High-Density Lipoproteins Induce Transforming Growth Factor-;2 Expression in Endothelial Cells
http://www.100md.com
《循环学杂志》
the Department of Pharmacological Sciences, University of Milan, Milan, Italy (G.D.N., E.C., M.M., G.C., A.L.C.)
King Gustaf V Research Institute, Department of Medicine, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden (P.E.)
Centro per lo Studio, la Terapia e la Prevenzione delle Vasculopatie Periferiche, Ospedale Bassini, Cinisello Balsamo, Italy (A.L.C.).
Abstract
Background— HDL is endowed with cardiovascular protective activities. In addition to its role in reverse cholesterol transport, HDL influences different functions of endothelial cells. In the present study, we investigated in endothelial cells the genes involved in inflammation modulated by HDL.
Methods and Results— Through cDNA array analysis, transforming growth factor (TGF)-;2 appeared to be a gene responsive to HDL treatment in endothelial cells. Quantitative real-time polymerase chain reaction confirmed that HDL subfraction 3 selectively induces TGF-;2 mRNA expression and protein release, whereas TGF-;1 and TGF-;3 were not affected. This effect was mainly PI3K/Akt dependent. Lysosphingolipids present in HDL such as sphingosine 1 phosphate and sphingosylphosphorylcholine mimicked the effects of the whole HDL. These results were confirmed in vivo in transgenic mice overexpressing human apolipoprotein (apo) A-I. Compared with apoA-I–knockout mice, phospho-Akt, phospho-ERK1/2, and TGF-;2 expression was increased in the aorta of transgenic mice overexpressing human apoA-I. In addition, the expression of phospho-Smad2/3, the transcription factor activated by TGF-;, is increased in transgenic mice compared with knockout mice.
Conclusions— Because TGF-; possesses antiinflammatory properties and stabilizes the plaque, the results of the present work suggest a novel target for the antiatherosclerotic effect of HDL.
Key Words: apolipoproteins ; endothelium ; lipoproteins ; signal transduction
Introduction
Atherosclerosis is recognized as an inflammatory disease.1 Recent work indicates that vascular inflammatory responses can be limited by antiinflammatory counterregulatory mechanisms that maintain the integrity and homeostasis of the vascular wall.2,3 These mechanisms include antiinflammatory signals such as transforming growth factor-; (TGF-;).4
The TGF-; family plays a critical role in the modulation of vascular inflammatory responses and remodeling.5–7 Upregulation of TGF-; production is an invariant response to vascular injury, indicating that regulation of gene expression of the TGF-; proteins by quiescent and injured endothelium is likely to be a critical factor affecting the progression of vascular inflammation. This hypothesis is supported by the finding of defective vasculogenesis and increased, and eventually lethal, vascular inflammation in mice null for TGF-;,8 their membrane receptors,9 or their downstream substrates, the Smad proteins.10
Three mammalian isoforms of TGF-; (TGF-;1, TGF-;2, and TGF-;3)8 are secreted by endothelial cells in a latent form in which latency-associated peptide and its 25-kDa carboxy-terminal dimer, which is the mature TGF-;,11 remain noncovalently bound. In vivo release of the mature dimer from latency-associated peptide occurs via its degradation or conformational changes11 and allows binding of the mature (bioactive) peptide to its specific membrane receptors. Activated TGF-; receptor phosphorylates intracellular Smad proteins, which propagate downstream signals of TGF-;s.12 Of note, it has been shown that TGF-;2 is selectively induced in human endothelial cells during hypoxia13 and that adenovirus infection14 mitigates inflammatory signals and augments the adaptive response of the endothelium. In addition, cDNA array analysis of endothelial cells incubated with TGF-;2 has shown decreased expression of interleukin 6, monocyte chemoattractant protein-1 (MCP-1), granulocyte-colony–stimulating factor, and other genes,15 confirming an antiinflammatory role of the isoform ;2 of TGF. In vivo inhibition of TGF-; signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice.16,17 These observations indicate that TGF-; is a key factor in promoting plaque stability and decreasing vascular wall inflammation.18
Several cardiovascular protective functions have been attributed to HDL. In addition to their role in reverse cholesterol transport, HDL exerts antioxidant effects by inhibiting LDL oxidation, and by acting as a scavenger of oxidized lipids, HDL reduces platelet aggregation and stimulates fibrinolysis.19 Furthermore, HDL influences different functions of endothelial cells,20–22 including angiogenesis, vasorelaxation, and cell proliferation, via modulation of the expression and of the activity of different genes such as endothelial nitric oxide synthase,22 cyclooxygenase, prostacyclin synthase,20,21 adhesion molecules,23 and proteases like ADAMTS-1.24
In the present study, we investigated the effects of native HDL3 and its components on the gene expression of several proinflammatory and antiinflammatory cytokines in human endothelial cells (HUVECs). Because the expression of TGF-;2 increased significantly, we investigated the molecular mechanisms involved in these effects.
Methods
Materials
HDL subfraction 3 (HDL3) was obtained from freshly isolated human plasma by preparative ultracentrifugation and dialyzed versus PBS containing 0.01% EDTA.24,25 HDL3 was used within 6 hours of preparation.
HUVECs were isolated and cultured as previously described.26 In all experiments, cells were preincubated with serum-free medium for 6 hours and incubated with HDL3 for 4 hours. Control cells were incubated for 4 hours, with the experimental medium containing the same percentage of PBS that was added with the stimulus.
Sphingosine 1 phosphate (S1P) and sphingosylphophorylcholine (SPC) (both from Sigma) were used at 10 μmol/L; apolipoprotein A-I (apoA-I; Sigma) was used at 10 μg/mL. The MEK inhibitor U0126 (New England Biolabs), the p38 MAPK inhibitor SB 203580 (Sigma), the PI3-K inhibitor Ly 294002, and the Akt inhibitor SH-5 (both from Alexis) were used at final concentrations of 10, 0.5, 50, and 10 μmol/L, respectively. At these concentrations, the inhibitors effectively decreased the phosphorylation of the downstream targets (data not shown).
The antibodies to phospho-Akt (Thr 308) and phospho-p44/42 MAPK (thr202/tyr204) were obtained from New England Biolabs. The antibody to phospho-Smad 2/3 (ser433/435) was obtained from Santa Cruz.
cDNA Array and Data Analysis
Total RNA extraction was performed as described.26 [-32P]dCTP-labeled cDNA was prepared and hybridized to the human inflammatory cytokines gene array (GEArray Q series, Superarray). Arrays were exposed on a phosphor plate and quantified as described.24,26 Normalized intensities and gene expression ratios were calculated as described.24,26
Quantitative Real-Time Polymerase Chain Reaction
TGF-;2 Protein Expression
TGF-;2 protein expression was determined by ELISA (Promega) according to the manufacturer’s instructions.
Animal Studies
The generation of transgenic mice expressing human apoA-I that were deficient in the endogenous murine apoA-I (hA-I) has been described previously27,28. Littermates of hA-I transgenic mice and murine A-I–knockout mice (A-I–/–) (3 males and 4 females in each group) (HDL cholesterol levels at the time of death, 124.2±31.1 and 11.9±4.9 mg/dL, respectively) were fed a standard diet for 18 weeks and killed by exposure to carbon dioxide according to the protocol of the internal ethics committee. After perfusion with saline, the aortas were isolated and placed in a storing solution (RNA Later, Ambion) at –20°C or paraffin embedded. For RNA isolation, the samples were homogenized in a dismembrator (B. Braun, Melsungen AG) and then processed as described.21 For immunofluorescence studies, aorta specimens (5 μm thick) sectioned and collected as described29 were incubated with primary antibody (1:50) overnight at 4°C, followed by incubation with anti-mouse IgG Alexa 633–conjugated (1:100, Molecular Probes) for 30 minutes and then propidium iodide (2.5 μg/mL) for 30 minutes. Coverslips were analyzed with a confocal microscope as described.21 Colocalization analysis was performed with the Laserpix software (Bio-Rad); the white pixels showed the phosphorylated transcription factor complex Smad2/3 when located in the nucleus.
Statistical Analysis
Data are given as mean±SD and are based on 4 separate experiments. Statistical analysis was performed by an unpaired Student t test with the use of the Statsoft Statistica Package.
Results
We analyzed the endothelial cell expression of inflammatory cytokines in the presence or absence of HDL3 (200 μg/mL protein) for 4 hours. Radioactively labeled cDNA probes generated from total RNA of control cells and cells incubated with HDL3 were hybridized in parallel to identical cDNA arrays (n=3). Under these conditions, few genes responded to HDL treatment; among them, TGF-;2 was induced by HDL3 (6.21±2.71-fold versus control cells). The effect was specific for TGF-;2 because no changes in TGF-;1 and TGF-;3 on HDL3 incubation were detected (Table 2).
To validate these findings, the expression of TGF-;1, TGF-;2, TGF-;3, and MCP-1 (as negative control) was investigated by quantitative real-time PCR. The induction of TGF-;2 was 2.31±0.66-fold for HDL3-treated cells (P<0.01). In agreement with the array data, no significant effect of HDL3 was detected on TGF-;1 (1.18±0.55-fold), TGF-;3 (1.25±0.70-fold), and MCP-1 (0.93±0.12-fold) expression.
Moreover, the effect of HDL3 on TGF-;2 was dose dependent (Figure 1A) in a range from 10 to 500 μg/mL; at these concentrations, again no effect of HDL3 on TGF-;1 and TGF-;3 expression was detected (Figure 1A). The maximal effect on TGF-;2 expression was observed at 4 hours. No effect of HDL3 on TGF-;1 and TGF-;3 expression was detected during up to 18 hours of incubation (Figure 1B).
Preincubation for 1 hour with U0126, LY294002, and SH-5 decreased the effect of HDL3 (from 2.31±0.16- to 1.25±0.19-, 1.19±0.41-, 1.24±0.16-fold with U0126, LY294002, and SH-5, respectively; all P<0.01), while a minor effect of SB 203580 was observed (1.91±0.15, P=NS) (Figure 2A).
HDL significantly increased TGF-;2 protein release (2.16±0.83-fold), an effect that was dependent on PI3K/Akt activation (Figure 2B).
To investigate the nature of the stimulatory component present in HDL, HUVECs were incubated with S1P, SPC, or apoA-I. S1P and SPC induced TGF-;2 mRNA and protein expression; apoA-I affected TGF-;2 mRNA and protein expression, but this effect did not reach statistical significance (Figure 3).
To confirm ex vivo the role of HDL on TGF-;2 expression, we analyzed the mRNA levels of TGF-; and the presence of the phosphorylated (ie, active) forms of ERK 1/2 and Akt in the aortas of hA-I mice compared with those of A-I–/– mice. These 2 mouse models with identical genetic backgrounds were chosen to allow comparison of an in vivo model of low HDL with a model of high HDL. Preliminary studies have shown that TGF-; levels and kinase activation were similar between A-I–/– mice and C57/B6 mice (data not shown).
TGF-;2 expression was significantly increased in transgenic hA-I (10.10±2.45-fold versus A-I–/–) (P=0.02), whereas no differences in TGF-;1 and TGF-;3 expression were observed between the 2 mice models (Figure 4). Increased expression of phospho-Akt and phospho-ERK 1/2 was detected in the aortas of hA-I mice compared with A-I–/– mice (Figure 5A and 5B). Immunofluorescence confirmed these findings, showing increased expression of phospho-Akt and phospho-ERK 1/2 (blue signal) in the endothelium (Figure 5D and 5E). The background of the secondary antibody (blue signal) is shown in Figure 5C with the elastic laminas (green) and nuclei (red).
Because to the best of our knowledge no antibody for selective detection of TGF-;2 in mice aortas is available, we studied the expression of the active form of the intracellular effector of TGF-; (phospho-Smad-2/3)12 to investigate whether TGF-;2 induced in the aortas of hA-I mice is functionally active. Increased expression of phospho-Smad-2/3 was detected in the aortas of hA-I mice compared with A-I–/– mice (Figure 6A through 6C). Furthermore, colocalization studies showed that phospho-Smad-2/3 is present in the nuclei of cells of the arterial wall of hA-I mice to a higher extent than in A-I–/– mice (Figure 6D). Our data also indicate that similar effects on intracellular pathways and downstream targets occur in the media of the vascular wall (Figure 6D).
Discussion
The inflammatory status in the vascular wall results from the imbalance between proinflammatory and antiinflammatory factors.4 TGF-; possesses several antiinflammatory properties on the endothelium.4,18,30 Inhibition of TGF-; signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice.16,17 These observations indicate that TGF-; is a key factor in promoting plaque stability and decreasing vascular wall inflammation.18
We show that HDL3 induces the expression of the antiinflammatory cytokine TGF-;2 in vitro and ex vivo. Our data add new insights, suggesting a further mechanism by which HDL3 may play an atheroprotective effect.
In endothelial cells, the effect of HDL3 is specific for TGF-;2 because TGF-;1 and TGF-;3 expression is not affected and is dependent mainly on PI3K/Akt activation. This observation is in agreement with the known role of Akt and ERK pathways in mediating HDL-induced endothelial gene expression and angiogenesis20,21 and supports a protective role for HDL on the endothelium.20,21 Two lysosphingolipids, S1P and SPC, which are carried by HDL and responsible for HDL-induced nitric oxide–dependent vasorelaxation31 and antiapoptotic effects,32 induced TGF-;2 expression, whereas a minor effect was observed with apoA-I. These findings support the role of both apo-AI and lysosphingolipids proposed by Nofer et al31 in the effects of HDL in vivo and provide a preliminary identification of the HDL components responsible for this effect.
Phospho-ERK1/2 and phospho-Akt expression is increased in the endothelium and vascular wall of mice expressing human apoA-I, suggesting that in vivo these 2 signaling pathways also play a major role in determining the vascular wall responses to HDL. Previous studies have shown that TGF-;1 and TGF-;3 expression is increased in advanced atherosclerotic plaques,33 in contrast to the findings in animal models in which inhibition of TGF-; promotes the switch to unstable plaque phenotype.16,17,30 These data may be reconciled by our observation of a specific effect of HDL on TGF-;2, suggesting that the antiatherosclerotic role of TGF-; may be confined to this isoform. These data also are supported by the observation that TGF-;2 downregulates the expression of proinflammatory genes induced by IL115 and LPS (Norata et al, unpublished observations) in endothelial cells. Alternatively, the increased TGF-;1 and TGF-;3 expression may represent a protective response in advanced plaques.
The ex vivo data on transgenic animals are substantiated by the increased expression of phospho-Smad-2/3, the intracellular effector of TGF-; signaling,12 in the aorta of hA-I mice, confirming that HDL-induced TGF-; expression results in increased TGF-; signaling pathway activity. Notably, this increased expression was observed in all cells of the arterial wall. Whether this is a direct effect of HDL3 or is an effect of TGF-;2 produced by endothelial cells remains to be addressed. Of note, a recent article reported that in mesangial cells S1P cross-activates the Smad signaling cascade and reduces IL-1;–induced expression of iNOS and MMP-9,34 suggesting a cross-talk of S1P with the TGF-; signaling pathways.
In summary, HDL induces TGF-;2 expression in vitro and in vivo mainly via PI3K/Akt activation; this effect results in increased TGF-;–dependent signaling in the endothelium and vascular wall cells. Because TGF-; possesses antiinflammatory properties and stabilizes the atherosclerotic plaque, our findings will help identify a novel mechanism by which HDL may exert protective effects on endothelial cells and vascular wall function.
Acknowledgments
This work was supported by grants from Consorzio Interuniversitario Ricerca Cardiovascolare, FIRB 2001 (RBNE01HLAK–006), Società Italiana Studio Aterosclerosi sezione Lombardia, and AFA Insurance and Swedish Medical Research Council (12660). We are grateful to Roberto Zecca for software assistance, Giulio Simonutti for technical assistance with the confocal microscopy, and Fabio Pellegatta for providing the HUVECs.
References
Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.
Uyemura K, Demer LL, Castle SC, Jullien D, Berliner JA, Gately MK, Warrier RR, Pham N, Fogelman AM, Modlin RL. Cross-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis. J Clin Invest. 1996; 97: 2130–2138.
Mallat Z, Besnard S, Duriez M, Deleuze V, Emmanuel F, Bureau MF, Soubrier F, Esposito B, Duez H, Fievet C, Staels B, Duverger N, Scherman D, Tedgui A. Protective role of interleukin-10 in atherosclerosis. Circ Res. 1999; 85: e17–e24.
Tedgui A, Mallat Z. Anti-inflammatory mechanisms in the vascular wall. Circ Res. 2001; 88: 877–887.
Pintavorn P, Ballermann BJ. TGF-; and the endothelium during immune injury. Kidney Int. 1997; 51: 1401–1412.
Pepper MS. Transforming growth factor-;: vasculogenesis, angiogenesis, and vessel wall integrity. Cytokine Growth Factor Rev. 1997; 8: 21–43.
Robertson AK, Rudling M, Zhou X, Gorelik L, Flavell RA, Hansson GK. Disruption of TGF-; signaling in T cells accelerates atherosclerosis. J Clin Invest. 2003; 112: 1342–1350.
Sanford LP, Ormsby I, Gittenberger-de Groot AC, Sariola H, Friedman R, Boivin GP, Cardell EL, Doetschman T. TGF;2 knockout mice have multiple developmental defects that are non-overlapping with other TGF; knockout phenotypes. Development. 1997; 124: 2659–2670.
Oh SP, Seki T, Goss KA, Imamura T, Yi Y, Donahoe PK, Li L, Miyazono K, ten Dijke P, Kim S, Li E. Activin receptor–like kinase 1 modulates transforming growth factor-beta 1 signaling in the regulation of angiogenesis. Proc Natl Acad Sci U S A. 2000; 97: 2626–2631.
Galvin KM, Donovan MJ, Lynch CA, Meyer RI, Paul RJ, Lorenz JN, Fairchild-Huntress V, Dixon KL, Dunmore JH, Gimbrone MA Jr, Falb D, Huszar D. A role for smad6 in development and homeostasis of the cardiovascular system. Nat Genet. 2000; 24: 171–174.
Schultz-Cherry S, Chen H, Mosher DF, Misenheimer TM, Krutzsch HC, Roberts DD, Murphy-Ullrich JE. Regulation of transforming growth factor-beta activation by discrete sequences of thrombospondin 1. J Biol Chem. 1995; 270: 7304–7310.
Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-; family signaling. Nature. 2003; 425: 577–584.
Akman HO, Zhang H, Siddiqui MA, Solomon W, Smith EL, Batuman OA. Response to hypoxia involves transforming growth factor-;2 and Smad proteins in human endothelial cells. Blood. 2001; 98: 3324–3331.
Ramalingam R, Rafii S, Worgall S, Brough DE, Crystal RG. E1(–)E4(+) adenoviral gene transfer vectors function as a "pro-life" signal to promote survival of primary human endothelial cells. Blood. 1999; 93: 2936–2944.
Yamagami S, Yokoo S, Mimura T, Amano S. Effects of TGF-;2 on immune response-related gene expression profiles in the human corneal endothelium. Invest Ophthalmol Vis Sci. 2004; 45: 515–521.
Mallat Z, Gojova A, Marchiol-Fournigault C, Esposito B, Kamate C, Merval R, Fradelizi D, Tedgui A. Inhibition of transforming growth factor-; signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice. Circ Res. 2001; 89: 930–934.
Lutgens E, Gijbels M, Smook M, Heeringa P, Gotwals P, Koteliansky VE, Daemen MJ. Transforming growth factor-; mediates balance between inflammation and fibrosis during plaque progression. Arterioscler Thromb Vasc Biol. 2002; 22: 975–982.
Grainger DJ. Transforming growth factor beta and atherosclerosis: so far, so good for the protective cytokine hypothesis. Arterioscler Thromb Vasc Biol. 2004; 24: 399–404.
Chiesa G, Sirtori CR. Use of recombinant apolipoproteins in vascular diseases: the case of apoA-I. Curr Opin Investig Drugs. 2002; 3: 420–426.
Calabresi L, Gomaraschi M, Franceschini G. Endothelial protection by high-density lipoproteins: from bench to bedside. Arterioscler Thromb Vasc Biol. 2003; 23: 1724–1731.
Norata GD, Callegari E, Inoue H, Catapano AL. HDL3 induces cyclooxygenase-2 expression and prostacyclin release in human endothelial cells via a p38 MAPK/CRE-dependent pathway: effects on COX-2/PGI-synthase coupling. Arterioscler Thromb Vasc Biol. 2004; 24: 871–877.
Mineo C, Yuhanna IS, Quon MJ, Shaul PW. High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J Biol Chem. 2003; 278: 9142–9149.
Ashby DT, Rye KA, Clay MA, Vadas MA, Gamble JR, Barter PJ. Factors influencing the ability of HDL to inhibit expression of vascular cell adhesion molecule-1 in endothelial cells. Arterioscler Thromb Vasc Biol. 1998; 18: 1450–1455.
Norata GD, Bjork H, Hamsten A, Catapano AL, Eriksson P. High-density lipoprotein subfraction 3 decreases ADAMTS-1 expression induced by lipopolysaccharide and tumor necrosis factor- in human endothelial cells. Matrix Biol. 2004; 22: 557–560.
Norata GD, Pirillo A, Callegari E, Hamsten A, Catapano AL, Eriksson P. Gene expression and intracellular pathways involved in endothelial dysfunction induced by VLDL and oxidized VLDL. Cardiovasc Res. 2003; 59: 169–180.
Norata GD, Pellegatta F, Hamsten A, Catapano AL, Eriksson P. Effects of HDL3 on the expression of matrix-degrading proteases in human endothelial cells. Int J Mol Med. 2003; 12: 73–78.
Chiesa G, Parolini C, Canavesi M, Colombo N, Sirtori CR, Fumagalli R, Franceschini G, Bernini F. Human apolipoproteins A-I and A-II in cell cholesterol efflux: studies with transgenic mice. Arterioscler Thromb Vasc Biol. 1998; 18: 1417–1423.
Williamson R, Lee D, Hagaman J, Maeda N. Marked reduction of high density lipoprotein cholesterol in mice genetically modified to lack apolipoprotein A-I. Proc Natl Acad Sci U S A. 1992; 89: 7134–7138.
Norata GD, Tonti L, Roma P, Catapano AL. Apoptosis and proliferation of endothelial cells in early atherosclerotic lesions: possible role of oxidized LDL. Nutr Metab Cardiovasc Dis. 2002; 12: 297–305.
Mallat Z, Tedgui A. The role of transforming growth factor ; in atherosclerosis: novel insights and future perspectives. Curr Opin Lipidol. 2002; 13: 523–529.
Nofer JR, van der Giet M, Tolle M, Wolinska I, von Wnuck Lipinski K, Baba HA, Tietge UJ, Godecke A, Ishii I, Kleuser B, Schafers M, Fobker M, Zidek W, Assmann G, Chun J, Levkau B. HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J Clin Invest. 2004; 113: 569–581.
Nofer JR, Levkau B, Wolinska I, Junker R, Fobker M, von Eckardstein A, Seedorf U, Assmann G. Suppression of endothelial cell apoptosis by high density lipoproteins (HDL) and HDL-associated lysosphingolipids. J Biol Chem. 2001; 276: 34480–34485.
Bobik A, Agrotis A, Kanellakis P, Dilley R, Krushinsky A, Smirnov V, Tararak E, Condron M, Kostolias G. Distinct patterns of transforming growth factor-; isoform and receptor expression in human atherosclerotic lesions: colocalization implicates TGF-; in fibrofatty lesion development. Circulation. 1999; 99: 2883–2891.
Xin C, Ren S, Kleuser B, Shabahang S, Eberhardt W, Radeke H, Schafer-Korting M, Pfeilschifter J, Huwiler A. Sphingosine 1-phosphate cross-activates the Smad signaling cascade and mimics transforming growth factor-;–induced cell responses. J Biol Chem. 2004; 279: 35255–35262.(Giuseppe D. Norata, PhD; )
King Gustaf V Research Institute, Department of Medicine, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden (P.E.)
Centro per lo Studio, la Terapia e la Prevenzione delle Vasculopatie Periferiche, Ospedale Bassini, Cinisello Balsamo, Italy (A.L.C.).
Abstract
Background— HDL is endowed with cardiovascular protective activities. In addition to its role in reverse cholesterol transport, HDL influences different functions of endothelial cells. In the present study, we investigated in endothelial cells the genes involved in inflammation modulated by HDL.
Methods and Results— Through cDNA array analysis, transforming growth factor (TGF)-;2 appeared to be a gene responsive to HDL treatment in endothelial cells. Quantitative real-time polymerase chain reaction confirmed that HDL subfraction 3 selectively induces TGF-;2 mRNA expression and protein release, whereas TGF-;1 and TGF-;3 were not affected. This effect was mainly PI3K/Akt dependent. Lysosphingolipids present in HDL such as sphingosine 1 phosphate and sphingosylphosphorylcholine mimicked the effects of the whole HDL. These results were confirmed in vivo in transgenic mice overexpressing human apolipoprotein (apo) A-I. Compared with apoA-I–knockout mice, phospho-Akt, phospho-ERK1/2, and TGF-;2 expression was increased in the aorta of transgenic mice overexpressing human apoA-I. In addition, the expression of phospho-Smad2/3, the transcription factor activated by TGF-;, is increased in transgenic mice compared with knockout mice.
Conclusions— Because TGF-; possesses antiinflammatory properties and stabilizes the plaque, the results of the present work suggest a novel target for the antiatherosclerotic effect of HDL.
Key Words: apolipoproteins ; endothelium ; lipoproteins ; signal transduction
Introduction
Atherosclerosis is recognized as an inflammatory disease.1 Recent work indicates that vascular inflammatory responses can be limited by antiinflammatory counterregulatory mechanisms that maintain the integrity and homeostasis of the vascular wall.2,3 These mechanisms include antiinflammatory signals such as transforming growth factor-; (TGF-;).4
The TGF-; family plays a critical role in the modulation of vascular inflammatory responses and remodeling.5–7 Upregulation of TGF-; production is an invariant response to vascular injury, indicating that regulation of gene expression of the TGF-; proteins by quiescent and injured endothelium is likely to be a critical factor affecting the progression of vascular inflammation. This hypothesis is supported by the finding of defective vasculogenesis and increased, and eventually lethal, vascular inflammation in mice null for TGF-;,8 their membrane receptors,9 or their downstream substrates, the Smad proteins.10
Three mammalian isoforms of TGF-; (TGF-;1, TGF-;2, and TGF-;3)8 are secreted by endothelial cells in a latent form in which latency-associated peptide and its 25-kDa carboxy-terminal dimer, which is the mature TGF-;,11 remain noncovalently bound. In vivo release of the mature dimer from latency-associated peptide occurs via its degradation or conformational changes11 and allows binding of the mature (bioactive) peptide to its specific membrane receptors. Activated TGF-; receptor phosphorylates intracellular Smad proteins, which propagate downstream signals of TGF-;s.12 Of note, it has been shown that TGF-;2 is selectively induced in human endothelial cells during hypoxia13 and that adenovirus infection14 mitigates inflammatory signals and augments the adaptive response of the endothelium. In addition, cDNA array analysis of endothelial cells incubated with TGF-;2 has shown decreased expression of interleukin 6, monocyte chemoattractant protein-1 (MCP-1), granulocyte-colony–stimulating factor, and other genes,15 confirming an antiinflammatory role of the isoform ;2 of TGF. In vivo inhibition of TGF-; signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice.16,17 These observations indicate that TGF-; is a key factor in promoting plaque stability and decreasing vascular wall inflammation.18
Several cardiovascular protective functions have been attributed to HDL. In addition to their role in reverse cholesterol transport, HDL exerts antioxidant effects by inhibiting LDL oxidation, and by acting as a scavenger of oxidized lipids, HDL reduces platelet aggregation and stimulates fibrinolysis.19 Furthermore, HDL influences different functions of endothelial cells,20–22 including angiogenesis, vasorelaxation, and cell proliferation, via modulation of the expression and of the activity of different genes such as endothelial nitric oxide synthase,22 cyclooxygenase, prostacyclin synthase,20,21 adhesion molecules,23 and proteases like ADAMTS-1.24
In the present study, we investigated the effects of native HDL3 and its components on the gene expression of several proinflammatory and antiinflammatory cytokines in human endothelial cells (HUVECs). Because the expression of TGF-;2 increased significantly, we investigated the molecular mechanisms involved in these effects.
Methods
Materials
HDL subfraction 3 (HDL3) was obtained from freshly isolated human plasma by preparative ultracentrifugation and dialyzed versus PBS containing 0.01% EDTA.24,25 HDL3 was used within 6 hours of preparation.
HUVECs were isolated and cultured as previously described.26 In all experiments, cells were preincubated with serum-free medium for 6 hours and incubated with HDL3 for 4 hours. Control cells were incubated for 4 hours, with the experimental medium containing the same percentage of PBS that was added with the stimulus.
Sphingosine 1 phosphate (S1P) and sphingosylphophorylcholine (SPC) (both from Sigma) were used at 10 μmol/L; apolipoprotein A-I (apoA-I; Sigma) was used at 10 μg/mL. The MEK inhibitor U0126 (New England Biolabs), the p38 MAPK inhibitor SB 203580 (Sigma), the PI3-K inhibitor Ly 294002, and the Akt inhibitor SH-5 (both from Alexis) were used at final concentrations of 10, 0.5, 50, and 10 μmol/L, respectively. At these concentrations, the inhibitors effectively decreased the phosphorylation of the downstream targets (data not shown).
The antibodies to phospho-Akt (Thr 308) and phospho-p44/42 MAPK (thr202/tyr204) were obtained from New England Biolabs. The antibody to phospho-Smad 2/3 (ser433/435) was obtained from Santa Cruz.
cDNA Array and Data Analysis
Total RNA extraction was performed as described.26 [-32P]dCTP-labeled cDNA was prepared and hybridized to the human inflammatory cytokines gene array (GEArray Q series, Superarray). Arrays were exposed on a phosphor plate and quantified as described.24,26 Normalized intensities and gene expression ratios were calculated as described.24,26
Quantitative Real-Time Polymerase Chain Reaction
TGF-;2 Protein Expression
TGF-;2 protein expression was determined by ELISA (Promega) according to the manufacturer’s instructions.
Animal Studies
The generation of transgenic mice expressing human apoA-I that were deficient in the endogenous murine apoA-I (hA-I) has been described previously27,28. Littermates of hA-I transgenic mice and murine A-I–knockout mice (A-I–/–) (3 males and 4 females in each group) (HDL cholesterol levels at the time of death, 124.2±31.1 and 11.9±4.9 mg/dL, respectively) were fed a standard diet for 18 weeks and killed by exposure to carbon dioxide according to the protocol of the internal ethics committee. After perfusion with saline, the aortas were isolated and placed in a storing solution (RNA Later, Ambion) at –20°C or paraffin embedded. For RNA isolation, the samples were homogenized in a dismembrator (B. Braun, Melsungen AG) and then processed as described.21 For immunofluorescence studies, aorta specimens (5 μm thick) sectioned and collected as described29 were incubated with primary antibody (1:50) overnight at 4°C, followed by incubation with anti-mouse IgG Alexa 633–conjugated (1:100, Molecular Probes) for 30 minutes and then propidium iodide (2.5 μg/mL) for 30 minutes. Coverslips were analyzed with a confocal microscope as described.21 Colocalization analysis was performed with the Laserpix software (Bio-Rad); the white pixels showed the phosphorylated transcription factor complex Smad2/3 when located in the nucleus.
Statistical Analysis
Data are given as mean±SD and are based on 4 separate experiments. Statistical analysis was performed by an unpaired Student t test with the use of the Statsoft Statistica Package.
Results
We analyzed the endothelial cell expression of inflammatory cytokines in the presence or absence of HDL3 (200 μg/mL protein) for 4 hours. Radioactively labeled cDNA probes generated from total RNA of control cells and cells incubated with HDL3 were hybridized in parallel to identical cDNA arrays (n=3). Under these conditions, few genes responded to HDL treatment; among them, TGF-;2 was induced by HDL3 (6.21±2.71-fold versus control cells). The effect was specific for TGF-;2 because no changes in TGF-;1 and TGF-;3 on HDL3 incubation were detected (Table 2).
To validate these findings, the expression of TGF-;1, TGF-;2, TGF-;3, and MCP-1 (as negative control) was investigated by quantitative real-time PCR. The induction of TGF-;2 was 2.31±0.66-fold for HDL3-treated cells (P<0.01). In agreement with the array data, no significant effect of HDL3 was detected on TGF-;1 (1.18±0.55-fold), TGF-;3 (1.25±0.70-fold), and MCP-1 (0.93±0.12-fold) expression.
Moreover, the effect of HDL3 on TGF-;2 was dose dependent (Figure 1A) in a range from 10 to 500 μg/mL; at these concentrations, again no effect of HDL3 on TGF-;1 and TGF-;3 expression was detected (Figure 1A). The maximal effect on TGF-;2 expression was observed at 4 hours. No effect of HDL3 on TGF-;1 and TGF-;3 expression was detected during up to 18 hours of incubation (Figure 1B).
Preincubation for 1 hour with U0126, LY294002, and SH-5 decreased the effect of HDL3 (from 2.31±0.16- to 1.25±0.19-, 1.19±0.41-, 1.24±0.16-fold with U0126, LY294002, and SH-5, respectively; all P<0.01), while a minor effect of SB 203580 was observed (1.91±0.15, P=NS) (Figure 2A).
HDL significantly increased TGF-;2 protein release (2.16±0.83-fold), an effect that was dependent on PI3K/Akt activation (Figure 2B).
To investigate the nature of the stimulatory component present in HDL, HUVECs were incubated with S1P, SPC, or apoA-I. S1P and SPC induced TGF-;2 mRNA and protein expression; apoA-I affected TGF-;2 mRNA and protein expression, but this effect did not reach statistical significance (Figure 3).
To confirm ex vivo the role of HDL on TGF-;2 expression, we analyzed the mRNA levels of TGF-; and the presence of the phosphorylated (ie, active) forms of ERK 1/2 and Akt in the aortas of hA-I mice compared with those of A-I–/– mice. These 2 mouse models with identical genetic backgrounds were chosen to allow comparison of an in vivo model of low HDL with a model of high HDL. Preliminary studies have shown that TGF-; levels and kinase activation were similar between A-I–/– mice and C57/B6 mice (data not shown).
TGF-;2 expression was significantly increased in transgenic hA-I (10.10±2.45-fold versus A-I–/–) (P=0.02), whereas no differences in TGF-;1 and TGF-;3 expression were observed between the 2 mice models (Figure 4). Increased expression of phospho-Akt and phospho-ERK 1/2 was detected in the aortas of hA-I mice compared with A-I–/– mice (Figure 5A and 5B). Immunofluorescence confirmed these findings, showing increased expression of phospho-Akt and phospho-ERK 1/2 (blue signal) in the endothelium (Figure 5D and 5E). The background of the secondary antibody (blue signal) is shown in Figure 5C with the elastic laminas (green) and nuclei (red).
Because to the best of our knowledge no antibody for selective detection of TGF-;2 in mice aortas is available, we studied the expression of the active form of the intracellular effector of TGF-; (phospho-Smad-2/3)12 to investigate whether TGF-;2 induced in the aortas of hA-I mice is functionally active. Increased expression of phospho-Smad-2/3 was detected in the aortas of hA-I mice compared with A-I–/– mice (Figure 6A through 6C). Furthermore, colocalization studies showed that phospho-Smad-2/3 is present in the nuclei of cells of the arterial wall of hA-I mice to a higher extent than in A-I–/– mice (Figure 6D). Our data also indicate that similar effects on intracellular pathways and downstream targets occur in the media of the vascular wall (Figure 6D).
Discussion
The inflammatory status in the vascular wall results from the imbalance between proinflammatory and antiinflammatory factors.4 TGF-; possesses several antiinflammatory properties on the endothelium.4,18,30 Inhibition of TGF-; signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice.16,17 These observations indicate that TGF-; is a key factor in promoting plaque stability and decreasing vascular wall inflammation.18
We show that HDL3 induces the expression of the antiinflammatory cytokine TGF-;2 in vitro and ex vivo. Our data add new insights, suggesting a further mechanism by which HDL3 may play an atheroprotective effect.
In endothelial cells, the effect of HDL3 is specific for TGF-;2 because TGF-;1 and TGF-;3 expression is not affected and is dependent mainly on PI3K/Akt activation. This observation is in agreement with the known role of Akt and ERK pathways in mediating HDL-induced endothelial gene expression and angiogenesis20,21 and supports a protective role for HDL on the endothelium.20,21 Two lysosphingolipids, S1P and SPC, which are carried by HDL and responsible for HDL-induced nitric oxide–dependent vasorelaxation31 and antiapoptotic effects,32 induced TGF-;2 expression, whereas a minor effect was observed with apoA-I. These findings support the role of both apo-AI and lysosphingolipids proposed by Nofer et al31 in the effects of HDL in vivo and provide a preliminary identification of the HDL components responsible for this effect.
Phospho-ERK1/2 and phospho-Akt expression is increased in the endothelium and vascular wall of mice expressing human apoA-I, suggesting that in vivo these 2 signaling pathways also play a major role in determining the vascular wall responses to HDL. Previous studies have shown that TGF-;1 and TGF-;3 expression is increased in advanced atherosclerotic plaques,33 in contrast to the findings in animal models in which inhibition of TGF-; promotes the switch to unstable plaque phenotype.16,17,30 These data may be reconciled by our observation of a specific effect of HDL on TGF-;2, suggesting that the antiatherosclerotic role of TGF-; may be confined to this isoform. These data also are supported by the observation that TGF-;2 downregulates the expression of proinflammatory genes induced by IL115 and LPS (Norata et al, unpublished observations) in endothelial cells. Alternatively, the increased TGF-;1 and TGF-;3 expression may represent a protective response in advanced plaques.
The ex vivo data on transgenic animals are substantiated by the increased expression of phospho-Smad-2/3, the intracellular effector of TGF-; signaling,12 in the aorta of hA-I mice, confirming that HDL-induced TGF-; expression results in increased TGF-; signaling pathway activity. Notably, this increased expression was observed in all cells of the arterial wall. Whether this is a direct effect of HDL3 or is an effect of TGF-;2 produced by endothelial cells remains to be addressed. Of note, a recent article reported that in mesangial cells S1P cross-activates the Smad signaling cascade and reduces IL-1;–induced expression of iNOS and MMP-9,34 suggesting a cross-talk of S1P with the TGF-; signaling pathways.
In summary, HDL induces TGF-;2 expression in vitro and in vivo mainly via PI3K/Akt activation; this effect results in increased TGF-;–dependent signaling in the endothelium and vascular wall cells. Because TGF-; possesses antiinflammatory properties and stabilizes the atherosclerotic plaque, our findings will help identify a novel mechanism by which HDL may exert protective effects on endothelial cells and vascular wall function.
Acknowledgments
This work was supported by grants from Consorzio Interuniversitario Ricerca Cardiovascolare, FIRB 2001 (RBNE01HLAK–006), Società Italiana Studio Aterosclerosi sezione Lombardia, and AFA Insurance and Swedish Medical Research Council (12660). We are grateful to Roberto Zecca for software assistance, Giulio Simonutti for technical assistance with the confocal microscopy, and Fabio Pellegatta for providing the HUVECs.
References
Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.
Uyemura K, Demer LL, Castle SC, Jullien D, Berliner JA, Gately MK, Warrier RR, Pham N, Fogelman AM, Modlin RL. Cross-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis. J Clin Invest. 1996; 97: 2130–2138.
Mallat Z, Besnard S, Duriez M, Deleuze V, Emmanuel F, Bureau MF, Soubrier F, Esposito B, Duez H, Fievet C, Staels B, Duverger N, Scherman D, Tedgui A. Protective role of interleukin-10 in atherosclerosis. Circ Res. 1999; 85: e17–e24.
Tedgui A, Mallat Z. Anti-inflammatory mechanisms in the vascular wall. Circ Res. 2001; 88: 877–887.
Pintavorn P, Ballermann BJ. TGF-; and the endothelium during immune injury. Kidney Int. 1997; 51: 1401–1412.
Pepper MS. Transforming growth factor-;: vasculogenesis, angiogenesis, and vessel wall integrity. Cytokine Growth Factor Rev. 1997; 8: 21–43.
Robertson AK, Rudling M, Zhou X, Gorelik L, Flavell RA, Hansson GK. Disruption of TGF-; signaling in T cells accelerates atherosclerosis. J Clin Invest. 2003; 112: 1342–1350.
Sanford LP, Ormsby I, Gittenberger-de Groot AC, Sariola H, Friedman R, Boivin GP, Cardell EL, Doetschman T. TGF;2 knockout mice have multiple developmental defects that are non-overlapping with other TGF; knockout phenotypes. Development. 1997; 124: 2659–2670.
Oh SP, Seki T, Goss KA, Imamura T, Yi Y, Donahoe PK, Li L, Miyazono K, ten Dijke P, Kim S, Li E. Activin receptor–like kinase 1 modulates transforming growth factor-beta 1 signaling in the regulation of angiogenesis. Proc Natl Acad Sci U S A. 2000; 97: 2626–2631.
Galvin KM, Donovan MJ, Lynch CA, Meyer RI, Paul RJ, Lorenz JN, Fairchild-Huntress V, Dixon KL, Dunmore JH, Gimbrone MA Jr, Falb D, Huszar D. A role for smad6 in development and homeostasis of the cardiovascular system. Nat Genet. 2000; 24: 171–174.
Schultz-Cherry S, Chen H, Mosher DF, Misenheimer TM, Krutzsch HC, Roberts DD, Murphy-Ullrich JE. Regulation of transforming growth factor-beta activation by discrete sequences of thrombospondin 1. J Biol Chem. 1995; 270: 7304–7310.
Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-; family signaling. Nature. 2003; 425: 577–584.
Akman HO, Zhang H, Siddiqui MA, Solomon W, Smith EL, Batuman OA. Response to hypoxia involves transforming growth factor-;2 and Smad proteins in human endothelial cells. Blood. 2001; 98: 3324–3331.
Ramalingam R, Rafii S, Worgall S, Brough DE, Crystal RG. E1(–)E4(+) adenoviral gene transfer vectors function as a "pro-life" signal to promote survival of primary human endothelial cells. Blood. 1999; 93: 2936–2944.
Yamagami S, Yokoo S, Mimura T, Amano S. Effects of TGF-;2 on immune response-related gene expression profiles in the human corneal endothelium. Invest Ophthalmol Vis Sci. 2004; 45: 515–521.
Mallat Z, Gojova A, Marchiol-Fournigault C, Esposito B, Kamate C, Merval R, Fradelizi D, Tedgui A. Inhibition of transforming growth factor-; signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice. Circ Res. 2001; 89: 930–934.
Lutgens E, Gijbels M, Smook M, Heeringa P, Gotwals P, Koteliansky VE, Daemen MJ. Transforming growth factor-; mediates balance between inflammation and fibrosis during plaque progression. Arterioscler Thromb Vasc Biol. 2002; 22: 975–982.
Grainger DJ. Transforming growth factor beta and atherosclerosis: so far, so good for the protective cytokine hypothesis. Arterioscler Thromb Vasc Biol. 2004; 24: 399–404.
Chiesa G, Sirtori CR. Use of recombinant apolipoproteins in vascular diseases: the case of apoA-I. Curr Opin Investig Drugs. 2002; 3: 420–426.
Calabresi L, Gomaraschi M, Franceschini G. Endothelial protection by high-density lipoproteins: from bench to bedside. Arterioscler Thromb Vasc Biol. 2003; 23: 1724–1731.
Norata GD, Callegari E, Inoue H, Catapano AL. HDL3 induces cyclooxygenase-2 expression and prostacyclin release in human endothelial cells via a p38 MAPK/CRE-dependent pathway: effects on COX-2/PGI-synthase coupling. Arterioscler Thromb Vasc Biol. 2004; 24: 871–877.
Mineo C, Yuhanna IS, Quon MJ, Shaul PW. High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J Biol Chem. 2003; 278: 9142–9149.
Ashby DT, Rye KA, Clay MA, Vadas MA, Gamble JR, Barter PJ. Factors influencing the ability of HDL to inhibit expression of vascular cell adhesion molecule-1 in endothelial cells. Arterioscler Thromb Vasc Biol. 1998; 18: 1450–1455.
Norata GD, Bjork H, Hamsten A, Catapano AL, Eriksson P. High-density lipoprotein subfraction 3 decreases ADAMTS-1 expression induced by lipopolysaccharide and tumor necrosis factor- in human endothelial cells. Matrix Biol. 2004; 22: 557–560.
Norata GD, Pirillo A, Callegari E, Hamsten A, Catapano AL, Eriksson P. Gene expression and intracellular pathways involved in endothelial dysfunction induced by VLDL and oxidized VLDL. Cardiovasc Res. 2003; 59: 169–180.
Norata GD, Pellegatta F, Hamsten A, Catapano AL, Eriksson P. Effects of HDL3 on the expression of matrix-degrading proteases in human endothelial cells. Int J Mol Med. 2003; 12: 73–78.
Chiesa G, Parolini C, Canavesi M, Colombo N, Sirtori CR, Fumagalli R, Franceschini G, Bernini F. Human apolipoproteins A-I and A-II in cell cholesterol efflux: studies with transgenic mice. Arterioscler Thromb Vasc Biol. 1998; 18: 1417–1423.
Williamson R, Lee D, Hagaman J, Maeda N. Marked reduction of high density lipoprotein cholesterol in mice genetically modified to lack apolipoprotein A-I. Proc Natl Acad Sci U S A. 1992; 89: 7134–7138.
Norata GD, Tonti L, Roma P, Catapano AL. Apoptosis and proliferation of endothelial cells in early atherosclerotic lesions: possible role of oxidized LDL. Nutr Metab Cardiovasc Dis. 2002; 12: 297–305.
Mallat Z, Tedgui A. The role of transforming growth factor ; in atherosclerosis: novel insights and future perspectives. Curr Opin Lipidol. 2002; 13: 523–529.
Nofer JR, van der Giet M, Tolle M, Wolinska I, von Wnuck Lipinski K, Baba HA, Tietge UJ, Godecke A, Ishii I, Kleuser B, Schafers M, Fobker M, Zidek W, Assmann G, Chun J, Levkau B. HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J Clin Invest. 2004; 113: 569–581.
Nofer JR, Levkau B, Wolinska I, Junker R, Fobker M, von Eckardstein A, Seedorf U, Assmann G. Suppression of endothelial cell apoptosis by high density lipoproteins (HDL) and HDL-associated lysosphingolipids. J Biol Chem. 2001; 276: 34480–34485.
Bobik A, Agrotis A, Kanellakis P, Dilley R, Krushinsky A, Smirnov V, Tararak E, Condron M, Kostolias G. Distinct patterns of transforming growth factor-; isoform and receptor expression in human atherosclerotic lesions: colocalization implicates TGF-; in fibrofatty lesion development. Circulation. 1999; 99: 2883–2891.
Xin C, Ren S, Kleuser B, Shabahang S, Eberhardt W, Radeke H, Schafer-Korting M, Pfeilschifter J, Huwiler A. Sphingosine 1-phosphate cross-activates the Smad signaling cascade and mimics transforming growth factor-;–induced cell responses. J Biol Chem. 2004; 279: 35255–35262.(Giuseppe D. Norata, PhD; )