidney after I/R injury. We identified transcription factors P65 and Snai1 that drive inflammation and fibrosis as potential therapeutic targets. Taking advantage of the established REVLTH, siRNAs targeting P65 and Snai1 were efficiently delivered to ischemic kidney and consequently blocked the expression of P-p65 and Snai1 in tubules. Moreover, dual suppression of P65 and Snai1 significantly improved I/R- and UUO-induced kidney injury by alleviating tubulointerstitial inflammation and fibrosis, and potently abrogated the transition to CKD. CONCLUSIONS: A red blood cell-derived extracellular vesicle platform targeted Kim-1 in acutely injured mouse kidney and delivered siRNAs for transcription factors P65 and Snai1, alleviating inflammation and fibrosis in the tubules.

An Efficient Screen for Cell-Intrinsic Factors Identifies the Chaperonin CCT and Multiple Conserved Mechanisms as Mediating Dendrite Morphogenesis.

Dendritic morphology is inextricably linked to neuronal function. Systematic large-scale screens combined with genetic mapping have uncovered several mechanisms underlying dendrite morphogenesis. However, a comprehensive overview of participating molecular mechanisms is still lacking. Here, we conducted an efficient clonal screen using a collection of mapped P-element insertions that were previously shown to cause lethality and eye defects in Drosophila melanogaster. Of 280 mutants, 52 exhibited dendritic defects. Further database analyses, complementation tests, and RNA interference validations verified 40 P-element insertion genes as being responsible for the dendritic defects. Twenty-eight mutants presented severe arbor reduction, and the remainder displayed other abnormalities. The intrinsic regulators encoded by the identified genes participate in multiple conserved mechanisms and pathways, including the protein folding machinery and the chaperonin-containing TCP-1 (CCT) complex that facilitates tubulin folding. Mutant neurons in which expression of CCT4 or CCT5 was depleted exhibited severely retarded dendrite growth. We show that CCT localizes in dendrites and is required for dendritic microtubule organization and tubulin stability, suggesting that CCT-mediated tubulin folding occurs locally within dendrites. Our study also reveals novel mechanisms underlying dendrite morphogenesis. For example, we show that Drosophila Nogo signaling is required for dendrite development and that Mummy and Wech also regulate dendrite morphogenesis, potentially via Dpp- and integrin-independent pathways. Our methodology represents an efficient strategy for identifying intrinsic dendrite regulators, and provides insights into the plethora of molecular mechanisms underlying dendrite morphogenesis.

Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury.

BACKGROUND: Exosomes derived from mesenchymal stem cells (MSC-exos) have been demonstrated with great potential in the treatment of multiple human diseases including acute kidney injury (AKI) by virtue of their intrinsic cargoes. However, there are major challenges of low yield and the lack of an established biomanufacturing platform to efficiently produce MSC-exos, thereby limiting their therapeutic application. Here, we aimed to establish a novel strategy to produce MSC-exos with a hollow fiber bioreactor-based three-dimensional (3D) culture system and evaluate the therapeutic efficacy of 3D-exosomes (3D-exos) on AKI. METHODS: Mesenchymal stem cells (MSCs) were isolated from fresh human umbilical cord and cultured in two-dimensional (2D) flasks. 2 × 108 MSCs were inoculated into the hollow fiber bioreactor for 3D culture. The culture supernatants were collected every 1 or 2 days for isolating exosomes. Exosomes from 2D (2D-exos) and 3D cultures were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting analysis of exosome markers. The yield of exosomes from 2 × 108 MSCs seeded in 2D and 3D culture system was compared, based on protein quantification. The therapeutic efficacy of 2D-exos and 3D-exos was investigated in a murine model of cisplatin-induced AKI in vivo and in vitro. RESULTS: 3D culture did not significantly change the surface markers of MSCs, as well as the morphology, size, and exosomal markers of 3D-exos when compared to those of 2D-exos. Compared with conventional 2D culture, the 3D culture system increased total exosome production up to 19.4-fold. 3D-exos were more concentrated in the harvested supernatants (15.5-fold) than 2D-exos, which led to a higher exosome collection efficiency of 3D culture system. In vivo, both 2D-exos and 3D-exos significantly alleviated cisplatin-induced murine AKI evidenced by improved renal function, attenuated pathological changes of renal tubules, reduced inflammatory factors, and repressed T cell and macrophage infiltration. Impressively, 3D-exos were more effective than 2D-exos. Moreover, 3D-exos were taken up by tubular epithelial cells (TECs) with improved efficiency, thereby exhibiting superior anti-inflammatory effect and improved viability of TECs in vitro. CONCLUSIONS: In summary, our findings demonstrate that the hollow fiber 3D culture system provides an efficient strategy for the continuous production of MSC-exos which has enhanced therapeutic potential for cisplatin-induced AKI.

Foxg1 Directly Represses Dbx1 to Confine the POA and Subsequently Regulate Ventral Telencephalic Patterning.

During early development, signaling centers, such as the cortical hem and the preoptic area (POA), are critical for telencephalic patterning. However, the mechanisms underlying the maintenance of signal centers are poorly understood. Here, we report that the transcription factor Foxg1 is required to confine the POA, a resource of Sonic Hedgehog (Shh) that is pivotal for ventral telencephalic development. Cell-specific deletion of Foxg1 achieved by crossing Foxg1fl/fl with Dbx1-cre or Nestin-CreER combined with tamoxifen induction results in a dramatic expansion of the POA accompanied by the significantly increased activity of the Shh signaling pathway. Ventral pattern formation was severely impaired. Moreover, we demonstrated that Foxg1 directly represses Dbx1 to restrict the POA. Furthermore, we found that the ventral pallium was expanded, which might also contribute to the observed patterning defects. These findings will improve our understanding of the maintenance of signal centers and help to elucidate the mechanisms underlying ventral telencephalic patterning.

Outer nuclear membrane protein Kuduk modulates the LINC complex and nuclear envelope architecture.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes spanning the nuclear envelope (NE) contribute to nucleocytoskeletal force transduction. A few NE proteins have been found to regulate the LINC complex. In this study, we identify one, Kuduk (Kud), which can reside at the outer nuclear membrane and is required for the development of Drosophila melanogaster ovarian follicles and NE morphology of myonuclei. Kud associates with LINC complex components in an evolutionarily conserved manner. Loss of Kud increases the level but impairs functioning of the LINC complex. Overexpression of Kud suppresses NE targeting of cytoskeleton-free LINC complexes. Thus, Kud acts as a quality control mechanism for LINC-mediated nucleocytoskeletal connections. Genetic data indicate that Kud also functions independently of the LINC complex. Overexpression of the human orthologue TMEM258 in Drosophila proved functional conservation. These findings expand our understanding of the regulation of LINC complexes and NE architecture.

Glial cell adhesive molecule unzipped mediates axon guidance in Drosophila.

Axon guidance needs help from the glial cell system during embryogenesis. In the Drosophila embryonic central nervous system (CNS), longitudinal glia (LG) have been implicated in axon guidance but the mechanism remains unclear. We identified the protein encoded by the Drosophila gene unzipped (uzip) as a novel cell adhesion molecule (CAM). Uzip expressed in Drosophila S2 cells triggered cell aggregation through homophilic binding. In the embryonic CNS, Uzip was mainly produced by the LG but was also located at axons, which is consistent with the secretion of Uzip expressed in cultured cells. Although uzip mutants displayed no axonal defect, loss of uzip enhanced the axonal defects in the mutant of N-cadherin (CadN) and the Wnt gene family member wnt5. Overexpression of uzip could rescue the phenotype in the CadNuzip(D43) mutant. Thus, Uzip is a novel CAM from the LG regulating axon guidance.