ExplorationVolume 4, Issue 2 20240202 INSIDE FRONT COVEROpen Access Inside Front Cover: Cerebrospinal fluid efflux through dynamic paracellular pores on venules as a missing piece of the brain drainage system (EXP2 2/2024) Yaqiong Dong, Yaqiong DongSearch for more papers by this authorTing Xu, Ting XuSearch for more papers by this authorLan Yuan, Lan YuanSearch for more papers by this authorYahan Wang, Yahan WangSearch for more papers by this authorSiwang Yu, Siwang YuSearch for more papers by this authorZhi Wang, Zhi WangSearch for more papers by this authorShizhu Chen, Shizhu ChenSearch for more papers by this authorChunhua Chen, Chunhua ChenSearch for more papers by this authorWeijiang He, Weijiang HeSearch for more papers by this authorTessandra Stewart, Tessandra StewartSearch for more papers by this authorWeiguang Zhang, Weiguang ZhangSearch for more papers by this authorXiaoda Yang, Xiaoda YangSearch for more papers by this author Yaqiong Dong, Yaqiong DongSearch for more papers by this authorTing Xu, Ting XuSearch for more papers by this authorLan Yuan, Lan YuanSearch for more papers by this authorYahan Wang, Yahan WangSearch for more papers by this authorSiwang Yu, Siwang YuSearch for more papers by this authorZhi Wang, Zhi WangSearch for more papers by this authorShizhu Chen, Shizhu ChenSearch for more papers by this authorChunhua Chen, Chunhua ChenSearch for more papers by this authorWeijiang He, Weijiang HeSearch for more papers by this authorTessandra Stewart, Tessandra StewartSearch for more papers by this authorWeiguang Zhang, Weiguang ZhangSearch for more papers by this authorXiaoda Yang, Xiaoda YangSearch for more papers by this author First published: 17 April 2024 https://doi.org/10.1002/EXP.20240202AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Graphical Abstract While asleep, the cerebrospinal fluid (CSF) flush over the brain parenchyma via pathways generated by neuroglia cells. The interstitial fluid (ISF) solutes containing metabolic wastes/toxins such as Aβ are collected in the paravenous space, where the CSF-ISF fluid eventually exit into blood through dynamically assembled, trumpet-shaped pores formed by vascular endothelial cells. Volume4, Issue2April 202420240202 RelatedInformation

Pyroptosis is an identified programmed cell death that has been highly linked to endoplasmic reticulum (ER) dynamics. However, the crucial proteins for modulating dynamic ER membrane curvature change that trigger pyroptosis are currently not well understood. In this study, a biotin-labeled chemical probe of potent pyroptosis inducer α-mangostin (α-MG) was synthesized. Through protein microarray analysis, reticulon-4 (RTN4/Nogo), a crucial regulator of ER membrane curvature, was identified as a target of α-MG. We observed that chemically induced proteasome degradation of RTN4 by α-MG through recruiting E3 ligase UBR5 significantly enhances the pyroptosis phenotype in cancer cells. Interestingly, the downregulation of RTN4 expression significantly facilitated a dynamic remodeling of ER membrane curvature through a transition from tubules to sheets, consequently leading to rapid fusion of the ER with the cell plasma membrane. In particular, the ER-to-plasma membrane fusion process is supported by the observed translocation of several crucial ER markers to the "bubble" structures of pyroptotic cells. Furthermore, α-MG-induced RTN4 knockdown leads to pyruvate kinase M2 (PKM2)-dependent conventional caspase-3/gasdermin E (GSDME) cleavages for pyroptosis progression. In vivo, we observed that chemical or genetic RTN4 knockdown significantly inhibited cancer cells growth, which further exhibited an antitumor immune response with anti-programmed death-1 (anti-PD-1). In translational research, RTN4 high expression was closely correlated with the tumor metastasis and death of patients. Taken together, RTN4 plays a fundamental role in inducing pyroptosis through the modulation of ER membrane curvature remodeling, thus representing a prospective druggable target for anticancer immunotherapy.

Obesity is a major public burden on the working population and induces chronic diseases. Its treatment often requires long-term medication, which makes patient compliance difficult. In this study, we reported the value of HORN-MN, which comprised a fast-soluble hyaluronic acid microneedle matrix and a weak acid-degradable oleanolic acid dimer of rosiglitazone nanoparticles. The results showed that the microneedles easily punctured the stratum corneum and dissolved in the dermis of the abdominal wall within 5 min, followed by the release of rosiglitazone nanoparticles. Thereafter, the nanoparticles were endocytosed by macrophages and white adipocytes, then degraded to oleanolic acid in the lysosomes, thereby, releasing rosiglitazone. Oleanolic acid significantly improved the inflammatory status of obese adipose tissue and promoted white adipocyte browning, and rosiglitazone significantly potentiated WAC browning. Accordingly, the patch demonstrated a remarkable obesity-reducing efficacy in mice. In conclusion, this study developed a quick paster type of soluble rosiglitazone nanoparticle microneedle for the treatment of obesity. This patch can be suitable for working people, with an evident obesity-reducing efficacy but no effect on skin integrity despite multiple administrations.

Simultaneous detection of different biomarkers related to the spatiotemporally dynamic immune events is of particular importance for the accurate evaluation of antitumor immune effects. Here, we have developed an AND-gate logic dual resonance energy transfer nanoprobe (named DRET) for dynamic monitoring of programmed CD8+ T cell activation and tumor cell apoptosis. Immunotherapy-induced granzyme B secretion from CD8+ T cells and the subsequent caspase-3 release from apoptotic tumor cells individually activate one of the tiers of the "AND-gate" logic DRET. The resulting fluorescence recovery and magnetic resonance T1 enhancement can be used for precise immunomodulatory drug screening, early efficacy prediction, and immune stratification. Particularly, not only "Responders" can be distinguished from "Non-responders", but also "Acquired resistance" can be identified from "Maintain responders", providing a novel approach to put forward the accurate evaluation of antitumor immunity.

OBJECTIVE:To unveil the pathological changes associated with demyelination in schizophrenia (SZ) and its consequential impact on interstitial fluid (ISF) drainage, and to investigate the therapeutic efficacy of ursolic acid (UA) in treating demyelination and the ensuing abnormalities in ISF drainage in SZ. METHODS:Female C57BL/6J mice, aged 6-8 weeks and weighing (20±2) g, were randomly divided into three groups: control, SZ model, and UA treatment. The control group received intraperitoneal injection (ip) of physiological saline and intragastric administration (ig) of 1% carboxymethylcellulose sodium (CMC-Na). The SZ model group was subjected to ip injection of 2 mg/kg dizocilpine maleate (MK-801) and ig administration of 1% CMC-Na. The UA treatment group underwent ig administration of 25 mg/kg UA and ip injection of 2 mg/kg MK-801. The treatment group received UA pretreatment via ig administration for one week, followed by a two-week drug intervention for all the three groups. Behavioral assessments, including the open field test and prepulse inhibition experiment, were conducted post-modeling. Subsequently, changes in the ISF partition drainage were investigated through fluorescent tracer injection into specific brain regions. Immunofluorescence analysis was employed to examine alterations in aquaporin 4 (AQP4) polarity distribution in the brain and changes in protein expression. Myelin reflex imaging using Laser Scanning Confocal Microscopy (LSCM) was utilized to study modifications in myelin within the mouse brain. Quantitative data underwent one-way ANOVA, followed by TukeyHSD for post hoc pairwise comparisons between the groups. RESULTS:The open field test revealed a significantly longer total distance [(7 949.39±1 140.55) cm vs. (2 831.01±1 212.72) cm, P < 0.001] and increased central area duration [(88.43±22.06) s vs. (56.85±18.58) s, P=0.011] for the SZ model group compared with the controls. The UA treatment group exhibited signifi-cantly reduced total distance [(2 415.80±646.95) cm vs. (7 949.39±1 140.55) cm, P < 0.001] and increased central area duration [(54.78±11.66) s vs. (88.43±22.06) s, P=0.007] compared with the model group. Prepulse inhibition test results demonstrated a markedly lower inhibition rate of the startle reflex in the model group relative to the controls (P < 0.001 for both), with the treatment group displaying significant improvement (P < 0.001 for both). Myelin sheath analysis indicated significant demyelination in the model group, while UA treatment reversed this effect. Fluorescence tracing exhibited a significantly larger tracer diffusion area towards the rostral cortex and reflux area towards the caudal thalamus in the model group relative to the controls [(13.93±3.35) mm2 vs. (2.79±0.94) mm2, P < 0.001 for diffusion area; (2.48±0.38) mm2 vs. (0.05±0.12) mm2, P < 0.001 for reflux area], with significant impairment of drainage in brain regions. The treatment group demonstrated significantly reduced tracer diffusion and reflux areas [(7.93±2.48) mm2 vs. (13.93±3.35) mm2, P < 0.001 for diffusion area; (0.50±0.30) mm2 vs. (2.48±0.38) mm2, P < 0.001 for reflux area]. Immunofluorescence staining revealed disrupted AQP4 polarity distribution and reduced AQP4 protein expression in the model group compared with the controls [(3 663.88±733.77) μm2 vs. (13 354.92±4 054.05) μm2, P < 0.001]. The treatment group exhibited restored AQP4 polarity distribution and elevated AQP4 protein expression [(11 104.68±3 200.04) μm2 vs. (3 663.88±733.77) μm2, P < 0.001]. CONCLUSION:UA intervention ameliorates behavioral performance in SZ mice, Thus alleviating hyperactivity and anxiety symptoms and restoring sensorimotor gating function. The underlying mechanism may involve the improvement of demyelination and ISF drainage dysregulation in SZ mice.

Cardiac hypertrophy characterized by abnormal cardiomyocyte viscosity is a typical sign of heart failure (HF) with vital importance for early diagnosis. However, current biochemical and imaging diagnostic methods are unable to detect this subclinical manifestation. In this work, we developed a series of NIR-I fluorescence probes for detecting myocardial viscosity based on the pyridazinone scaffold. The probes showed weak fluorescence due to free intramolecular rotation under low-viscosity conditions, while they displayed strong fluorescence with limited intramolecular rotation in response to a high-viscosity environment. Among them, CarVis2 exhibited higher stability and photobleaching resistance than commercial dyes. Its specific response to viscosity was not influenced by the pH and biological species. Furthermore, CarVis2 showed rapid and accurate responses to the viscosity of isoproterenol (ISO)-treated H9C2 cardiomyocytes with good biocompatibility. More importantly, CarVis2 demonstrated excellent sensitivity in monitoring myocardial viscosity variation in HF mice in vivo, potentially enabling earlier noninvasive identification of myocardial abnormalities compared to traditional clinical imaging and biomarkers. These findings revealed that CarVis2 can serve as a powerful tool to monitor myocardial viscosity, providing the potential to advance insights into a pathophysiological mechanism and offering a new reference strategy for early visual diagnosis of HF.

Intracellular delivery crossing the endomembrane barrier is the "last mile to target" for nano delivery systems carrying biomacromolecules, including genetic medicines. Nevertheless, a mass of nanomedicines is currently restricted by their equivocal safety and delivery efficiency. Here, we establish a universal strategy independent of nanomaterials. Such a policy broadly facilitates the intracellular delivery of all kinds of tested nanomedicines, subtly by inducing ARF6 GTPases to their overactivated GTP-bound state. ARF6, one member of ARF subfamily in small GTPases, is verified to regulate intracellular vesicle transport and lipid metabolism through GTP/GDP conversion. ARF6 biased to GTP-bound state causes the increased endocytosis and reduced exocytosis of eleven types of nanoparticles. This universal effect is derived from the formation of a hybrid type of endosomes triggered by overactivated ARF6 via regulating cholesterol-associated vesicles and lipid raft/caveolae pathways. Due to the mild microenvironment in hybrid endosomes, the internalized protein and nanoparticles are steadily delivered to the cytoplasm, avoiding the intensive degradation in lysosomes. Based on these findings, we identify QS11, a safe small molecule inhibitor of ARF GTPase-activating proteins, significantly enhances the antitumor efficacy of siEGFR-loaded nanoparticles by inducing ARF6 overactivation. In sum, these findings reveal that the tactics of tuning ARF6 GTPases to GTP-bound form will widely benefit cellular nano delivery.

目的 设计合成双荧光素修饰的6A,6D-双脱氧-α-环糊精.方法 从α-环糊精出发,经过苄基化保护得到过苄基化-α-环糊精(1),特定温度下利用DIBAL-H对过苄基化-α-环糊精进行选择性脱保护,得到中间体2,中间体2经磺酰化、叠氮化后将6A,6D位点上伯醇转化为叠氮,再经氢化铝锂还原为伯胺.再在钯碳/氢气条件脱除苄基保护,获得6A,6D-双脱氧-6A,6D-二氨基-α-环糊精(6).碱性条件下,中间体6与两倍当量的异硫氰基荧光素发生亲核加成反应,得到6A,6D-双脱氧-6A,6D-双荧光素-α-环糊精(7).结果 6个中间体和1个目标产物的结构经过核磁共振氢谱和质谱确证.结论 成功设计合成双荧光素修饰的6A,6D-双脱氧-α-环糊精(7),并且获得新的环糊精衍生物6A,6D-氧-对甲苯磺酰基-2A-F,3A-F,6B,6c,6E,6F-十六氧苄基-α-环糊精(3).

The glymphatic system plays a key role in the clearance of waste from the parenchyma, and its dysfunction has been associated with the pathogenesis of Alzheimer's disease (AD). However, questions remain regarding its complete mechanisms. Here, we report that efflux of cerebrospinal fluid (CSF)/interstitial fluid (ISF) solutes occurs through a triphasic process that cannot be explained by the current model, but rather hints at the possibility of other, previously undiscovered routes from paravenous spaces to the blood. Using real-time, in vivo observation of efflux, a novel drainage pathway was discovered, in which CSF molecules enter the bloodstream directly through dynamically assembled, trumpet-shaped pores (basolateral ϕ<8 μm; apical ϕ < 2 μm) on the walls of brain venules. As Zn2+ could facilitate the brain clearance of macromolecular ISF solutes, Zn2+-induced reconstruction of the tight junctions (TJs) in vascular endothelial cells may participate in pore formation. Thus, an updated model for glymphatic clearance of brain metabolites and potential regulation is postulated. In addition, deficient clearance of Aβ through these asymmetric venule pores was observed in AD model mice, supporting the notion that impaired brain drainage function contributes to Aβ accumulation and pathogenic dilation of the perivascular space in AD.

Abstract Intracellular delivery crossing the endomembrane barrier is the “last mile to target” for nano delivery systems carrying biomacromolecules, including genetic medicines. Nevertheless, a mass of nanomedicines is currently restricted by their equivocal safety and delivery efficiency. Here, we trimmed nano delivery at the cell perspective and established a general strategy independent of nanomaterials. Such a policy broadly facilitates the intracellular delivery of all kinds of tested nanomedicines, subtly by inducing ARF6 GTPases to their overactivated GTP-bound state. We discovered that ARF6, one member of ARF subfamily in small GTPases, regulated intracellular vesicle transport and lipid metabolism through GTP/GDP conversion. More importantly, we demonstrated that ARF6 biased to GTP-bound form induced GTPase overactivation, promoting endocytosis and reducing exocytosis of cargoes, including transferrin proteins and eleven types of nanogranules. This universal effect was mechanistically derived from forming a particular category of hybrid endosomes triggered by overactivated ARF6 via regulating cholesterol-associated vesicles and lipid raft/caveolae pathways. Cargoes were steadily and slowly delivered to the cytoplasm due to the mild microenvironment in hybrid endosomes. Based on these findings, we identified that QS11, a safe small molecule inhibitor of ARF GTPase-activating proteins, enhanced the antitumor efficacy of siEGFR-loaded nanoparticles by inducing ARF6 overactivation. In sum, it demonstrates that the tactics of tuning ARF6 GTPases to GTP-bound form will widely benefit cellular nano delivery.

Ulcerative colitis (UC) is a significant burden on human health, and the elucidation of the mechanism by which it develops has potential for the prevention and treatment of UC. It has been reported that acteoside (ACT) exhibits strong anti-inflammatory activity. In the present study, it was hypothesized that ACT may exert a protective effect against UC. The effects of ACT on inflammation, oxidative stress and apoptosis were evaluated using dextran sulphate sodium (DSS)-treated mice and DSS-treated human colorectal adenocarcinoma Caco-2 cells, which have an epithelial morphology. The results demonstrated that the ACT-treated mice with DSS-induced UC exhibited significantly reduced colon inflammation, as demonstrated by a reversal in body weight loss, colon shortening, disease activity index score, inflammation, oxidative stress and colonic barrier dysfunction. Further in vivo experiments demonstrated that ACT inhibited DSS-induced apoptosis in colon tissues, as demonstrated by the results of the TUNEL assay and the altered protein expression levels of Bax, cleaved caspase-3 and Bcl-2. Furthermore, DSS significantly stimulated the protein expression levels of high mobility group box 1 protein (HMGB1), which serves a central role in the initiation and progression of UC, an effect which was markedly inhibited by ACT. Finally, DSS significantly decreased the protein expression levels of heme oxygenase-1 (HO-1) in colon tissues and the effect of ACT on GSH, apoptotic proteins and HMGB1 was markedly attenuated in the presence of the HO-1 inhibitor tin protoporphyrin. In conclusion, ACT ameliorated colon inflammation through HMGB1 inhibition in a HO-1-dependent manner.

The RAF/MEK/ERK pathway is a crucial signal path which is closely associated with the proliferation, differentiation, and apoptosis of tumors. MEK1/2 is a key kinase target in the pathway, with ERK1/2 acting as the main substrate of it. Despite the rapid development of MEK1/2 inhibitors, acquired resistance still happens and remains a significant problem. Most of the inhibitors possess a similar diarylamine scaffold. Here we designed and synthesized a series of MEK1/2 degraders based on a coumarin derivative which was a potent non-diarylamine allosteric MEK1/2 inhibitor. P6b among them showed the most potent degradation effect, with DC50 values of 0.3 μM and 0.2 μM in MEK1 and MEK2 degradation, respectively. An antiproliferation assay showed that it more significantly inhibits the growth of A375 cells (IC50= 2.8 μM) compared to A549 cells (IC50 = 27.3 μM). To sum up, we discovered P6b with a non-diarylamine scaffold for the first time as a potent MEK PROTAC effective in human cancer cells.

Fluorescent proteins (FPs) are commonly used probes for coding genes that enable specific protein or whole-cell labeling. Their fluorescence intensity is used for molecular quantitation and intermolecular interaction analysis. Since FPs are usually small soluble proteins, they easily cross the membranes if cell integrity is disrupted, resulting in FP signal attenuation/loss. Specimen prefixation to preserve FP localization within cells/tissues is therefore useful. However, specific fixatives can weaken or eliminate FP signals. We studied the effects of five common fixatives on FP fluorescence intensity and biological functions to determine their suitability for FP signal and FRET efficiency preservation in cells and tissues. FP (GFP, YFP, CFP and RFP)-expressing HEK293T cells with methanol, 95% ethanol, 4% PFA, acetone and glutaraldehyde, and brain tissue sections of EGFP- and tdTomato-labeled transgenic fluorescent mice was fixed with 4% PFA. The FP signals in HEK293T cells and brain tissue from transgenic fluorescent mice were weakened or even eliminated after fixation with these fixatives. The fixatives affected FP biological function, and the FP FRET efficiency significantly differed between prefixation and postfixation (all p<0.01). Thus, fixatives impair FP fluorescence to some extent, leading to attenuation/loss of signals or even biological functions. Fixatives should be applied carefully in FP-related experiments to avoid bias.

Lanthanide ions (Ln3+) doped hydroxyapatite (HAP) particles are well established in biomedical areas. Although Ln elements are closely located in the periodic table and have plenty of similar characteristics, the minor differences in the effective ionic radii could cause alterations in the physicochemical and biological properties of HAP substitutes. The present study synthesized lanthanum-(La-) and gadolinium-(Gd-) doped HAP particles (La-HAP and Gd-HAP). And the effects of two types of particles on bone marrow stem cells (BMSCs) viability were also measured and compared in vitro. The results indicated that the Gd-HAP adsorbed more serum proteins from culture media and inhibited the new layer of apatite formation on its surface when comparing to La-HAP with a similar crystalline structure, particle size, and Zeta potential. These surface modifications can significantly reduce the cell adhesion of Gd-HAP, simultaneously decreasing the Gd-HAP particle uptake efficiency. Moreover, the cell viability of Gd-HAP remained higher than that of La-HAP in culture periods. We concluded that a slight variation in the effective ionic radii between Gd3+ and La3+ could alter the adsorption of serum proteins on the particles' surface, modulating subsequent cellular responses. The present work provides an interesting view that Gd-HAP is endowed with better cellular biocompatibility than La-HAP.

A tremendous number of proteins participate in the delivery and transport process of nanomedicines. Nanoprotein interactions not only mediate drug delivery but also determine drug safety. In the field of biomedical sciences, the epithelial barrier is a huge challenge for gastrointestinal, intratracheal, intranasal, vaginal, and intrauterine delivery of nanomedicines. However, the molecular mechanisms by which nanomedicines cross tissue or cell barriers are not well understood. Here, we explored the nanoprotein interactions during the transcytosis of nanoparticles across the epithelial barrier by focusing on the transport pathway and mechanisms. Due to the limitations of traditional methods in resolving nanoprotein interactions, we developed a backward analysis strategy. By simultaneously analyzing the protein corona on the particle surface and the cellular response after transcytosis, we integrated the information on both directly and indirectly interacting proteins, establishing a holistic nanoprotein interaction atlas. It revealed the dominant role of the EV/ER/Golgi/SV pathway in the transcytosis of nanoparticles. More importantly, based on the established atlas, we discovered the association of Wnt/β-catenin signaling with nanoparticle transportation. The endocytosis for entering cells and exocytosis/transcytosis for leaving cells were differently regulated by the Wnt pathway. Notably, this regulatory effect was dependent on the particle size. Bigger nanoparticles departed from cells through the exocytosis pathway faster because of the specific bridging effect on the Wnt-Frizzled interaction and the feedback loop construction based on the exosomes. This mechanism gives an interpretation at the molecular level to the transcytosis dilemma of larger nanoparticles. Moreover, the size-dependent Wnt/β-catenin signaling pathway provides a promising regulatory and screening platform for the transportation of different nanomedicines through the epithelial barrier.

In vivo imaging of cerebral hydrogen peroxide (H2O2) may facilitate early diagnosis of cerebral ischemia reperfusion injury (CIRI) and a revelation of its pathological progression. In this study, we report our rational design of a brain-targeting fluorescent probe using the basis of a pyridazinone scaffold. A structure-activity relationship study reveals that PCAB is the best candidate (Ki = 15.8 nM) for a histamine H3 receptor (H3R), which is highly expressed in neurons of the central nervous system. As a two-photon fluorescent probe, PCAB exhibits a fast, selective reaction toward both extra- and intracellular H2O2 in SH-SY5Y cells under oxygen glucose deprivation and resupply. In vivo fluorescent imaging of a middle cerebral artery occlusion mouse confirms that PCAB is an ultrasensitive probe with potent blood-brain barrier penetration, precise brain targeting, and fast detection of CIRI.

Atherosclerosis, as a silent killer, remains one of the most common causes of human morbidity and mortality worldwide due to the lack of efficient strategy for early detection and targeted therapy. In this work, a self-driven bioinspired nanovehicle is developed, which can accurately manage early atherosclerosis with simultaneously multiple-targeting, dual-modality therapy as well as noninvasive magnetic resonance imaging (MRI). The magnetic nanoclusters (MNCs) with satisfactory superparamagnetism are camouflaged with leukocyte membranes, thus acquiring inherently targeting and transmigrating capabilities to intimal foam cells in early atherosclerotic lesions, which is validated using tailor-made microfluidic devices and transwell assays. Upon sequentially embedding an anti-inflammatory drug simvastatin (ST) and decorating a targetable apolipoprotein A-I mimetic 4F peptide (AP), the as-fabricated MNC@M-ST/AP exhibits excellent anti-atherosclerotic effects by alleviating inflammation and oxidative stress as well as promoting cholesterol efflux via RCT pathways. This bioinspired leukocyte membrane-hitchhiking strategy will open new perspectives for the future clinical translations of biocompatible nanosystem in early detection and treatment of atherosclerosis.

Angiogenesis is an essential pathological feature of vulnerable atherosclerotic plaque. Exosome-derived microRNAs (miRNAs or miRs) have been proven to be important regulators of angiogenesis. However, the role of exosomes, which are secreted by endothelial cells (ECs) under conditions of oxidative stress, in angiogenesis remain unclear. The present study aimed to investigate the effects and mechanism of oxidative stress-activated endothelial-derived exosomes in angiogenesis. Exosomes were isolated from H2O2-stimulated human umbilical vein ECs (HUVECs; termed Exo(-H2O2)) by differential centrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. Exo(-H2O2) enhanced HUVEC proliferation, migration and tube formation, as determined by EdU incorporation assay, scratch wound migration assay and tube formation assay, respectively. miR-92a-3p was identified as the predominantly downregulated miRNA in the Exo(-H2O2)-treated HUVECs by small RNA sequencing, and the expression of primary miR-92a (pri-miR-92a-1) was also decreased, as shown by RT-qPCR. Similarly, the inhibition of miR-92a-3p promoted angiogenesis in vitro and in vivo. miR-92a-3p overexpression blocked the pro-angiogenic effects of Exo(-H2O2) on target ECs. Tissue factor (TF), a molecule involved in angiogenesis, was increased in HUVECs in which miR-92a-3p expression was downregulated, as shown by mRNA sequencing. TF was also predicted as a target of miR-92a-3p by using the RNA-hybrid program. The overexpression or suppression of miR-92a-3p modified TF expression at both the mRNA and protein level, as measured by RT-qPCR and western blot analysis, respectively. Luciferase reporter assays suggested that miR-92a-3p inhibited TF expression by binding to the 3' untranslated region of TF. On the whole, the findings of the present study demonstrate that exosomes released from oxidative stress-activated ECs stimulate angiogenesis by inhibiting miR-92a-3p expression in recipient ECs, and TF may be involved in the regulatory effects of miR-92a-3p on angiogenesis.

目的 探讨脑胶质瘤微环境内组织间隙(extracellular space ,ECS)的结构特征以及瘤内脑组织间液( interstitial fluid,ISF)引流的变化. 方法 48只SD大鼠随机分为丘脑对照组、丘脑肿瘤组、尾状核对照组、尾状核肿瘤组,每组12只,每组又分为光学示踪亚组和磁示踪亚组各6只.应用光、磁示踪法,分别以Alexa Flour 594和钆喷酸葡铵(Gd-DTPA)作为示踪剂,对SD大鼠的丘脑、尾状核区C6胶质瘤内ECS结构及ISF的扩散过程进行示踪分析,并与相应对照组进行比较.应用MRI检测示踪剂在脑ECS中的扩散和分布,计算获取Gd-DTPA在脑ECS内的有效扩散系数( DECS)、清除速率( k’)、迂曲度(λ)和半衰期(t1/2)等扩散参数.应用共聚焦显微镜对注射示踪剂2 h后的离体脑切片成像,并分析得到扩散分布的最大面积.对同一脑区对照组与肿瘤组的光、磁示踪结果 进行对比分析. 结果 与丘脑对照组比较,丘脑肿瘤组的k’显著增加[(7.27 ±1.08)×10 －4 mm2/s vs.(3.69 ±0.46)×10－4 mm2/s, t＝7.474, P＝0.000],t1/2显著缩短[(21.36 ±2.67)min vs. (53.86 ±3.18)min, t＝－19.165, P＝0.000],DECS显著减小[(2.27 ±0.22)×10 －4 mm2/s vs.(3.14 ±0.41)×10 －4 mm2/s, t＝－4.536, P＝0.001],λ显著增加[(2.11 ±0.10)%vs.(1.06 ±0.01)%, t＝25.201, P＝0.000].与尾状核对照组比较,尾状核肿瘤组的k’显著增加[(6.87 ±1.09)×10 －4 mm2/s vs.(3.25 ±0.31)×10 －4 mm2/s, t＝7.867, P＝0.000],t1/2显著缩短[(23.77 ±7.31) min vs.(87.20 ±4.31) min, t＝－18.309, P＝0.000],DECS显著减小[(2.38 ±0.79)×10 －4 mm2/s vs. (3.35 ±0.12)×10 －4 mm2/s, t＝－2.986, P＝0.014],λ显著增加[(2.12 ±0.31)% vs.(1.73 ±0.03)%, t＝3.067, P＝0.012]. 结论 丘脑和尾状核区胶质瘤内ISF的k’、λ显著增加,t1/2 、DECS显著减少.

On the basis of the pyridazinone scaffold and photoinduced electron transfer (PET) mechanism, we designed a smart nitric oxide (NO) probe, PYSNO, with high sensitivity and selectivity. PYSNO exhibited a rapid response to both exogenous and endogenous NO. This probe can also be used in tracking and investigating NO generation in animal tissue. In the myocardial fibrosis model for mice, PYSNO exhibited a powerful imaging property in vivo as a result of unravelling the progressive relationship between the generation of myocardial NO and the occurrence of myocardial fibrosis.