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NF-κB signaling is essential for resistance to heat stress-induced early stage apoptosis in human umbilical vein endothelial cells.

ABSTRACT: Cell apoptosis induced by heat stress is regulated by a complex signaling network. We previously reported that a p53-dependent pathway is involved. Here, we present evidence that NF-κB signaling plays a crucial role in preventing heat stress-induced early apoptosis. Human umbilical vein endothelial cells (HUVECs) were examined and increased phosphorylation of p65 and IκBα were detected, without IκBα degradation. When NF-κB signaling was inhibited by BAY11-7082, or a small interference RNA (siRNA) targeting p65, a significant increase in cell apoptosis and caspase-3 activity was observed, as well as reduced expression and translocation of HSP27 into the nucleus, an accumulation of reactive oxygen species, and prolonged phosphorylation of mitogen-activated protein kinases (MAPKs). In addition, an association between HSP27 and p65 was identified which may enhance NF-κB activation. When HSP27 was overexpressed, pretreatment of HUVECs with the antioxidant, apocynin, or N-acetyl cysteine, suppressed apoptosis. Similarly, inhibition of JNK and p38 with SP600125 and SB203580, respectively, also suppressed apoptosis, whereas siRNA-mediated HSP27 knockdown and treatment with the ERK 1/2 inhibitor PD98059 did otherwise. In conclusion, these findings suggest a novel role for an NF-κB signaling pathway involving HSP27, ROS, and MAPKs that confers a protective effect against heat stress-induced cell apoptosis.

SUBMITTER: Liu Y

PROVIDER: S-EPMC4559749 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Macrophages enhance lipopolysaccharide induced apoptosis via Ang1 and NF-κB pathways in human umbilical vein endothelial cells.

Project description:Lipopolysaccharide (LPS) could induce apoptosis and dysfunction of endothelial cells. We aimed to reveal the effects of macrophages on cell proliferation and apoptosis in LPS induced human umbilical vein endothelial cells (HUVECs). THP-1 derived macrophages and HUVECs were co-cultured in the presence of LPS. Cell viability was measured by Cell Counting Kit-8 and apoptosis was analyzed by flow cytometry. Expression of Ang1, the NF-κB component p65 was evaluated by western blot and quantitative PCR. Small interfering RNAs (siRNAs) were used to knockdown the expression of proinflammatory cytokines and p65 in HUVECs. Plasmid transfection-mediated overexpression of Ang1 was employed to see its effects on cell proliferation and apoptosis in HUVECs. Macrophages enhanced LPS-induced proliferation impairments and apoptosis in HUVECs, which could be attenuated by siRNA-mediated knockdown of cytokines TNF-α, IL-1β, IL-6 and IL-12p70 in macrophages. The dysfunction of HUVECs was tightly associated with reduced Ang1 expression and increased phosphorylated p65 (p-65). Overexpression of Ang1 in HUVECs significantly decreased p-p65, suggesting negatively regulation of p-p65 by Ang1. Overexpression of Ang1, adding recombinant Ang1 or silencing of p65 substantially attenuated the dysfunction of HUVECs in terms of cell proliferation and apoptosis. In conclusions, THP-1-derived macrophages enhance LPS induced dysfunction of HUVECs via Ang1 and NF-κB pathways, suggesting new therapeutic targets for sepsis.

| S-EPMC7858588 | biostudies-literature

Heat stress induces apoptosis through a Ca²⁺-mediated mitochondrial apoptotic pathway in human umbilical vein endothelial cells.

Project description:BackgroundHeat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined.MethodsThe percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O2-, H2O2, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry.ResultsIn this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca2+, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca2+ into the cytoplasm after heat stress.ConclusionOur findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca2+, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process.

| S-EPMC4280109 | biostudies-literature

NF‑κB/IκBα signaling pathways are essential for resistance to heat stress‑induced ROS production in pulmonary microvascular endothelial cells.

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| S-EPMC8477608 | biostudies-literature

Hypericin-photodynamic therapy induces human umbilical vein endothelial cell apoptosis.

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| S-EPMC4682094 | biostudies-literature

Heat stress induces apoptosis through transcription-independent p53-mediated mitochondrial pathways in human umbilical vein endothelial cell.

Project description:Cells apoptosis induced by intense heat stress is the prominent feature of heat-related illness. However, little is known about the biological effects of heat stress on cells apoptosis. Herein, we presented evidence that intense heat stress could induce early apoptosis of HUVEC cells through activating mitochondrial pathway with changes in mitochondrial membrane potential(ΔΨm), release of cytochrome c, and activation of caspase-9 and -3. We further revealed that p53 played a crucial role in heat stress-induced early apoptosis, with p53 protein rapidly translocated into mitochondria. Using pifithrin-α(PFT), a p53's mitochondrial translocation inhibitor, we found that pretreated with PFT, heat stress induced mitochondrial p53 translocation was significantly suppressed, accompanied by a significant alleviation in the loss of ΔΨm, cytochrome c release and caspase-9 activation. Furthermore, we also found that generation of reactive oxygen species (ROS) was a critical mediator in heat stress-induced apoptosis. In addition, the antioxidant MnTMPyP significantly decreased the heat stress-induced p53's mitochondrial translocation, followed by the loss of ΔΨm, cytochrome c release, caspase-9 activation and heat stress-mediated apoptosis. Conclusively, these findings indicate the contribution of the transcription-independent mitochondrial p53 pathway to early apoptosis in HUVEC cells induced by oxidative stress in response to intense heat stress.

| S-EPMC3966036 | biostudies-other

Regulation of NF-κB activation through a novel PI-3K-independent and PKA/Akt-dependent pathway in human umbilical vein endothelial cells.

Project description:The transcription factor NF-κB regulates numerous inflammatory diseases, and proteins involved in the NF-κB-activating signaling pathway are important therapeutic targets. In human umbilical vein endothelial cells (HUVECs), TNF-α-induced IκBα degradation and p65/RelA phosphorylation regulate NF-κB activation. These are mediated by IKKs (IκB kinases) viz. IKKα, β and γ which receive activating signals from upstream kinases such as Akt. Akt is known to be positively regulated by PI-3K (phosphoinositide-3-kinase) and differentially regulated via Protein kinase A (PKA) in various cell types. However, the involvement of PKA/Akt cross talk in regulating NF-κB in HUVECs has not been explored yet. Here, we examined the involvement of PKA/Akt cross-talk in HUVECs using a novel compound, 2-methyl-pyran-4-one-3-O-β-D-2',3',4',6'-tetra-O-acetyl glucopyranoside (MPTAG). We observed that MPTAG does not directly inhibit IKK-β but prevents TNF-α-induced activation of IKK-β by blocking its association with Akt and thereby inhibits NF-κB activation. Interestingly, our results also revealed that inhibitory effect of MPTAG on Akt and NF-κB activation was unaffected by wortmannin, and was completely abolished by H-89 treatment in these cells. Thus, MPTAG-mediated inhibition of TNF-α-induced Akt activation was independent of PI-3K and dependent on PKA. Most importantly, MPTAG restores the otherwise repressed activity of PKA and inhibits the TNF-α-induced Akt phosphorylation at both Thr308 and Ser473 residues. Thus, we demonstrate for the first time the involvement of PKA/Akt cross talk in NF-κB activation in HUVECs. Also, MPTAG could be useful as a lead molecule for developing potent therapeutic molecules for diseases where NF-κB activation plays a key role.

| S-EPMC3465347 | biostudies-literature

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Upregulation of P53 promoted G1 arrest and apoptosis in human umbilical cord vein endothelial cells from preeclampsia.

Project description:Preeclampsia is a leading cause of maternal and perinatal morbidity and mortality. Current research has focused on endothelial dysfunction regarding pathogenesis of preeclampsia. However, very limited or no studies so far have been performed to assess possible damaged endothelial cell growth/development in the placenta-umbilical cord circulation system in human preeclampsia.We isolated and cultured human umbilical cord vein endothelial cells (HUVECs) from normal and preeclampsia pregnancies in vitro. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay to measure cell growth and flow cytometric analysis to determine cell-cycle distribution. Annexin V-fluorescein isothiocyanate/propidium iodide double staining was employed for cell apoptosis experiments.The study showed that the cell growth was significantly suppressed, accompanied by the increased G1 arrest and apoptosis in cultured HUVECs from preeclampsia pregnancies comparing with normotensive controls. Protein P53 was upregulated in the cultured HUVECs from preeclampsia pregnancies, which induced G1 arrest, followed by upregulating P21 expression, and downregulating cyclin E expression and CDK2-cyclin E complexes. On the other hand, upregulation of P53 also activated Bax gene and repressed Bcl-2 and BIRC5 genes, resulting in an increase of the Bax/Bcl-2 ratio and subsequently activating caspase cascade, ultimately led to an initiation of the apoptotic machinery.These results indicated that in preeclampsia, vascular endothelial cells could be damaged and cellular proliferation was depressed in human placenta-umbilical cord circulation, adding new information on endothelial cell injury for better understanding the pathogenesis of preeclampsia.

| S-EPMC5649442 | biostudies-literature

Zika Virus Infection Induces Elevation of Tissue Factor Production and Apoptosis on Human Umbilical Vein Endothelial Cells.

Project description:Zika virus (ZIKV) infection is typically characterized by a mild disease presenting with fever, maculopapular rash, headache, fatigue, myalgia, and arthralgia. A recent animal study found that ZIKV-infected pregnant Ifnar -/-mice developed vascular damage in the placenta and reduced amount of fetal capillaries. Moreover, ZIKV infection causes segmental thrombosis in the umbilical cord of pregnant rhesus macaques. Furthermore, several case reports suggest that ZIKV infection cause coagulation disorders. These results suggest that ZIKV could cause an alteration in the host hemostatic response, however, the mechanism has not been investigated thus far. This paper aims to determine whether ZIKV infection on HUVECs induces apoptosis and elevation of tissue factor (TF) that leads to activation of secondary hemostasis. We infected HUVECs with two ZIKV strains and performed virus titration, immunostaining, and flow cytometry to confirm and quantify infection. We measured TF concentrations with flow cytometry and performed thrombin generation test (TGT) as a functional assay to assess secondary hemostasis. Furthermore, we determined the amount of cell death using flow cytometry. We also performed enzyme-linked immunosorbent assay (ELISA) to determine interleukin (IL)-6 and IL-8 production and conducted blocking experiments to associate these cytokines with TF expression. Both ZIKV strains infected and replicated to high titers in HUVECs. We found that infection induced elevation of TF expressions. We also showed that increased TF expression led to shortened TGT time. Moreover, the data revealed that infection induced apoptosis. In addition, there was a significant increase of IL-6 and IL-8 production in infected cells. Here we provide in vitro evidence that infection of HUVECs with ZIKV induces apoptosis and elevation of TF expression that leads to activation of secondary hemostasis.

| S-EPMC6491739 | biostudies-literature

Ketamine ameliorates hypoxia-induced endothelial injury in human umbilical vein endothelial cells.

Project description:OBJECTIVES:Hypoxia leads to endothelial cell inflammation, apoptosis, and damage, which plays an important role in the complications associated with ischemic cardiovascular disease. As an oxidoreductase, p66Shc plays an important role in the regulation of reactive oxygen species (ROS) production and apoptosis. Ketamine is widely used in clinics. This study was designed to assess the potential protective effect of ketamine against hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs). Moreover, we explored the potential mechanism by which ketamine protected against hypoxia-induced endothelial injury. METHODS:The protective effects of ketamine against hypoxia-induced injury was assessed using cell viability and adhesion assays, quantitative polymerase chain reaction, and western blotting. RESULTS:Our data showed that hypoxia reduced HUVEC viability, increased the adhesion between HUVECs and monocytes, and upregulated the expression of endothelial adhesion molecules at the protein and mRNA levels. Moreover, hypoxia increased ROS accumulation and upregulated p66Shc expression. Furthermore, hypoxia downregulated sirt1 expression in HUVECs. Alternatively, ketamine was shown to reverse the hypoxia-mediated reduction of cell viability and increase in the adhesion between HUVECs and monocytes, ameliorate hypoxia-induced ROS accumulation, and suppress p66Shc expression. Moreover, EX527, a sirt1 inhibitor, reversed the protective effects of ketamine against the hypoxia-mediated reduction of cell viability and increase in adhesion between HUVECs and monocytes. CONCLUSION:Ketamine reduces hypoxia-induced p66Shc expression and attenuates ROS accumulation via upregulating sirt1 in HUVECs, thus attenuating hypoxia-induced endothelial cell inflammation and apoptosis.

| S-EPMC7470431 | biostudies-literature

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