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BKCa channels (large-conductance Ca2+-activated K+ channels) play a critical role in regulating vascular tone and blood pressure. These channels are present in the smooth muscle cells of blood vessels and are activated by voltage and increased intracellular Ca2+ concentration. More recently, the expression and activity of BKCa have been proposed to be relevant in endothelial cells, too, specifically in human umbilical vein endothelial cells (HUVECs), the more studied cell type in the fetoplacental circulation. The role of BKCa in endothelial cells is not well understood, but in HUVECs or placental endothelium, these channels could be crucial for vascular tone regulation during pregnancy as part of endothelium-derived hyperpolarization (EDH), a key mechanism for an organ that lacks nervous system innervation like the placenta.In this review, we will discuss the evidence about the role of BKCa (and other Ca2+-activated K+ channels) in HUVECs and the placenta to propose a physiological mechanism for fetoplacental vascular regulation and a pathophysiological role of BKCa, mainly associated with pregnancy pathologies that present maternal hypertension and/or placental hypoxia, like preeclampsia.

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BACKGROUND: Isolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100. RESULTS: First, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1-1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1-1% Triton X-100 for our method were determined via western blotting of eight extra-/intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei. CONCLUSIONS: The in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

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BACKGROUND: Isolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100. RESULTS: First, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1-1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1-1% Triton X-100 for our method were determined via western blotting of eight extra-/ intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei. CONCLUSIONS: The in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for the severe pandemic of acute respiratory disease, coronavirus disease 2019 (COVID-19), experienced in the 21st century. The clinical manifestations range from mild symptoms to abnormal blood coagulation and severe respiratory failure. In severe cases, COVID-19 manifests as a thromboinflammatory disease. Damage to the vascular compartment caused by SARS-CoV-2 has been linked to thrombosis, triggered by an enhanced immune response. The molecular mechanisms underlying endothelial activation have not been fully elucidated. We aimed to identify the proteins correlated to the molecular response of human umbilical vein endothelial cells (HUVECs) after exposure to SARS-CoV-2, which might help to unravel the molecular mechanisms of endothelium activation in COVID-19. In this direction, we exposed HUVECs to SARS-CoV-2 and analyzed the expression of specific cellular receptors, and changes in the proteome of HUVECs at different time points. We identified that HUVECs exhibit non-productive infection without cytopathic effects, in addition to the lack of expression of specific cell receptors known to be essential for SARS-CoV-2 entry into cells. We highlighted the enrichment of the protein SUMOylation pathway and the increase in SUMO2, which was confirmed by orthogonal assays. In conclusion, proteomic analysis revealed that the exposure to SARS-CoV-2 induced oxidative stress and changes in protein abundance and pathways enrichment that resembled endothelial dysfunction.

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Breast cancer is the most frequent malignancy among women in developed countries and the main cause of death related to cancer in women worldwide. Extracellular vesicles (EVs) are vesicles with a variable size enclosed within a phospholipid bilayer that contain a variety of molecules with biological activity. Cancer cells release EVs that induce proliferation, escape from apoptosis, reprogramming energy metabolism, invasion and metastasis. In this study we studied whether EV fractions deprived of platelet EVs from breast cancer women (BC EVs) can mediate cell processes related with angiogenesis in human umbilical vein endothelial cells (HUVECs). Our findings demonstrate that BC EVs enhance migration, invasion and formation of new tubules in HUVECs, compared with EV fractions deprived of platelet EVs from healthy women (Ctrl EVs). In summary, we demonstrate, for the first time, that BC EVs induce cellular processes in HUVECs that participate in angiogenesis.

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Although polyphenols have great pharmacological potential, the main disadvantage is that they have low bioavailability at the desired site. Thus, the use of biocompatible systems for drug delivery is a strategy that is currently gaining great interest. The objective of this study is to evaluate the effect of microencapsulation of caffeic acid and pinocembrin on the antioxidant and antiangiogenic activity of both polyphenols, by the use of nPSi-ßCD composite microparticles. For this HUVEC, cells were exposed to H2O2 and to treatments with polyphenols in solution and loaded in the composite microparticle. The polyphenols were incorporated into a microparticle using nanoporous silicon, chitosan and a ß-cyclodextrin polymer as the biomaterial. The evaluation of the antiangiogenic effect of the treatments with polyphenols in solution and microencapsulated was carried out through functional tests, and the changes in the expression of target genes associated with the antioxidant pathway and angiogenesis was performed through qPCR. The results obtained show that the caffeic acid and pinocembrin have an antioxidant and antiangiogenic activity, both in solution as microencapsulated. In the caffeic acid, a greater biological effect was observed when it was incorporated into the nPSi-ßCD composite microparticle. Our results suggest that the nPSi-ßCD composite microparticle could be used as an alternative oral drug administration system.

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for the severe pandemic of acute respiratory disease, coronavirus disease 2019 (COVID-19), experienced in the 21st century. The clinical manifestations range from mild symptoms to abnormal blood coagulation and severe respiratory failure. In severe cases, COVID-19 manifests as a thromboinflammatory disease. Damage to the vascular compartment caused by SARS-CoV-2 has been linked to thrombosis, triggered by an enhanced immune response. The molecular mechanisms underlying endothelial activation have not been fully elucidated. We aimed to identify the proteins correlated to the molecular response of human umbilical vein endothelial cells (HUVECs) after exposure to SARS-CoV-2, which might help to unravel the molecular mechanisms of endothelium activation in COVID-19. In this direction, we exposed HUVECs to SARS-CoV-2 and analyzed the expression of specific cellular receptors, and changes in the proteome of HUVECs at different time points. We identified that HUVECs exhibit non-productive infection without cytopathic effects, in addition to the lack of expression of specific cell receptors known to be essential for SARS-CoV-2 entry into cells. We highlighted the enrichment of the protein SUMOylation pathway and the increase in SUMO2, which was confirmed by orthogonal assays. In conclusion, proteomic analysis revealed that the exposure to SARS-CoV-2 induced oxidative stress and changes in protein abundance and pathways enrichment that resembled endothelial dysfunction.

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Extracellular vesicles (EVs) are vesicles secreted by normal and malignant cells that are implicated in tumor progression. Linoleic acid (LA) is an essential polyunsaturated fatty acid that induces migration, invasion and an increase in phospholipase D activity in breast cancer cells. In this study, we determined whether stimulation of MDA-MB-231 breast cancer cells with LA induces the secretion of EVs, which can mediate cell processes related with angiogenesis in human umbilical vein endothelial cells (HUVECs). Our findings demonstrate that treatment of MDA-MB-231 cells with 90 µM LA for 48 h induce an increase in the number of EVs released. Moreover, EVs from MDA-MB-231 stimulated with 90 µM LA induce FAK and Src activation and migration via FAK and Src activity, whereas the secretion of these EVs is through FFAR1 and FFAR4 activation in HUVECs. The EVs from MDA-MB-231 cells treated with LA also increase proliferation, invasion, MMP-9 secretion, an increase of MMP-2 secretion and formation of new tubules in HUVECs. In summary, we demonstrate, for the first time, that treatment with LA induces the release of EVs from MDA-MB-231 cells that induce cellular processes involved with angiogenesis in HUVECs.

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Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling pathway.

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BACKGROUND: LincRNAs have been revealed to be tightly associated with various tumorigeneses and cancer development, but the roles of specific lincRNA on tumor-related angiogenesis was hardly studied. Here, we aimed to investigate whether linc-OIP5 in breast cancer cells affects the angiogenesis of HUVECs and whether the linc-OIP5 regulations are involved in angiogenesis-related Notch and Hippo signaling pathways. METHODS: A trans-well system co-cultured HUVECs with linc-OIP5 knockdown breast cancer cell MDA-MB-231 was utilized to study the proliferation, migration and tube formation abilities of HUVECs and alterations of related signaling indicators in breast cancer cells and their conditioned medium through a series of cell and molecular experiments. RESULTS: Overexpressed linc-OIP5, YAP1, and JAG1 were found in breast cancer cell lines MCF7 and MDA-MB-231 and the expression levels of YAP1 and JAG1 were proportional to the breast cancer tissue grades. MDA-MB-231 cells with linc-OIP5 knockdown led to weakened proliferation, migration, and tube formation capacity of co-cultured HUVECs. Besides, linc-OIP5 knockdown in co-cultured MDA-MB-231 cells showed downregulated YAP1 and JAG1 expression, combined with a reduced JAG1 level in conditioned medium. Furthermore, a disrupted DLL4/Notch/NRP1 signaling in co-cultured HUVECs were also discovered under this condition. CONCLUSION: Hence, linc-OIP5 in MDA-MB-231 breast cancer cells may act on the upstream of the YAP1/Notch/NRP1 signaling circuit to affect proliferation, migration, and tube formation of co-cultured HUVECs in a non-cellular direct contact way through JAG1 in conditioned medium. These findings at least partially provide a new angiogenic signaling circuit in breast cancers and suggest linc-OIP5 could be considered as a therapeutic target in angiogenesis of breast cancers.

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BACKGROUND: LincRNAs have been revealed to be tightly associated with various tumorigeneses and cancer development, but the roles of specific lincRNA on tumor-related angiogenesis was hardly studied. Here, we aimed to investigate whether linc-OIP5 in breast cancer cells affects the angiogenesis of HUVECs and whether the linc-OIP5 regulations are involved in angiogenesis-related Notch and Hippo signaling pathways. METHODS: A trans-well system co-cultured HUVECs with linc-OIP5 knockdown breast cancer cell MDA-MB-231 was utilized to study the proliferation, migration and tube formation abilities of HUVECs and alterations of related signaling indicators in breast cancer cells and their conditioned medium through a series of cell and molecular experiments. RESULTS: Overexpressed linc-OIP5, YAP1, and JAG1 were found in breast cancer cell lines MCF7 and MDA-MB-231 and the expression levels of YAP1 and JAG1 were proportional to the breast cancer tissue grades. MDA-MB-231 cells with linc-OIP5 knockdown led to weakened proliferation, migration, and tube formation capacity of co-cultured HUVECs. Besides, linc-OIP5 knockdown in co-cultured MDA-MB-231 cells showed downregulated YAP1 and JAG1 expression, combined with a reduced JAG1 level in conditioned medium. Furthermore, a disrupted DLL4/Notch/NRP1 signaling in co-cultured HUVECs were also discovered under this condition. CONCLUSION: Hence, linc-OIP5 in MDA-MB-231 breast cancer cells may act on the upstream of the YAP1/Notch/NRP1 signaling circuit to affect proliferation, migration, and tube formation of co-cultured HUVECs in a non-cellular direct contact way through JAG1 in conditioned medium. These findings at least partially provide a new angiogenic signaling circuit in breast cancers and suggest linc-OIP5 could be considered as a therapeutic target in angiogenesis of breast cancers.

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Propolis is widely recognized for its various therapeutic properties. These are attributed to its rich composition in polyphenols, which exhibit multiple biological properties (e.g., antioxidant, anti-inflammatory, anti-angiogenic). Despite its multiple benefits, oral administration of polyphenols results in low bioavailability at the action site. An alternative to face this problem is the use of biomaterials at nano-micro scale due to its high versatility as carriers and delivery systems of various drugs and biomolecules. The aim of this work is to determine if nPSi-ßCD microparticles are a suitable material for the load and controlled release of caffeic acid (CA) and pinocembrin (Pin), two of the main components of a Chilean propolis with anti-atherogenic and anti-angiogenic activity. Polyphenols and nPSi-ßCD microparticles cytocompatibility studies were carried out with human umbilical vein endothelial cells (HUVECs). Results from physicochemical characterization demonstrated nPSi-ßCD microparticles successfully retained and controlled release CA and Pin. Furthermore, nPSi-ßCD microparticles presented cytocompatibility with HUVECs culture at concentrations of 0.25 mg/mL. These results suggest that nPSi-ßCD microparticles could safely be used as an alternate oral delivery system to improve controlled release and bioavailability of CA or Pin-and eventually other polyphenols-thus enhancing its therapeutic effect for the treatment of different diseases.

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We examined the effect of plasma incubation from preeclampsia pregnant on the antiangiogenic miR-195-5p expression. Higher miR-195-5p expression was found in cultures incubated with preeclampsia plasma compared to those incubated with healthy pregnant plasma. Next, as VEGF is a target of miR-195-5p we have quantified its expression by real-time qPCR and ELISA. We found reduced VEGF levels in culture incubated with preeclampsia plasma. Therefore, we have concluded that the higher expression of miR-195-5p in endothelial cell cultures incubated with preeclampsia plasma may contribute to decreased expression of VEGFA (gene and protein) and increased antiangiogenic status in preeclampsia. Therefore, this miR may be an important target in preeclampsia.

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