Cell Migration Of Human Umbilical Vein Endothelial Cells Research Articles

Unveiling the Regulatory Role of LncRNA MYU in Hypoxia-Induced Angiogenesis via the miR-23a-3p Axis in Endothelial Cells

Background: Angiogenesis is essential for various physiological and pathological processes, such as embryonic development and cancer cell proliferation, migration, and invasion. Long noncoding RNAs (lncRNAs) play pivotal roles in normal homeostasis and disease processes by regulating gene expression through various mechanisms, including competing endogenous RNAs (ceRNAs) of target microRNAs (miRNAs). The lncRNA MYU is known to promote prostate cancer proliferation via the miR-184/c-Myc regulatory axis and to be upregulated in vascular endothelial cells under hypoxic conditions, which often occurs in solid tumors. In the present study, we investigated whether MYU might affect cancer growth by regulating angiogenesis in vascular endothelial cells under hypoxia. Methods: The expression of MYU-regulated miR-23a-3p and interleukin-8 (IL-8) in HUVEC cell lines was examined using qRT-PCR. The CCK-8 assay, EdU assay, wound-healing assay, and tube-formation assay were used to assess the effects of MYU on cell proliferation, migration, and tube formation of HUVEC cells in vitro. The dual-luciferase reporter assay was performed to examine the effects of miR-23a-3p on MYU and IL-8 expression. Results: We found that the overexpression of MYU and knockdown of miR-23a-3p in human umbilical vein endothelial cells (HUVECs) under hypoxia promoted cell proliferation, migration, and tube formation. Mechanistically, MYU was shown to bind competitively to miR-23a-3p, thereby preventing miR-23a-3p binding to the 3′ untranslated region of IL-8 mRNA. In turn, increased production of pro-angiogenic IL-8 promoted HUVEC proliferation, migration, and tube formation under hypoxia. Conclusion: This study identified a new role for lncRNA MYU as a ceRNA for miR-23a-3p and uncovered a novel MYU–miR-23a-3p–IL-8 regulatory axis for angiogenesis. MYU and/or miR-23a-3p may thus represent new targets for the treatment of hypoxia-related diseases by promoting angiogenesis.

METTL3-mediated NDUFB5 m6A modification promotes cell migration and mitochondrial respiration to promote the wound healing of diabetic foot ulcer

BackgroundDiabetic foot ulcer (DFU) is the most devastating complication of diabetes mellitus (DM) and plays a major role in disability and death in DM patients. NADH: ubiquinone oxidoreductase subunit B5 (NDUFB5) plays an important role in maintaining mitochondrial respiration, but whether it is involved in regulating the progression of advanced glycation end products (AGEs)-mediated DFU is still unclear.MethodsFirstly, the role of AGEs on cell viability, migration, and mitochondrial respiration in human umbilical vein endothelial cells (HUVECs) was explored in vitro. Next, NDUFB5 expression was detected in human samples and AGEs-treated HUVECs, and NDUFB5’s effect on AGEs-induced HUVECs injury and skin wound in diabetic mice was further clarified. In addition, the role of m6A modification mediated by methyltransferase-like 3 (METTL3) in regulating NDUFB5 expression and AGEs-induced HUVECs injury was investigated.ResultsNDUFB5 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs, whereas mitochondrial fusion promoter M1 facilitated cell viability, migration, and mitochondrial oxiadative respiration in NDUFB5 knockdown HUVECs. Meanwhile, NDUFB5 promotes skin wound healing in diabetic mice. Besides, METTL3-mediated m6A modification and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) enhanced NDUFB5 expression in HUVECs. Furthermore, METTL3 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs by increasing NDUFB5.ConclusionMETTL3-mediated NDUFB5 m6A modification inhibits AGEs-induced cell injury in HUVECs. METTL3 and NDUFB5 might serve as potential targets for DFU therapy in the future.

METTL3-mediated NDUFB5 m6A modification promotes cell migration and mitochondrial respiration to promote the wound healing of diabetic foot ulcer

BackgroundDiabetic foot ulcer (DFU) is the most devastating complication of diabetes mellitus (DM) and plays a major role in disability and death in DM patients. NADH: ubiquinone oxidoreductase subunit B5 (NDUFB5) plays an important role in maintaining mitochondrial respiration, but whether it is involved in regulating the progression of advanced glycation end products (AGEs)-mediated DFU is still unclear.MethodsFirstly, the role of AGEs on cell viability, migration, and mitochondrial respiration in human umbilical vein endothelial cells (HUVECs) was explored in vitro. Next, NDUFB5 expression was detected in human samples and AGEs-treated HUVECs, and NDUFB5’s effect on AGEs-induced HUVECs injury and skin wound in diabetic mice was further clarified. In addition, the role of m6A modification mediated by methyltransferase-like 3 (METTL3) in regulating NDUFB5 expression and AGEs-induced HUVECs injury was investigated.ResultsNDUFB5 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs, whereas mitochondrial fusion promoter M1 facilitated cell viability, migration, and mitochondrial oxiadative respiration in NDUFB5 knockdown HUVECs. Meanwhile, NDUFB5 promotes skin wound healing in diabetic mice. Besides, METTL3-mediated m6A modification and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) enhanced NDUFB5 expression in HUVECs. Furthermore, METTL3 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs by increasing NDUFB5.ConclusionMETTL3-mediated NDUFB5 m6A modification inhibits AGEs-induced cell injury in HUVECs. METTL3 and NDUFB5 might serve as potential targets for DFU therapy in the future.

Hypoxic adipose stem cell-derived exosomes carrying high-abundant USP22 facilitate cutaneous wound healing through stabilizing HIF-1α and upregulating lncRNA H19.

Hypoxic preconditioning has been recognized as a promotive factor for accelerating cutaneous wound healing. Our previous study uncovered that exosomal lncRNA H19, derived from adipose-derived stem cells (ADSCs), plays a crucial role in orchestrating cutaneous wound healing. Herein, we aimed to explore whether there is a connection between hypoxia and ADSC-derived exosomes (ADSCs-exos) in cutaneous wound healing. Exosomes extracted from ADSCs under normoxic and hypoxic conditions were identified using transmission electron microscope (TEM) and particle size analysis. The effects of ADSCs-exos on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8, EdU, wound healing, and tube formation assays. Expression patterns of H19, HIF-1α, and USP22 were measured. Co-immunoprecipitation, chromatin immunoprecipitation, ubiquitination, and luciferase reporter assays were conducted to confirm the USP22/HIF-1α/H19 axis, which was further validated in a mice model of skin wound. Exosomes extracted from hypoxia-treated ADSCs (termed as H-ADSCs-exos) significantly increased cell proliferation, migration, and angiogenesis in H2O2-exposed HUVECs, and promoted cutaneous wound healing invivo. Moreover, H-ADSCs and H-ADSCs-exos, which exhibited higher levels of H19, were found to be transcriptionally activated by HIF-1α. Mechanically, H-ADSCs carrying USP22 accounted for deubiquitinating and stabilizing HIF-1α. Additionally, H-ADSCs-exos improved cell proliferation, migration, and angiogenesis in H2O2-triggered HUVECs by activating USP22/HIF-1α axis and promoting H19 expression, which may provide a new clue for the clinical treatment of cutaneous wound healing.

The requirement of the mitochondrial protein NDUFS8 for angiogenesis

Mitochondria are important for the activation of endothelial cells and the process of angiogenesis. NDUFS8 (NADH:ubiquinone oxidoreductase core subunit S8) is a protein that plays a critical role in the function of mitochondrial Complex I. We aimed to investigate the potential involvement of NDUFS8 in angiogenesis. In human umbilical vein endothelial cells (HUVECs) and other endothelial cell types, we employed viral shRNA to silence NDUFS8 or employed the CRISPR/Cas9 method to knockout (KO) it, resulting in impaired mitochondrial functions in the endothelial cells, causing reduction in mitochondrial oxygen consumption and Complex I activity, decreased ATP production, mitochondrial depolarization, increased oxidative stress and reactive oxygen species (ROS) production, and enhanced lipid oxidation. Significantly, NDUFS8 silencing or KO hindered cell proliferation, migration, and capillary tube formation in cultured endothelial cells. In addition, there was a moderate increase in apoptosis within NDUFS8-depleted endothelial cells. Conversely, ectopic overexpression of NDUFS8 demonstrated a pro-angiogenic impact, enhancing cell proliferation, migration, and capillary tube formation in HUVECs and other endothelial cells. NDUFS8 is pivotal for Akt-mTOR cascade activation in endothelial cells. Depleting NDUFS8 inhibited Akt-mTOR activation, reversible with exogenous ATP in HUVECs. Conversely, NDUFS8 overexpression boosted Akt-mTOR activation. Furthermore, the inhibitory effects of NDUFS8 knockdown on cell proliferation, migration, and capillary tube formation were rescued by Akt re-activation via a constitutively-active Akt1. In vivo experiments using an endothelial-specific NDUFS8 shRNA adeno-associated virus (AAV), administered via intravitreous injection, revealed that endothelial knockdown of NDUFS8 inhibited retinal angiogenesis. ATP reduction, oxidative stress, and enhanced lipid oxidation were detected in mouse retinal tissues with endothelial knockdown of NDUFS8. Lastly, we observed an increase in NDUFS8 expression in retinal proliferative membrane tissues obtained from human patients with proliferative diabetic retinopathy. Our findings underscore the essential role of the mitochondrial protein NDUFS8 in regulating endothelial cell activation and angiogenesis.

NEDD4L is a promoter for angiogenesis and cell proliferation in human umbilical vein endothelial cells.

Dysregulated angiogenesis leads to neovascularization, which can promote or exacerbate various diseases. Previous studies have proved that NEDD4L plays an important role in hypertension and atherosclerosis. Hence, we hypothesized that NEDD4L may be a critical regulator of endothelial cell (EC) function. This study aimed to define the role of NEDD4L in regulating EC angiogenesis and elucidate their underlying mechanisms. Loss- and gain-of-function of NEDD4L detected the angiogenesis and mobility role in human umbilical vein endothelial cells (HUVECs) using Matrigel tube formation assay, cell proliferation and migration. Pharmacological pathway inhibitors and western blot were used to determine the underlying mechanism of NEDD4L-regulated endothelial functions. Knockdown of NEDD4L suppressed tube formation, cell proliferation and cell migration in HUVECs, whereas NEDD4L overexpression promoted these functions. Moreover, NEDD4L-regulated angiogenesis and cell progression are associated with the phosphorylation of Akt, Erk1/2 and eNOS and the expression of VEGFR2 and cyclin D1 and D3. Mechanically, further evidence was confirmed by using Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Overexpression NEDD4L-promoted angiogenesis, cell migration and cell proliferation were restrained by these inhibitors. In addition, overexpression NEDD4L-promoted cell cycle-related proteins cyclin D1 and D3 were also suppressed by Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Our results demonstrated a novel finding that NEDD4L promotes angiogenesis and cell progression by regulating the Akt/Erk/eNOS pathways.

TNF-α stimulated exosome derived from fibroblast-like synoviocytes isolated from rheumatoid arthritis patients promotes HUVEC migration, invasion and angiogenesis by targeting the miR-200a-3p/KLF6/VEGFA axis

The pathogenesis of rheumatoid arthritis (RA) is heavily impacted by the inflammation and activation of fibroblast-like synoviocytes (FLS). The objective of this investigation is to clarify the involvement of exosomes derived from FLS stimulated by tumour necrosis factor α (TNF-α) in angiogenesis and the underlying mechanisms. FLS cells were obtained from synovial fluid of RA patients and exosomes were obtained from FLS cell supernatant with TNF-α stimulation by ultracentrifugation. Exosomes were subsequently analysed using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. The functional effects of exosomes with TNF-α stimulation on human umbilical vein endothelial cells (HUVEC) migration, invasion, and angiogenesis was evaluated using wound scratch healing test, transwell invasion assay, and tube formation assay. DNA nanoball-seq (DNBSEQ) sequencing platform was utilised to analysis different expression miRNA from exosomes, miRNA and mRNA from HUVEC. The expression level of miR-200a-3p was determined through quantitative real-time polymerase chain reaction (qRT-PCR). The quantification of KLF6 and VEGFA expression levels were performed by qRT-PCR and western blot analysis. The validation of the association between miR-200a-3p and KLF6 was established through a fluorescence enzyme reporting assay. In comparison to exosome induced by PBS, exosome induced by TNF-α exhibited a substantial exacerbation of invasion, migration, and angiogenesis in HUVEC. 4 miRNAs in exosomes and HUVEC cells, namely miR-1246, miR-200a-3p, miR-30a-3p, and miR-99b-3p was obtained. MiR-200a-3p maintained high consistency with the sequencing results. We obtained 5 gene symbols, and KLF6 was chose for further investigation. The expression of miR-200a-3p in exosomes induced by TNF-α and in HUVEC treated with these exosomes demonstrated a significantly increase. Additionally, HUVEC cells displayed a notable decrease in KLF6 expression and a significant elevation in VEGFA expression. This was further confirmed by the fluorescence enzyme report assay, which provided evidence of the direct targeting of KLF6 by miR-200a-3p. Exosomes induced by TNF-α have the ability to enhance the migration, invasion, and angiogenesis of HUVEC cells via the miR-200a-3p/KLF6/VEGFA axis.

External Application of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Hyaluronic Acid Gel Repairs Foot Wounds of Types I and II Diabetic Rats Through Paracrine Action Mode.

Diabetic foot ulcer (DFU) is a main diabetic complication with unmet treatment needs. This study applied human umbilical cord-derived mesenchymal stem cells-hyaluronic acid (hucMSCs-HA) gel to treat DFU in a noninvasive external way and investigated its paracrine action and mechanism. In this study, after analyzing the physical and biological properties of HA gel, hucMSCs-HA gel was applied in 2 in vivo models (types I and II DFU), and a molecular mechanism was investigated. To evaluate the paracrine action of hucMSCs, hucMSCs-conditional medium (MSC-CM) was collected to treat 1 in vivo model (type I DFU) and 2 in vitro models (high glucose (HG)-injured human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs)). The results indicated that HA gel with a porous microstructure underwent over 90% degradation and swelled to the maximum value within 48 h. In vivo, hucMSCs-HA gel accelerated wound healing of DFU rats by improving re-epithelialization, collagen deposition, and angiogenesis, in which a paracrine action of hucMSCs was confirmed and the phosphorylation of p38, ERK1/2, JNK, and Akt was increased. In vitro, MSC-CM improved cell viability, wound healing, migration, tube formation, cell senescence, and abnormal expressions (TNF-α, IL-1β, IL-6, ET-1, p16 genes, and PCNA protein) of HUVECs, also improved cell viability, wound healing, antioxidant stress, and abnormal expressions (COL1, COL3, COL4, SOD1, SOD2 genes, and PCNA protein) of HSFs. Summarily, noninvasive external application of hucMSCs-HA gel shows great perspective against DFU and exerts wound healing effects through the MAPK and Akt pathways-mediated paracrine mechanism.

Tartary buckwheat protein-derived peptide AFYRW alleviates H2O2-induced vascular injury via the PI3K/AKT/NF-κB pathway

Tartary buckwheat protein-derived peptide (Ala-Phe-Tyr-Arg-Trp, AFYRW) is a natural active peptide that hampers the atherosclerosis process, but the underlying role of AFYRW in angiogenesis remains unknown. Here, we present a system-based study to evaluate the effects of AFYRW on H2O2-induced vascular injury in human umbilical vein endothelial cells (HUVECs). HUVECs were co-incubated with H2O2 for 2 h in the vascular injury model, and AFYRW was added 24 h in advance to investigate the protective mechanism of vascular injury. We identified that AFYRW inhibits oxidative stress, cell migration, cell invasion, and angiogenesis in H2O2-treated HUVECs. In addition, we found H2O2-induced upregulation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), phosphorylation of nuclear factor-κB (NF-κB) p65 and nuclear translocation of NF-κB decreased by AFYRW. Taken together, AFYRW attenuated H2O2-induced vascular injury through the PI3K/AKT/NF-κB pathway. Thereby, AFYRW may serve as a therapeutic option for vascular injuries.

Exosomes from hypoxia-conditioned apical papilla stem cells accelerate angiogenesis in vitro through Notch/JAG1/VEGF signaling

Dental pulp angiogenesis is a committed step in pulp regeneration therapy, and exosomes provide a new cell-free choice for tissue regeneration. This study revealed the underlying regulatory mechanism of exosomes from stem cells of the apical papilla (SCAPs) under hypoxic state on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. Exosomes extracted from normoxia or hypoxia-pretreated SCAPs were co-cultured with HUVECs, and hypoxia pretreatment increased the release of exosomes and the internalization of exosomes by HUVECs. Compared to normoxic SCAPs-derived exosomes, exosomes from hypoxic SCAPs were found to promote cell proliferation and migration in HUVECs, as it was respectively determined by Cell Counting Kit-8, RT-qPCR and Transwell assay. Besides, hypoxia-educated SCAPs-exosomes especially enhanced the angiogenesis abilities of HUVECs in vitro, which were confirmed by tube formation assay and RT-qPCR detection of angiogenesis-related molecular markers. Interestingly, we found that the hypoxia inducible factor-1α (HIF-1α)/Notch1 signaling pathway was activated in hypoxic SCAPs, and protein jagged-1 (JAG1) was delivered by hypoxic SCAPs-derived exosomes to increase vascular endothelial growth factor (VEGF) production in HUVECs. Moreover, exogenous interference of JAG1 expression in HUVECs partially neutralized the activities of hypoxic SCAPs-exosomes in promoting cell proliferation, migration and tube formation of HUVECs. In summary, this study elucidates that exosomes from hypoxic SCAPs shows high potential to promote angiogenesis in vitro through the HIF-1α/JAG1/VEGF signaling cascade, which may provide a new perspective for the development of vascular reconstruction measures during dental regeneration engineering.

Downregulation of VEGFR2 signaling by cedrol abrogates VEGF‑driven angiogenesis and proliferation of glioblastoma cells through AKT/P70S6K and MAPK/ERK1/2 pathways.

Cedrol is a sesquiterpene alcohol isolated from Cedrus atlantica, which has been traditionally used in aromatherapy and has anticancer, antibacterial and antihyperalgesic effects. One characteristic of glioblastoma (GB) is the overexpression of vascular endothelial growth factor (VEGF), which induces a high degree of angiogenesis. Although previous studies have reported that cedrol inhibits GB growth by inducing DNA damage, cell cycle arrest and apoptosis, its role in angiogenesis remains unclear. The aim of the present study was to investigate the effects of cedrol on VEGF-induced angiogenesis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 0-112 µM cedrol and 20 ng/ml VEGF for 0-24 h, and then anti-angiogenic activation of cedrol was determined by MTT assay, wound healing assay, Boyden chamber assay, tube formation assay, semi-quantitative reverse transcription-PCR and western blotting. These results demonstrated that cedrol treatment inhibited VEGF-induced cell proliferation, migration and invasion in HUVECs. Furthermore, cedrol prevented VEGF and DBTRG-05MG GB cells from inducing capillary-like tube formation in HUVECs and decreased the number of branch points formed. Moreover, cedrol downregulated the phosphorylation of VEGF receptor 2 (VEGFR2) and the expression levels of its downstream mediators AKT, ERK, VCAM-1, ICAM-1 and MMP-9 in HUVECs and DBTRG-05MG cells. Taken together, these results demonstrated that cedrol exerts anti-angiogenic effects by blocking VEGFR2 signaling, and thus could be developed into health products or therapeutic agents for the prevention or treatment of cancer and angiogenesis-related diseases in the future.

Umbilical cord blood-derived exosomes from healthy term pregnancies protect against hyperoxia-induced lung injury in mice.

Bronchopulmonary dysplasia (BPD) is a chronic, devastating disease primarily occurring in premature infants. To date, intervention strategies to prevent or treat BPD are limited. We aimed to determine the effects of umbilical cord blood-derived exosomes (UCB-EXOs) from healthy term pregnancies on hyperoxia-induced lung injury and to identify potential targets for BPD intervention. A mouse model of hyperoxia-induced lung injury was created by exposing neonatal mice to hyperoxia after birth until the 14th day post birth. Age-matched neonatal mice were exposed to normoxia as the control. Hyperoxia-induced lung injury mice were intraperitoneally injected with UCB-EXO or vehicle daily for 3 days, starting on day 4 post birth. Human umbilical vein endothelial cells (HUVECs) were insulted with hyperoxia to establish an in vitro model of BPD to investigate angiogenesis dysfunction. Our results showed that UCB-EXO alleviated lung injuries in hyperoxia-insulted mice by reducing histopathological grade and collagen contents in the lung tissues. UCB-EXO also promoted vascular growth and increased miR-185-5p levels in the lungs of hyperoxia-insulted mice. Additionally, we found that UCB-EXO elevated miR-185-5p levels in HUVECs. MiR-185-5p overexpression inhibited cell apoptosis, whereas promoted cell migration in HUVECs exposed to hyperoxia. The luciferase reporter assay results revealed that miR-185-5p directly targeted cyclin-dependent kinase 6 (CDK6), which was downregulated in the lungs of hyperoxia-insulted mice. Together, these data suggest that UCB-EXO from healthy term pregnancies protect against hyperoxia-induced lung injuries via promoting neonatal pulmonary angiogenesis partially by elevating miR-185-5p.

Self-Assembled DNA Nanocages Promote Cell Migration and Differentiation of Human Umbilical Vein Endothelial Cells.

DNA nanocages have been explored for abilities to influence cellular behavior and functions. Recent times have seen the development of new emergent functionalities of DNA nanodevices as a class of biomaterials with an immense capacity to interface with biological systems and with vast potential in disease diagnosis and therapeutics. Being chemically robust and biocompatible in nature, DNA nanocages have been surface modified and structurally fine-tuned to find emerging applications in the field of stem-cell therapy and tissue regeneration. DNA nanocages can be used for therapeutic angiogenesis that involves the induction of blood vessel formation and can be used to treat ischemic diseases like stroke or heart failure. This work addresses the effect of DNA nanocages' structural topology on their capacity to stimulate endothelial cell angiogenesis. We tested a panel of four DNA nanocage geometries and checked their potential on the differentiation of human umbilical vein endothelial cells (HUVECs). While different DNA nanocage geometries showed successful induction of angiogenesis and cell migration in HUVECs, tetrahedral DNA cages showed the maximum uptake and angiogenesis potential, thus indicating that not only the composition of materials, but also the 3D arrangement of ligands might play role in stimulating angiogenesis.

Aidi injection enhances the anti-tumor impact of doxorubicin in H22 tumor-containing mice

Ethnopharmacological relevanceAidi injection (AD) is a traditional medical preparation that has a Chinese origin. It is extensively used particularly in combination with doxorubicin (DOX) for the management of hepatocellular carcinoma (HCC). However, the combination's synergistic mechanism has not yet been clarified. Aim of the studyTo investigate the anti-tumor impact of AD in combination with DOX and their synergistic mechanism in HCC. Materials and methodsAn H22 mouse xenograft model was utilized to study the impact of AD, DOX, and their combination on HCC in vivo. Their effects on cell vitality, apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) production, caspase-3, and cleaved caspase-3 protein expression were also investigated in H22 cells in vitro. Subsequently, human umbilical vein endothelial cells (HUVECs) were utilized to investigate the impacts of AD, DOX, and their combination on cell viability, migration, invasion, tube formation, and vascular endothelial growth factor (VEGF) protein expression. ResultsThe study established that the tumor inhibition rate of AD combined with DOX reached 79.51%, which was significantly higher than that of AD (25.14%) or DOX (49.48%) alone. Additionally, the Q-value characterizing the synergy between AD and DOX was 1.72, demonstrating a strong synergistic effect. Furthermore, compared to AD or DOX administration alone, the combined administration group significantly decreased the alpha-fetoprotein (AFP) level in the serum, increased the tumor necrosis area, increased the Bax/Bcl-2, Cyt-c, caspase-9, Fas, Fasl, caspase-8, and caspase-3 protein expression, and significantly increased the CD31 and Ki67 protein expression in tumor tissue. Compared to AD or DOX alone, AD combined with DOX treatment had a synergistic effect on H22 cells (combination index values < 0.9), which inhibited cell viability, reduced mitochondrial membrane potential (MMP), induced apoptosis, promoted MMP loss, and increased ROS generation, cleaved caspase-3/caspase-3 levels, and caspase-3 activity. Moreover, combined administration showed a more pronounced inhibition of cell viability, migration, invasion, tube formation, and VEGF protein expression in HUVECs. ConclusionsAD enhances the anti-tumor effect of DOX by promoting apoptosis and inhibiting angiogenesis and cell proliferation. The findings of this study lay experimental foundations for the clinical combination of AD and DOX.

IL-1b in the Secretomes of MSCs Seeded on Human Decellularized Allogeneic Bone Promotes Angiogenesis

Angiogenesis plays an important role in the development of bone and bone regeneration to provide the required molecules. Mesenchymal stem cells (MSCs) are pluripotent, self-renewing, and spindle-shaped cells, which can differentiate into multiple lineages such as chondrocytes, osteocytes, and adipocytes. MSCs derived from bone marrow (BMMSCs), adipose tissue (ADMSCs), and Wharton's jelly (UCMSCs) are popular in the field of tissue regeneration. MSCs have been proposed that can promote bone regeneration by enhancing vascularization. In this study, the angiogenic potential of secretomes of undifferentiated and osteo-differentiated BMMSCs, ADMSCs, and UCMSCs seeded on human decellularized allogeneic bone were compared. Human umbilical vein endothelial cells (HUVECs) were treated with MSC secretomes. Cell growth, cell migration, and angiogenesis of HUVECs were analyzed by MTT, wound healing, and tube formation assays. Angiogenic gene expression levels of MSCs were evaluated using real-time quantitative PCR. Antibody neutralization was performed to validate the candidate target. Our study demonstrates that the angiogenic gene expression profile is tissue-dependent and the angiogenic ability of secretomes is independent of the state of differentiation. We also explore that IL-1b is important for MSC angiogenic potential. Taken together, this study proves that IL-1b in the secretomes plays a vital role in angiogenesis.

Fabrication and biological assessment of halloysite-doped micro/nano structures on titanium surface

Titanium (Ti) is widely used as a biomaterial for dental implants, but its insufficient angiogenic and osteogenic properties prolong the restoration period. In this study, halloysite nanotubes (HNTs) were embedded on the surface of Ti via micro arc oxidation (MAO) treatment to enhance its early osseointegration. The surface physiochemical properties were examined, and the results confirmed that HNTs were successfully incorporated into the MAO coating. It was also discovered that the surface wettability and negative charge of the corresponding samples increased. In cytocompatibility tests involving human umbilical vein endothelial cells (HUVECs) and MC3T3-E1 cells, the HNT group exhibited no cytotoxicity compared with the MAO group and the best performance for cell adhesion and spreading among the three groups (Ti, MAO and HNT). Regarding angiogenesis, the HNT group outperformed the Ti and MAO groups in cell migration, tube formation, and angiogenic gene expression of HUVECs. Furthermore, with regard to osteogenesis, the HNT group exhibited the highest levels of alkaline phosphatase activity, collagen secretion, mineralized calcium nodules, and osteogenic gene expression of MC3T3-E1 cells among the three groups with significant differences. These findings indicated that the HNT specimens could significantly promote angiogenesis as well as osteogenesis at both the cellular and molecular levels.

UCMSCs-derived exosomal circHIPK3 promotes ulcer wound angiogenesis of diabetes mellitus via miR-20b-5p/Nrf2/VEGFA axis.

Experiments confirmed that circular RNAs contributed to the pathogenesis of diabetic foot ulcers (DFUs). CircHIPK3 was upregulated in type 2 diabetes mellitus (T2DM), but its role in DFU remained unknown. Our study aimed to investigate the regulatory functions of exosomal circHIPK3 and its potential mechanisms in DFU. Exosomal size and distribution, marker proteins, and circHIPK3 levels were evaluated by transmission electron microscope, ExoView R200, western blot, and qRT-PCR. Flow cytometry, MTT, Wound healing assays, and tube formation assays were used to assess the roles of exosomal circHIPK3 in high glucose (HG)-treated human umbilical vein endothelial cells (HUVECs). The relationships between Nrf2/VEGFA/circHIPK3 and miR-20b-5p, and between Nrf2 and VEGFA were determined by luciferase reporter assay and RNA immunoprecipitation. We used cell and mice models to investigate the mechanisms of exosomal circHIPK3 under diabetic conditions. CircHIPK3 was significantly upregulated in exo-circHIPK3 rather than exo-vector. Exo-circHIPK3 remarkably inhibited cell apoptosis but promoted cell proliferation, migration, and tube formation in HG-treated HUVECs. Luciferase reporter and RIP assays showed that miR-20b-5p targeted and inhibited Nrf2 and VEGFA, and circHIPK3 acted as a ceRNA of miR-20b-5p to inhibit the binding to its downstream genes Nrf2 and VEGFA. Mechanistically, circHIPK3 promoted cell proliferation, migration, and angiogenesis via downregulating miR-20b-5p to upregulate Nrf2 and VEGFA. However, the overexpressed miR-20b-5p could abolish the promoting effects of circHIPK3 overexpression on cell proliferation, migration, and tube formation under HG conditions. UCMSCs-derived exosomal circHIPK3 protected HG-treated HUVECs via miR-20b-5p/Nrf2/VEGFA axis. The exosomal circHIPK3 might be a therapeutic candidate to treat DFU.

TNFAIP3 mediates FGFR1 activation-induced breast cancer angiogenesis by promoting VEGFA expression and secretion.

To investigate the role and mechanism of TNF-inducible protein 3(TNFAIP3) in breast cancer angiogenesis induced by fibroblast growth factor receptor1 (FGFR1) activation. The immunohistochemical assay was used to detect the expression of vascular endothelial cell marker CD31 and CD105 in mice DCIS.COM-iFGFR1 transplanted tumor (previously established by our group). The effects of TNFAIP3 knockout/knockdown breast cancer cell lines on angiogenesis, migration, and invasion of Human Umbilical Vein Endothelial Cells (HUVEC) were detected by the tubulogenesis and Trewells assay. RNA-seq analysis of TNFAIP3 downstreams differential genes after TNFAIP3 knockdown. The expression and secretion of VEGFA after FGFR1 activation in breast cancer cells were detected by qPCR, Western blot, and ELISA. Immunohistochemistry showed that TNFAIP3 knockout inhibited the expression of CD31 and CD105 in DCIS grafted tumors promoted by FGFR1 activation. Tubulogenesis and Trewells experiments showed that TNFAIP3 gene knockout/knockdown inhibited the angiogenesis, migration, and invasion of HUVEC cells promoted by FGFR1 activation. qPCR assay showed that VEGFA mRNA level in the TNFAIP3 knockdown cell line was significantly down-regulated (p < 0.05). qPCR, Western blot and ELISA results showed that TNFAIP3 gene knockout/knockdown could inhibit the expression and secretion of VEGFA in breast cancer cells induced by FGFR1 activation. TNFAIP3 promotes breast cancer angiogenesis induced by FGFR1 activation through the expression and secretion of VEGFA.

Caspase‐4/11 is critical for angiogenesis by repressing Notch1 signalling via inhibiting γ‐secretase activity

Notch1 activation mediated by γ-secretase is critical for angiogenesis. GeneCards database predicted that Caspase-4 (CASP4, with murine ortholog CASP11) interacts with presenilin-1, the catalytic core of γ-secretase. Therefore, we investigated the role of CASP4/11 in angiogenesis. In vivo, we studied the role of Casp11 in several angiogenesis mouse models using Casp11 wild-type and knockout mice. In vitro, we detected the effects of CASP4 on endothelial functions and Notch signalling by depleting or overexpressing CASP4 in human umbilical vein endothelial cells (HUVECs). The functional domain responsible for the binding of CASP4 and presenilin-1 was detected by mutagenesis and co-immunoprecipitation. Casp11 deficiency impaired adult angiogenesis in ischaemic hindlimbs, melanoma xenografts and Matrigel plugs, but not the developmental angiogenesis of retina. Bone marrow transplantation revealed that the pro-angiogenic effect depended on CASP11 derived from non-haematopoietic cells. CASP4 expression was induced by inflammatory factors and CASP4 knockdown decreased cell viability, proliferation, migration and tube formation in HUVECs. Mechanistically, CASP4/11 deficiency increased Notch1 activation in vivo and in vitro, while CASP4 overexpression repressed Notch1 signalling in HUVECs. Moreover, CASP4 knockdown increased γ-secretase activity. The γ-Secretase inhibitor DAPT restored the effects of CASP4 siRNA on Notch1 activation and angiogenesis in HUVECs. Notably, the catalytic activity of CASP4/11 was dispensable. CASP4 directly interacted with presenilin-1 through the caspase recruitment domain (CARD). These findings reveal a critical role of CASP4/11 in adult angiogenesis and make this molecule a promising therapeutic target for angiogenesis-related diseases in the future.

Differential Proteomic Profiles of Coronary Serum Exosomes in Acute Myocardial Infarction Patients with or Without Diabetes Mellitus: ANGPTL6 Accelerates Regeneration of Endothelial Cells Treated with Rapamycin via MAPK Pathways.

Delayed re-endothelialization after coronary drug-eluting stent implantation is associated with an increased incidence of late in-stent thrombosis. Serum exosomes exhibit controversial effects on promoting endothelialization. This study aimed to compare the angiogenic effects of serum exosomes derived from patients with acute myocardial infarction (AMI) and AMI plus diabetes mellitus (DM) and to explore the underlying mechanisms. Serum exosomes derived from patients in the control (Con-Exos), AMI (AMI-Exos), and AMI plus DM (AMI+DM-Exos) groups were isolated and identified using standard assays. CCK-8, wound healing, and tube formation assays were performed to detect the angiogenic abilities of serum exosomes on rapamycin-conditioned human umbilical vein endothelial cells (HUVECs). Differential proteomic profiles between AMI-Exos and AMI+DM-Exos were analyzed by mass spectrometry. The effects and potential mechanisms of exosomal angiopoietin-like 6 (ANGPTL6) were investigated. Functional assays indicated that compared with Con-Exos, AMI-Exos enhanced, whereas AMI+DM-Exos inhibited the cell proliferation, migration, and tube formation of rapamycin-conditioned HUVECs. Subsequently, 28 differentially expressed proteins between AMI-Exos and AMI+DM-Exos were identified, which were correlated with material transportation, immunity, and inflammatory reaction. Moreover, ANGPTL6 was highly enriched in AMI-Exos. Overexpression and knockdown of ANGPTL6 enhanced and inhibited angiogenesis, respectively. Furthermore, the effect of ANGPTL6 on angiogenesis was mediated via the activation of ERK 1/2, JNK, and p38 pathways. The inhibition of ERK 1/2 signaling markedly attenuated the migration abilities of overexpressing ANGPTL6. Diabetes impairs the regenerative capacities of serum exosomes. Exosomal ANGPTL6 contributes to endothelial repair and is a novel therapeutic target for enhanced stent endothelization.