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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:e302.)
© 2007 American Heart Association, Inc.

Vascular Biology

Leptin Induces C-Reactive Protein Expression in Vascular Endothelial Cells

Prachi Singh; Michal Hoffmann; Robert Wolk; Abu S.M. Shamsuzzaman; Virend K. Somers

From the Division of Cardiovascular Diseases, Department of Internal Medicine (P.S., M.H., R.W., A.S.M.S., V.K.S.), Mayo Clinic College of Medicine, Rochester, Minn; the Hypertension Unit (M.H.), Medical University of Gdansk, Poland; and Cardiovascular/Metabolic Diseases (R.W.), Pfizer Global Research & Development, Pfizer Inc, Groton, Conn.

Correspondence to Dr Virend K. Somers, MD, DPhil, Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, MN, 55905. E-mail somers.virend{at}mayo.edu

	Abstract

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Objective— There is increasing evidence of an association between leptin and increased cardiovascular risk. Higher leptin levels are associated with increased levels of C-reactive protein (CRP), which itself elicits proatherogenic effects in the vascular endothelium. We tested the hypothesis that leptin induces CRP expression in human coronary artery endothelial cells (HCAECs).

Methods and Results— We confirmed the presence of both long and short isoforms of the leptin receptor in cultured HCAECs. Leptin but not IFNA/D nor tumor necrosis factor (TNF) , induced expression of CRP. A dose dependent increase of CRP mRNA and protein was observed with increasing concentration of leptin (0 to 400 ng/mL). This increased CRP expression was attenuated in the presence of anti-leptin receptor antibodies and also by inhibition of ERK1/2 by PD98059 (20 to 40 µmol/L). Time (0 to 60 minutes) and leptin concentration (0 to 200 ng/mL)-dependence of ERK1/2 phosphorylation were evident in response to leptin treatment. Leptin also elicited ROS generation. Inhibition of ROS by catalase (200 µg/mL) prevented ERK1/2 phosphorylation and CRP mRNA transcription.

Conclusion— Leptin induces CRP expression in HCAECs via activation of the leptin receptor, increased ROS production, and phosphorylation of ERK1/2. These studies suggest a mechanism for the proatherogenic effects of leptin.

High leptin levels are associated with increased cardiovascular risk. In this study we provide evidence for leptin-dependent C-reactive protein (CRP) induction in vascular endothelial cells and investigated the signaling pathway involved. Thus, we demonstrate an additional molecular mechanism for the proatherogenic activity of leptin.

Key Words: leptin • endothelium • C-reactive protein • obesity • atherosclerosis

	Introduction

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Leptin, a protein encoded by the Ob gene, is produced mainly by adipocytes and is primarily involved in regulation of food intake and energy expenditure. Several studies have shown an independent interaction between high leptin and atherosclerosis,^1,2 myocardial infarction, stroke, and coronary artery intima-media thickness, suggesting that high levels of leptin imply increased cardiovascular risk.³ Detrimental effects of leptin may include sympathetic activation, pressor responses, insulin resistance, platelet activation and aggregation, inflammation, oxidative stress, and proliferation and migration of vascular smooth muscle cells.^3,4 The molecular mechanisms underlying the association of leptin with poor cardiovascular outcomes are not fully elucidated.

C-reactive protein (CRP), an acute phase protein, is also an indicator of cardiovascular (CV) risk.⁵ Localization of CRP in atherosclerotic lesions^6–8 and its role in complement activation, cell adhesion, and thrombosis make it likely an important mediator in the development and progression of the atherosclerotic lesion.⁹ We and others have shown positive correlations between CRP and plasma leptin in normal weight and obese subjects.^10,11 Exogenous administration of leptin increases plasma CRP in normal weight and fasting obese subjects.^12,13 Decreases in leptin during weight loss and fasting directly correlate with decreased CRP levels.¹⁴ Induction of CRP by leptin in vascular endothelial cells would have important implications for understanding interactions between leptin and CRP in promoting CV risk. We tested the hypothesis that leptin increases CRP expression in human coronary artery endothelial cells and sought to determine the signaling pathways involved.

	Methods

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Experiments were performed on human coronary artery endothelial cells (HCAECs, from Cambrex, Walkersville, MD). Cells were grown in endothelial growth media-2 (EGM-2) media supplemented with growth factors and 2% FBS. All experiments were performed at 3 to 5 passages with 70% to 80% confluence after overnight incubation in serum and growth factor–free media.¹⁵

The cells were incubated with either leptin (50 to 400 ng/mL; Sigma), recombinant interferon-A/D (IFN-A/D; 200 U/mL; Sigma), or tumor necrosis factor- (TNF-; 50 ng/mL; Promega) for 24 or 48 hours for RNA or protein analysis. Interleukin (IL)-1 (20 ng/mL) and IL-6 (20 ng/mL (R&D systems) were used as positive induction control and buffer used to make leptin stock was used as vehicle (0 leptin) control. The role of the leptin receptor in CRP induction was determined in the presence of human anti-leptin receptor antibodies (0.01 to 0.02 dilutions, Linco Research).

The effect of inhibiting activation of p38 and ERK1/2 pathway and ROS generation on CRP induction was determined in the presence of specific inhibitors such as SB203580 (5 to 20 µmol/L; Sigma), PD98059 (20 to 40 µmol/L; Sigma), and catalase (200 µg/mL; Sigma), respectively. All inhibitory molecules were incubated with the cells for 30 minutes before leptin (100 ng/mL) treatment. To study the signaling pathway, the cells were incubated with leptin (100 ng/mL) for varying durations (0 to 60 minutes). The effect of increasing leptin concentration (0 to 200 ng/mL; 20 minutes treatment) and ROS inhibition on activation of MAPK was also determined.

ROS generation in response to leptin treatment was determined using MitoSOX red mitochondrial superoxide indicator (Molecular Probes) for live cell imaging. The cells were washed with Hank’s balanced salt solution (HBSS, Mediatech Inc) and loaded with MitoSOX (2 µmol/L) for 20 minutes before leptin (100 ng/mL) treatment. The cells were then observed in a fluorescence microscope after 20 minutes of leptin treatment to determine the ROS levels. Catalase treatment (200 µg/mL) was administered 20 minutes before the leptin treatment when needed. ROS quantification was performed using fluorescence activated cell sorting (FACS) analysis. The cells were initially treated with versene-EDTA (Cambrex), washed with HBSS, and loaded with 2',7'-dichlorodihydrofluorescein (1 µmol/L, H₂DCFDA; Molecular Probes) for 10 minutes at 37°C. The cells were then treated with leptin (100 ng/mL) for 15 minutes and then analyzed by FACS.

RNA was extracted from the cells after 24 hours of leptin treatment using an RNA isolation kit (Invitrogen, Carlsbad, Calif). cDNA was synthesized using total RNA (1 µg/reaction) with high capacity cDNA Archive kit (Applied Biosystems, Foster City, Calif). CRP TaqMan probe and 18sRNA TaqMan probe (endogenous control; Applied Biosystems) were used in standard conditions (as recommended by the manufacturer) to determine the level of CRP transcription in the treated cells. Absolute values of CRP and 18sRNA transcripts were calculated using standards. The CRP value obtained from the standard curve was divided by the value for endogenous control to obtain a normalized target value ratio. Results are expressed as fold increases as compared with vehicle (0 leptin) control. To further verify our results we used an additional endogenous control, human TATA binding protein TaqMan probe (Hu-TBP TaqMan probe, Applied Biosystems).

Western blot analysis was done to quantify CRP and the proteins of the signaling pathway (phosphorylated ERK1/2, ERK1/2, phosphorylated p38, p38) in the various experimental cell lysates. The cells were lysed immediately after the experiment, and 20 to 50 µg of protein per well was loaded and transferred to membrane. The membrane was blocked with 5% milk and incubated with specific primary antibody (anti-human CRP, Calbiochem; anti-human β-actin, Sigma; pERK/extracellular signal regulated kinase [ERK] and pp38/p38 antibodies, Cell Signaling). After washing and incubating with appropriate HRP-conjugated secondary antibodies the membrane was developed with enhanced chemiluminescence (Amersham Biosciences). Western blot analysis was also done to show the presence of the leptin receptor (Ob-R) (Santa Cruz Biotech) in HCAECs.

All experiments were performed at least 3 times. The data are presented as mean±SD. Statistical significance was determined using ANOVA followed by unpaired Student t test. The level of significance was set at P<0.05.

	Results

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Incubation of HCAECs with leptin resulted in a significant increase in CRP mRNA (Figure 1). Ligands such as IFN- A/D and TNF- did not elicit any significant changes in CRP mRNA transcription levels. However, a nonsignificant increase in CRP mRNA was observed after incubation with IL-1β and IL-6, and a significant increase was observed when the cells were incubated with both IL-1β and IL-6 simultaneously.

View larger version (9K): [in this window] [in a new window]   Figure 1. Effect of different ligands on CRP mRNA transcription in HCAECs. The cells were incubated with ligands such as leptin (100 ng/mL), IL-1β (20 ng/mL), IL-6 (20 ng/mL), IFN-A/D (200 U/mL), and TNF- (50 ng/mL). Quantitative mRNA analysis was done after 24-hour treatment. A significant increase in CRP mRNA was observed with leptin treatment. Induction by cytokines (IL-1β + IL-6) was used as a positive control. All data are represented as mean±SD of 3 independent experiments, each in triplicate. *P<0.05, in comparison to control.

The presence of the leptin receptor (Ob-R) in HCAECs was confirmed by Western blot (Figure 2A). Both long and short isoforms of the receptor were observed. HCAECs incubated with increasing doses of leptin (0 to 400 ng/mL) showed increased expression of CRP mRNA (Figure 2B) and protein (Figure 2C). The increases in CRP mRNA were significant (P=0.005) and comparable to the cytokine-induced increase in CRP expression. We further verified our results using a Human TATA binding protein (Hu-TBP) TaqMan probe in addition to 18sRNA TaqMan probe as an endogenous control with similar findings (data not shown). Furthermore, in the presence of increasing concentrations of human anti–Ob-R antibodies, an inhibitory effect on leptin-induced CRP mRNA expression was observed (Figure 2D), indicating that the leptin-dependent CRP response was mediated via the leptin receptor.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of leptin administration in HCAECs. A, Western blot showing the presence of the leptin receptor. Both long (132 kDa) and short isoforms (102, 103 kDa) are seen. B, Quantitative mRNA analysis showing increases in CRP mRNA in response to treatment with increasing leptin concentrations ranging from 50 to 400 ng/mL. C, Western blot showing increase in CRP protein and graph showing CRP: β-Actin density ratio in response to treatment with increasing leptin concentrations. D, Inhibition of CRP mRNA in the presence of anti-leptin receptor antibodies. The cells were treated with the antibody 30 minutes before leptin (100 ng/mL) treatment. With increasing concentration of antibody an increased inhibition is observed. All data are represented as mean±SD of 4 independent experiments, each in triplicate. *P<0.05, **P<0.005, in comparison to control.

Leptin is known to activate a number of signaling pathways including mitogen-activated protein kinases (MAPKs) in different cell types. Hence we sought to determine the role of MAPKs in regulation of leptin-induced CRP production in HCAECs. Leptin showed a time-dependent (0 to 60 minutes) activation and deactivation of ERK1/2 (Figure 3A). Maximum phosphorylation of ERK1/2 was seen at 20 minutes after leptin treatment and declined to basal levels in 60 minutes. The increase in phosphorylation of ERK1/2 after 20 minutes of leptin treatment was dose-dependent (Figure 3B). Inhibition of leptin-induced CRP mRNA expression was observed after incubation with ERK inhibitor PD98059 in increasing doses (20 to 40 µg/mL; Figure 3C), suggesting that the effects of leptin on CRP are mediated through ERK. Increased ERK1/2 phosphorylation was seen only at 50 ng/mL or higher leptin concentrations, which is consistent with our finding that lower leptin concentration (20 ng/mL) did not elicit any significant increase in CRP mRNA expression as compared with 0 leptin control (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 3. Signaling pathway involved in leptin-dependent CRP expression in HCAECs. A, Western blot showing increased phosphorylation of ERK1/2 in response to leptin treatment (100 ng/mL) with time. Maximum phosphorylation is seen at 20 minutes after treatment. B, Western blot showing increased ERK1/2 phosphorylation in response to increased leptin concentrations. The ERK1/2 measurements were done 20 minutes after the leptin treatment. Increased ERK1/2 phosphorylation is seen only after leptin treatment at concentrations 50 ng/mL or higher. C, Quantitative mRNA analysis showing inhibition of CRP mRNA in response to increasing concentrations of ERK1/2 inhibitor PD98059. D, Western blot showing absence of any increased phosphorylation of p38 in response to leptin treatment (100 ng/mL). E, Western blot showing absence of any increased phosphorylation of p38 in response to increasing concentrations of leptin. F, Quantitative mRNA analysis showing absence of any effect of p38 inhibitor SB203580 on CRP mRNA. The Western blots are representative of the 3 independent experiments. The quantitative RNA data are represented as mean±SD of 4 independent experiments, each in triplicate. *P<0.05, **P<0.005, as compared with 0 inhibition control.

In contrast to ERK, leptin treatment did not alter the phosphorylation levels of p38 with time (Figure 3D). Also, increasing leptin concentrations had no effect on phosphorylation of p38 (Figure 3E). Finally, inhibition of p38 by increasing concentrations of SB203580 (5 to 20 µg/mL) did not affect CRP mRNA expression (Figure 3F).

Considering that the maximum activation of ERK1/2 was observed at 20 minutes instead of 10 minutes as previously reported,¹⁶ we sought to determine whether ERK1/2 phosphorylation was secondary to ROS generation. Leptin (100 ng/mL) treatment caused an increase in reactive oxygen species as indicated by increased fluorescence in leptin-treated cells as compared with vehicle control (Figure 4A). ROS generation in response to leptin treatment was inhibited by catalase (200 µg/mL) treatment. This is also evident from our FACS analysis showing increased gated events (Figure 4B) and shift in the fluorescence peak (Figure 4C) during leptin treatment, both of which tended to reverse during catalase treatment. The leptin-treated cells in presence of catalase failed to show any increased ERK1/2 phosphorylation (Figure 4D) and CRP mRNA transcription (Figure 4E), indicating that ERK1/2 phosphorylation during leptin treatment may be secondary to ROS generation.

View larger version (45K): [in this window] [in a new window]   Figure 4. Leptin dependent ERK1/2 phosphorylation is mediated by reactive oxygen species. A, Photomicrographs of cells showing ROS production represented by fluorescence. B, Graphical representation of % gated events during FACS analysis. C, Graphical representation of shift in fluorescence peak during FACS analysis. The green line represents control, pink line represents leptin treatment, and blue line represents catalase + leptin treatment. In all the experiments, increased ROS production is observed in response to leptin treatment. Also the ROS production gets attenuated in presence of catalase. D, Western blot showing the effect of ROS inhibition on ERK1/2 phosphorylation. Lane 1, 0 leptin (Vehicle control); lane 2, leptin treatment; lane 3, catalase + leptin treatment; lane 4, catalase treatment. In the presence of catalase, no increase in ERK1/2 phosphorylation was observed in response to leptin treatment (20 minutes). E, Quantitative mRNA analysis showing inhibition of CRP mRNA in response to catalase treatment. F, Schematic representation of the proposed signaling pathway of leptin-dependent CRP induction. During all the experiments the cells were treated with catalase (200 µg/mL) 30 minutes before leptin (100 ng/mL) treatment. The results are representative of 4 independent experiments and data are represented as mean±SD.

	Discussion

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This study has 2 important and novel findings. First, leptin induces CRP expression in vascular endothelial cells, and second, increased CRP expression is evident only at high physiological concentrations of leptin (>30 ng/mL). Our studies further suggest that leptin induces CRP expression in HCAECs via activation of the leptin receptor, increased ROS production, and phosphorylation of ERK1/2 (Figure 4F).

These data are consistent with the independent positive correlation between plasma leptin and CRP,^10,11 and with increases in plasma CRP after exogenous leptin administration in healthy human subjects.^12,13 It was recently shown that leptin at normal physiological levels (0.5 to 20 ng/mL) induces CRP expression in primary human hepatocyte culture.¹⁷ Leptin does not appear to induce CRP expression in primary human adipocyte culture.¹⁸ However, this latter study included only CRP measurements from cell culture supernatants and did not involve any RNA studies or intracellular CRP measurements. We have been able to demonstrate in HCAECs a leptin-dependent increase in CRP at both transcriptional and translational levels, but only at physiologically higher leptin levels. Hence there appears to be a tissue-specific and dose-dependent CRP response to leptin.

Several human studies suggest an independent association between high leptin and worse cardiovascular prognosis.^1,2 These clinical results are consistent with several in vivo studies in leptin deficient ob/ob mice, leptin receptor–deficient db/db mice, and ApoE mice, which suggest that lack of leptin protects against atherosclerosis, whereas high leptin levels are proatherogenic.^3,19 Administration of leptin has been shown to enhance atherosclerotic lesion formation in wild-type and ob/ob mice despite favorable changes in body weight, lipid profiles, and insulin sensitivity.¹⁹ However, the exact mechanisms whereby leptin may promote atherosclerosis remain unclear.

Vascular endothelial cells are an important component of the atherosclerotic process.²⁰ This is especially true for coronary artery endothelial cells where endothelial cell dysfunction, inflammation, and prothrombotic states may contribute to coronary atherosclerosis, ischemia, infarction, and death. CRP is known to localize in atherosclerotic lesions^6–8 and is also expressed in vascular smooth muscle cells²¹ and endothelial cells.¹⁵ CRP exerts many proatherogenic effects on the vascular endothelium, including activation of the complement system, induction of intercellular adhesion molecule-1 (ICAM-1), vCAM-1, E-selectin, MCP-1, ET-1, plasminogen activator inhibitor (PAI)-1, decreases in e-NOS, tPA activity, and thrombomodulin expression.⁹ CRP also promotes proliferation and migration of smooth muscle cells in vitro and in vivo. Thus, local expression of CRP in HCAEC likely plays an important role in the development and progression of atherosclerotic lesions.

Our present findings suggest that the detrimental effects of leptin on coronary vasculature may be mediated at least in part by induction of intracellular endothelial CRP expression. In several studies,^1,2 with both leptin and CRP in the model, CRP lost its predictive power whereas leptin remained significantly related to cardiovascular outcome, supporting the proposition that leptin is an important mediator of increased risk. The effect of leptin on CRP production in HCAECs only at higher leptin levels is also supportive of a proatherogenic effect in the setting of hyperleptinemia as seen in obesity.

In the present study, we suggest the pathway through which leptin upregulates CRP expression in endothelial cells, and we demonstrate the role of the leptin receptor. Our findings confirm that leptin induces ROS formation,²² which we further show as a cause of ERK1/2 activation. The role for leptin in induction of CRP in endothelial cells is unique and important. Other ligands such as IFN- A/D and TNF-, which are known to activate ERK1/2, failed to induce CRP mRNA transcription. A limitation to our study is that it is in vitro. In an in vivo environment, interaction with proteins such as soluble leptin receptor and CRP¹⁷ may modulate leptin regulation of CRP expression. We could also speculate that leptin, via upregulation of CRP, is involved in downregulating its own activity. However the data regarding leptin-CRP interaction and its ability to interfere with leptin signaling remain controversial and needs further investigation.²³

CRP is already being targeted for drug development. CRP inhibitors are capable of significantly reducing the size of myocardial infarction in the rat model.²⁴ Our present findings may be potentially relevant to inhibiting the proatherogenic effect of leptin, and may have direct implications for prevention and treatment of cardiovascular diseases. Leptin itself, which causes increased expression of intracellular CRP, may hence be a good therapeutic target.

	Acknowledgments

 
Sources of Funding

P.S. is supported by a Postdoctoral Fellowship from the American Heart Association. M.H. is supported by a Pickwick grant from the National Sleep Foundation. A.S.M.S. is supported by a Scientist Development Grant from the American Heart Association, and V.K.S. is supported by NIH grants R01HL65176, R01HL70302, R01HL73211, and M01-RR00585.

Disclosures

P.S., M.H., R.W., and V.K.S. are working with Mayo Health Solutions regarding intellectual property related to these studies. V.K.S. is a consultant for Respironics, Res Med, Cardiac Concepts, and Sepracor.

	Footnotes

 
Original received March 5, 2007; final version accepted June 15, 2007.

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: 27/9/e302    most recent ATVBAHA.107.148353v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Singh, P. Articles by Somers, V. K. Search for Related Content PubMed PubMed Citation Articles by Singh, P. Articles by Somers, V. K. Related Collections Risk Factors Gene regulation Mechanism of atherosclerosis/growth factors