Vascular Pharmacology 61 (2014) 72–79
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Propofol protects human umbilical vein endothelial cells from cisplatin-induced injury Minmin Zhu 1, Jiawei Chen 1, Hua Yin, Hui Jiang, Meilin Wen, Changhong Miao ⁎ Department of Anaesthesiology, Fudan University Shanghai Cancer Center, Shanghai, People's Republic of China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China
a r t i c l e
i n f o
Article history: Received 13 November 2013 Received in revised form 24 March 2014 Accepted 2 April 2014 Available online 13 April 2014 Keywords: Cisplatin Propofol eNOS ICAM-1 Endothelium
a b s t r a c t The anticancer drug cisplatin can up-regulate endothelial adhesion molecule expression, and trigger vascular endothelial injury. Propofol, an intravenous anesthetic, can inhibit endothelial adhesion molecule expression in some situations. Here, we explored whether and how propofol improved cisplatin-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells. Compared with control group, cisplatin reduced endothelial nitric oxide synthase dimer/monomer ratio, activated protein kinase C and enhanced endothelial nitric oxide synthase-Thr495 phosphorylation, decreased nitric oxide production, augmented intercellular adhesion molecule 1 expression and monocyte-endothelial adhesion. These cisplatin-mediated effects were attenuated by propofol treatment. Nω-Nitro-L-arginine methyl ester hydrochloride, a nitric oxide synthase inhibitor, inhibited the effect of propofol on cisplatin-induced intercellular adhesion molecule 1 expression. Propofol improved cisplatin-mediated tetrahydrobiopterin reduction and nitrotyrosine overexpression. Compared with control group, cisplatin and PMA, a protein kinase C activator, both increased endothelial nitric oxide synthaseThr495 phosphorylation, while propofol and GFX, a protein kinase C inhibitor, both decreased cisplatin-induced endothelial nitric oxide synthase-Thr495 phosphorylation. Our data indicated that propofol, via reducing cisplatin-induced endothelial nitric oxide synthase uncoupling and endothelial nitric oxide synthase-Thr495 phosphorylation, restored nitric oxide production, intercellular adhesion molecule 1 expression and monocyteendothelial interaction. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Cisplatin is a widely used chemotherapy agent in anti-cancer treatment with significant benefits in many patients. However, some safety concerns have been reported, such as nephrotoxicity [1–3], ototoxicity [4–6], neurotoxicity [7–10], and increased incidence of thrombosis [11]. These concerns have limited it from benefiting more patients in clinical practice. Previous studies have demonstrated that part of the cisplatin-mediated side effects may be attributed to potential vascular endothelium injury [12–16]. Consistent with this, recent studies reported that cisplatin-based chemotherapy was associated with higher incidence of atherosclerotic disease, coronary artery disease, and myocardial infarction [17]. The underlying mechanisms may be explained by cisplatin-induced up-regulation of adhesion molecules and leukocyteendothelial adhesion [18], which in turn triggers endothelial dysfunction and ischemic injury of tissues and organs.
⁎ Corresponding author at: Department of Anesthesiology, Fudan University Shanghai Cancer Center, No270 DongAn Road, Shanghai, 200032, People's Republic of China. Tel.: +86 21 64175590; fax: +86 21 6417 4774. E-mail address: [email protected] (C. Miao). 1 Equal contributors.
http://dx.doi.org/10.1016/j.vph.2014.04.001 1537-1891/© 2014 Elsevier Inc. All rights reserved.
Nitric oxide (NO) is an important signaling chemokine in vascular homeostasis. In vascular endothelium, NO is mainly produced by endothelial NO synthase (eNOS), and is a potent vasodilator [19] with the property of anti-platelet [20], anti-leukocyte adhesion [21], anti-inflammation, and cyto-protection [22]. Disruption of NO balance is a key feature of endothelial dysfunction in various vascular diseases. Large amount of evidence obtained from animal and human studies indicated that imbalance of synthesis and bioavailability of NO is involved in many cardiovascular diseases [23]. Previous studies have indicated that the reduction of NO production is associated with severe endothelial injury in in vitro and in animal studies [24–26]. Further, such endothelial injury could be improved by the restoration of NO production [24,26]. Nevertheless, the molecular mechanism is far from clear. It was reported that impaired NO production up-regulates expression of adhesion molecules and enhances the interaction between leukocytes and endothelium [27,28], thus increasing the incidence of endothelial injury [29]. A previous study has demonstrated that cisplatin induced endothelial dysfunction by attenuating NO production in human umbilical vein endothelial cells (HUVECs) [30]. Interventions which target monocyte-endothelial interaction could attenuate endothelial injury [31]. Recently, more and more patients who suffer from cancers are receiving cisplatin-based chemotherapy combined with surgical resection.
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Cisplatin-induced endothelial injury may be exaggerated perioperatively. These patients usually need general anesthesia. Propofol (2,6diisopropylphenol) is a widely used intravenous anesthetic agent with a satisfactory safety profile. Previous studies indicated that propofol could protect vascular endothelial injury via its effects of antiendothelial dysfunction [32] and anti-inflammation [33] in many situations. In this study, we therefore investigated whether propofol could protect vascular endothelium, which was exposed to cisplatin. If so, we could alleviate these cisplatin-induced noxious effects by choosing appropriate agents. Further, we investigated the underlying mechanisms. 2. Materials and methods 2.1. Cell culture HUVECs (Clonetics; Lonza, Basel, Switzerland) were cultured in DMEM with 5 mM glucose and 10% fetal bovine serum in incubator containing 5% CO2 at 37 °C. Cells were sub-cultured when reaching 90% confluence. The fourth passage of HUVECs was used in the present study. 2.2. Study design HUVECs were treated with different concentrations of cisplatin (Sigma, St. Louis, MO) (0.1, 0.5, 1.0, and 5.0 μg/ml) for different time courses (6, 12, 24 and 36 h). By measuring monocyte-endothelial adhesion, we determined the appropriate cisplatin treatment condition with maximal effect on monocyte-endothelial adhesion. During general anesthesia, plasma concentrations of propofol (Sigma, St. Louis, MO) range from 5 to 50 μM [34]. To mimic in vivo situation, after cisplatin treatment, HUVECs were incubated with different concentrations (5, 10, 20, and 40 μM) of propofol for 2 h. The optimal concentration of propofol with significant inhibitory effects on monocyte-endothelial adhesion was determined. These treatment conditions were used in the following studies in which HUVECs were cultured and divided into four groups to examine the underlying signaling pathways. Group 1: HUVECs were cultured in DMEM as control; Group 2: HUVECs were treated with 20 μM propofol for 2 h; Group 3: HUVECs were treated with 1 μg/ml cisplatin for 24 h; Group 4: HUVECs were treated with 1 μg/ml cisplatin for 22 h and co-incubated with 20 μM propofol for the last 2 h.
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Cruz, CA), and polyclonal antibody against intercellular adhesion molecule 1 (ICAM-1) (Cell Signaling Technology, Danvers, MA), eNOS (Santa Cruz Biotechnology, Santa Cruz, CA), p-eNOS-Thr495 (Santa Cruz Biotechnology, Santa Cruz, CA), p-eNOS-Ser1177 (Cell Signaling Technology, Danvers, MA), and nitrotyrosine (Abcam, Cambridge, UK). Thereafter, the primary antibodies were washed away, and the membranes were incubated with secondary antibodies for 1 h at room temperature. Subsequently, the membranes were washed with TBST for three times, and detected by the ECL system. The respective densities of the protein bands were analyzed by Scan-gel-it software. Peroxynitrite reacts with protein tyrosine residues to form nitrotyrosine, so this can be used as a marker of peroxynitrite production. In the present study, β-actin was used as loading control. The data were expressed as the ratio of specific protein expression and beta-actin expression.
2.5. NO production assay NO production was determined by a nitrate reductase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing City, P. R. China) according to the manufacturer's instructions. Briefly, cell culture medium, reagent 1 and reagent 2 were mixed and kept at 37 °C for 60 min. Then, reagent 3 and reagent 4 were added, mixed and kept at room temperature for 40 min. The supernatant was harvested after the samples were centrifuged at 3500 rpm for 10 min. Then, chromogenic agent was added and results were detected at 550 nM spectrophotometrically. The data were expressed as the percentage of the control group.
2.6. Total biopterin and tetrahydrobiopterin (BH4) level determination Biopterin levels were detected as described previously [32]. In brief, HUVECs were lysed in pre-cold extract buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM DTT). After protein concentration was determined, a 1:1 mixture of 1.5 M HClO4 and 2 M H3PO4 was used to remove protein. Total biopterin (BH4, dihydrobiopterin, biopterin) was measured after acid oxidation, while dihydrobiopterin and biopterin were measured after base oxidation. Biopterin was measured by liquid chromatographymass spectrometry. The BH4 level was counted by subtracting dihydrobiopterin and biopterin from total biopterin. The results were shown as the percentage of the control group.
2.3. Isolation and adhesion of monocytes to HUVECs
2.7. Protein kinase C (PKC) activity assay
Human monocyte isolation was accomplished with the use of Histopaque-1077 (Sigma, St. Louis, MO) according to the manufacturer's instructions as described previously [33]. Briefly, 8 ml heparinized blood from volunteers was layered onto 8 ml Histopaque1077. The monocytes were harvested after the samples were centrifuged at 400 g for 30 min. Isolated monocytes were washed with PBS, and re-suspended in the DMEM, and then added to the HUVECs. After being cultured at 37 °C for 30 min, cells were washed with PBS, and observed under a phase-contrast microscope. Adherent cells were counted in 10 different fields from five separate culture dishes. The data were expressed as folds increased compared with control group. The present study was performed in accordance with the declaration of Helsinki.
PKC activity was measured with SignaTECT PKC assay system (Promega, Madison, WI) according to the manufacturer's instructions as described previously [33]. Briefly, membrane extracts, reaction buffer and [γ-32P] ATP were mixed and kept at 30 °C for 10 min. PKC phosphorylation was measured by detecting the radioactivity. PKC activity was determined by subtracting the enzymatic activity without phospholipids from the enzymatic activity with phospholipids. The data were expressed as fold increase compared with control group.
2.4. Western blot analysis Equal amount (60 μg) of protein extracted from different groups of HUVECs was separated by 6% SDS-PAGE and transferred to nitrocellulose membranes. To study eNOS dimer/monomer expression, lowtemperature SDS-PAGE was performed as described previously [24]. After being blocked in 5% skim milk, the membranes were incubated with an antibody at 4 °C for overnight. The primary antibodies were monoclonal antibody against β-actin (Santa Cruz Biotechnology, Santa
2.8. Superoxide anion (O− 2 ) accumulation assay O− 2 accumulation was measured by the reduction of ferricytochrome c assay as described previously [32]. Briefly, cells were washed and cultured with Krebs-HEPES buffer containing 20 μM ferricytochrome c (Sigma, St. Louis, MO) in the presence or in the absence of superoxide dismutase (Sigma, St. Louis, MO). The absorbance was read at 550 nm spectrophotometrically. Reduction of ferricytochrome c in the presence of superoxide dismutase was subtracted from the data in the absence of superoxide dismutase. Arbitrary unit was used as the unit for absorbance difference. Then we counted the cell number, and expressed the data as arbitrary unit/106 cells.
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2.9. Statistical analysis Results were expressed as mean ± SD, and n represents the times of repeated experiments using different cell cultures. Statistical significance was determined in multiple comparisons among independent groups of results in which analysis of variance indicated the presence of significant difference. Bonferroni post hoc test was followed when appropriate. A value of P b 0.05 was considered significant.
3. Results 3.1. Cisplatin-induced monocyte-endothelial interaction, ICAM-1 expression and their modulation by propofol In HUVECs, cisplatin induced a marked augmentation of monocyteendothelial adhesion in both concentration- and time-dependent manner. Incubation of cells with 1 μg/ml cisplatin for 24 h caused a
significant augmentation of monocyte-endothelial adhesion (Fig. 1A, B). This treatment condition was used in the following experiments. Next, we found that treatment of cells with propofol for 2 h could attenuate cisplatin-induced monocyte-endothelial adhesion in a concentration-dependent manner. Compared with cisplatin treatment, incubation of cells with 20 μM propofol significantly attenuated cisplatin-induced monocyte-endothelial adhesion (P b 0.05 vs. cisplatin treatment; Fig. 1C). The propofol solvent dimethyl sulfoxide did not affect cisplatin-induced monocyte-endothelial adhesion. In addition, 20 μM propofol had no effect on basal monocyteendothelial adhesion in HUVECs (Fig. 1C). Thereafter, 20 μM propofol was used in the following experiments to investigate the potential signaling pathways responsible for the protective effects of propofol. Consistently, incubation of cells with 1 μg/ml cisplatin for 24 h caused a significant up-regulation of ICAM-1 expression (P b 0.05 vs. control; Fig. 1D, E), which was attenuated by 20 μM propofol treatment (P b 0.05 vs. cisplatin treatment; Fig. 1D, E).
Fig. 1. Cisplatin-induced monocyte-endothelial interaction, ICAM-1 expression and its modulation by propofol. (A) HUVECs were cultured in different concentrations of cisplatin (0, 0.1, 0.5, 1, 5 μg/ml) for 24 h. (B) HUVECs were cultured in 1 μg/ml cisplatin for different times (0, 6, 12, 24, 36 h). (C) HUVECs were treated with 1 μg/ml cisplatin for 22 h and co-incubated with different concentrations of propofol (10, 20, 40 μM) for 2 h. Compared with control group, cisplatin augmented monocyte-endothelial adhesion in both concentration- (A) and timedependent manner (B), and monocyte-endothelial adhesion could be attenuated by propofol in a concentration-dependent manner (C). (D) HUVECs were cultured in either 0 μg/ml or 1 μg/ml cisplatin for 24 h with corresponding treatment. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with antibodies to ICAM-1. (E) The protein expression ratio of ICAM-1 and β-actin. The ratio in the control group was set as 1. (*P b 0.05 vs. control, #P b 0.05 VS. cisplatin treatment, n = 5. Data are shown as mean ± SD.)
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3.2. The effects of cisplatin, propofol and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) on nitrite production and ICAM-1 expression. Compared with control group, cisplatin caused a marked reduction of nitrite production (P b 0.05 vs. control; Fig. 2A), which could be reversed by propofol treatment (P b 0.05 vs. cisplatin treatment; Fig. 2A). The propofol solvent dimethyl sulfoxide did not affect cisplatin-induced nitrite reduction. Compared with control group, nitrite production could be inhibited by L-NAME (100 μM), an NOS inhibitor (P b 0.05 vs. control; Fig. 2A). Compared with control group, cisplatin up-regulated ICAM-1 expression (P b 0.05 vs. control; Fig. 2 B, C), which could be inhibited by propofol treatment (P b 0.05 vs. cisplatin treatment; Fig. 2B, C). More importantly, L-NAME could inhibit the effect of propofol on cisplatininduced ICAM-1 expression (P b 0.05 vs. control; Fig. 2B, C). Moreover, L-NAME alone could increase ICAM-1 expression in HUVECs compared with control group (P b 0.05 vs. control; Fig. 2B, C). 3.3. Cisplatin-induced eNOS expression and its modulation by propofol Compared with control group, cisplatin reduced the ratio of eNOS dimer/monomer (P b 0.05 vs. control; Fig. 3A, B) and enhanced eNOSThr495 phosphorylation (P b 0.05 vs. control; Fig. 3A, C). Both effects were reversed by propofol (P b 0.05 both for eNOS dimer/monomer and eNOS-Thr495 phosphorylation vs. cisplatin treatment; Fig. 3A, B, C). Please note cisplatin and propofol had no effect on total eNOS expression and eNOS-Ser1177 phosphorylation (Fig. 3A, C). 3.4. The effect of cisplatin on total biopterin, BH4 and nitrotyrosine level and their modulation by propofol Furthermore, we determined the total biopterin and BH4 levels in response to cisplatin and propofol treatment. Compared with control
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group, cisplatin decreased total biopterin (P b 0.05 vs. control; Fig. 4A), BH4 levels (P b 0.05 vs. control; Fig. 4A), and the ratio of BH4/ total biopterin (P b 0.05 vs. control; Fig. 4B), which could all be reversed by propofol (P b 0.05 for total biopterin, BH4 levels and BH4/total biopterin ratio vs. cisplatin treatment; Fig. 4A, B). Compared with control group, cisplatin treatment increased nitrotyrosine expression (P b 0.05 vs. control; Fig. 4C), which was both attenuated by propofol and L-NAME (P b 0.05 vs. cisplatin treatment; Fig. 4C). 3.5. The effect of cisplatin on PKC activity and eNOS-Thr495 phosphorylation and its modulation by propofol and PKC inhibitor Compared with control group, cisplatin induced a marked PKC activation (P b 0.05 vs. control), which was attenuated by propofol treatment (P b 0.05 vs. cisplatin treatment; Fig. 5A). Compared with control group, cisplatin and PMA (300 nM), a PKC activator, both increased eNOS-Thr495 phosphorylation (P b 0.05 vs. control; Fig. 5 B, C), while propofol and GFX (10 μM), a PKC inhibitor, both decreased cisplatin-induced eNOS-Thr495 phosphorylation (P b 0.05 vs. cisplatin treatment; Fig. 5 B, C). Compared with control group, cisplatin induced a significant O− 2 accumulation (P b 0.05 vs. control group; Fig. 5D), which was attenuated by propofol and L-NAME (P b 0.05 vs. cisplatin treatment; Fig. 5D). 4. Discussion The main findings of the present study are that propofol could protect HUVECs against cisplatin-induced vascular endothelial injury by inhibiting ICAM-1 expression and monocyte-endothelial interaction. Our data also suggested that the protective effects of propofol might be achieved via improving NO-eNOS pathways in HUVECs.
Fig. 2. The effect of L-NAME and propofol on cisplatin-induced ICAM-1 expression. (A) Cisplatin reduced nitrite production in HUVECs, which could be improved by propofol. Compared with control group, L-NAME treatment decreased nitrite production. (B) HUVECs were cultured in either 0 μg/ml or 1 μg/ml cisplatin for 24 h with corresponding treatment. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with antibodies to ICAM-1. (C) The protein expression ratio of ICAM-1 and β-actin. The ratio in the control group was set as 1. (*P b 0.05 vs. control, #P b 0.05 VS. cisplatin treatment, n = 5. Data are shown as mean ± SD.)
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Fig. 3. Cisplatin-induced eNOS expression and its modulation by propofol. (A) HUVECs were cultured in either 0 μg/ml or 1 μg/ml cisplatin for 24 h with corresponding treatment. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with antibodies to eNOS, p-eNOS-Ser1177 and p-eNOS-Thr495. (B) Quantification of eNOS band density: ratio of dimer/monomer. (C) The protein expression ratio of eNOS or p-eNOS and β-actin. The ratio in the control group was set as 1. (*P b 0.05 vs. control, #P b 0.05 vs. cisplatin treatment, n = 5. Data are shown as mean ± SD.)
Although cisplatin has been an important component in anti-cancer chemotherapy for decades, its side effects have limited it from benefiting more patients. Cisplatin-mediated side effects have been suggested to be associated with potential vascular endothelial injury [12–16]. Consistently, researchers indicated that cisplatin-based regimens are associated with vascular injury and complications [35–37]. However, the mechanism is still not well studied. It was reported that cisplatin can up-regulate the expression of adhesion molecules in endothelial cells and augment leukocyte-endothelial adhesion [19], which may in turn trigger endothelial injury [29]. Nitric oxide (NO) is an important signaling chemokine in vascular homeostasis. Previous studies indicated that impaired release of NO from vascular endothelium increased endothelial adhesion molecules expression and leukocyte-endothelium interaction [27,28]. In our study we found cisplatin could decrease NO production and increase ICAM-1 expression and monocyte-endothelial adhesion (Figs. 1, 2). We also examined other adhesion molecules, including vascular cell adhesion molecule 1 and endothelial selectin. They were not significantly elevated after cisplatin treatment (data not shown), which was consistent with a previous study [19]. In HUVECs, the main source of NO is eNOS. At the cellular level, eNOS activity is regulated by total eNOS expression [38], the ratio of eNOS dimer/monomer [39], and eNOS phosphorylation [40]. In the present study, we found cisplatin decreased the ratio of eNOS dimer/monomer and increased eNOSThr495 phosphorylation (Fig. 3), which results in NO reduction. Under physiological situations, eNOS produces NO, whereas in some pathophysiological situations, eNOS produces O− 2 rather than NO [41,42]. This phenomenon is known as eNOS “uncoupling” [43], during which eNOS switched from a protective enzyme into a source of oxidative stress [44]. BH4, a cofactor for eNOS, is essential for the dimerization of
eNOS [45,46], while peroxynitrite could reduce BH4 levels by oxidation [47]. A previous study reported that cisplatin increased nitrotyrosine expression in the renal medulla of rats [48]. Similarly, we found cisplatin increased nitrotyrosine expression in HUVECS in the present study. As − a result, eNOS becomes uncoupled and generates O− 2 . Redundant O2 would react with NO to yield more peroxynitrite, thus forming a vicious circle. eNOS is also regulated by eNOS phosphorylation. Previous studies have shown that eNOS-Ser1177 phosphorylation activates eNOS [49–51] whereas eNOS-Thr495 and eNOS-Ser114 phosphorylation inhibits its activity [49]. In our study, we showed cisplatin had no effect on eNOSSer1177 phosphorylation (Fig. 3), which is similar to a previous study [30]. We also found cisplatin had no effect on eNOS-Ser114 phosphorylation (data not shown). However, we discovered that cisplatin induced eNOS-Thr495 phosphorylation which may also be involved in NO reduction. Moreover, we found cisplatin could enhance PKC activity. We also reported that PMA, a PKC activator, could increase eNOS-Thr495 phosphorylation (Fig. 5), which is similar to the effect of cisplatin. So we propose that cisplatin increased eNOS-Thr495 phosphorylation via activating PKC. PKC activation was associated with vascular reactive oxygen species accumulation [52] and NO reduction [53]. In HUVECs, eNOS uncoupling is a main source for oxygen species accumulation and NO reduction in some pathophysiological situations [32], so we believe that eNOS uncoupling is an important mechanism responsible for cisplatin-induced PKC activation. Although iNOS has been proposed to be a main resource of NO production in HUVECs, in the present study, iNOS expression and the ratio of dimer/monomer were not altered by cisplatin and propofol treatment (data not shown). We therefore believe that iNOS is not or only minimally involved in the cisplatininduced endothelial injury. Besides eNOS, other mediators such as NAPDH oxidase [54], xanthine oxidase [55] and mitochondria [56]
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Fig. 4. The effect of cisplatin on total biopterin, BH4 and nitrotyrosine level and its modulation by propofol. (A) Cisplatin decreased total biopterin and BH4 levels, which could be reversed by propofol. (B) Cisplatin decreased the ratio of BH4/total biopterin, which could be improved by propofol. (C) HUVECs were cultured in either 0 μg/ml or 1 μg/ml cisplatin for 24 h with corresponding treatment. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with antibodies to nitrotyrosine. (D) The protein expression ratio of nitrotyrosine and β-actin. The ratio in the control group was set as 1. (*P b 0.05 vs. control, #P b 0.05 vs. cisplatin treatment, n = 5. Data are shown as mean ± SD.)
may also play important roles in cisplatin-induced O− 2 generation. In the present study, we mainly focused on the eNOS pathway, because it is mainly expressed in vascular endothelial cells, and its function alters the balance between NO production and O− 2 accumulation. It is true that we cannot rule out the possibility that NAPDH oxidase, xanthine oxidase or mitochondria may also be involved in endothelial injury. This needs to be examined in future studies. Propofol is a widely used intravenous anesthetic and its properties have been well studied in the last decades. Propofol has been reported to activate eNOS and increase NO production by reducing eNOS uncoupling via peroxynitrite elimination in HUVECs [32]. Propofol could eliminate peroxynitrite by two mechanisms: (i) eliminating peroxynitrite directly [57] and (ii) eliminating peroxynitrite indirectly via scavenging O− 2 [58]. Peroxynitrite could oxidatively reduce BH4 levels [47] and result in eNOS uncoupling, thus forming a positive feedback. In the present study, we found propofol, via reducing peroxynitrite generation (Fig. 4 C, D), increased BH4 levels (Fig. 4A, B), reduced cisplatin-induced eNOS uncoupling (Fig. 3, A and B) and restored NO production (Fig. 2 A) as well as O− 2 accumulation (Fig. 5). As a result, it inhibited the vicious circle. We therefore propose that propofol mediated-eNOS re-coupling is an important mechanism responsible for the protective effect of propofol against cisplatin-induced endothelial injury. Propofol has also been reported to activate eNOS and increase NO production by inducing eNOS phosphorylation [59]. The activity of eNOS can be regulated by changes in its phosphorylation at Ser1177, Ser114 and Thr495. Phosphorylation at Ser1177 and dephosphorylation at Thr495 and Ser114 of eNOS leads to enhanced activity of the enzyme, and thus augments NO production. In the present study, we did not detect altered phosphorylation of p-eNOS-Ser1177
and p-eNOS-Ser114 (data not shown) by cisplatin or propofol. However, we found propofol could decrease cisplatin-induced eNOS-Thr495 phosphorylation. We therefore believe that propofol mediated-decrease of p-eNOS-Thr495 is another important mechanism responsible for the protective effect of propofol against cisplatin-induced endothelial injury. Previously published work indicated that propofol concentrationdependently increases eNOS Ser1177 phosphorylation and NO production in HUVEC [59]. In that study, 50 uM propofol for 10 min caused a maximum effect on eNOS Ser1177 phosphorylation and NO production. In the present study, we used 20 uM propofol for 2 h. The discrimination could be explained by the different concentration and duration of propofol treatment. Furthermore, the present study indicated that propofol inhibited cisplatin-induced PKC activation (Fig. 5A), whereas propofol and GFX, a PKC inhibitor, decreased cisplatin-induced p-eNOSThr495 phosphorylation similarly (Fig. 5 B, C). These findings strongly suggest that propofol decreased p-eNOS-Thr495 phosphorylation via inhibiting PKC activity. PKC can be activated by vascular reactive oxygen species accumulation [52] and NO reduction [53]. eNOS uncoupling is an important cause for NO reduction and O− 2 accumulation in HUVECs [32]. Therefore, we speculate that the mechanism by which propofol inhibits PKC activity may mainly be the restoration of eNOS uncoupling. Propofol is chemically similar to endogenous antioxidant a-tocopherol (Vitamin E) and theoretically should demonstrate similar properties [60]. Indeed, previous studies have indicated that Vitamin E could increase NO production in HUVECs [61] and in hypercholesterolemic patients [62]. These studies strongly indicated that agents which are chemically similar to propofol and Vitamin E may protect cisplatininduced endothelial injury via the property of antioxidant and restoration of NO production.
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Fig. 5. The effect of cisplatin on PKC activity and p-eNOS-Thr495 phosphorylation and its modulation by propofol and PKC inhibitor. (A) Cisplatin induced PKC activation, which could be attenuated by propofol. (B) HUVECs were cultured in either 0 μg/ml or 1 μg/ml cisplatin for 24 h with corresponding treatment. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with antibodies to p-eNOS-Thr495. (C) The protein expression ratio of p-eNOS-Thr495 and β-actin. The ratio in the control group was set as 1. (D) Cisplatin induced O− 2 accumulation, which could be attenuated by propofol and L-NAME. Data were expressed as arbitrary unit/106 cells. (*P b 0.05 vs. control, #P b 0.05 vs. cisplatin treatment, n = 5. Data are shown as mean ± SD.)
This study has some limitations. It was performed in HUVECs, which is different from in vivo settings in studying drug effectiveness and toxicity. Since propofol increased NO production, and reduced oxidative stress as well as monocyte-endothelial interaction, we propose that propofol will inhibit cisplatin-induced vascular injury in vivo, but this needs to be verified in future studies. In summary, the present study suggested that cisplatin induced eNOS uncoupling by inducing peroxynitrite generation and reducing BH4 levels, and induced eNOS-Thr495 phosphorylation by activating PKC activity, leading to NO reduction and up-regulation of ICAM-1 expression. More importantly, our study demonstrated that propofol, via scavenging peroxynitrite and inhibiting PKC activity, reduced cisplatininduced eNOS uncoupling and eNOS-Thr495 phosphorylation, and restored ICAM-1 expression and endothelial function.
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Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph
Propofol protects human umbilical vein endothelial cells from cisplatin-induced injury Minmin Zhu 1, Jiawei Chen 1, Hua Yin, Hui Jiang, Meilin Wen, Changhong Miao ⁎ Department of Anaesthesiology, Fudan University Shanghai Cancer Center, Shanghai, People's Republic of China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China
a r t i c l e
i n f o
Article history: Received 13 November 2013 Received in revised form 24 March 2014 Accepted 2 April 2014 Available online 13 April 2014 Keywords: Cisplatin Propofol eNOS ICAM-1 Endothelium
a b s t r a c t The anticancer drug cisplatin can up-regulate endothelial adhesion molecule expression, and trigger vascular endothelial injury. Propofol, an intravenous anesthetic, can inhibit endothelial adhesion molecule expression in some situations. Here, we explored whether and how propofol improved cisplatin-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells. Compared with control group, cisplatin reduced endothelial nitric oxide synthase dimer/monomer ratio, activated protein kinase C and enhanced endothelial nitric oxide synthase-Thr495 phosphorylation, decreased nitric oxide production, augmented intercellular adhesion molecule 1 expression and monocyte-endothelial adhesion. These cisplatin-mediated effects were attenuated by propofol treatment. Nω-Nitro-L-arginine methyl ester hydrochloride, a nitric oxide synthase inhibitor, inhibited the effect of propofol on cisplatin-induced intercellular adhesion molecule 1 expression. Propofol improved cisplatin-mediated tetrahydrobiopterin reduction and nitrotyrosine overexpression. Compared with control group, cisplatin and PMA, a protein kinase C activator, both increased endothelial nitric oxide synthaseThr495 phosphorylation, while propofol and GFX, a protein kinase C inhibitor, both decreased cisplatin-induced endothelial nitric oxide synthase-Thr495 phosphorylation. Our data indicated that propofol, via reducing cisplatin-induced endothelial nitric oxide synthase uncoupling and endothelial nitric oxide synthase-Thr495 phosphorylation, restored nitric oxide production, intercellular adhesion molecule 1 expression and monocyteendothelial interaction. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Cisplatin is a widely used chemotherapy agent in anti-cancer treatment with significant benefits in many patients. However, some safety concerns have been reported, such as nephrotoxicity [1–3], ototoxicity [4–6], neurotoxicity [7–10], and increased incidence of thrombosis [11]. These concerns have limited it from benefiting more patients in clinical practice. Previous studies have demonstrated that part of the cisplatin-mediated side effects may be attributed to potential vascular endothelium injury [12–16]. Consistent with this, recent studies reported that cisplatin-based chemotherapy was associated with higher incidence of atherosclerotic disease, coronary artery disease, and myocardial infarction [17]. The underlying mechanisms may be explained by cisplatin-induced up-regulation of adhesion molecules and leukocyteendothelial adhesion [18], which in turn triggers endothelial dysfunction and ischemic injury of tissues and organs.
⁎ Corresponding author at: Department of Anesthesiology, Fudan University Shanghai Cancer Center, No270 DongAn Road, Shanghai, 200032, People's Republic of China. Tel.: +86 21 64175590; fax: +86 21 6417 4774. E-mail address: [email protected] (C. Miao). 1 Equal contributors.
http://dx.doi.org/10.1016/j.vph.2014.04.001 1537-1891/© 2014 Elsevier Inc. All rights reserved.
Nitric oxide (NO) is an important signaling chemokine in vascular homeostasis. In vascular endothelium, NO is mainly produced by endothelial NO synthase (eNOS), and is a potent vasodilator [19] with the property of anti-platelet [20], anti-leukocyte adhesion [21], anti-inflammation, and cyto-protection [22]. Disruption of NO balance is a key feature of endothelial dysfunction in various vascular diseases. Large amount of evidence obtained from animal and human studies indicated that imbalance of synthesis and bioavailability of NO is involved in many cardiovascular diseases [23]. Previous studies have indicated that the reduction of NO production is associated with severe endothelial injury in in vitro and in animal studies [24–26]. Further, such endothelial injury could be improved by the restoration of NO production [24,26]. Nevertheless, the molecular mechanism is far from clear. It was reported that impaired NO production up-regulates expression of adhesion molecules and enhances the interaction between leukocytes and endothelium [27,28], thus increasing the incidence of endothelial injury [29]. A previous study has demonstrated that cisplatin induced endothelial dysfunction by attenuating NO production in human umbilical vein endothelial cells (HUVECs) [30]. Interventions which target monocyte-endothelial interaction could attenuate endothelial injury [31]. Recently, more and more patients who suffer from cancers are receiving cisplatin-based chemotherapy combined with surgical resection.
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Cisplatin-induced endothelial injury may be exaggerated perioperatively. These patients usually need general anesthesia. Propofol (2,6diisopropylphenol) is a widely used intravenous anesthetic agent with a satisfactory safety profile. Previous studies indicated that propofol could protect vascular endothelial injury via its effects of antiendothelial dysfunction [32] and anti-inflammation [33] in many situations. In this study, we therefore investigated whether propofol could protect vascular endothelium, which was exposed to cisplatin. If so, we could alleviate these cisplatin-induced noxious effects by choosing appropriate agents. Further, we investigated the underlying mechanisms. 2. Materials and methods 2.1. Cell culture HUVECs (Clonetics; Lonza, Basel, Switzerland) were cultured in DMEM with 5 mM glucose and 10% fetal bovine serum in incubator containing 5% CO2 at 37 °C. Cells were sub-cultured when reaching 90% confluence. The fourth passage of HUVECs was used in the present study. 2.2. Study design HUVECs were treated with different concentrations of cisplatin (Sigma, St. Louis, MO) (0.1, 0.5, 1.0, and 5.0 μg/ml) for different time courses (6, 12, 24 and 36 h). By measuring monocyte-endothelial adhesion, we determined the appropriate cisplatin treatment condition with maximal effect on monocyte-endothelial adhesion. During general anesthesia, plasma concentrations of propofol (Sigma, St. Louis, MO) range from 5 to 50 μM [34]. To mimic in vivo situation, after cisplatin treatment, HUVECs were incubated with different concentrations (5, 10, 20, and 40 μM) of propofol for 2 h. The optimal concentration of propofol with significant inhibitory effects on monocyte-endothelial adhesion was determined. These treatment conditions were used in the following studies in which HUVECs were cultured and divided into four groups to examine the underlying signaling pathways. Group 1: HUVECs were cultured in DMEM as control; Group 2: HUVECs were treated with 20 μM propofol for 2 h; Group 3: HUVECs were treated with 1 μg/ml cisplatin for 24 h; Group 4: HUVECs were treated with 1 μg/ml cisplatin for 22 h and co-incubated with 20 μM propofol for the last 2 h.
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Cruz, CA), and polyclonal antibody against intercellular adhesion molecule 1 (ICAM-1) (Cell Signaling Technology, Danvers, MA), eNOS (Santa Cruz Biotechnology, Santa Cruz, CA), p-eNOS-Thr495 (Santa Cruz Biotechnology, Santa Cruz, CA), p-eNOS-Ser1177 (Cell Signaling Technology, Danvers, MA), and nitrotyrosine (Abcam, Cambridge, UK). Thereafter, the primary antibodies were washed away, and the membranes were incubated with secondary antibodies for 1 h at room temperature. Subsequently, the membranes were washed with TBST for three times, and detected by the ECL system. The respective densities of the protein bands were analyzed by Scan-gel-it software. Peroxynitrite reacts with protein tyrosine residues to form nitrotyrosine, so this can be used as a marker of peroxynitrite production. In the present study, β-actin was used as loading control. The data were expressed as the ratio of specific protein expression and beta-actin expression.
2.5. NO production assay NO production was determined by a nitrate reductase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing City, P. R. China) according to the manufacturer's instructions. Briefly, cell culture medium, reagent 1 and reagent 2 were mixed and kept at 37 °C for 60 min. Then, reagent 3 and reagent 4 were added, mixed and kept at room temperature for 40 min. The supernatant was harvested after the samples were centrifuged at 3500 rpm for 10 min. Then, chromogenic agent was added and results were detected at 550 nM spectrophotometrically. The data were expressed as the percentage of the control group.
2.6. Total biopterin and tetrahydrobiopterin (BH4) level determination Biopterin levels were detected as described previously [32]. In brief, HUVECs were lysed in pre-cold extract buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM DTT). After protein concentration was determined, a 1:1 mixture of 1.5 M HClO4 and 2 M H3PO4 was used to remove protein. Total biopterin (BH4, dihydrobiopterin, biopterin) was measured after acid oxidation, while dihydrobiopterin and biopterin were measured after base oxidation. Biopterin was measured by liquid chromatographymass spectrometry. The BH4 level was counted by subtracting dihydrobiopterin and biopterin from total biopterin. The results were shown as the percentage of the control group.
2.3. Isolation and adhesion of monocytes to HUVECs
2.7. Protein kinase C (PKC) activity assay
Human monocyte isolation was accomplished with the use of Histopaque-1077 (Sigma, St. Louis, MO) according to the manufacturer's instructions as described previously [33]. Briefly, 8 ml heparinized blood from volunteers was layered onto 8 ml Histopaque1077. The monocytes were harvested after the samples were centrifuged at 400 g for 30 min. Isolated monocytes were washed with PBS, and re-suspended in the DMEM, and then added to the HUVECs. After being cultured at 37 °C for 30 min, cells were washed with PBS, and observed under a phase-contrast microscope. Adherent cells were counted in 10 different fields from five separate culture dishes. The data were expressed as folds increased compared with control group. The present study was performed in accordance with the declaration of Helsinki.
PKC activity was measured with SignaTECT PKC assay system (Promega, Madison, WI) according to the manufacturer's instructions as described previously [33]. Briefly, membrane extracts, reaction buffer and [γ-32P] ATP were mixed and kept at 30 °C for 10 min. PKC phosphorylation was measured by detecting the radioactivity. PKC activity was determined by subtracting the enzymatic activity without phospholipids from the enzymatic activity with phospholipids. The data were expressed as fold increase compared with control group.
2.4. Western blot analysis Equal amount (60 μg) of protein extracted from different groups of HUVECs was separated by 6% SDS-PAGE and transferred to nitrocellulose membranes. To study eNOS dimer/monomer expression, lowtemperature SDS-PAGE was performed as described previously [24]. After being blocked in 5% skim milk, the membranes were incubated with an antibody at 4 °C for overnight. The primary antibodies were monoclonal antibody against β-actin (Santa Cruz Biotechnology, Santa
2.8. Superoxide anion (O− 2 ) accumulation assay O− 2 accumulation was measured by the reduction of ferricytochrome c assay as described previously [32]. Briefly, cells were washed and cultured with Krebs-HEPES buffer containing 20 μM ferricytochrome c (Sigma, St. Louis, MO) in the presence or in the absence of superoxide dismutase (Sigma, St. Louis, MO). The absorbance was read at 550 nm spectrophotometrically. Reduction of ferricytochrome c in the presence of superoxide dismutase was subtracted from the data in the absence of superoxide dismutase. Arbitrary unit was used as the unit for absorbance difference. Then we counted the cell number, and expressed the data as arbitrary unit/106 cells.
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2.9. Statistical analysis Results were expressed as mean ± SD, and n represents the times of repeated experiments using different cell cultures. Statistical significance was determined in multiple comparisons among independent groups of results in which analysis of variance indicated the presence of significant difference. Bonferroni post hoc test was followed when appropriate. A value of P b 0.05 was considered significant.
3. Results 3.1. Cisplatin-induced monocyte-endothelial interaction, ICAM-1 expression and their modulation by propofol In HUVECs, cisplatin induced a marked augmentation of monocyteendothelial adhesion in both concentration- and time-dependent manner. Incubation of cells with 1 μg/ml cisplatin for 24 h caused a
significant augmentation of monocyte-endothelial adhesion (Fig. 1A, B). This treatment condition was used in the following experiments. Next, we found that treatment of cells with propofol for 2 h could attenuate cisplatin-induced monocyte-endothelial adhesion in a concentration-dependent manner. Compared with cisplatin treatment, incubation of cells with 20 μM propofol significantly attenuated cisplatin-induced monocyte-endothelial adhesion (P b 0.05 vs. cisplatin treatment; Fig. 1C). The propofol solvent dimethyl sulfoxide did not affect cisplatin-induced monocyte-endothelial adhesion. In addition, 20 μM propofol had no effect on basal monocyteendothelial adhesion in HUVECs (Fig. 1C). Thereafter, 20 μM propofol was used in the following experiments to investigate the potential signaling pathways responsible for the protective effects of propofol. Consistently, incubation of cells with 1 μg/ml cisplatin for 24 h caused a significant up-regulation of ICAM-1 expression (P b 0.05 vs. control; Fig. 1D, E), which was attenuated by 20 μM propofol treatment (P b 0.05 vs. cisplatin treatment; Fig. 1D, E).
Fig. 1. Cisplatin-induced monocyte-endothelial interaction, ICAM-1 expression and its modulation by propofol. (A) HUVECs were cultured in different concentrations of cisplatin (0, 0.1, 0.5, 1, 5 μg/ml) for 24 h. (B) HUVECs were cultured in 1 μg/ml cisplatin for different times (0, 6, 12, 24, 36 h). (C) HUVECs were treated with 1 μg/ml cisplatin for 22 h and co-incubated with different concentrations of propofol (10, 20, 40 μM) for 2 h. Compared with control group, cisplatin augmented monocyte-endothelial adhesion in both concentration- (A) and timedependent manner (B), and monocyte-endothelial adhesion could be attenuated by propofol in a concentration-dependent manner (C). (D) HUVECs were cultured in either 0 μg/ml or 1 μg/ml cisplatin for 24 h with corresponding treatment. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with antibodies to ICAM-1. (E) The protein expression ratio of ICAM-1 and β-actin. The ratio in the control group was set as 1. (*P b 0.05 vs. control, #P b 0.05 VS. cisplatin treatment, n = 5. Data are shown as mean ± SD.)
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3.2. The effects of cisplatin, propofol and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) on nitrite production and ICAM-1 expression. Compared with control group, cisplatin caused a marked reduction of nitrite production (P b 0.05 vs. control; Fig. 2A), which could be reversed by propofol treatment (P b 0.05 vs. cisplatin treatment; Fig. 2A). The propofol solvent dimethyl sulfoxide did not affect cisplatin-induced nitrite reduction. Compared with control group, nitrite production could be inhibited by L-NAME (100 μM), an NOS inhibitor (P b 0.05 vs. control; Fig. 2A). Compared with control group, cisplatin up-regulated ICAM-1 expression (P b 0.05 vs. control; Fig. 2 B, C), which could be inhibited by propofol treatment (P b 0.05 vs. cisplatin treatment; Fig. 2B, C). More importantly, L-NAME could inhibit the effect of propofol on cisplatininduced ICAM-1 expression (P b 0.05 vs. control; Fig. 2B, C). Moreover, L-NAME alone could increase ICAM-1 expression in HUVECs compared with control group (P b 0.05 vs. control; Fig. 2B, C). 3.3. Cisplatin-induced eNOS expression and its modulation by propofol Compared with control group, cisplatin reduced the ratio of eNOS dimer/monomer (P b 0.05 vs. control; Fig. 3A, B) and enhanced eNOSThr495 phosphorylation (P b 0.05 vs. control; Fig. 3A, C). Both effects were reversed by propofol (P b 0.05 both for eNOS dimer/monomer and eNOS-Thr495 phosphorylation vs. cisplatin treatment; Fig. 3A, B, C). Please note cisplatin and propofol had no effect on total eNOS expression and eNOS-Ser1177 phosphorylation (Fig. 3A, C). 3.4. The effect of cisplatin on total biopterin, BH4 and nitrotyrosine level and their modulation by propofol Furthermore, we determined the total biopterin and BH4 levels in response to cisplatin and propofol treatment. Compared with control
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group, cisplatin decreased total biopterin (P b 0.05 vs. control; Fig. 4A), BH4 levels (P b 0.05 vs. control; Fig. 4A), and the ratio of BH4/ total biopterin (P b 0.05 vs. control; Fig. 4B), which could all be reversed by propofol (P b 0.05 for total biopterin, BH4 levels and BH4/total biopterin ratio vs. cisplatin treatment; Fig. 4A, B). Compared with control group, cisplatin treatment increased nitrotyrosine expression (P b 0.05 vs. control; Fig. 4C), which was both attenuated by propofol and L-NAME (P b 0.05 vs. cisplatin treatment; Fig. 4C). 3.5. The effect of cisplatin on PKC activity and eNOS-Thr495 phosphorylation and its modulation by propofol and PKC inhibitor Compared with control group, cisplatin induced a marked PKC activation (P b 0.05 vs. control), which was attenuated by propofol treatment (P b 0.05 vs. cisplatin treatment; Fig. 5A). Compared with control group, cisplatin and PMA (300 nM), a PKC activator, both increased eNOS-Thr495 phosphorylation (P b 0.05 vs. control; Fig. 5 B, C), while propofol and GFX (10 μM), a PKC inhibitor, both decreased cisplatin-induced eNOS-Thr495 phosphorylation (P b 0.05 vs. cisplatin treatment; Fig. 5 B, C). Compared with control group, cisplatin induced a significant O− 2 accumulation (P b 0.05 vs. control group; Fig. 5D), which was attenuated by propofol and L-NAME (P b 0.05 vs. cisplatin treatment; Fig. 5D). 4. Discussion The main findings of the present study are that propofol could protect HUVECs against cisplatin-induced vascular endothelial injury by inhibiting ICAM-1 expression and monocyte-endothelial interaction. Our data also suggested that the protective effects of propofol might be achieved via improving NO-eNOS pathways in HUVECs.
Fig. 2. The effect of L-NAME and propofol on cisplatin-induced ICAM-1 expression. (A) Cisplatin reduced nitrite production in HUVECs, which could be improved by propofol. Compared with control group, L-NAME treatment decreased nitrite production. (B) HUVECs were cultured in either 0 μg/ml or 1 μg/ml cisplatin for 24 h with corresponding treatment. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with antibodies to ICAM-1. (C) The protein expression ratio of ICAM-1 and β-actin. The ratio in the control group was set as 1. (*P b 0.05 vs. control, #P b 0.05 VS. cisplatin treatment, n = 5. Data are shown as mean ± SD.)
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Fig. 3. Cisplatin-induced eNOS expression and its modulation by propofol. (A) HUVECs were cultured in either 0 μg/ml or 1 μg/ml cisplatin for 24 h with corresponding treatment. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with antibodies to eNOS, p-eNOS-Ser1177 and p-eNOS-Thr495. (B) Quantification of eNOS band density: ratio of dimer/monomer. (C) The protein expression ratio of eNOS or p-eNOS and β-actin. The ratio in the control group was set as 1. (*P b 0.05 vs. control, #P b 0.05 vs. cisplatin treatment, n = 5. Data are shown as mean ± SD.)
Although cisplatin has been an important component in anti-cancer chemotherapy for decades, its side effects have limited it from benefiting more patients. Cisplatin-mediated side effects have been suggested to be associated with potential vascular endothelial injury [12–16]. Consistently, researchers indicated that cisplatin-based regimens are associated with vascular injury and complications [35–37]. However, the mechanism is still not well studied. It was reported that cisplatin can up-regulate the expression of adhesion molecules in endothelial cells and augment leukocyte-endothelial adhesion [19], which may in turn trigger endothelial injury [29]. Nitric oxide (NO) is an important signaling chemokine in vascular homeostasis. Previous studies indicated that impaired release of NO from vascular endothelium increased endothelial adhesion molecules expression and leukocyte-endothelium interaction [27,28]. In our study we found cisplatin could decrease NO production and increase ICAM-1 expression and monocyte-endothelial adhesion (Figs. 1, 2). We also examined other adhesion molecules, including vascular cell adhesion molecule 1 and endothelial selectin. They were not significantly elevated after cisplatin treatment (data not shown), which was consistent with a previous study [19]. In HUVECs, the main source of NO is eNOS. At the cellular level, eNOS activity is regulated by total eNOS expression [38], the ratio of eNOS dimer/monomer [39], and eNOS phosphorylation [40]. In the present study, we found cisplatin decreased the ratio of eNOS dimer/monomer and increased eNOSThr495 phosphorylation (Fig. 3), which results in NO reduction. Under physiological situations, eNOS produces NO, whereas in some pathophysiological situations, eNOS produces O− 2 rather than NO [41,42]. This phenomenon is known as eNOS “uncoupling” [43], during which eNOS switched from a protective enzyme into a source of oxidative stress [44]. BH4, a cofactor for eNOS, is essential for the dimerization of
eNOS [45,46], while peroxynitrite could reduce BH4 levels by oxidation [47]. A previous study reported that cisplatin increased nitrotyrosine expression in the renal medulla of rats [48]. Similarly, we found cisplatin increased nitrotyrosine expression in HUVECS in the present study. As − a result, eNOS becomes uncoupled and generates O− 2 . Redundant O2 would react with NO to yield more peroxynitrite, thus forming a vicious circle. eNOS is also regulated by eNOS phosphorylation. Previous studies have shown that eNOS-Ser1177 phosphorylation activates eNOS [49–51] whereas eNOS-Thr495 and eNOS-Ser114 phosphorylation inhibits its activity [49]. In our study, we showed cisplatin had no effect on eNOSSer1177 phosphorylation (Fig. 3), which is similar to a previous study [30]. We also found cisplatin had no effect on eNOS-Ser114 phosphorylation (data not shown). However, we discovered that cisplatin induced eNOS-Thr495 phosphorylation which may also be involved in NO reduction. Moreover, we found cisplatin could enhance PKC activity. We also reported that PMA, a PKC activator, could increase eNOS-Thr495 phosphorylation (Fig. 5), which is similar to the effect of cisplatin. So we propose that cisplatin increased eNOS-Thr495 phosphorylation via activating PKC. PKC activation was associated with vascular reactive oxygen species accumulation [52] and NO reduction [53]. In HUVECs, eNOS uncoupling is a main source for oxygen species accumulation and NO reduction in some pathophysiological situations [32], so we believe that eNOS uncoupling is an important mechanism responsible for cisplatin-induced PKC activation. Although iNOS has been proposed to be a main resource of NO production in HUVECs, in the present study, iNOS expression and the ratio of dimer/monomer were not altered by cisplatin and propofol treatment (data not shown). We therefore believe that iNOS is not or only minimally involved in the cisplatininduced endothelial injury. Besides eNOS, other mediators such as NAPDH oxidase [54], xanthine oxidase [55] and mitochondria [56]
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Fig. 4. The effect of cisplatin on total biopterin, BH4 and nitrotyrosine level and its modulation by propofol. (A) Cisplatin decreased total biopterin and BH4 levels, which could be reversed by propofol. (B) Cisplatin decreased the ratio of BH4/total biopterin, which could be improved by propofol. (C) HUVECs were cultured in either 0 μg/ml or 1 μg/ml cisplatin for 24 h with corresponding treatment. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with antibodies to nitrotyrosine. (D) The protein expression ratio of nitrotyrosine and β-actin. The ratio in the control group was set as 1. (*P b 0.05 vs. control, #P b 0.05 vs. cisplatin treatment, n = 5. Data are shown as mean ± SD.)
may also play important roles in cisplatin-induced O− 2 generation. In the present study, we mainly focused on the eNOS pathway, because it is mainly expressed in vascular endothelial cells, and its function alters the balance between NO production and O− 2 accumulation. It is true that we cannot rule out the possibility that NAPDH oxidase, xanthine oxidase or mitochondria may also be involved in endothelial injury. This needs to be examined in future studies. Propofol is a widely used intravenous anesthetic and its properties have been well studied in the last decades. Propofol has been reported to activate eNOS and increase NO production by reducing eNOS uncoupling via peroxynitrite elimination in HUVECs [32]. Propofol could eliminate peroxynitrite by two mechanisms: (i) eliminating peroxynitrite directly [57] and (ii) eliminating peroxynitrite indirectly via scavenging O− 2 [58]. Peroxynitrite could oxidatively reduce BH4 levels [47] and result in eNOS uncoupling, thus forming a positive feedback. In the present study, we found propofol, via reducing peroxynitrite generation (Fig. 4 C, D), increased BH4 levels (Fig. 4A, B), reduced cisplatin-induced eNOS uncoupling (Fig. 3, A and B) and restored NO production (Fig. 2 A) as well as O− 2 accumulation (Fig. 5). As a result, it inhibited the vicious circle. We therefore propose that propofol mediated-eNOS re-coupling is an important mechanism responsible for the protective effect of propofol against cisplatin-induced endothelial injury. Propofol has also been reported to activate eNOS and increase NO production by inducing eNOS phosphorylation [59]. The activity of eNOS can be regulated by changes in its phosphorylation at Ser1177, Ser114 and Thr495. Phosphorylation at Ser1177 and dephosphorylation at Thr495 and Ser114 of eNOS leads to enhanced activity of the enzyme, and thus augments NO production. In the present study, we did not detect altered phosphorylation of p-eNOS-Ser1177
and p-eNOS-Ser114 (data not shown) by cisplatin or propofol. However, we found propofol could decrease cisplatin-induced eNOS-Thr495 phosphorylation. We therefore believe that propofol mediated-decrease of p-eNOS-Thr495 is another important mechanism responsible for the protective effect of propofol against cisplatin-induced endothelial injury. Previously published work indicated that propofol concentrationdependently increases eNOS Ser1177 phosphorylation and NO production in HUVEC [59]. In that study, 50 uM propofol for 10 min caused a maximum effect on eNOS Ser1177 phosphorylation and NO production. In the present study, we used 20 uM propofol for 2 h. The discrimination could be explained by the different concentration and duration of propofol treatment. Furthermore, the present study indicated that propofol inhibited cisplatin-induced PKC activation (Fig. 5A), whereas propofol and GFX, a PKC inhibitor, decreased cisplatin-induced p-eNOSThr495 phosphorylation similarly (Fig. 5 B, C). These findings strongly suggest that propofol decreased p-eNOS-Thr495 phosphorylation via inhibiting PKC activity. PKC can be activated by vascular reactive oxygen species accumulation [52] and NO reduction [53]. eNOS uncoupling is an important cause for NO reduction and O− 2 accumulation in HUVECs [32]. Therefore, we speculate that the mechanism by which propofol inhibits PKC activity may mainly be the restoration of eNOS uncoupling. Propofol is chemically similar to endogenous antioxidant a-tocopherol (Vitamin E) and theoretically should demonstrate similar properties [60]. Indeed, previous studies have indicated that Vitamin E could increase NO production in HUVECs [61] and in hypercholesterolemic patients [62]. These studies strongly indicated that agents which are chemically similar to propofol and Vitamin E may protect cisplatininduced endothelial injury via the property of antioxidant and restoration of NO production.
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Fig. 5. The effect of cisplatin on PKC activity and p-eNOS-Thr495 phosphorylation and its modulation by propofol and PKC inhibitor. (A) Cisplatin induced PKC activation, which could be attenuated by propofol. (B) HUVECs were cultured in either 0 μg/ml or 1 μg/ml cisplatin for 24 h with corresponding treatment. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with antibodies to p-eNOS-Thr495. (C) The protein expression ratio of p-eNOS-Thr495 and β-actin. The ratio in the control group was set as 1. (D) Cisplatin induced O− 2 accumulation, which could be attenuated by propofol and L-NAME. Data were expressed as arbitrary unit/106 cells. (*P b 0.05 vs. control, #P b 0.05 vs. cisplatin treatment, n = 5. Data are shown as mean ± SD.)
This study has some limitations. It was performed in HUVECs, which is different from in vivo settings in studying drug effectiveness and toxicity. Since propofol increased NO production, and reduced oxidative stress as well as monocyte-endothelial interaction, we propose that propofol will inhibit cisplatin-induced vascular injury in vivo, but this needs to be verified in future studies. In summary, the present study suggested that cisplatin induced eNOS uncoupling by inducing peroxynitrite generation and reducing BH4 levels, and induced eNOS-Thr495 phosphorylation by activating PKC activity, leading to NO reduction and up-regulation of ICAM-1 expression. More importantly, our study demonstrated that propofol, via scavenging peroxynitrite and inhibiting PKC activity, reduced cisplatininduced eNOS uncoupling and eNOS-Thr495 phosphorylation, and restored ICAM-1 expression and endothelial function.
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