技术资料

Cell therapy is a novel promising option for treatment of ischemic diseases. Administered endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. However,the number of cells that home to ischemic tissues is restricted. The GTPase Rap1 plays an important role in the regulation of adhesion and chemotaxis. We investigated whether pharmacologic activation of Epac1,a nucleotide exchange protein for Rap1,which is directly activated by cAMP,can improve the adhesive and migratory capacity of distinct progenitor cell populations. Stimulation of Epac by a cAMP-analog increased Rap1 activity and stimulated the adhesion of human EPCs,CD34(+) hematopoietic progenitor cells,and mesenchymal stem cells (MSCs). Specifically,short-term stimulation with a specific Epac activator increased the beta2-integrin-dependent adhesion of EPCs to endothelial cell monolayers,and of EPC and CD34(+) cells to ICAM-1. Furthermore,the Epac activator enhanced the beta1-integrin-dependent adhesion of EPCs and MSCs to the matrix protein fibronectin. In addition,Epac1 activation induced the beta1- and beta2-integrin-dependent migration of EPCs on fibronectin and fibrinogen. Interestingly,activation of Epac rapidly increased lateral mobility of beta1- and beta2-integrins,thereby inducing integrin polarization,and stimulated beta1-integrin affinity,whereas the beta2-integrin affinity was not increased. Furthermore,prestimulation of EPCs with the Epac activator increased homing to ischemic muscles and neovascularization-promoting capacity of intravenously injected EPCs in the model of hind limb ischemia. These data demonstrate that activation of Epac1 increases integrin activity and integrin-dependent homing functions of progenitor cells and enhances their in vivo therapeutic potential. These results may provide a platform for the development of novel therapeutic approaches to improve progenitor cell homing. View Publication

The canonical Wnt signaling pathway can determine human bone marrow stromal (mesenchymal) stem cell (hMSC) differentiation fate into osteoblast or adipocyte lineages. However,its downstream targets in MSC are not well characterized. Thus,using DNA microarrays,we compared global gene expression patterns induced by Wnt3a treatment in two hMSC lines: hMSC-LRP5(T253) and hMSC-LRP5(T244) cells carrying known mutations of Wnt co-receptor LRP5 (T253I or T244M) that either enhances or represses canonical Wnt signaling,respectively. Wnt3a treatment of hMSC activated not only canonical Wnt signaling,but also the non-canonical Wnt/JNK pathway through upregulation of several non-canonical Wnt components e.g. naked cuticle 1 homolog (NKD1) and WNT11. Activation of the non-canonical Wnt/JNK pathway by anisomycin enhanced osteoblast differentiation whereas its inhibition by SP600125 enhanced adipocyte differentiation of hMSC. In conclusion,canonical and non-canonical Wnt signaling cooperate in determining MSC differentiation fate. View Publication

Lung transplantation is the primary treatment for end-stage lung diseases. However,ischemia-reperfusion injury (IRI) significantly impacts transplant outcomes. 4-Octyl itaconate (4-OI) has shown potential in mitigating organ IRI,although its effects in lung transplantation require further exploration. BEAS-2B cells were used to model transplantation,assessing the effects of 4-OI through viability,apoptosis,and ROS assays. qRT-PCR analyzed cytokine transcription post-cold ischemia/reperfusion (CI/R). RNA sequencing and Gene Ontology analysis elucidated 4-OI’s mechanisms of action,confirmed by Western blotting. ALI-airway and lung transplantation organoid models evaluated improvements in bronchial epithelial morphology and function due to 4-OI. ELISA measured IL-6 and IL-8 levels. Rat models of extended cold preservation and non-heart-beating transplantation assessed 4-OI’s impact on lung function,injury,and inflammation. Our findings indicate that 4-OI (100 µM) during cold preservation effectively maintained cell viability,decreased apoptosis,and reduced ROS production in BEAS-2B cells under CI/R conditions. It also downregulated pro-inflammatory cytokine transcription,including IL1B,IL6,and TNF. Inhibition of Nrf2 partially reversed these protective effects. In cold preservation solutions,4-OI upregulated Nrf2 target genes such as NQO1,HMOX1,and SLC7A11. In ALI airway models,4-OI enhanced bronchial epithelial barrier integrity and ciliary beat function after CI/R. In rat models,4-OI administration improved lung function and reduced pulmonary edema,tissue injury,apoptosis,and systemic inflammation following extended cold preservation or non-heart-beating lung transplantation. Incorporating 4-OI into cold preservation solutions appears promising for alleviating CI/R-induced bronchial epithelial injury and enhancing lung transplant outcomes via Nrf2 pathway activation. The online version contains supplementary material available at 10.1186/s12931-025-03151-7. View Publication

Although hematopoietic stem cells (HSC) are the best characterized and the most clinically used adult stem cells,efforts are still needed to understand how to best ex vivo expand these cells. Here we present our unexpected finding that OCT4 is involved in the enhancement of cytokine-induced expansion capabilities of human cord blood (CB) HSC. Activation of OCT4 by Oct4-activating compound 1 (OAC1) in CB CD34(+) cells enhanced ex vivo expansion of HSC,as determined by a rigorously defined set of markers for human HSC,and in vivo short-term and long-term repopulating ability in NSG mice. Limiting dilution analysis revealed that OAC1 treatment resulted in 3.5-fold increase in the number of SCID repopulating cells (SRCs) compared with that in day 0 uncultured CD34(+) cells and 6.3-fold increase compared with that in cells treated with control vehicle. Hematopoietic progenitor cells,as assessed by in vitro colony formation,were also enhanced. Furthermore,we showed that OAC1 treatment led to OCT4-mediated upregulation of HOXB4. Consistently,siRNA-mediated knockdown of HOXB4 expression suppressed effects of OAC1 on ex vivo expansion of HSC. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC. View Publication

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS),but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight,we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin beta7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore,phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFalpha secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFalpha production,while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms,but not markers of immune activation,decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation,but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms. View Publication

Mesenchymal stem cells (MSC) can differentiate into osteoblasts,adipocytes,chondrocytes and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSC into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of the nuclear protein deacetylase Sirt1 has recently been shown to decrease adipocyte development from preadipocytes via inhibition of PPARgamma2. In vitro,MSC differentiate to osteoblasts when exposed to bone-inducing medium. However,adipocytes are also developed. In the present study we have targeted Sirt1 to control adipocyte development during differentiation of MSC into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation demonstrates an interesting alternative to PPARgamma antagonists. These results are important for the evolving field of cell-based tissue engineering,but may also be relevant in the search for new treatments of osteoporosis. View Publication

TFF1,a secreted protein,plays an essential role in keeping the integrity of gastric mucosa and its barrier function. Loss of TFF1 expression in the TFF1-knockout (KO) mouse leads to a pro-inflammatory phenotype with a cascade of gastric lesions that include low-grade dysplasia,high-grade dysplasia,and adenocarcinomas. In this study,we demonstrate nuclear localization of p-STATY705,with significant overexpression of several STAT3 target genes in gastric glands from the TFF1-KO mice. We also show frequent loss of TFF1 with nuclear localization of STAT3 in human gastric cancers. The reconstitution of TFF1 protein in human gastric cancer cells and 3D gastric glands organoids from TFF1-KO mice abrogates IL6-induced nuclear p-STAT3Y705 expression. Reconstitution of TFF1 inhibits IL6-induced STAT3 transcription activity,suppressing expression of its target genes. TFF1 blocks IL6R$\alpha$-GP130 complex formation through interfering with binding of IL6 to its receptor IL6R$\alpha$. These findings demonstrate a functional role of TFF1 in suppressing gastric tumorigenesis by impeding the IL6-STAT3 pro-inflammatory signaling axis. View Publication

Pilocytic astrocytoma is commonly viewed as a benign lesion. However,disease onset is most prevalent in the first two decades of life,and children are often left with residual or recurrent disease and significant morbidity. The Hedgehog (Hh) pathway regulates the growth of higher WHO grade gliomas,and in this study,we have evaluated the activation and operational status of this regulatory pathway in pilocytic astrocytomas. Expression levels of the Hh pathway transcriptional target PTCH were elevated in 45% of tumor specimens analyzed (ages 1-22 years) and correlated inversely with patient age. Evaluation of a tissue array revealed oligodendroglioma-like features,pilomyxoid features,infiltration,and necrosis more commonly in specimens from younger patients (below the median patient age of 10 years). Immunohistochemical staining for the Hh pathway components PTCH and GLI1 and the proliferation marker Ki67 demonstrated that patients diagnosed before the age of 10 had higher staining indices than those diagnosed after the age of 10. A significant correlation between Ki67 and PTCH and GLI1 staining indices was measured,and 86% of Ki67-positive cells also expressed PTCH. The operational status of the Hh pathway was confirmed in primary cell culture and could be modulated in a manner consistent with a ligand-dependent mechanism. Taken together,these findings suggest that Hh pathway activation is common in pediatric pilocytic astrocytomas and may be associated with younger age at diagnosis and tumor growth. View Publication

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene,which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding,induction of endoplasmic reticulum (ER) stress,activation of the unfolded protein response (UPR),and ultimately a block in granulocytic differentiation. To test this model,we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice,though without significant basal UPR activation,are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover,inaction of protein kinase RNA-like ER kinase (Perk),one of the major sensors of ER stress,either alone or in combination with G193X Elane,had no effect on basal granulopoiesis. However,inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN. View Publication

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here,we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal,proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive),neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells,as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs,an effect blocked by Notch pathway inhibition. Moreover,R-m-AEA treatment for 48 h,increased proliferation as assessed by BrdU incorporation assay,an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly,stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation),at 7 days,as shown by counting the number of NeuN-positive neurons in the cultures. Moreover,by monitoring intracellular calcium concentrations ([Ca(2+)]i) in single cells following KCl and histamine stimuli,a method that allows the functional evaluation of neuronal differentiation,we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251,for 7 days,thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation,R-m-AEA also increased neurite growth,as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together,these results demonstrate that CB1R activation induces proliferation,self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures. View Publication

The use of human pluripotent cell progeny for cardiac disease modeling,drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyogenic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success,recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR,which in turn induces the Activin-A and Bmp pathways,is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis,and to ultimately promote myocyte maturation,we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq,induction with CHIR during Day 1 (Days 0-1) was followed by immediate expression of Nodal ligands and receptors,followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic Activin-A (50-100 ng/ml) during Day 0-1 efficiently induced definitive endoderm,whereas CHIR supplemented with Activin-A at low levels (10 ng/ml) consistently improved cardiomyogenic efficiency,even when CHIR alone was ineffective. Moreover,co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis,as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast,co-induction with CHIR plus low levels (3-10 ng/ml) of Bmp4 during Day 0-1 consistently and strongly inhibited cardiomyogenesis. These findings,which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression,are consistent with the idea that Nodal (Activin-A) levels toggle the entry of cells into the endodermal or mesodermal lineages,while Bmp levels regulate subsequent allocation into mesodermal cell types. View Publication

The fine control of synaptic function requires robust trans-synaptic molecular interactions. However,it remains poorly understood how trans-synaptic bridges change to reflect the functional states of the synapse. Here,we develop optical tools to visualize in firing synapses the molecular behavior of two trans-synaptic proteins,LGI1 and ADAM23,and find that neuronal activity acutely rearranges their abundance at the synaptic cleft. Surprisingly,synaptic LGI1 is primarily not secreted,as described elsewhere,but exo- and endocytosed through its interaction with ADAM23. Activity-driven translocation of LGI1 facilitates the formation of trans-synaptic connections proportionally to the history of activity of the synapse,adjusting excitatory transmission to synaptic firing rates. Accordingly,we find that patient-derived autoantibodies against LGI1 reduce its surface fraction and cause increased glutamate release. Our findings suggest that LGI1 abundance at the synaptic cleft can be acutely remodeled and serves as a critical control point for synaptic function. View Publication