Hypoxic bone marrow mesenchymal stem cell exosomes promote angiogenesis and enhance endometrial injury repair through the miR-424-5p-mediated DLL4/Notch signaling pathway

Cell culture

Human umbilical vein endothelial cells (HUVECs) were purchased from Otwo Biotench (HTX3606; Shenzhen, China), and BMSCs were purchased from Otwo Biotench (HTX1946; Shenzhen, China). The cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in a 5% CO₂ incubator until the cell density reached 80%. A coculture system of HUVECs and exosomes was established using a Transwell system. HUVECs and 100 μg/ml exosomes were cultured in the upper and lower chambers of a Transwell system for 24, 48, 72 and 96 h at 37 °C in a 5% CO₂ incubator. To verify that miR-424-5p facilitates angiogenesis through the DLL4/Notch pathway, 500 ng/ml Jagged1, an activator of the Notch pathway, or 10 μmol/L DAPT, an inhibitor of the Notch pathway, was added to the cell culture dish at 37 °C. The cells were cultured in a 5% CO₂ incubator for 24 h and then harvested for analysis.

Cell transfection

BMSCs were collected in the logarithmic growth phase, flushed with PBS, digested with 0.25% trypsin solution, seeded in 24-well plates, and then cultured for 24 h in 5% CO₂ at 37 °C. The cells were then transfected with Lipofectamine^® 3000 reagent (Invitrogen, Waltham, MA, USA), the miR-424-5p inhibitor, the miR-424-5p mimic and the corresponding negative controls (NC-inhibitor, NC-mimic), pc-DNA, and pc-DLL4. The transfection efficiency was measured 48 h after transfection, and the cells were used for subsequent experiments after successful transfection.

Animal model

In this study, female SD rats aged 8 weeks and weighing 180–220 g (bought from the Animal Experimental Center of Kunming Medical University) were selected as the experimental animals and kept in the same cage. This study was authorized by the Animal Ethics Committee of Kunming Medical University (Approval number: kmmu20220879), and all methods conformed to the stipulations of the National Institutes of Health Guidelines for the Care of Laboratory Animals. The rats were adaptively fed for 1 week under standard SPF quality conditions in an environment with a temperature of 22–26 °C, a relative humidity of 52–58%, and a 12 h/12 h light-dark cycle. Thirty-six mice were randomly divided into three groups: the normal control group (Group 1), the IUA group (Group 2), and the IUA+PBS group (Group 3). Group 1 rats (n = 6) were healthy and did not undergo any manipulation. The Group 2 and Group 3 rats were healthy, and an IUA rat model was established by anesthetizing the rats. Then, the skin (approximately 2 cm long) was longitudinally incised approximately 1 cm above the urethral orifice, and the endometrium was scraped with a small ear-picking spoon until the uterine wall became rough and pale. Finally, the uterus was flushed with normal saline, and the wound was sutured. Group 3 (five rats) received intrauterine injections of 0.2 ml of PBS in addition to completing the curettage procedure.

On the 14^th day after surgery, Group 2 (25 rats) were randomly divided into five groups with five rats in each group and treated according to the treatment group. Group A (IUAs) rats received no treatment. Group B rats (IUAs+BMSC-hypo-exos) were treated with BMSC-hyp-exos. Group C rats (IUAs+miR-424-5p mimic) were treated with the miR-424-5p mimic. Group D rats (IUAs+miR-424-5p mimic+pc-DLL4) were treated with the miR-424-5p mimic and overexpressing DLL4. Group E rats (IUAs+miR-424-5p mimic+Jagged1) were treated with a miR-424-5p mimic and a Notch pathway activator (Jagged1).

Purification and characterization of exosomes

BMSC-exos were purified by differential centrifugation. BMSCs were cultured in serum-free DMEM for 48 h. The supernatant was then centrifuged using a differential centrifugation method, after which the cells were collected and concentrated. The concentrated supernatant was loaded onto a 30% sucrose/D₂O buffer pad (5 ml, density 1.210 G/cm³), followed by ultracentrifugation at 100,000 × g for 3 h. After the test tube was centrifuged, the liquid components at the bottom of the test tube were collected, flushed with PBS, and centrifuged at 1,500 × g and 100 kDa MWCO for 30 min. The supernatant and exosomes were subsequently filtered through a 0.22 μm filter and stored at −70 °C. CD9, CD63, CD81 and HSP70 were identified via western blotting. The morphology of the purified exosomes was observed via transmission electron microscopy (Hitachi H-7650; Hitachi, Tokyo, Japan).

Exosome labeling and internalization

BMSC-exo was traced with PKH67 (HR8659; Beijing Biotech Co., Ltd., Beijing, China) dye. The labeled exosomes were then suspended in a 100-kDa MWCO hollow fiber membrane, and PBS was used as the negative control. HUVECs (5 × 10³ per well) were seeded in 96-well plates, hatched with traced exosomes for 4 h at 37 °C, flushed with PBS and immobilized in 4% paraformaldehyde. Finally, a confocal image was obtained with an Array Scan VTI (Thermo Fisher Scientific, Waltham, MA, US).

Identification by flow cytometry

The cells were resuspended into single cells, after which 10 μL of fluorescein isothiocyanate (FITC)-labeled anti-CD9, anti-CD63 and anti-CD81 antibodies (Abcam, Cambridge, UK) were added. The cells were then incubated at 4 °C for 20 min in the dark, flushed twice with PBS, and resuspended in 1 ml of PBS. Flow cytometry was used to measure fluorescence intensity, and CD9, CD63, and CD81 levels were analyzed using Flow Jo 7.2 software.

Anoxic treatment

After counting, BMSCs were seeded into 10 cm dishes at a mass of 1 × 10⁷ after they were cultured in complete substrate supplemented with 10% FBS for 12 h, after which the medium was replaced with 10 ml of serum-free medium per dish (DMEM/F12) after full adherence. The cells were then cultured in anoxic incubators (0.5% O₂, 5% CO₂) or normoxic incubators (21% O₂, 5% CO₂) for 48 h.

CCK-8 cell viability assay

Cell activity was measured by CCK-8 according to the manufacturer’s instructions. Cells were seeded into 96-well plates at 5 × 10³ cells/well and cultured for the indicated durations. Then, 10 µl of CCK-8 reagent was added to the substrate in each well. After 1 h of incubation, the optical density (OD) values were read by a microplate reader at a wavelength of 450 nm. This process was repeated for three wells at each time point, and the experiment was independently executed three times.

Western blot

The total protein was extracted from the cells and tissues, and the protein concentration was determined using a BCA test kit. The samples were separated with 10% SDS‒PAGE, transferred to PVDF membranes, soaked in 5% skim milk powder, and blocked at room temperature for 2 h. Prediluted primary antibodies against CD9 (1:500), HSP70 (1:300), CD63 (1:300), CD81 (1:300), DLL4 (1:500), Notch1 (1:500), Notch2 (1:2,000), and GAPDH (1:500) were added, and the samples were subsequently incubated overnight at 4 °C. After the membrane was flushed, the samples were incubated with an enzyme-labeled secondary antibody (1:2,000) at room temperature for 2 h. Quantitative tests were performed using an enhanced chemiluminescence (ECL) kit, and the band gray values were analyzed via ImageJ.

Immunofluorescence assay

HUVECs were grown on glass covers overnight to prepare cell slides, immobilized with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 15 min, blocked with 5% bovine serum albumin at room temperature for 1 h, flushed with PBS, incubated with a primary antibody against CD34 (Ab81289, 1:50) overnight at 37 °C, incubated with a fluorescein isothiocyanate-labeled secondary antibody for 2 h, and finally incubated with Hoechst 33342 (1:200; Sigma‒Aldrich, St. Louis, MO, US) for 15 min in the dark for counterstaining. The cells were then observed using a fluorescence microscope.

RT–qPCR

Total RNA was extracted using TRIzol reagent and reverse transcribed into cDNA. RT‒qPCR was performed using SYBR Green mix, with U6 and GAPDH serving as internal controls. The detailed RT‒qPCR primer sequences are shown in Table 1, and the relative gene expression was calculated using the 2^−ΔΔCt method.

Tissue staining

The rat uteruses were flushed with PBS, and paraffin sections of the tissue were prepared. The sections were routinely dewaxed and dyed with hematoxylin for 1 min for HE staining. The plants were then flushed with tap water, differentiated with 1% hydrochloric acid for 10 s, blued with 1% ammonia water for 5 s, dyed with eosin for 2 min, and sealed after dehydration for observation and analysis. For the immunohistochemical reaction, the sections were routinely dewaxed, incubated with primary antibodies against CD31 (ab28364, 1:50) and VEGFA (ab52917, 1:100) overnight at 4 °C, flushed with PBS, and incubated with secondary antibodies (1:1,000) overnight at 4 °C. After the excess PBS was removed from the sections, the sections were chemically stained, dehydrated, and sealed for observation and analysis.

Angiogenesis test

Matrigel (50 μL) was added to each well, after which the cells were allowed to polymerize for the endothelial tube formation assay. The cells in each group were then seeded on Matrigel in a 96-well plate at a mass of 1 × 10⁵ cells, observed by microscopy, and then photographed after hatching at 37 °C for 12 h.

Target binding site prediction dual-luciferase gene reporter experiment

The wild-type (WT) 3′-UTR of DLL4 cDNA was synthesized and cloned using the starBase database to identify the site at which miR-424-5p binds to DLL4, which was subsequently input into the pMIR luciferase miRNA target expression reporter to generate the WT-DLL4-3′-UTR. Based on the name of the WT-DLL4-3′-UTR, the nucleotide that may bind to miR-424-5p to generate a mutant DLL4-3′-UTR, the resulting vector was named MUT-DLL4-3′-UTR. These vectors were then transiently transfected with the miR-424-5 p mimic or NC mimic into 293T cells using Liposome 3000 reagent. Luciferase activity was then assessed after 48 h of transfection, and the Renilla luciferase vector was used as the internal control.

Statistical analysis

GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA) was used for analyzing the experimental data and for plotting the graphics. Student’s t test and one-way analysis of variance were used for statistical analysis. P < 0.05 was considered to indicate statistical significance.

Hypoxic BMSC-derived exosomes significantly promoted the proliferation and angiogenesis of HUVECs

The effects of normal oxygen BMSC exosomes and hypoxic BMSC exosomes on the proliferation and angiogenesis of HUVECs were observed. First, the extracted exosomes were identified. The morphology of the purified exosomes observed by transmission electron microscopy was characterized by the presence of circular vesicles (Fig. 1A). Flow cytometry was used to detect the exosome markers CD9, CD63 and CD81 (which are involved in the transport of exosomes on the membrane), and the results showed that the cells were all positive for the exosome markers CD9, CD63 and CD81 (Fig. 1B). Western blotting also detected the expression of the exosome markers CD9, CD63, CD81 and HSP70 (Fig. 1C). This finding indicated that we successfully extracted the exosomes. We cocultured exosomes with HUVECs to observe the proliferation and angiogenesis of HUVECs induced by the exosomes. It was first confirmed that HUVECs can take up exosomes by PKH67 fluorescence staining; that is, BMSCs-exos with green fluorescence were observed in the nuclei of blue HUVECs (Fig. 1D). Then, HUVECs that were not cocultured with exosomes were taken as the normal control group (NC), and HUVECs that were cocultured with BMSC exosomes treated with normal oxygen (the BMSCs-norm-exo group) and BMSC exosomes treated with hypoxia (the BMSCS-hyp-exo group) were taken as the experimental group. The proliferation of HUVECs detected by a CCK-8 assay showed that, compared with that in the NC group, the viability of the cells in the BMSC-norm-exo and BMSC-hyp-exo groups was significantly greater, and the ability of the BMSC-hyp-exo group to promote cell proliferation was greater than that of the BMSC-norm-exo group (Fig. 1E). The expression of Ang1 (maintaining vascular stability), Flk1 (encoding vascular endothelial growth Factor A receptor), Vash1 (inhibiting angiogenesis), and TSP1 (inhibiting angiogenesis) was detected by RT‒qPCR. The results showed that the expression levels of Ang1 and Flk1 were significantly upregulated after coculture with exosomes. The expression levels of Vash1 and TSP1 were significantly downregulated, and the regulatory effect of exosomes on angiogenesis-related genes was more significant after hypoxia treatment (Figs. 1F–1I). Vascular formation experiments showed that HUVECs had a denser and more extended tubular network after coculture with exosomes, and the effect of the BMSC-hyp-exo group was greater than that of the BMSC-norm-exo group (Fig. 1J). These results indicate that BMSC-derived exosomes can promote the proliferation and angiogenesis of HUVECs and that the stimulatory effect of hypoxic BMSC-derived exosomes is more significant than that of normally oxygenated BMSC-derived exosomes.

Hypoxic BMSC-exos promote cell proliferation and angiogenesis through miR-424-5p

Through RT‒qPCR, we observed that miR-424-5p was highly expressed in the exosome group compared with the NC group, and its expression in the BMSC-hyp-exo group was significantly greater than that in the BMSC-norm-exo group (Fig. 2A). These results indicated that hypoxia promoted the expression of the BMSC-derived exosomal miR-424-5p. To further clarify the mechanism by which hypoxia-related BMSC-exos regulate cell proliferation and angiogenesis via miR-424-5p, a miR-424-5p inhibitor and miR-424-5p mimic were transfected into BMSCs, and exosomes were extracted from the BMSCs after hypoxia treatment. The exosomes were then cocultured with HUVECs. First, the transfection efficiency was confirmed. The results showed that the expression of miR-424-5p was significantly downregulated in the co-hyp-exo+miR-424-5p inhibitor group and significantly upregulated in the co-hyp-exo+miR-424-5p mimic group, indicating successful transfection (Fig. 2B). The proliferation of HUVECs was detected by CCK-8, and the results showed that the proliferation of HUVECs in the co-hyp-exo group was greater than that in the NC group. However, proliferation decreased in the co-hyp-exo+miR-424-5p inhibitor group compared with the co-hyp-exo+NC-inhibitor group. Compared with that in the co-hyp-exo+NC-mimic group, the proliferative activity in the co-hyp-exo+miR-424-5p mimic group was increased (Fig. 2C). These results indicated that the ability of exosomes to promote HUVEC proliferation was weakened by transfection of the miR-424-5p inhibitor, while the opposite effect was observed with transfection of the miR-424-5p mimic. The expression of the proangiogenic genes Ang1 and Flk1 and the antiangiogenic genes Vash1 and TSP1 was detected by RT‒qPCR. The results showed that the expression of Ang1 and Flk1 was upregulated and that the expression of Vash1 and TSP1 was downregulated after coculture with exosomes. Transfection of the miR-424-5p inhibitor inhibited the expression of Ang1 and Flk1 and promoted the expression of Vash1 and TSP1, while transfection of the miR-424-5p mimic had the opposite effect (Figs. 2D–2J). Finally, angiogenesis experiments showed that angiogenesis was enhanced in the co-hyp-exo group compared with the control group, and after transfection with the miR-424-5p inhibitor, angiogenesis was weakened compared with that in the co-hyp-exo+NC-inhibitor group. After transfection with the miR-424-5p mimic, angiogenesis was further enhanced compared with that in the co-hyp-exo+NC-mimic group (Fig. 2H). These findings suggested that hypoxic BMSC-exos promote cell proliferation and angiogenesis through miR-424-5p.

Hypoxic exosome-mediated angiogenesis is related to the DLL4/Notch signaling pathway

Studies have shown that the DLL4/Notch signaling pathway plays an important role in angiogenesis; therefore, we explored whether hypoxic exosomes affect cell angiogenesis through the DLL4/Notch signaling pathway. First, the expression of DLL4/Notch signaling pathway-related proteins (DLL4, Notch1 and Notch2) after coculture of hypoxic exosomes with HUVECs and overexpression of DLL4 (pc-DLL4) was detected. Western blot analysis of the cells cocultured with hypoxic exosomes and HUVECs revealed that the expression levels of DLL4, Notch1 and Notch2 were downregulated, while the expression levels of DLL4, Notch1 and Notch2 were upregulated after the overexpression of DLL4 (Fig. 3A). These findings suggested that the processing of hypoxic exosomes induces changes in the DLL4/Notch signaling pathway and that DLL4 is the key protein involved. Moreover, the formation of HUVECs was detected. We detected the expression of the marker CD34 in endothelial tip cells by immunofluorescence and flow cytometry. The results showed that the expression of the marker CD34 in endothelial tip cells was upregulated after coculture of hypoxic exosomes with HUVECs but downregulated after overexpression of DLL4 (Figs. 3B and 3C). These results suggest that the promotive effect of hypoxic exosomes on CD34 expression can be reversed by overexpression of DLL4. In addition, RT‒qPCR and angiogenesis experiments showed that after hypoxic exosome treatment, the expression of the proangiogenic genes Ang1 and Flk1 was upregulated, the expression of the antiangiogenic genes Vash1 and TSP1 was downregulated, and angiogenesis was significantly enhanced. However, after overexpression of DLL4, the above phenomenon was reversed (Figs. 3D–3H). These findings suggested that hypoxic exosome-mediated angiogenesis is related to the DLL4/Notch signaling pathway.

miR-424-5p targets the expression of DLL4 and affects angiogenesis

Using starBase, we predicted that DLL4 is a miR-424-5p-binding protein (Fig. 4A). A dual-luciferase reporter gene experiment showed that transfection of the miR-424-5p mimic significantly reduced the fluorescence activity of the WT-DLL4 group but had no effect on the fluorescence activity of the MUT-DLL4 group, which further confirmed the targeted binding relationship between miR-424-5p and DLL4 (Fig. 4B). Western blot analysis of the miR-424-5p mimic group revealed that the expression of DLL4 was downregulated compared with that in the NC-mimic group and upregulated in the miR-424-5p inhibitor group compared with that in the NC-inhibitor group (Fig. 4C). These findings suggested that miR-424-5p can target and negatively regulate the expression of DLL4. To further clarify that miR-424-5p affects angiogenesis by regulating the DLL4 protein, we also overexpressed DLL4 (pc-DLL4) after transfecting cells with the miR-424-5p mimic and evaluated the effects of DLL4 on the effects of miR-424-5p. First, Western blot analysis showed that transfection of the miR-424-5p mimic inhibited the expression of DLL4, Notch1 and Notch2. However, after overexpression of DLL4, the effect of the miR-424-5p mimic was reversed to a certain extent, and the expression of DLL4, Notch1, and Notch2 was promoted (Fig. 4D). Immunofluorescence and flow cytometry showed that the miR-424-5p mimic promoted the expression of CD34, and overexpression of DLL4 reversed the effect of the miR-424-5p mimic (Figs. 4E and 4F). The RT‒qPCR results showed that the expression of Ang1 and Flk1 was upregulated and that the expression of Vash1 and TSP1 was downregulated in the miR-424-5p mimic group compared with the NC-mimic group. Compared with those in the miR-424-5p mimic+pc-DNA group, the expression of Ang1 and Flk1 was downregulated, and the expression of Vash1 and TSP1 was upregulated in the miR-424-5p mimic+pc-DLL4 group (Figs. 4G–4J). These results indicated that the effects of the miR-424-5p mimic on the expression of Ang1, Flk1, Vash1 and TSP1 were also reversed by overexpression of DLL4. Finally, angiogenesis experiments also showed that the promoting effect of the miR-424-5p mimic on angiogenesis was reversed by overexpression of DLL4 (Fig. 4K). This finding suggested that the effect of miR-424-5p on HUVEC angiogenesis occurs through the regulation of DLL4.

Hypoxic exosomes promote angiogenesis by mediating the DLL4/Notch signaling pathway through miR-424-5p

To clarify the molecular mechanism through which miR-424-5p promotes angiogenesis through the DLL4/Notch signaling pathway, Jagged1 (a Notch pathway activator) and DAPT (a Notch pathway inhibitor) were added after transfection of the cells with the miR-424-5p mimic. The effect of Jagged1 and DAPT on angiogenesis was assessed via RT‒qPCR and angiogenesis assays. The RT‒qPCR results showed that the expression of Ang1 and Flk1 was upregulated and that the expression of Vash1 and TSP1 was downregulated in the miR-424-5p mimic group compared with the NC-mimic group. Compared with those in the miR-424-5p mimic group, the expression of Ang1 and Flk1 was downregulated, the expression of Vash1 and TSP1 was upregulated in the miR-424-5p mimic+ Jagged1 group, the expression of Ang1 and Flk1 was further upregulated, and the expression of Vash1 and TSP1 was further downregulated in the miR-424-5p mimic+DAPT group (Figs. 5A–5D). Angiogenesis experiments showed that angiogenesis was greater in the miR-424-5p mimic group than in the NC-mimic group. Compared with that in the miR-424-5p mimic group, the angiogenesis ability of the miR-424-5p mimic+Jagged1 group was decreased, and the angiogenesis ability of the miR-424-5p mimic+DAPT group was further strengthened (Fig. 5E). These results indicated that activation of the DLL4/Notch signaling pathway could weaken the effect of the miR-424-5p mimic on promoting blood vessel formation. In general, hypoxic exosomes promote blood vessel formation through the DLL4/Notch signaling pathway mediated by miR-424-5p.

Hypoxic bone marrow mesenchymal stem cell exosomes improve endometrial injury in IUA rats

To test the influence of BMSC-exos on angiogenesis in vivo, we established an IUA model of uterine adhesions in rats. H&E staining revealed that the endometrial cavity surface of the NC group was covered with regularly arranged columnar epithelium, and abundant endometrial glands were observed. Compared with those in the NC group, the gross uterine morphology in the IUA and IUA + PBS groups was contracted, the endometrial glands were noticeably reduced, and tissue hyperplasia was observed. Pathology improved, and the number of endometrial glands increased in the IUA + BMSC-hyp-exo group (Fig. 6A). Immunohistochemical results showed that VEGFA and CD31 levels were lower in the IUA and IUA + PBS groups than in the NC group. VEGFA and CD31 levels were elevated in the IUA + BMSC-hyp-exo group compared to those in the IUA + PBS group (Fig. 6B). These results suggested that treatment with hypoxic BMSC exosomes promoted the expression of angiogenesis-related proteins. RT‒qPCR and Western blotting revealed that miR-424-5p was expressed at lower levels and that DLL4, Notch1, and Notch2 were expressed at higher levels in the IUA and IUA + PBS groups than in the NC group. miR-424-5p was upregulated and DLL4, Notch1, and Notch2 were downregulated in the IUA + BMSC-hyp-exo group compared with those in the IUA + PBS group (Figs. 6C and 6D). In animal experiments, the therapeutic effect of hypoxic BMSC-derived exosomes on IUAs was confirmed to be related to miR-424-5p and the DLL4/Notch pathway. These results indicate that BMSC-derived exosomes facilitate IUA vascularization and ameliorate endometrial damage in vivo.

miR-424-5p improves endometrial injury in IUA rats by mediating the DLL4/Notch signaling pathway

The mechanism by which miR-424-5p improves IUAs by mediating the DLL4/Notch signaling pathway to promote angiogenesis was further verified in vivo. H&E staining revealed that uterine tissue hyperplasia and endometrial gland density were significantly lower in the IUA group than in the NC group. Compared with that in the IUA group, the pathological status of rats in the IUA+miR-424-5p mimic group was improved. Tissue hyperplasia and endometrial glands from the IUA+miR-424-5p mimic+pc-DLL4 group and IUA+miR-424-5p mimic+Jagged1 group appeared again and were significantly reduced compared with those from the IUA+miR-424-5p mimic group (Fig. 7A). Treatment with the miR-424-5p mimic effectively alleviated endometrial injury in IUA rats, but treatment with the DLL4/Notch pathway activator reversed this effect. Immunohistochemical results showed that VEGFA and CD31 levels were significantly lower in the IUA group than in the NC group. Compared with those in the IUA group, the VEGFA and CD31 levels were greater in the IUA + miR-424-5p mimic group. VEGFA and CD31 levels in the IUA + miR-424-5p mimic + pc-DLL4 group and the IUA + miR-424-5p mimic + Jagged1 group were lower than those in the IUA + miR-424-5p mimic group (Figs. 7B and 7C). The RT‒qPCR and Western blot results showed that, compared with that in the NC group, the expression of miR-424-5p was downregulated, while the expression of DLL4, Notch1 and Notch2 was upregulated in the IUA group. Treatment with the miR-424-5p mimic promoted the expression of miR-424-5p and inhibited the expression of DLL4, Notch1, and Notch2, while pc-DLL4 and Jagged1 reversed the effects of the miR-424-5p mimic (Figs. 7C and 7D). These results suggest that miR-424-5p facilitates IUA vascularization in vivo by mediating DLL4/Notch signaling, ameliorating endometrial injury.