Propofol ameliorates endothelial inflammation induced by hypoxia/reoxygenation in human umbilical vein endothelial cells: Role of phosphatase A2

doi:10.1016/j.vph.2015.06.002

Vascular Pharmacology

Volume 73, October 2015, Pages 149-157

https://doi.org/10.1016/j.vph.2015.06.002 Get rights and content

Abstract

Hypoxia/reoxygenation (H/R) induces endothelial inflammation with augmentation of endothelial adhesion molecules over-expression. Propofol was reported to attenuate endothelial adhesion molecule expression in some situations. Here, we examined the molecular mechanism for how propofol restored H/R-mediated up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs). Compared with the control group, H/R up-regulated expression of Pin-1 and PP2A, increased p66^Shc–Ser³⁶ phosphorylation, induced p66^Shc mitochondrial translocation, O₂⁻ accumulation and NF-κB activation, and decreased eNOS–Ser¹¹⁷⁷ phosphorylation and nitric oxide (NO) production, thus up-regulating expression of endothelial adhesion molecules and increasing mononuclear-endothelial interaction. More importantly, except that propofol had no effect on H/R-induced p66^Shc–Ser³⁶ phosphorylation, most of H/R-mediated changes were alleviated by propofol, resulting in the reduction of endothelial adhesion molecules expression and mononuclear-endothelial adhesion. Moreover, we demonstrated the protective effect of propofol on H/R-induced endothelial inflammation was similar to that of calyculin A, an inhibitor of PP2A. In contrast, FTY720, an activator of PP2A, antagonized the effect of propofol. Our data indicated that propofol down-regulated PP2A expression, leading to reduced dephosphorylation of p66^Shc–Ser³⁶ and eNOS–Ser¹¹⁷⁷, which is associated with ROS accumulation and NO reduction, resulting in inhibition of endothelial adhesion molecule expression and mononuclear-endothelial interaction.

Graphical abstract

Introduction

Hypoxia/reoxygenation (H/R) in an in vitro situation mimics the ischemia/reperfusion (I/R) model in vivo. It leads to the accumulation of inflammatory cytokines [1], [2] and activation of inflammatory signaling pathways in endothelial cells [3]. Endothelial inflammation consists of the augmentation of endothelial adhesion molecule expression, such as intercellular adhesion molecule 1(ICAM-1) and endothelial selectin (E-selectin). The increased expression of endothelial adhesion molecules induces mononuclear-endothelial adhesion, which leads to endothelial injury [4].

The nuclear factor kappa B (NF-κB) signal pathway was reported to be involved in H/R-induced endothelial adhesion molecules expression [5]. Previous data have indicated that reactive oxygen species (ROS) could activate NF-κB, which plays an important role in ROS-mediated endothelial injury due to H/R [6]. Mitochondrion is the major organelle where ROS is generated [7], [8]. Furthermore, the p66^Shc adaptor protein is important in regulating mitochondrial ROS generation [9], [10], [11]. The adjustment of p66^Shc function is a complex process. First, p66^Shc is phosphorylated at the site of Ser³⁶, then Ser³⁶–phosphorylated p66^Shc is isomerized with a prolylisomerase Pin1 and dephosphorylated by the phosphatase A2 (PP2A) [12]. Only isomerized and dephosphorylated p66^Shc could translocate to the mitochondria and subsequently lead to ROS generation [13].

It was also reported that reduced nitric oxide (NO) production from vascular endothelium induced endothelial adhesion molecule expression and leukocyte-endothelium adhesion [14], [15], resulting in endothelial inflammation. Moreover, improvement of NO production could protect against H/R-induced endothelial injury [16]. And this effect was reversed by L-NANE, an endothelial NO synthase (eNOS) inhibitor [16].

Propofol (2, 6-diisopropylphenol) is a widely used intravenous anesthetic agent. It was reported that propofol could inhibit the expression of endothelial adhesion molecules in H/R-treated endothelial cells [17]. However, the mechanism by which propofol protects against H/R-induced adhesion molecules expression is still elusive. In the present study, we examined whether and how propofol protects HUVECs against H/R-induced endothelial adhesion molecules expression.

Section snippets

Cell culture and reagents

HUVECs (Clonetics) were incubated in Dulbecco's Modified Eagle's Medium (DMEM) and 10% fetal bovine serum in incubator containing 5% CO₂ at 37 °C. Cells were sub-cultured when reaching around 90% confluence. The fourth passage of HUVECs was employed in the present study.

Propofol (sigma), calyculin A (sigma) and FTY720 (sigma) were dissolved in dimethyl sulfoxide (DMSO) (sigma). The final concentration of DMSO was regulated to 0.01% for each medium to minimize any potential toxicity or

H/R-induced mononuclear-endothelial adhesion and endothelial adhesion molecules expression, and its modulation by propofol

In HUVECs, compared with control group, H/R induced more mononuclear-endothelial adhesion. Pre-incubation of cells with 1 μM propofol for 30 min had no effect on baseline mononuclear-endothelial adhesion (Fig. 1A). When pre-treatment of propofol reached 5 or 25 μM, the mononuclear-endothelial adhesion was inhibited. Therefore propofol worked in a dose-dependent manner. To be noted, the solvent 0.01% of dimethyl sulfoxide (DMSO) did not show any significant impact on H/R-induced

Discussion

The major finding of the present study is that H/R induced p66^Shc–Ser³⁶ phosphorylation and p66^Shc mitochondrial translocation, which resulted in ROS accumulation, NF-κB activation and translocation to nucleus; H/R also inhibited eNOS–Ser¹¹⁷⁷ phosphorylation and NO production, thus augmenting endothelial adhesion molecules expression and increasing mononuclear-endothelial adhesion. More importantly, propofol was observed to protect HUVECs against H/R-induced endothelial adhesion molecules

Conflict of interest

None declared.

Acknowledgments

This project is supported by the Natural Science Foundation of Shanghai (No. 12ZR1406700).

References (38)

C. Kupatt et al.
Nitric oxide attenuates reoxygenation-induced ICAM-1 expression in coronary microvascular endothelium: role of NFkappaB
J. Mol. Cell. Cardiol.
(1997)
J.C. Martinou et al.
Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics
Dev. Cell
(2011)
M. Giorgio et al.
Electron transfer between cytochrome c and p66^Shc generates reactive oxygen species that trigger mitochondrial apoptosis
Cell
(2005)
C.V. Diogo et al.
Cardiac mitochondrial dysfunction during hyperglycemia-the role of oxidative stress and p66^Shc signaling
Int. J. Biochem. Cell Biol.
(2013)
J.E. Cartwright et al.
Endothelial cell adhesion molecule expression and lymphocyte adhesion to endothelial cells: effect of nitric oxide
Exp. Cell Res.
(1997)
R.C. Ziegelstein et al.
Hypoxia/reoxygenation stimulates Ca^2 +-dependent ICAM-1 mRNA expression in human aorticendothelial cells
Biochem. Biophys. Res. Commun.
(2004)
C.S. Callicutt et al.
Diminished lung injury with vascular adhesion molecule-1 blockade in choline-deficient ethionine diet-induced pancreatitis
Surgery
(2003)
I.M. Grumbach et al.
A negative feedback mechanism involving nitric oxide and nuclear factor kappa-B modulates endothelial nitric oxide synthase transcription
J. Mol. Cell. Cardiol.
(2005)
B.J. Michell et al.
Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase
J. Biol. Chem.
(2001)
J. Li et al.
The effects of propofol on mitochondrial dysfunction following focal cerebral ischemia-reperfusion in rats
Neuropharmacology
(2014)

M. Zhu et al.

Propofol protects human umbilical vein endothelial cells from cisplatin-induced injury

Vasc. Pharmacol.

(2014)

R. Shreeniwas et al.

Hypoxia-mediated induction of endothelial cell interleukin-1: an autocrine mechanism promoting expression of leukocyte adhesion molecules on the vessel surface

J. Clin. Invest.

(1992)

Y. Ala et al.

Hypoxia/reoxygenation stimulates endothelial cells to promote interleukin-1 and interleukin-6 production: effects of free radical scavengers

Agents Actions

(1992)

H. Ichikawa et al.

Molecular mechanisms of anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells

Circ. Res.

(1997)

W. Sluiter et al.

Leukocyte adhesion molecules on the vascular endothelium: their role in the pathogenesis of cardiovascular disease and the mechanisms underlying their expression

J. Cardiovasc. Pharmacol.

(1993)

H. Xie et al.

NF-κB activation plays a role in superoxide-mediated cerebral endothelial dysfunction after hypoxia/reoxygenation

Stroke

(2005)

D.E. Handy et al.

Redox regulation of mitochondrial function

Antioxid. Redox Signal.

(2012)

E. Migliaccio et al.

The p66^Shc adaptor protein controls oxidative stress response and life span in mammals

Nature

(1999)

M. Trinei et al.

A p53-p66^Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis

Oncogene

(2002)

Cited by (16)

The metabolic crosstalk between PIN1 and the tumour microenvironment
2023, Seminars in Cancer Biology
Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) is a member of a family of peptidyl-prolyl isomerases that specifically recognizes and binds phosphoproteins, catalyzing the rapid cis–trans isomerization of phosphorylated serine/threonine-proline motifs, which leads to changes in the structures and activities of the targeted proteins. Through this complex mechanism, PIN1 regulates many hallmarks of cancer including cell autonomous metabolism and the crosstalk with the cellular microenvironment. Many studies showed that PIN1 is largely overexpressed in cancer turning on a set of oncogenes and abrogating the function of tumor suppressor genes. Among these targets, recent evidence demonstrated that PIN1 is involved in lipid and glucose metabolism and accordingly, in the Warburg effect, a characteristic of tumor cells. As an orchestra master, PIN1 finely tunes the signaling pathways allowing cancer cells to adapt and take advantage from a poorly organized tumor microenvironment. In this review, we highlight the trilogy among PIN1, the tumor microenvironment and the metabolic program rewiring.
Multitarget neuroprotection by quercetin: Changes in gene expression in two perinatal asphyxia models
2021, Neurochemistry International
Citation Excerpt :
It would be important therefore to further analyze the effective change in this phosphatase's activity in the rat model. If a decreased activity could be demonstrated after quercetin treatment we could hypothesize that, as was described for propofol protective action (Zhu et al., 2015), a decrease in PP2A expression could control endothelial inflammation induced by HIE. On the other hand, as addressed by Kruse et al. (2020), the regulatory subunit affects the phosphorylation site preference of the holoenzyme catalytic subunit, suggesting that quercetin could be redirecting phosphatase activity to different substrates.
Hypoxic-ischemic encephalopathy (HIE) causes mortality and long-term neurologic morbidities in newborns, affecting pathways related to energy failure, excitotoxicity and oxidative stress that often lead to cell death. The whole process of HIE injury is coupled to changes in the expression of a great array of proteins. A nanoliposomal preparation of the flavonoid quercetin has been shown to exert neuroprotective effects in perinatal asphyxia models.
This study aimed to identify neonatal HIE markers and explore the effect of quercetin administration in two perinatal asphyxia models: newborn rats and piglets. In the rat model, nanoliposomal quercetin administration reduced mortality after asphyxia. In the piglet model, quercetin partially overrode the reduction of HIF-1α mRNA levels in the cortex induced by asphyxia. Quercetin administration also reduced increased level of HO-1 mRNA in asphyctic piglets. These results suggest that quercetin neuroprotection might be involved in the regulation of HIF-1α, HO-1 and their targets.
A proteomic approach revealed that the glycolytic pathway is strongly regulated by quercetin in both species. We also identified a set of proteins differentially expressed that could be further considered as markers. In piglets, this set includes Acidic Leucine-rich nuclear phosphoprotein 32 (ANP32A), associated with nervous system differentiation, proteins related with death pathways and alpha-enolase which can be converted to neuron-specific enolase, a glycolytic enzyme that may promote neuroprotection. In newborn rats, other promising proteins associated with neurogenesis and neuroprotection emerged, such as dihydropyrimidinase-related proteins, catalytic and regulatory subunits of phosphatases and heterogeneous nuclear ribonucleoprotein K (hnRNPK).
Our results show that a nanoliposomal preparation of quercetin, with protective effect in two HIE mammal models, modulates the expression of proteins involved in energy metabolism and other putative neuroprotective signals in the cortex. Identification of these signals could reveal potential molecular pathways involved in disease onset and the novel quercetin neuroprotective strategy.
The mystery of mitochondria-ER contact sites in physiology and pathology: A cancer perspective
2020, Biochimica et Biophysica Acta - Molecular Basis of Disease
Citation Excerpt :
Shc adaptor proteins bind to activated receptor tyrosine kinases (RTKs) in response to various growth factors [202] and participate in the regulation of cellular proliferation and differentiation [203]. The Ser36 residue located in the unique p66Shc collagen homology domain (CH2) is phosphorylated by either protein kinase Cβ II (PKCβII) [204] or c-Jun N-terminal kinase (JNK) [205] in response to various environmental and intracellular stress stimuli, such as H2O2 or Taxol treatment [206], hypoxia/reoxygenation injury [207–210], UV radiation [211], and high-glucose environments [212–214], as well as in response to metabolic stresses [215]. After isomerization by peptidyl-prolyl cis-trans isomerase 1 (PIN1) and dephosphorylation by PP2A, p66Shc is translocated into mitochondria [204]; however, MAM-localized p66Shc may also be able to affect mitochondria.
Mitochondria-associated membranes (MAM), physical platforms that enable communication between mitochondria and the endoplasmic reticulum (ER), are enriched with many proteins and enzymes involved in several crucial cellular processes, such as calcium (Ca²⁺) homeostasis, lipid synthesis and trafficking, autophagy and reactive oxygen species (ROS) production. Accumulating studies indicate that tumor suppressors and oncogenes are present at these intimate contacts between mitochondria and the ER, where they influence Ca²⁺ flux between mitochondria and the ER or affect lipid homeostasis at MAM, consequently impacting cell metabolism and cell fate. Understanding these fundamental roles of mitochondria-ER contact sites as important domains for tumor suppressors and oncogenes can support the search for new and more precise anticancer therapies. In the present review, we summarize the current understanding of basic MAM biology, composition and function and discuss the possible role of MAM-resident oncogenes and tumor suppressors.
Propofol post-conditioning lessens renal ischemia/reperfusion-induced acute lung injury associated with autophagy and apoptosis through MAPK signals in rats
2020, Gene
Citation Excerpt :
These properties lead to inflammatory mediators being released from lung inflammatory cells, and their serum concentrations were parallel to the degree of pulmonary injury. Pro offers defense by attenuating reactive oxygen species and inflammatory mediators (Zhu et al., 2015). To date, mechanisms by which Pro reduces inflammatory responses have not yet been elucidated, but may involve inhibition of MAPK signaling.
Renal Ischemia/Reperfusion (rI/R)-induced acute lung injury (ALI) is a major problem in rI/R. The objective of the current study was to explore the defensive roles of propofol (Pro), an intravenous anesthetic, on rI/R-induced ALI through mitogen-activated protein kinase (MAPK) signaling. Rats were divided into Sham, Pro (10 mg/kg), rI/R, rI/R + Pro (5 mg/kg), and rI/R + Pro (10 mg/kg) groups. Rats were treated with Pro at 1 h after rI/R treatment. Serum and lung tissues at 24 h after rI/R were collected to evaluate morphological changes and the expression of myeloperoxidase (MPO), inflammatory cytokines, and crucial proteins in the MAPK pathway. Pro attenuated the production of mediators, resulting in reduced levels of autophagy and apoptosis by restricting the MAPK pathway in rI/R-induced ALI model. Pro represses rI/R-induced pulmonary autophagy and apoptosis by decreasing the production of inflammatory molecules, and the effects of Pro are involved in the inhibition of the MAPK pathway.
Ketamine ameliorates hypoxia-induced endothelial injury in human umbilical vein endothelial cells
2020, Clinics
Citation Excerpt :
Ischemia-induced endothelial injury that occurs after hypoxia results in the increased expression of cell adhesion molecules, such as ICAM-1 and E-selectin, which interact with circulating neutrophils, inducing them to migrate to the sites of endothelium, to further aggravate endothelial cell inflammation and damage (21). Moreover, ROS also play a vital role in hypoxia-mediated endothelial inflammation (22). An increase in ROS accumulation under hypoxic conditions can lead to endothelial cell apoptosis and necrosis (6).
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.
The protective effects of ketamine against hypoxia-induced injury was assessed using cell viability and adhesion assays, quantitative polymerase chain reaction, and western blotting.
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.
Ketamine reduces hypoxia-induced p66Shc expression and attenuates ROS accumulation via upregulating sirt1 in HUVECs, thus attenuating hypoxia-induced endothelial cell inflammation and apoptosis.
Regulation of the phosphoprotein phosphatase 2A system and its modulation during oxidative stress: A potential therapeutic target?
2019, Pharmacology and Therapeutics
Citation Excerpt :
Several groups have also shown hypoxia to upregulate PP2A. In human colorectal cancer cells, rat primary alveolar epithelial cells and mouse heart, hypoxia increases PP2A activity (Caraballo et al., 2011; Larsen et al., 2008; Lin, Lee, et al., 2012), PP2A mRNA and protein abundance (Chen et al., 2014; Lu et al., 2017; Zhu et al., 2015). While hyperoxia (95% O2, 1-24 h) decreases PPA activity, independently of altering PP2A abundance in macrophages (Nyunoya, Monick, Powers, Yarovinsky, & Hunninghake, 2005).
Phosphoprotein phosphatases are of growing interest in the pathophysiology of many diseases and are often the neglected partner of protein kinases. One family member, PP2A, accounts for dephosphorylation of ~55-70% of all serine/threonine phosphosites. Interestingly, dysregulation of kinase signalling is a hallmark of many diseases in which an increase in oxidative stress is also noted. With this in mind, we assess the evidence to support oxidative stress-mediated regulation of the PP2A system
In this article, we first present an overview of the PP2A system before providing an analysis of the regulation of PP2A by endogenous inhibitors, post translational modification, and miRNA. Next, a detailed critique of data implicating reactive oxygen species, ischaemia, ischaemia-reperfusion, and hypoxia in regulating the PP2A holoenzyme and associated regulators is presented. Finally, the pharmacological targeting of PP2A, its endogenous inhibitors, and enzymes responsible for its post-translational modification are covered.
There is extensive evidence that oxidative stress modulates multiple components of the PP2A system, however, most of the data pertains to the catalytic subunit of PP2A. Irrespective of the underlying aetiology, free radical-mediated attenuation of PP2A activity is an emerging theme. However, in many instances, a dichotomy exists, which requires clarification and mechanistic insight. Nevertheless, this raises the possibility that pharmacological activation of PP2A, either through small molecule activators of PP2A or CIP2A/SET antagonists may be beneficial in modulating the cellular response to oxidative stress. A better understanding of which, will have wide ranging implications for cancer, heart disease and inflammatory conditions.

View all citing articles on Scopus

¹: Equal contributors.

View full text