Human Umbilical Vein Endothelial Cells Invasion Research Articles

Exosomes from adipose-derived stem cells overexpressing microRNA-671–3p promote fat graft angiogenesis and adipogenic differentiation

Exosomes from adipose-derived stem cells (ADSCs) have been demonstrated to benefit angiogenesis, wound healing, and fat grafting. Small noncoding RNAs such as microRNA (miRNA) and circular RNA play critical roles in mediating the function of ADSCs-derived exosomes. However, the underlying mechanisms have not been fully elucidated. In this study, we investigated the function and mechanism of human ADSCs-derived exosomes (hADSCs-Exo) in promoting fat graft angiogenesis and adipogenic differentiation. hADSCs-Exo were isolated and identified, and treatment with hADSCs-Exo enhanced fat graft angiogenesis and adipogenic differentiation in a mouse fat graft implantation model. We found that hADSCs-Exo overexpressed miR-671–3p and promoted human umbilical vein endothelial cell (HUVEC) proliferation, migration, and invasion. Bioinformatics analysis and luciferase reporter assay validated that TMEM127 is a direct target of miR-671–3p. Rescue experiments demonstrated that TMEM127 overexpression partially antagonized the function of hADSCs-Exo in vitro, such as suppressing HUVEC cell proliferation, migration, and invasion. Moreover, TMEM127 overexpression abrogated the function of hADSCs-Exo on fat graft angiogenesis and adipogenic differentiation. Taken together, our findings demonstrate that miR-671–3p-overexpressing exosomes from ADSC promote fat graft angiogenesis and adipogenic differentiation, which highlights the potential of targeting the ADSC-Exosomes-miR-671–3p/TMEM127 axis to improve outcome of fat graft and tissue engineering regenerative medicine.

TCEB2/HIF1A signaling axis promotes chemoresistance in ovarian cancer cells by enhancing glycolysis and angiogenesis

Ovarian cancer is an extremely malignant gynaecological tumour with a poor patient prognosis and is often associated with chemoresistance. Thus, exploring new therapeutic approaches to improving tumour chemosensitivity is important. The expression of transcription elongation factor B polypeptide 2 (TCEB2) gene is reportedly upregulated in ovarian cancer tumour tissues with acquired resistance, but the specific mechanism involved in tumour resistance remains unclear. In this study, we found that TCEB2 was abnormally highly expressed in cisplatin-resistant tumour tissues and cells. TCEB2 silencing also inhibited the growth and glycolysis of SKOV-3/cisplatin (DDP) and A2780/DDP cells. We further incubated human umbilical vein endothelial cells (HUVECs) with culture supernatants from cisplatin-resistant cells having TCEB2 knockdown. Results revealed that the migration, invasion, and angiogenesis of HUVECs were significantly inhibited. Online bioinformatics analysis revealed that the hypoxia-inducible factor-1A (HIF-1A) protein may bind to TCEB2, and TCEB2 silencing inhibited SKOV-3/DDP cell growth and glycolysis by downregulating HIF1A expression. Similarly, TCEB2 promoted HUVEC migration, invasion, and angiogenesis by upregulating HIF1A expression. In vivo experiments showed that TCEB2 silencing enhanced the sensitivity of ovarian cancer nude mice to cisplatin and that TCEB2 knockdown inhibited the glycolysis and angiogenesis of tumour cells. Our findings can serve as a reference for treating chemoresistant ovarian cancer.

Targeting RhoA expression with formononetin and salvianolic acid B to mitigate pancreatic cancer-associated endothelial cells changes

Ethnopharmacological relevanceAccording to the theory of Qi and blood in Traditional Chinese Medicine (TCM), the combination of Qi-reinforcing herbs and blood-activating herbs has a synergistic effect in improving blood stasis syndrome, especially in tumor treatment. The classic “Radix Astragali - Salvia miltiorrhiza” duo exemplifies this principle, renowned for invigorating Qi and activating blood flow, employed widely in tumor therapies. Our prior research underscores the potent inhibition of pancreatic tumor xenografts by the combination of Formononetin (from Radix Astragali) and Salvianolic acid B (from Salvia miltiorrhiza) in vitro. However, it remains unclear whether this combination can inhibit the abnormal vascularization of pancreatic tumors to achieve its anti-cancer effect. Aim of the studyAbnormal vasculature, known to facilitate tumor growth and metastasis. Strategies to normalize tumor-associated blood vessels provide a promising avenue for anti-tumor therapy. This study aimed to unravel the therapeutic potential of Formononetin combined with Salvianolic acid B (FcS) in modulating pancreatic cancer's impact on endothelial cells, illuminate the underlying mechanisms that govern this therapeutic interaction, thereby advancing strategies to normalize tumor vasculature and combat cancer progression. Materials and methodsA co-culture system involving Human Umbilical Vein Endothelial Cells (HUVECs) and PANC-1 cells was established to investigate the potential of targeting abnormal vasculature as a novel anti-tumor therapeutic strategy. We systematically compared HUVEC proliferation, migration, invasion, and lumenogenesis in both mono- and co-culture conditions with PANC-1 (H-P). Subsequently, FcS treatment of the H-P system was evaluated for its anti-angiogenic properties. Molecular docking was utilized to predict the interactions between Formononetin and Salvianolic acid B with RhoA, and the post-treatment expression of RhoA in HUVECs was assessed. Furthermore, we utilized shRhoA lentivirus to elucidate the role of RhoA in FcS-mediated effects on HUVECs. In vivo, a zebrafish xenograft tumor model was employed to assess FcS's anti-tumor potential, focusing on cancer cell proliferation, migration, apoptosis, and vascular development. ResultsFcS treatment demonstrated a significant, dose-dependent inhibition of PANC-1-induced alterations in HUVECs, including proliferation, migration, invasion, and tube formation capabilities. Molecular docking analyses indicated potential interactions between FcS and RhoA. Further, FcS treatment was found to downregulate RhoA expression and modulated the PI3K/AKT signaling pathway in PANC-1-induced HUVECs. Notably, the phenotypic inhibitory effects of FcS on HUVECs were attenuated by RhoA knockdown. In vivo zebrafish studies validated FcS's anti-tumor activity, inhibiting cancer cell proliferation, metastasis, and vascular sprouting, while promoting tumor cell apoptosis. ConclusionsThis study underscores the promising potential of FcS in countering pancreatic cancer-induced endothelial alterations. FcS exhibits pronounced anti-abnormal vasculature effects, potentially achieved through downregulation of RhoA and inhibition of the PI3K/Akt signaling pathway, thereby presenting a novel therapeutic avenue for pancreatic cancer management.

USP7-stabilised HIPK2 promotes high glucose-induced endothelial cell dysfunctions to accelerate diabetic foot ulcers

Background: This study aimed to explore the molecular mechanism of homeodomain-interacting protein kinase 2 (HIPK2) in diabetic foot ulcers (DFU). Methods: High glucose (HG)-induced human umbilical vein endothelial cells (HUVECs) were used to construct DFU cell models. Cell functions were determined using CCK8 assay, EdU assay, flow cytometry, transwell assay, wound healing assay and tube formation assay. Quantitative real-time PCR and western blot were applied to measure the gene expression. Results: HG treatment suppressed HUVECs proliferation, invasion, migration, and angiogenesis, while enhanced apoptosis. HIPK2 was overexpressed in DFU patients, and its knockdown alleviated HG-induced HUVECs dysfunctions. USP7 stabilised HIPK2 protein by reducing its ubiquitination. USP7 overexpression promoted HG-induced HUVECs dysfunctions, and HIPK2 upregulation also reversed the regulation of USP7 knockdown on HG-induced HUVECs dysfunctions. USP7/HIPK2 axis inhibited the activity of PI3K/AKT pathway. Conclusion: Our study revealed that USP7-stabilised HIPK2 contributed to HG-induced HUVECs dysfunctions, thus accelerating DFU process.

Maternal administration of APAP induces angiogenesis disorders in mouse placenta via SOCS3/JAK1/STAT3 pathway

Acetaminophen (APAP, also known as paracetamol) is a commonly used antipyretic and analgesic that is considered safe to use during pregnancy. However, a growing body of research indicates that gestational administration of APAP increased the risk of neurodevelopmental, reproductive and genitourinary disorders in offspring, alongside impairments in placental development. Notably, over-dosed APAP exhibits direct toxicity to endothelial cells, but there is very limited research investigating the impact of APAP on placental angiogenesis, a gap we aim to address in this study. Pregnant mice were gavaged with APAP (15, 50 and 150 mg/kg/d) from embryonic day 11.5 (E11.5) to E13.5. Administration of 150 mg/kg/d APAP leads to low birth weight (LBW) of the offspring and disordered vascular structures within the labyrinthine (Lab) layer of the placenta. This disruption is accompanied by a significant increase in Suppressor of Cytokine Signaling 3 (SOCS3) level, a negative regulator of the Janus kinase signal transducer 1 and activator of the transcription 3 (JAK1/STAT3) signaling. Meanwhile, Human umbilical vein endothelial Cells (HUVECs) with the treatment of 3 mM APAP exhibited reduced cell viability, whereas 1 mM APAP significantly affected the proliferation, migration, invasion and angiogenic capacities of HUVECs. Further, SOCS3 was up-regulated in HUVECs, accompanied by inhibition of JAK1/STAT3 pathways. Knocking-down SOCS3 in HUVECs restored the nuclear translocation of STAT3 and efficiently promoted cellular capacity of tube formation. Overall, short-term maternal administration of overdosed APAP impairs angiogenic capacities of fetal endothelial cells via SOCS3/JAK1/STAT3 pathway in the mouse placenta. This study reveals that overdose of APAP during pregnancy may adversely affect placental angiogenesis, emphasizing the importance of adhering to the safe principles of smallest effective dose for the shortest required durations.

MEG3-Mediated Oral Squamous-Cell-Carcinoma-Derived Exosomal miR-421 Activates Angiogenesis by Targeting HS2ST1 in Vascular Endothelial Cells.

Exosomal microRNAs (miRNAs) from cancer cells play a key role in mediating the oral squamous cell carcinoma (OSCC) microenvironment. The objective of this study was to investigate how the long non-coding RNA (lncRNA) MEG3 affects OSCC angiogenesis through exosomal miR-421. Global miRNA microarray analysis and quantitative real-time PCR (qRT-PCR) were performed to determine the level of miRNAs in OSCC cell-derived exosomes. Cell migration, invasion, tube formation, immunohistochemistry, and hemoglobin concentrations were used to study the effects of exosomal miR-421 in angiogenesis. Western blotting was used to determine the expression level of HS2ST1 and VEGFR2-related downstream proteins. MiRNA array and qRT-PCR identified the upregulation of miR-421 in OSCC cell-derived exosomes. Furthermore, exosomal miR-421 can be taken up by human umbilical vein endothelial cells (HUVECs) and then target HS2ST1 through VEGF-mediated ERK and AKT phosphorylation, thereby promoting HUVEC migration, invasion, and tube formation. Additionally, forced expression of the lncRNA MEG3 in OSCC cells reduced exosomal miR-421 levels and then increased HS2ST1 expression, thereby reducing the VEGF/VEGFR2 pathway in HUVECs. Our results demonstrate a novel mechanism by which lncRNA MEG3 can act as a tumor suppressor and regulate endothelial angiogenesis through the exosomal miR-421/HS2ST1 axis, which provides a potential therapeutic strategy for OSCC angiogenesis.

Eugenol suppresses VEGF-dependent angiogenesis by JAK2/STAT3 pathway in non-small cell lung cancer

Background: Non-small cell lung cancer (NSCLC) is a highly vascularized solid tumor, and tumor angiogenesis is closely associated with the metastasis and progression of NSCLC. Antiangiogenic drugs can target the tumor microenvironment to degrade existing tumor blood vessels while inhibiting tumor angiogenesis and have become one of the indispensable treatments for patients with advanced NSCLC. Although various new drugs have been tested in different settings of NSCLC, none of them have shown the desired therapeutic effects so far. Therefore, the search for new and effective therapeutic modalities has become a new goal for treating NSCLC. Objective: Eugenol is a phenolic aromatic compound derived from Eugenia caryophyllata, Cinnamomum cassia, etc., which has historically been used for various medical purposes. Studies have shown that eugenol exhibits significant anticancer effects against several types of cancer; however, its therapeutic effect on angiogenesis remains a mystery. In this study, the in vitro and in vivo antiangiogenic effects of eugenol in NSCLC and the underlying molecular mechanism were explored, which could provide a promising strategy for the treatment of NSCLC. Methods: The effects of eugenol on the proliferative capacity of human umbilical vein endothelial cells (HUVECs) and A549 cells were examined by methyl thiazolyl tetrazolium assay. The migration and invasion of eugenol-treated HUVECs were evaluated by wounding-healing and transwell assay, and the angiogenesis was measured by tube formation assay. The expression of angiogenesis-related genes and proteins, as well as the JAK2/STAT3 pathway, was evaluated by real-time quantitative PCR and western blot. Flow cytometry was performed to detect the effect of eugenol on the apoptotic profile of A549 cells. Finally, the A549 tumor-bearing nude mice were constructed to evaluate the in vivo anti-NSCLC activity of eugenol. Results: Eugenol inhibited the migration, invasion, and tube formation of HUVECs. Meanwhile, eugenol blocked the phosphorylation of vascular endothelial growth factor reporter-2 and inhibited the expression of other angiogenesis-related proteins. In addition, eugenol suppressed the expression of p-JAK2 and p-STAT3 in HUVECs and A549 cells. Eugenol also suppressed the proliferation of A549 cells by promoting apoptosis and inhibited tumor growth and microvessel formation in A549 cell xenograft-bearing nude mice. Conclusions: Eugenol could be a potential lead compound for the treatment of NSCLC by blocking the vascular endothelial growth factor/vascular endothelial growth factor reporter-2 and JAK2/STAT3 signaling pathways.

Gene expression profiling of venous malformations identifies the role of SDC1 in venous endothelial cells

ObjectiveTo obtain insight into the molecular process implicated in venous malformations (VMs) and identify potential targets for treatment of VMs, this study profiled the gene expression pattern in VMs, investigated alterations of syndecan-1 (SDC1) expression in VMs, and tested the hypothesis that aberrant SDC1 expression triggers abnormal angiogenesis and VM development. MethodsMicroarray analysis was performed to identify differentially expressed genes (DEGs) on a transcriptome-wide level in VMs and conjunctive normal. Gene Ontology molecular functional analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were carried out to establish enhancement of biological signaling pathways involved in VMs. Among the DEGs, we focused on SDC1, which is involved in matrix remodeling, cell proliferation and invasion, and angiogenesis. SDC1 expression in VMs was verified by qRT-PCR, western blotting, and immunohistochemistry. Loss-of-function of SDC1 was achieved in human umbilical vein endothelial cells (HUVECs) by siRNA to investigate the roles of SDC1 in cell migration, invasion, and angiogenesis. ResultsCompared with control tissue, the transcriptome study identified 274 upregulated DEGs and 3 downregulated DEGs. The transcript and protein levels of SDC1 were significantly decreased in VMs compared with normal tissue. Inhibition of SDC1 enhanced HUVEC migration, invasion, and angiogenesis. ConclusionOur genome-wide microarray analysis suggests the involvement of numerous genes in VMs. Among them, SDC1 plays a substantial role in the process of angiogenesis and development of VMs. SDC1 may represent a potential target for a molecular therapy for VMs.

Emodin suppresses alkali burn-induced corneal inflammation and neovascularization by the vascular endothelial growth factor receptor 2 signaling pathway.

To investigate the effects of emodin on alkali burn-induced corneal inflammation and neovascularization. The ability of emodin to target vascular endothelial growth factor receptor 2 (VEGFR2) was predicted by molecular docking. The effects of emodin on the invasion, migration, and proliferation of human umbilical vein endothelial cells (HUVEC) were determined by cell counting kit-8, Transwell, and tube formation assays. Analysis of apoptosis was performed by flow cytometry. CD31 levels were examined by immunofluorescence. The abundance and phosphorylation state of VEGFR2, protein kinase B (Akt), signal transducer and activator of transcription 3 (STAT3), and P38 were examined by immunoblot analysis. Corneal alkali burn was performed on 40 mice. Animals were divided randomly into two groups, and the alkali-burned eyes were then treated with drops of either 10 μM emodin or phosphate buffered saline (PBS) four times a day. Slit-lamp microscopy was used to evaluate inflammation and corneal neovascularization (CNV) in all eyes on Days 0, 7, 10, and 14. The mice were killed humanely 14 d after the alkali burn, and their corneas were removed and preserved at －80 ℃ until histological study or protein extraction. Molecular docking confirmed that emodin was able to target VEGFR2. The findings revealed that emodin decreased the invasion, migration, angiogenesis, and proliferation of HUVEC in a dose-dependent manner. In mice, emodin suppressed corneal inflammatory cell infiltration and inhibited the development of corneal neovascularization induced by alkali burn. Compared to those of the PBS-treated group, lower VEGFR2 expression and CD31 levels were found in the emodin-treated group. Emodin dramatically decreased the expression of VEGFR2, p-VEGFR2, p-Akt, p-STAT3, and p-P38 in VEGF-treated HUVEC. This study provides a new avenue for evaluating the molecular mechanisms underlying corneal inflammation and neovascularization. Emodin might be a promising new therapeutic option for corneal alkali burns.

CPD-002, a novel VEGFR2 inhibitor, relieves rheumatoid arthritis by reducing angiogenesis through the suppression of the VEGFR2/PI3K/AKT signaling pathway

Synovial angiogenesis is a key player in the development of rheumatoid arthritis (RA), and anti-angiogenic therapy is considered a promising approach for treating RA. CPD-002 has demonstrated efficacy in suppressing tumor angiogenesis as a VEGFR2 inhibitor, but its specific impacts on RA synovial angiogenesis and possible anti-RA effects need further study. We examined the influences of CPD-002 on the migration and invasion of human umbilical vein endothelial cells (HUVECs) and its impacts on HUVECs' tube formation and vessel sprouting ex vivo. The therapeutic potential of CPD-002 in adjuvant-induced arthritis (AIA) rats and its suppression of synovial angiogenesis were examined. The involvement of the VEGFR2/PI3K/AKT pathway was assessed both in HUVECs and AIA rat synovium. Here, CPD-002 inhibited the migration and invasion of VEGF-stimulated HUVECs, decreased their chemotactic response to RA fibroblast-like synoviocyte-released chemoattractants, and exhibited anti-angiogenic effects in vitro and ex vivo. CPD-002′s targeting of VEGFR2 was confirmed with molecular docking and cellular thermal shift assays, supported by the abolishment of CPD-002′s effects upon using VEGFR2 siRNA. CPD-002 relieved paw swelling, arthritis index, joint damage, and synovial angiogenesis, indicating its anti-arthritic and anti-angiogenic effects in AIA rats. Moreover, the anti-inflammatory effects in vivo and in vitro of CPD-002 contributed to its anti-angiogenic effects. Mechanistically, CPD-002 hindered the activation of VEGFR2/PI3K/AKT pathway in VEGF-induced HUVECs and AIA rat synovium, as evidenced by reduced p-VEGFR2, p-PI3K, and p-AKT protein levels alongside elevated PTEN protein levels. Totally, CPD-002 showed anti-rheumatoid effects via attenuating angiogenesis through the inhibition of the VEGFR2/PI3K/AKT pathway.

LSD1 inhibition by tranylcypromine hydrochloride reduces alkali burn-induced corneal neovascularization and ferroptosis by suppressing HIF-1α pathway.

Corneal neovascularization (CNV) is a sight-threatening condition that necessitates epigenetic control. The role of lysine-specific demethylase 1 (LSD1) in CNV remains unclear, despite its established significance in tumor angiogenesis regulation. An alkali burn-induced CNV mouse model was used in vivo. The effects of LSD1 inhibitor tranylcypromine hydrochloride (TCP) were examined through slit lamp, histological staining, and immunofluorescence. The expression of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) levels were assessed in corneal tissues. Oxidative stress and ferrous ion expression during CNV were determined using 4-HNE, GPX4, and FerroOrange staining. In vitro, a hypoxia-reoxygenation (H/R) model was established using human umbilical vein endothelial cells (HUVECs) to study LSD1 or hypoxia-inducible factor (HIF-1α) knockdown and lentiviral overexpression of HIF-1α. The effects on HUVECs migration, invasion, and angiogenesis were evaluated through cell scratching assay, transwell migration assay and tube formation assay. The role of ferroptosis was investigated using ROS staining, FerroOrange staining, and key ferroptosis proteins. Further, The JAK2/STAT3 pathway's involvement in CNV regulation was explored through in vivo experiments with subconjunctival injection of AG490. The results showed a substantial correlation between corneal damage and LSD1 levels. In addition, HIF-1α expression was also elevated after alkali burns, and subconjunctival injection of TCP reduced corneal inflammation and neovascularization. Corneal alkali burns increased ROS levels and reduced antioxidative stress indicators, accompanied by elevated ferrous ion levels, which were reversed by TCP injection. In vitro, TCP or siRNAs inhibited H/R-induced ferroptosis and angiogenesis in HUVECs by affecting specific protein expressions and MDA, SOD, and GSH levels. HIF-1α levels, associated with ROS production, ferroptosis, and angiogenesis, increased during H/R, but were reversed by TCP or siRNA administration. HIF-1α overexpression counteracted the effects of LSD1 inhibition. Additionally, AG490 injection effectively reduced HIF-1α and VEGFA expression in the CNV model. These findings suggest that LSD1 inhibition via the HIF-1α-driven pathway prevents angiogenesis, oxidative stress, and ferroptosis in corneal alkali burn-induced CNV, highlighting LSD1 as a potential therapeutic target.

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.

TRPM2-L Participates in the Interleukin-6 Pathway to Enhance Tumor Growth in Prostate Cancer by Hypoxia-Inducible Factor-1α.

Interleukin-6 (IL-6) can promote cell proliferation in prostate cancer (PCa). Full-length transient receptor potential melastatin 2 (TRPM2-L) is highly expressed in PCa. However, the association between IL-6 and TRPM2-L in PCa is unclear. Here, human PCa cell lines, PC-3 and DU-145, were treated with 10 μg/mL tocilizumab, an IL-6 receptor (IL-6R) inhibitor, and the TRPM2-L protein expression in cells was significantly decreased. Cells were stably transfected with TRPM2 short-interfering RNA (siRNA) and cell survival clearly declined. Recombinant IL-6 treatment weakened the effects of TRPM2-siRNA on cell survival. TRPM2-L binds directly to IL-6R in PC-3 and DU-145 cells. The protein expression of hypoxia-inducible factor-1α was suppressed by reduction with TRPM2-L in PC-3 and DU-145 cells. Human umbilical vein endothelial cells (HUVECs) were indirectly cocultured with PCa cells, and the invasion and angiogenic activity of HUVECs were enhanced after coculture with PCa cells. However, TRPM2-L reduction in PCa cells significantly decreased the invasion and angiogenic activity of HUVECs compared to the control coculture. In vivo, xenograft tumors were induced using PC-3 cells. Tocilizumab treatment or TRPM2-L reduction clearly suppressed tumor growth. Meanwhile, the injection of mouse recombinant IL-6 weakened the antitumor effects of TRPM2-L reduction. These data demonstrate that the IL-6/TRPM2-L axis in PCa tumor growth is important, and interference of the IL-6/TRPM2-L axis may be a novel approach for PCa therapy.

Astragaloside IV Suppresses the Effects of Hepatocellular Carcinoma Cells on Proliferation, Angiogenesis, and Invasion in Human Umbilical Vein Endothelial Cells by Controlling Exosomes by Inhibiting Rab27a

Hepatocellular carcinoma (HCC) is the third most common cancer worldwide, and invasion and metastasis are the leading causes of death. Astragaloside IV (AS-IV), a kind of traditional Chinese medicine, has been widely used in cancer therapy. Cell Counting Kit-8, tube formation, and Transwell analyses were performed to determine cell proliferation, invasion, and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the treatment of Hep3B or SK-Hep1 cells-derived exosomes or Hep3B or SK-Hep1 cells with or without AS-IV treatment. Then, Rab27a overexpression in Hep3B cells was selected to determine the function of AS-IV on the regulation of cell proliferation, invasion, and angiogenesis of HUVECs. Ultimately, the injection of Hep3B cell-derived exosomes or treatment with AS-IV on nude mice was used to comprehensively investigate AS-IV functions in vivo. Hep3B or SK-Hep1 cells promoted proliferation, invasion, and angiogenesis of HUVECs, which were inhibited by AS-IV therapy. The concentrations of Hep3B and SK-Hep1 cells-derived exosomes were markedly reduced after AS-IV therapy. Meanwhile, Rab27a expression was significantly decreased in SK-Hep1 or Hep3B cells after AS-IV therapy, and it inhibited the effects of AS-IV on Hep3B-induced proliferation, invasion, and angiogenesis of HUVECs. Tumor growth was also suppressed by AS-IV, which was accompanied by the decreased expression levels of Ki67 and CD34. Overall, this study demonstrated that AS-IV inhibits the effects of HCC cell-derived exosomes on proliferation, invasion, and angiogenesis of HUVECs via inhibiting Rab27a expression. Practical Applications. To our knowledge, this is the first study to explore the efficacy of AS-IV against HCC cell-induced proliferation, invasion, and angiogenesis of HUVECs to reveal the underlying mechanism. In this study, CCK-8, Transwell, and tube formation assay detection show that AS-IV has an inhibitory effect on HCC cell-induced proliferation, invasion, and angiogenesis of HUVECs by inhibiting Rab27a, which helps in the development of novel nutraceutical/functional food against HCC progression and thus could improve the quality of life in patients with HCC.

Exosomal B7-H3 facilitates colorectal cancer angiogenesis and metastasis through AKT1/mTOR/VEGFA pathway

B7-H3 (CD276), an immune checkpoint molecule, is aberrantly overexpressed in many types of cancer, and plays important roles in tumor immune evasion, carcinogenesis and metastasis, as well as angiogenesis. However, the mechanisms underlying B7-H3-promoted angiogenesis are still largely unknown. In this study, based on the observation of overexpression of B7-H3 on the tumor cells and vascular endothelial cells (VECs) in colorectal cancer (CRC) tissues, we investigated the roles of cancer cell-drived exosomal B7-H3 in tumor angiogenesis and metastasis through crosstalk between cancer cells and VECs. We found that CRC cell-drived exosomal B7-H3 was uptaken by human umbilical vein endothelial cells (HUVECs) and consequently activated the AKT serine/threonine kinase 1 (AKT1) / mechanistic target of rapamycin kinase (mTOR) / vascular endothelial growth factor A (VEGFA) signaling pathway, thus augmenting the abilities of migration, invasion and tube formation of HUVECs. Furthermore, administration of CRC cell-drived exosomes with reinforced B7-H3 promoted the pulmonary angiogenesis and metastasis of CRC cells in mice. In addition, high expression of B7-H3 was observed in urinary exosomes isolated from CRC patients. Our findings reveal that CRC-derived exosomal B7-H3 promotes tumor angiogenesis and metastasis by activating the AKT1/mTOR/VEGFA signaling pathway. It provides novel insights into the roles of CRC-drived exosomes in CRC progression.

EDIL3 and VEGF Synergistically Affect Angiogenesis in Endothelial Cells.

Angiogenesis is one of the histologically predominant characteristics of psoriasis. Vascular endothelial growth factor (VEGF) and epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3) have critical effects on angiogenesis. Both these proteins are vital proangiogenic factors in tumor occurrence and progression; however, the relationship between EDIL3 and VEGF with psoriasis remains unclear. We aimed to elucidate the role of EDIL3 and VEGF and the involved mechanisms in psoriasis-associated angiogenesis. EDIL3 and VEGF expression in cutaneous tissue was determined by immunohistochemical assay. The effects of EDIL3 on VEGF, VEGFR2, and the growth, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were analyzed by Western blotting assay, cell counting kit-8 assay, Transwell assay, and Matrigel tube formation assay. EDIL3 and VEGF levels in psoriatic lesions significantly increased as compared to those in normal individuals and showed a positive relationship with the Psoriasis Area and Severity Index. The downregulation of EDIL3 decreased VEGF and VEGFR2 expression in HUVECs. Moreover, the decreased expression of EDIL3 and VEGF reduced the growth, invasion, and tube formation abilities of HUVECs, while EDIL3 resistance to VEGF and VEGFR2 was restored by using the EDIL3 recombinant protein. These results suggest that psoriasis is also characterized by EDIL3 and VEGF-mediated angiogenesis. Thus, EDIL3 and VEGF could serve as novel targets for treating psoriasis.

Abstract 2438: Loss of galectin 3 reveals its potential roles in the aggressive pathology of uterine serous carcinoma

Abstract Objective: Uterine serous cancer (USC) represents about 10% of all uterine cancers, yet it accounts for up to 40% of all uterine cancer deaths. This lethality is due in part to the highly aggressive clinical course of this disease. Recently, galectin 3 (Gal3), a glycoprotein, was shown to contribute to malignant features in other tumors including ovarian cancer. In this study, we test the hypothesis that Gal3 facilitates malignant features in USC and might provide an opportunity for therapeutic intervention. Methods: The TCGA database was used to define the relationship between LGALS3 mRNA expression and prognosis. Gal3 knockout cells were established via CRISPR/Cas9-mediated deletion from USC cell lines, ARK1 and ARK2 (Gal3-KO). Gal3’s role in cell proliferation, cell cycle, and cell death were assessed. Gal3’s influence on apoptotic priming was done by BH3 profiling. Gal3 small molecule inhibitors (SMIs) were used to as an orthogonal approach to genetic knockdown. The effect of Gal3 loss on migration, invasion, and angiogenesis was assessed with transwell, Matrigel-invasion and HUVEC (human umbilical vein endothelial cells) tube-forming assays. The impact of Gal3 loss on cancer stem cells (CSC), phenotypically characterized by CD44, CD117, and ALDH activity, was measured by flow cytometry, and functionally by colony-forming and sphere-forming assays. An in vivo limiting dilution tumorigenic assay was performed with ARK1 Gal3-CTRL and KO cells in immunocompromised mice. Statistical analysis was done and a significance level of p < 0.05 was used. Results: The TCGA database revealed a worse prognosis for patients with USC and high LGALS3 mRNA. Cell Counts revealed Gal3-KO cells proliferated at a slower rate compared to Gal3-CTRL cells, and the difference wasn’t attributed to an increase in cell death. Cell cycle analysis showed an extended G2/M arrest in Gal3-KO cells suggesting Gal3 may influence cell cycle checkpoints. Unlike SKOV3 ovarian cancer cells, loss of Gal3 did not affect apoptotic sensitivity of USC cells as determined by BH3 profiling. Gal3 knockout reduced the number of migrating and invading cells in vitro. Gal3 SMIs (GB1107 and TD139) often differed in their response when compared to the knockout strategy. Conditioned media (CM) from ARK2 Gal3-CTRL promoted tube formation and invasion of HUVECs. This effect was reduced with CM from ARK2 Gal3-KO cells, suggesting Gal3 influences the tumor vascular microenvironment. Gal3 loss markedly reduced CD117+ cells and cells displaying high ALDH activity. Functionally, these findings were supported by a reduction in the number and size of colonies and fewer spheres formed by Gal3-KO cells compared to Gal3-CTRL cells. ARK1 Gal3-KO cells formed fewer and smaller tumors compared to Gal3-CTRL in mice. Conclusion: Gal3 has the potential to promote the more aggressive features of USC and patients would likely benefit from an anti-Gal3 based strategy. Citation Format: Yusuke Matoba, Dominique T. Zarrella, Venkatesh Pooladanda, Eugene Kim, Shaan Kumar, Mengyao Xu, Xingping Qin, Raj Kumar, Artem Kononenko, Irva Veillard, Kristopher Sarosiek, Oladapo Yeku, David R. Spriggs, Bo R. Rueda. Loss of galectin 3 reveals its potential roles in the aggressive pathology of uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2438.

Antiangiogenic potential of Lepista nuda extract suppressing MAPK/p38 signaling-mediated developmental angiogenesis in zebrafish and HUVECs

The medicinal properties of natural/edible plant products and their use are popular in traditional practice owing to their nutritional contents with little to no side effects. Lepista nuda (L. nuda), an edible mushroom (Clitocybe nuda, commonly known as blewit), has attracted researchers to evaluate its contents and the mechanism of its activities. In the current study, we focused on evaluating the antiangiogenic effects of L. nuda water extract on zebrafish development and in vitro human umbilical vein endothelial cell (HUVEC) tube formation. Bioactive components such as ergothioneine, eritadenine, and adenosine were identified and quantified by HPLC analysis. The L. nuda extract showed antiangiogenic properties and inhibited intersegmental vessel (ISV), caudal vein plexus (CVP), hyaloid vessel (HV), and subintestinal vessel (SIV) development in Tg (fli1: EGFP) zebrafish embryos. The expression of angiogenesis-related genes (vegfaa, kdrl, vegfba, flt1, kdr) was affected following L. nuda extract treatment. L. nuda extract attenuated in vitro HUVEC tube formation, migration, and invasion. Furthermore, inhibition of MAPK/p38 signaling and depletion of proangiogenic genes, including growth factors (fgf, ang2, and vegfa); primary and accessory receptors (tie2, vegfr2, and eng); MMPs (mmp1 and mmp2); and cytokines (il-1α, il-1β, il-6, and tnf-α) was observed in HUVECs following L. nuda treatment. An in vivo zebrafish xenograft assay showed that L. nuda extract inhibited HuCCT1 cell-induced SIV sprouting in HuCCT1-injected embryos. Collectively, the results suggest that L. nuda could be a potential inhibitor of angiogenesis limiting cancer progression.

MPRα and PR co-operate in progesterone inhibition of endothelial cell focal adhesion.

Progesterone causes vascular smooth muscle cell relaxation through membrane progesterone receptors (mPRs), which are members of the progestin and adipoQ receptor (PAQR) family, and nuclear PRs (nPRs). However, beneficial vascular effects of progesterone in preventing pre-atherosclerosis and the involvement of mPRs and nPRs remain unclear. The results show short- to long-term treatments with 100 nM progesterone (P4) and specific agonists for mPRs, OD 02-0, and nPRs, R5020, inhibited pre-atherosclerotic events in human umbilical vein endothelial cells (HUVECs), decreasing focal adhesion (FA) by monocytes, FA signaling, HUVEC migration and invasion, and vinculin expression. Progesterone and OD 02-0, but not R5020, inhibited phosphorylation of Src and focal adhesion kinase, critical kinases of FA signaling, within 20 min and migration and invasion of HUVECs and monocyte adhesion after 3 h. These inhibitory P4 and 02-0 effects were attenuated with MAP kinase and Pi3k inhibitors, indicating involvement of these kinases in this mPR-mediated action. However, after 16 h, OD 02-0 was no longer effective in inhibiting FA signaling, while both progesterone and R5020 decreased the activity of the two kinases. Knockdown of receptor expression with siRNA confirmed that mPRα mediates short-term and nPR long-term inhibitory effects of progesterone on FA signaling. Thus, progesterone inhibition of FA signaling and pre-atherosclerosis is coordinated through mPRα and nPRs.

Accelerated Angiogenesis of Human Umbilical Vein Endothelial Cells Under Negative Pressure Was Associated With the Regulation of Gene Expression Involved in the Proliferation and Migration.

Negative pressure has been used as a preferred therapy for wound healing; however, the mechanisms by which negative pressure promotes tissue restoration remain unclear. In the present study, RNA-sequencing analysis was performed to identify differentially expressed genes in human umbilical vein endothelial cells (HUVECs) exposed to negative pressure. Cell viability and DNA synthesis were examined using the cell counting kit-8 assay and bromodeoxyuridine incorporation, respectively. Cell migration was assessed using tube formation, Transwell, and wound healing assays. Activity of the serine/threonine kinase (AKT) signaling pathway was also examined by measuring the levels of phospho-paxicillin, phospho-focal adhesion kinase (p-FAK), and p-AKT1. The exposure of HUVECs to negative pressure enhanced cell proliferation and DNA synthesis. Negative pressure enhanced the migration and invasion of HUVECs, which was accompanied by upregulation of genes involved in angiogenesis, extracellular matrix organization, and cytoskeletal organization. The mRNA levels of growth factors, including placental growth factor and platelet-derived growth factor B, also increased. In addition, phosphorylation of paxicillin, focal adhesion kinase, and AKT increased under negative pressure. Collectively, the findings of this study demonstrated that negative pressure stimulates the angiogenic activity of HUVECs by increasing their proliferation and migration via activation of the AKT signaling pathway.