Literature
首页医源资料库在线期刊动脉硬化血栓血管生物学杂志2007年第27卷第12期

IGF-1 Reduces Inflammatory Responses, Suppresses Oxidative Stress, and Decreases Atherosclerosis Progression in ApoE-Deficient Mice

来源:《动脉硬化血栓血管生物学杂志》
摘要:Whereasgrowthfactors,viatheirabilitytostimulatevascularsmoothmusclecell(VSMC)proliferationandmigration,havebeenthoughttoplayapermissiveroleinatherosclerosisinitiationandprogression,theroleofinsulin-likegrowthfactor-1(IGF-1)isunknown。Herewereportforthefi......

点击显示 收起

【摘要】  Objective— Whereas growth factors, via their ability to stimulate vascular smooth muscle cell (VSMC) proliferation and migration, have been thought to play a permissive role in atherosclerosis initiation and progression, the role of insulin-like growth factor-1 (IGF-1) is unknown. Here we report for the first time that IGF-1 infusion decreased atherosclerotic plaque progression in ApoE-deficient mice on a Western diet.

Methods and Results— ApoE-null mice (8 weeks) were infused with vehicle or recombinant human IGF-1 and fed a high-fat diet for 12 weeks. Analysis of aortic sinuses revealed that IGF-1 infusion decreased atherosclerotic plaque progression and macrophage infiltration into lesions. Furthermore, IGF-1 decreased vascular expression of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-, reduced aortic superoxide formation and urinary 8-isoprostane levels, and increased aortic pAkt and eNOS expression and circulating endothelial progenitor cells, consistent with an antiinflammatory, antioxidant, and prorepair effect on the vasculature.

Conclusions— Our data indicate that an increase in circulating IGF-1 reduces vascular inflammatory responses, systemic and vascular oxidant stress and decreases atherosclerotic plaque progression. These findings have major implications for the treatment of atherosclerosis.

Although insulin-like growth factor-1 (IGF-1) has been described to have pleiotropic effects in the vasculature, its role in the development and progression of atherosclerosis is obscure. Here we report that in ApoE-deficient mice fed a high-fat diet continuous infusion of IGF-1 suppressed progression of atherosclerosis potentially via antiinflammatory, antioxidant, and prorepair effects.

【关键词】  insulinlike growth factor atherosclerosis apolipoprotein E inflammatory response oxidative stress


Introduction


Atherosclerosis is the principal underlying cause of most cardiovascular disease-related deaths, the leading cause of mortality in the USA. 1 Long considered to result from progressive vascular lipid accumulation, atherosclerosis is now recognized as a chronic inflammatory disease, 2–5 in which oxidative stress plays a key initiating role. Thus, multiple oxidative stimuli including oxidation of low-density lipoprotein (LDL) in the subendothelial space can result in endothelial cell adhesion molecule expression, monocyte and T cell recruitment, and macrophage lipid accumulation and foam cell formation. 6 Growth factors, cytokines, chemokines, and proteases modulate many steps in the atherosclerotic process including the migration and proliferation of smooth muscle cells. Furthermore, vascular and extravascular progenitor cells contribute to atherogenesis, and these include circulating endothelial progenitor cells (EPCs) that are likely part of a vascular repair system. 7


Insulin-like growth factor-1 (IGF-1) is an endocrine and autocrine/paracrine growth factor that is the primary mediator of the effect of growth hormone (GH) on developmental growth. 8 IGF-1 is expressed in vascular cells 9 and in monocyte/macrophages, 10,11 but its role in atherogenesis is unknown. IGF-1 is a mitogen for endothelial cells 12 and vascular smooth muscle cells, 13 and can potentiate endothelial cell TNF- –induced c-Jun and NF- B activation. 14 Furthermore, IGF-1 has potent survival effects on vascular cells and prevents oxidized LDL–induced apoptosis of vascular smooth muscle cells. 15 We have previously shown that oxidized LDL downregulates IGF-1 and IGF-1 receptor expression in vascular smooth muscle cells 16,17 and that expression of these molecules is reduced in areas of advanced human plaque staining positive for oxidized LDL, 18 suggesting that decreased IGF-1 activity could contribute to the atherosclerotic process. Similar findings were reported in cultured plaque-derived smooth muscle cells. 19 To investigate the potential role of IGF-1 in atherogenesis we infused recombinant human IGF-1 in ApoE-deficient mice. Our findings demonstrate that increased circulating IGF-1 reduces systemic and vascular oxidant stress and vascular cytokine expression and decreases atherosclerosis progression. These findings provide fundamental new insights into biological effects of IGF-1.


Methods


Material


Recombinant human IGF-1 was from Tercica. Anti–phospho-Akt, Akt, phospho-eNOS, eNOS, Sca-1, and Flk-1 antibodies were from BD Biosciences. Dihydroethidium (DHE) was from Invitrogen. L-NAME and D-NAME were from Sigma-Aldrich.


Animals


All animal experiments were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals. ApoE-null mice (C57BL/6, 8 weeks of age) were infused with saline or IGF-1 (1.5 mg/kg/d) and fed a Western-type diet (42% of total cal from fat; 0.15% cholesterol) for 12 weeks. Mice were housed individually and maintained on a 12-hour light–dark cycle.


Preparation of Native and Oxidized LDL


Native LDL (nLDL) was separated from human plasma of healthy donors (purchased from The Blood Center, New Orleans, LA) by sodium bromide stepwise density gradient centrifugation. Oxidized LDL (OxLDL) was prepared as previously described. 17 Briefly, an aliquot of the nLDL fraction was passed through a 10-DG desalting column, and then incubated with 5 µmol/L CuSO 4 at 37°C for 3 hours. The value for thiobarbituric acid-reactive substances (TBARS) in OxLDL was 37.2±1.2 nmol/L malondialdehyde per mg protein. TBARS was not detectable in nLDL.


Atherosclerosis Quantification


Under anesthesia, IGF-1– or saline–infused mice were perfused with saline then 4% buffered paraformaldehyde plus 5% sucrose and the heart dissected, fixed overnight, and paraffin embedded. Serial sections (6 µm) were taken throughout the entire aortic valve area (AVA) as per Paigen et al, 20 and routinely stained with hematoxylin and eosin (H&E). To quantitate plaque size, images of 3 sections of each AVA were acquired with a DP70 digital camera connected to a microscope (Olympus), and lesion areas determined using Image-Pro plus v 6.0 software (Media Cybernetics Inc). The mean value of plaque cross-sectional areas from 3 sections was used to estimate the extent of atherosclerosis for each animal. We also quantified atherosclerosis using Oil Red O–stained frozen sections of aortic valves. Serial sections (20 µm) were cut from the site where the aorta valve cusps appear to the aorta root, and every other section was stained with Oil Red O (5 mg/mL for 12 minutes) and counterstained with hematoxylin before quantification. For a limited number of animals atherosclerosis was quantified by measuring surface area of Oil Red O–positive lesions on en face preparations of whole aortas. Under anesthesia, the mouse was perfused through the left ventricle as mentioned above then the entire aorta was dissected and placed overnight in formaldehyde. The adventitial fat was dissected, the aorta was stained with Oil Red O and opened longitudinally, pinned en face, and photographed. The total arterial surface area and total lesion area were determined using Image-Pro Plus. The extent of lesion development was defined as percentage of the total area of a given artery that was occupied by Oil Red O–positive atherosclerotic lesions.


Immunohistochemical Analysis


Serial 6-µm paraffin-embedded cross sections were taken throughout the entire AVA, and 3 sets of serial sections obtained at 60-µm intervals were used for measurement of macrophage lesion area. Every first section in each set was stained with H&E, and plaque area size was estimated as described above. The second and third sections in each set were incubated with rat anti-mouse Mac-3 monoclonal antibody (1:20, M3/84 clone, BD Pharmingen) or with isotype-matched rat IgG (Abcam) followed by incubation with biotinylated secondary antibody and avidin-peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories Inc). Sections were developed with DAB substrate kit, and counterstained with hematoxylin.


Aortic Superoxide Measurement


Aortic superoxide levels were measured with DHE on serial frozen sections (10 µm) obtained from the root of aorta. DHE specificity was previously validated using smooth muscle cells exposed to OxLDL. 21 Two serial sections each from IGF-1–infused and saline-infused mice were tested in parallel. One pair of sections from IGF-1 and saline-infused mice were pretreated with superoxide scavenger polyethylene glycol (PEG)-superoxide dismutase (PEG-SOD, 100 U, Sigma) 22 in buffer (50 mmol/L Tris-HCL, pH 7.4), and another pair of sections was preincubated with buffer only. All 4 sections were stained by DHE (2 µmol/L, 45 minutes, 37°C) in the dark in a humidified chamber, briefly washed, and quickly imaged with a fluorescent microscope keeping the same exposure for every section. DHE fluorescence was quantified by averaging the mean fluorescence intensity within 3 identical circles placed on a plaque-free area of aortic wall using Image-Pro Plus. The superoxide-induced DHE signal was expressed as PEG-SOD-inhibitable fluorescence after subtraction of the DHE signal obtained from scavenger-pretreated section. Three to 5 sections from each animal (n=4 for each saline-or IGF-1-infused group) were analyzed using this procedure and the average superoxide-induced DHE fluorescence was calculated.


Urine 8-Isoprostane Assay


Urine was collected for 24 hours in the presence of antioxidant butylated hydroxytoluene (BHT) (1 mmol/L), filtered with 0.2 µm filters, and frozen at –80°C. Commercially available ELISA (Cayman Chemical) was used to measure urinary 8-isoprostane (8-IP) levels, and the results were expressed relative to the urinary creatinine levels determined by colorimetric assay (Cayman Chemical). All assays were repeated 3 times.


Quantitative Real-Time RT-PCR


Total RNA extraction and real-time PCR was performed as previously described. 21 Briefly, total aortic RNA was isolated using the TriPure Isolation Reagent (Roche) followed by purification with the RNeasy mini kit (Qiagen). cDNA was synthesized using the First Strand cDNA Synthesis kit (Amersham) and used for the 40-cycle 2-step PCR with sequence-specific primer pairs in the iCycler IQ Real-Time Detection System (Bio-Rad).


Flow Cytometry


White blood cells in EDTA-treated whole blood were immunostained with R-phycoerythrin (PE)-conjugated anti-mouse Flk-1 monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated anti-mouse Sca-1 monoclonal antibody and were analyzed by flow cytometer immediately. Sca-1+/Flk-1+ cells were considered to be endothelial progenitors, and were counted to evaluate circulating EPC number.


Cell Culture


Human aortic endothelial cells (HAECs) were obtained from Lonza and maintained in EGM-2 medium with supplements. Cells at passages 2 to 10 were used for experiments. Cultured human aortic smooth muscle cells (HASMCs; Cambrex Bio Science, Walkersville, Md) were grown in SmBM medium supplemented with 5% fetal calf serum, antibiotics, and supplements. The cells were used for experiments at passages 4 to 10.


Quantification of Reactive Oxygen Species Production


Generation of intracellular superoxide was measured as described by Higashi et al, 17 using DHE fluorescence. HAECs were grown to confluence on a 96-well plate and were washed once with phenol red-free EGM medium. The cells were preincubated in the same medium containing 100 ng/mL IGF-1 and 100 µmol/L D-NAME or L-NAME for 1 hour, and subsequently treated with 60 µg/mL native LDL or OxLDL for 2 hours. Superoxide formation was determined by staining cells with 5 µmol/L DHE for 30 minutes. The fluorescent intensity was read directly from the culture plate at emission wavelength of 590 nm and excitation wavelength of 485 nm. HASMCs were preincubated for 24 hours with 0 to 100 ng/mL IGF-1 before exposure to 60 µg/mL native LDL or OxLDL or 100 nmol/L Ang II and reactive oxygen species (ROS) production was measured as described above.


Statistical Analysis


All numerical data are expressed as mean±SEM. Two-tailed unpaired Student t tests were performed to determine statistical significance. Differences were considered significant at P <0.05.


Results


IGF-1 Reduces Macrophage Infiltration, TNF- Levels, and Decreases Atherosclerosis Progression in ApoE –/– Mice


IGF-1 and saline-infused ApoE-deficient mice fed a Western diet developed early stages of atherosclerotic lesions (presence of foam cells were evident) and advanced lesions (cholesterol crystals and acellular areas were present). Morphometric analysis of atherosclerotic plaque accumulation on the intimal surface of the aortic valve revealed that IGF-1-infused mice had a 27% reduction in lesion size compared with control mice (0.259±0.018 mm 2 versus 0.356±0.031 mm 2; Figure 1 A). En face analysis of whole aortas revealed that IGF-1–infused mice had a similar 28% reduction in lesion size (IGF-1, 8.61±0.53% versus control 12.01±2.76%, n=7), although this effect was not statistically significant ( P =0.22). We estimated macrophage levels within the atherosclerotic lesions of IGF-1–infused and control mice as an index of atherosclerotic burden and also as an indicator of inflammatory responses. The plaque area staining positive for macrophages in IGF-1–infused mice was decreased by 36% compared with control (0.050±0.003 mm 2 versus 0.078±0.007 mm 2; Figure 1 B) with a slight reduction in macrophage area/lesion area ratio (20.2±1.7% versus 25.3±1.8%, P =0.07). TNF- –immunopositive plaque area was also decreased by 58.3% in IGF-1–infused mice compared with control ( P <0.05; Figure 1 C). These data demonstrate that IGF-1 reduced macrophage infiltration and atherosclerotic plaque progression over 12 weeks, consistent with an antiinflammatory effect.


Figure 1. IGF-1 suppresses atherosclerotic plaque progression, reduces macrophage accumulation, and decreases TNF- level in aortic valves of ApoE-deficient mice. A, Frozen or paraffin-embedded cross sections from saline- or IGF-1–infused ApoE-deficient mice were obtained throughout the aortic valve area and stained with Oil Red O and hematoxylin or H&E, respectively, then imaged and quantified with Image-Pro Plus. Magnification x 100. B and C, IGF-1 reduces macrophage accumulation (B) and decreases TNF- level (C) within atherosclerotic plaque. Paraffin-embedded serial sections were stained with H&E, rat anti-mouse Mac-3 antibody, and with rat non-specific IgG (B) or rabbit anti-mouse TNF- antibody and rabbit non-specific IgG, respectively (C). Magnification x 400. Aortic lesion size, Mac-3– and TNF- –positive lesion areas are shown for each mouse from saline-infused group (empty circles) or IGF-1-infused group (solid circles). Vertical bars are mean±SEM per group.


IGF-1 Suppresses Oxidative Stress in ApoE –/– Mice and in Cultured Endothelial Cells


8-isoprostane levels are an index of systemic oxidative stress that correlate with the severity of atherosclerosis in humans and in animal models. 23 We found that IGF-1 infusion decreased urine 8-IP levels by 39% ( Figure 2 A). To measure the effect of IGF-1 on aortic superoxide levels, frozen sections from the aortic roots of IGF-1–infused and control mice were stained with DHE with/without pretreatment with the superoxide-specific scavenger PEG-SOD ( Figure 2B and 2 C). IGF-1 markedly suppressed superoxide levels in the aortae of ApoE-deficient mice (85% decrease compared with saline-infused mice; Figure 2 C).


Figure 2. IGF-1 inhibits oxidative stress in vivo and in cultured endothelial cells. A, IGF-1 decreases urine 8-isoprostane levels, an index of systemic oxidative stress. Urine 8-isoprostane (8-IP) levels were normalized to creatinine levels. All assays were repeated 3 times and mean values are shown. B and C, IGF-1 decreases aortic superoxide levels. Aortic superoxides were measured by staining aortic root sections with DHE with or without pretreatment with PEG-SOD. B, Representative images; C, Superoxide-induced DHE signal is expressed as PEG-SOD-inhibitable mean fluorescence for each mouse from saline-infused group (empty circles) or IGF-1–infused group (solid circles). Vertical bars are mean±SEM per group. D, IGF-1 reduces oxLDL-induced ROS generation in human aortic endothelial cells. Human aortic endothelial cells were incubated with indicated concentration (ng/mL) of recombinant human IGF-1 and D-NAME (empty bar; 100 µmol/L) or L-NAME (solid bar; 100 µmol/L) for 1 hour and for another 2 hours with nLDL or OxLDL (60 µg/mL) in addition to IGF-1 and D-/L-NAME. Superoxide generation was determined by coincubating the cells with 5 µmol/L DHE for 30 minutes followed by fluorescent intensity determination. * P <0.001 vs nLDL-treated controls (n=4). # P <0.001 vs OxLDL and no IGF-1–treated cells (n=4).


We explored whether IGF-1 could reduce oxidative stress in cultured HAECs or HASMCs. Consistent with previous reports 24,25 oxidized LDL induced superoxide formation in endothelial cells after 2 hours of incubation. Intriguingly, coincubation with IGF-1 suppressed oxLDL-induced superoxide generation (66% decrease with 100 ng/mL; n=4, P <0.001; Figure 2 D). L-NAME, a nitric oxide (NO) synthase inhibitor, did not significantly blunt this antioxidant effect of IGF-1. Superoxide levels with L-NAME tended to be higher than those with the inactive isomer D-NAME, but not significantly so. OxLDL or angiotensin II (Ang II) markedly increased cellular superoxides in HASMCs, however unlike endothelial cells, pretreatment with IGF-1 had no effect on oxLDL- or Ang II–induced superoxides in this cell model (supplemental Figure IA and IB, available online at http://atvb.ahajournals.org). These data suggest that IGF-1–dependent suppression of aortic superoxide levels is primarily mediated via the endothelium.


IGF-1 Downregulates Vascular Proinflammatory Cytokine Gene Expression and Increases Endothelial NO Synthase Expression and Circulating Endothelial Progenitor Cells


IGF-1 infusion did not change body weight or total cholesterol levels ( Table 1 ), indicating that the atheroprotective effects of IGF-1 were not attributable to elimination of the hypercholesterolemic source of vascular injury in ApoE-deficient mice. Additionally, lipid profiles were not affected by IGF-1 as assessed by high-performance liquid chromatography (HPLC)-based serum lipoprotein fractionation (supplemental Figure II and supplemental Table). We also found no statistically significant difference in blood glucose levels between saline- and IGF-1–infused groups (266±45 mg/dL, n=7 and 233±43 mg/dL, n=5, respectively). However, mouse (endogenous) IGF-1 levels were significantly lower in IGF-1–infused mice ( P <0.001, n=7), probably because of a negative feed-back effect. The total concentration of IGF-1 (mouse IGF-1 plus human IGF-1) in IGF-1–infused mice was approximately twice that of saline-infused mice ( Table 1 ). Using 60% increase in IGF-1 binding protein(s) (IGFBPs) sized 30 kDa (n=7, P <0.01) for IGF-1 mice compared with control (data not shown), consistent with an increase in IGFBP-5. Furthermore, IGF-1 infusion produced a 2.5-fold increase in lung and aortic levels of phosphorylated Akt, a key downstream molecule in the IGF-1 signaling pathway ( Figure 3 ).


Table 1. Serum IGF-1, Total Cholesterol, and Proinflammatory Cytokines


Figure 3. IGF-1 increases Akt phosphorylation in mouse lung and aorta. Lung and aortic homogenates were subjected to immunoblotting analysis with rabbit anti–phospho-Akt and mouse anti-total Akt antibody. Representative Westerns (A) and densitometry data (n=3, B) are shown. * P <0.05 vs saline-infused mice.


To further determine mechanisms of the antiatherogenic effect of IGF-1, we measured tissue and circulating levels of IL-6 and TNF-, proinflammatory cytokines involved in atherosclerotic lesion development. No changes were detected in serum levels of these cytokines; however, aortic IL-6 and TNF- mRNA levels were reduced 2.9-fold and 4.2-fold, respectively ( Table 2 ), consistent with a reduction in macrophage and TNF- immunopositivity (see Figure 1B and 1 C).


Table 2. Aortic eNOS and Proinflammatory Cytokine Gene Expression


Circulating endothelial progenitor cells (EPCs) contribute to angiogenesis in normal vascular development and are part of a vascular repair system under pathological conditions including atherosclerosis. 26,27 We measured circulating EPC levels in IGF-1– and saline-infused mice by assessing Sca-1+/Flk-1+ cells by fluorescence-activated-cell sorter (FACS) analysis. There was a strong trend to more circulating EPCs in IGF-1–infused mice compared with control ( Figure 4, 3.58±0.82% versus 1.61±0.42%, P =0.06, n=8 in IGF-1–infused mice and n=7 in saline-infused mice). This result suggests that IGF-1 promotes endothelial repair by increasing circulating EPCs, potentially contributing to atheroprotection.


Figure 4. IGF-1 increases circulating endothelial progenitor cells. White blood cells were immunostained with anti–Sca-1 antibody conjugated with FITC and anti–Flk-1 antibody conjugated with PE, and the positively stained population was determined by flow cytometry. The left panel indicates a representative result from 7 independent analyses of the saline-infused mice, and the right from 8 independent analyses of the IGF-1-infused mice.


Endothelial nitric oxide synthase (eNOS) is the major NO-producing enzyme in the vasculature and as such can exert multiple beneficial effects including an antioxidant effect and atheroprotective effect. 28 It has been reported that eNOS activity is essential for IGF-1–stimulated EPC mobilization, 29 and eNOS expression is inhibited in advanced atherosclerosis. 30 We found that IGF-1 infusion markedly upregulated eNOS gene expression in the aorta ( Table 2 ) and IGF-1 increased eNOS Ser1177 phosphorylation in cultured endothelial cells (supplemental Figure IIIA). Because increased eNOS expression and phosphorylation could mediate an increase in NO levels, we measured plasma and urinary nitrate/nitrite (NOx) as a systemic index of NO bioavailability. The proatherogenic diet reduced urinary NOx levels by 2.5- to 3-fold in both saline- and IGF-1–infused mice compared with basal (pre-surgery) NOx levels (supplemental Figure IIIB). However, this reduction was significantly blunted by IGF-1 infusion (NOx levels, saline group, 14.12±1.35 µmol/d/g BW, n=8 versus IGF-1 group, 23.62±3.44 µmol/d/g BW, n=8, P <0.05). Consistently, plasma NOx levels were also higher in IGF-1 mice versus saline control (data not shown). These results provide initial evidence suggesting that eNOS and eNOS-derived nitric oxide may be involved in the antiatherogenic effect of IGF-1.


Discussion


Multiple growth factors may participate in the atherosclerotic process, including IGF-1, which circulates at high levels, is expressed in the arterial wall and has endocrine and autocrine/paracrine effects. 8 Growth factors such as platelet-derived growth factor and IGF-1 have traditionally been considered to contribute to the atherosclerotic process via their ability to stimulate smooth muscle cell migration and proliferation. 2 However, there is limited data to support this hypothesis as it relates to IGF-1. Of note, IGF-1 is overexpressed in the rat aorta after balloon injury. 31 However, in this model of restenosis, a stable D-peptide analogue of IGF-1 reduced intimal smooth muscle proliferation but did not significantly reduce intimal thickening. 32 Inhibition of the Vβ3 integrin reduces IGF-1–mediated signaling and atherosclerotic lesion formation in hypercholesterolemic pigs. 33 However, this model includes a component of mechanical injury, and Vβ3 inhibition is not specific for IGF-1 signaling. We and others have shown that IGF-1 has pleiotropic effects on vascular smooth muscle cells in vitro 15,16,18,34 including inhibition of oxidized LDL–induced apoptosis. 15 In the current study, we report that an approximate 2-fold increase in circulating levels of IGF-1 reduces atherosclerotic burden in ApoE-deficient mice, and that this reduction is associated with a constellation of potential beneficial effects including decreased expression of vascular proinflammatory cytokines, a decrease in macrophage accumulation within atherosclerotic lesions, suppression of oxidative stress, upregulation of vascular eNOS expression, and increased circulating EPCs. Of note, the reduction in atherosclerotic burden as assessed by en face analysis of whole aortas was not statistically significant, possibly because of limited sample size and significant interanimal variability of this measurement. However, we cannot exclude a preferential effect of IGF-1 on the aortic valve area.


To our knowledge, this is the first report of an antiatherogenic effect of IGF-1. Clearly, mechanisms are likely complex and represent the sum effects of IGF-1 on multiple cell types and potentially multiple signaling pathways involved in atherogenesis. Our current data suggests that one major mechanism underlying the antiatherogenic effect of IGF-1 could be an antioxidant effect targeting the endothelium and leading to decreases in aortic superoxide levels and macrophage infiltration. We demonstrated that IGF-1 protected aortic endothelial cells but not aortic smooth muscle cells from oxidized LDL-induced reactive oxygen species formation in vitro and that IGF-1 increased Ser1177 eNOS phosphorylation in endothelial cells. Because eNOS mRNA levels were increased in the aortae of ApoE-deficient mice in response to IGF-1, it is likely that increased eNOS expression or activation led to an increase in nitric oxide bioavailability and in plasma/urinary nitrate/nitrite levels. It has been reported that restoration of eNOS functional activity in animal models of atherosclerosis leads to reduced oxidative stress, inflammation, and atherosclerosis. 35,36 Furthermore, IGF-1 can increase eNOS activity through the PI 3-kinase/Akt pathway, 37 and as we demonstrated IGF-1 infusion markedly increases tissue Akt phosphorylation in ApoE-deficient mice (see Figure 3 ). Because eNOS appears to be essential for mobilization of EPCs, 38 our data indicating that IGF-1 increased circulating EPC levels could also potentially be explained via IGF-1–induced increase in eNOS expression or activity. Interestingly, recently Thum et al have shown that IGF-1 increased EPC levels and improved EPC function in mice and that inhibition of eNOS abolished the stimulatory effect of IGF-1 on migration of EPCs. 29 Because EPCs play an important role in vascular repair, 26,39,40 their upregulation by IGF-1 could contribute to the antiatherosclerotic effect of IGF-1. Interestingly, in cultured endothelial cells, the ability of IGF-1 to reduce ROS was not blunted by L-NAME, suggesting that at least in vitro, the antioxidant effect of IGF-1 may be in part NO-independent. Extrapolation of this data, however, to the in vivo situation, is tenuous at best, particularly in view of the potential role of eNOS uncoupling in inflammatory states that could contribute to ROS generation. 28 Further studies using transgenic approaches will be required to determine the contribution of eNOS to the antioxidant and antiatherogenic effect of IGF-1 in vivo. In this respect, the report that IGF-1 overexpression attenuated cardiomyocyte death in diabetic mice by interfering with angiotensin II–dependent superoxide formation 41 is relevant and supports the hypothesis that IGF-1 has antioxidant effects in vivo.


Our finding that IGF infusion decreased TNF- and IL-6 gene expression in the aorta is consistent with the report that local IGF-1 gene transfer attenuated TNF- expression in burn wounds 42 and that IGF-1/BP-3 infusion in burned children reduced IL-6 levels. 43,44 Furthermore, the IGF-1–induced reduction in atherosclerotic burden in ApoE-deficient mice is consistent with the growing evidence from epidemiological studies that IGF-1 may be atheroprotective. 45 Thus, most studies, but not all, 46 have suggested that lower serum IGF-1 is associated with an increased risk of ischemic heart disease and congestive heart failure. 47,48


In summary, we have demonstrated that IGF-1 infusion in ApoE-deficient mice decreases macrophage infiltration and atherosclerotic plaque burden. This beneficial effect is accompanied by a reduction in vascular IL-6 and TNF- gene expression without changes in circulating cytokine or lipid levels. Furthermore, IGF-1 markedly reduces vascular superoxide levels and systemic oxidative stress and upregulates vascular eNOS expression and circulating EPC. These findings establish a new paradigm for biological effects of IGF-1 and have major implications for treating atherosclerosis.


Acknowledgments


The authors thank Jean-Luc Delafontaine and John Wolpers for their assistance in atherosclerosis data acquisition.


Sources of Funding


This work was supported by grants from the National Institute of Health R01HL070241 and R01HL080682 (to P.D.) and a grant from the Tulane Research Enhancement Fund (P.D.).


Disclosures


None.


S.S. and Y.H. contributed equally to this study.


Original received May 25, 2007; final version accepted September 24, 2007.

【参考文献】
  Rosamond W, Flegal K, Friday G. Heart disease and stroke statistics: 2007 update. Circulation. 2007; 115: e69–e171.

Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999; 340: 115–126.

Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.

Libby P. Inflammation and cardiovascular disease mechanisms. Am J Clin Nutr. 2006; 83: 456S–460S.

Hansson GK, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol. 2006; 6: 508–519.

Parthasarathy S, Rankin SM. Role of oxidized low density lipoprotein in atherogenesis. Prog Lipid Res. 1992; 31: 127–143.

Werner N, Nickenig G. Clinical and therapeutical implications of EPC biology in atherosclerosis. J Cell Mol Med. 2006; 10: 318–332.

Delafontaine P, Song YH, Li Y. Expression, regulation, and function of IGF-1, IGF-1R, and IGF-1 binding proteins in blood vessels. Arterioscler Thromb Vasc Biol. 2004; 24: 435–444.

Delafontaine P, Lou H, Alexander RW. Regulation of insulin-like growth factor I messenger RNA levels in vascular smooth muscle cells. Hypertension. 1991; 18: 742–747.

Wynes MW, Riches DW. Transcription of macrophage IGF-I exon 1 is positively regulated by the 5'-untranslated region and negatively regulated by the 5'-flanking region. Am J Physiol Lung Cell Mol Physiol. 2005; 288: L1089–L1098.

Rom WN, Basset P, Fells GA, Nukiwa T, Trapnell BC, Crysal RG. Alveolar macrophages release an insulin-like growth factor I-type molecule. J Clin Invest. 1988; 82: 1685–1693.

Bar RS, Boes M, Dake BL, Booth BA, Henley SA, Sandra A. Insulin, insulin-like growth factors, and vascular endothelium. Am J Med. 1988; 85: 59–70.

Delafontaine P. Growth factors and vascular smooth muscle cell growth responses. Eur Heart J. 1998; 19 Suppl G: G18–G22.

Che W, Lerner-Marmarosh N, Huang Q, Osawa M, Ohta S, Yoshizumi M, Glassman M, Lee JD, Yan C, Berk BC, Abe J. Insulin-like growth factor-1 enhances inflammatory responses in endothelial cells: role of Gab1 and MEKK3 in TNF-alpha-induced c-Jun and NF-kappaB activation and adhesion molecule expression. Circ Res. 2002; 90: 1222–1230.

Li Y, Higashi Y, Itabe H, Song YH, Du J, Delafontaine P. Insulin-like growth factor-1 receptor activation inhibits oxidized LDL-induced cytochrome C release and apoptosis via the phosphatidylinositol 3 kinase/Akt signaling pathway. Arterioscler Thromb Vasc Biol. 2003; 23: 2178–2184.

Scheidegger KJ, James RW, Delafontaine P. Differential effects of low density lipoproteins on insulin-like growth factor-1 (IGF-1) and IGF-1 receptor expression in vascular smooth muscle cells. J Biol Chem. 2000; 275: 26864–26869.

Higashi Y, Peng T, Du J, Sukhanov S, Li Y, Itabe H, Parthasarathy S, Delafontaine P. A redox-sensitive pathway mediates oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor. J Lipid Res. 2005; 46: 1266–1277.

Okura Y, Brink M, Itabe H, Scheidegger KJ, Kalangos A, Delafontaine P. Oxidized low-density lipoprotein is associated with apoptosis of vascular smooth muscle cells in human atherosclerotic plaques. Circulation. 2000; 102: 2680–2686.

Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest. 1995; 95: 2266–2274.

Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis. 1987; 68: 231–240.

Sukhanov S, Higashi Y, Shai SY, Itabe H, Ono K, Parthasarathy S, Delafontaine P. Novel effect of oxidized low-density lipoprotein: cellular ATP depletion via downregulation of glyceraldehyde-3-phosphate dehydrogenase. Circ Res. 2006; 99: 191–200.

Faraci FM, Didion SP. Vascular protection: superoxide dismutase isoforms in the vessel wall. Arterioscler Thromb Vasc Biol. 2004; 24: 1367–1373.

Patrignani P, Tacconelli S. Isoprostanes and other markers of peroxidation in atherosclerosis. Biomarkers. 2005; 10 Suppl 1: S24–29.

Rueckschloss U, Galle J, Holtz J, Zerkowski HR, Morawietz H. Induction of NAD(P)H oxidase by oxidized low-density lipoprotein in human endothelial cells: antioxidative potential of hydroxymethylglutaryl coenzyme A reductase inhibitor therapy. Circulation. 2001; 104: 1767–1772.

Galle J, Lehmann-Bodem C, Hubner U, Heinloth A, Wanner C. CyA and OxLDL cause endothelial dysfunction in isolated arteries through endothelin-mediated stimulation of O(2)(-) formation. Nephrol Dial Transplant. 2000; 15: 339–346.

Xu Q, Zhang Z, Davison F, Hu Y. Circulating progenitor cells regenerate endothelium of vein graft atherosclerosis, which is diminished in ApoE-deficient mice. Circ Res. 2003; 93: e76–e86.

Werner N, Nickenig G. Endothelial progenitor cells in health and atherosclerotic disease. Ann Med. 2007; 39: 82–90.

Kawashima S, Yokoyama M. Dysfunction of endothelial nitric oxide synthase and atherosclerosis. Arterioscler Thromb Vasc Biol. 2004; 24: 998–1005.

Thum T, Hoeber S, Froese S, Klink I, Stichtenoth DO, Galuppo P, Jakob M, Tsikas D, Anker SD, Poole-Wilson PA, Borlak J, Ertl G, Bauersachs J. Age-dependent impairment of endothelial progenitor cells is corrected by growth-hormone-mediated increase of insulin-like growth-factor-1. Circ Res. 2007; 100: 434–443.

Oemar BS, Tschudi MR, Godoy N, Brovkovich V, Malinski T, Luscher TF. Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis. Circulation. 1998; 97: 2494–2498.

Cercek B, Fishbein MC, Forrester JS, Helfant RH, Fagin JA. Induction of insulin-like growth factor I messenger RNA in rat aorta after balloon denudation. Circ Res. 1990; 66: 1755–1760.

Hayry P, Myllarniemi M, Aavik E, Alatalo S, Aho P, Yilmaz S, Raisanen-Sokolowski A, Cozzone G, Jameson BA, Baserga R. Stabile D-peptide analog of insulin-like growth factor-1 inhibits smooth muscle cell proliferation after carotid ballooning injury in the rat. Faseb J. 1995; 9: 1336–1344.

Nichols TC, du Laney T, Zheng B, Bellinger DA, Nickols GA, Engleman W, Clemmons DR. Reduction in atherosclerotic lesion size in pigs by alphaVbeta3 inhibitors is associated with inhibition of insulin-like growth factor-I-mediated signaling. Circ Res. 1999; 85: 1040–1045.

Bornfeldt KE, Raines EW, Nakano T, Graves LM, Krebs EG, Ross R. Insulin-like growth factor-I and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation. J Clin Invest. 1994; 93: 1266–1274.

Alp NJ, McAteer MA, Khoo J, Choudhury RP, Channon KM. Increased endothelial tetrahydrobiopterin synthesis by targeted transgenic GTP-cyclohydrolase I overexpression reduces endothelial dysfunction and atherosclerosis in ApoE-knockout mice. Arterioscler Thromb Vasc Biol. 2004; 24: 445–450.

van Haperen R, de Waard M, van Deel E, Mees B, Kutryk M, van Aken T, Hamming J, Grosveld F, Duncker DJ, de Crom R. Reduction of blood pressure, plasma cholesterol, and atherosclerosis by elevated endothelial nitric oxide. J Biol Chem. 2002; 277: 48803–48807.

Isenovic ER, Divald A, Milivojevic N, Grgurevic T, Fisher SE, Sowers JR. Interactive effects of insulin-like growth factor-1 and beta-estradiol on endothelial nitric oxide synthase activity in rat aortic endothelial cells. Metabolism. 2003; 52: 482–487.

Aicher A, Heeschen C, Mildner-Rihm C, Urbich C, Ihling C, Technau-Ihling K, Zeiher AM, Dimmeler S. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med. 2003; 9: 1370–1376.

Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, Wang T, Gregg D, Ramaswami P, Pippen AM, Annex BH, Dong C, Taylor DA. Aging, progenitor cell exhaustion, and atherosclerosis. Circulation. 2003; 108: 457–463.

Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.

Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F, Nadal-Ginard B, Leri A, Anversa P. IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress. Diabetes. 2001; 50: 1414–1424.

Spies M, Nesic O, Barrow RE, Perez-Polo JR, Herndon DN. Liposomal IGF-1 gene transfer modulates pro- and anti-inflammatory cytokine mRNA expression in the burn wound. Gene Ther. 2001; 8: 1409–1415.

Jeschke MG, Barrow RE, Herndon DN. Insulinlike growth factor I plus insulinlike growth factor binding protein 3 attenuates the proinflammatory acute phase response in severely burned children. Ann Surg. 2000; 231: 246–252.

Spies M, Wolf SE, Barrow RE, Jeschke MG, Herndon DN. Modulation of types I and II acute phase reactants with insulin-like growth factor-1/binding protein-3 complex in severely burned children. Crit Care Med. 2002; 30: 83–88.

Kaplan RC, Strickler HD, Rohan TE, Muzumdar R, Brown DL. Insulin-like growth factors and coronary heart disease. Cardiol Rev. 2005; 13: 35–39.

Kawachi S, Takeda N, Sasaki A, Kokubo Y, Takami K, Sarui H, Hayashi M, Yamakita N, Yasuda K. Circulating insulin-like growth factor-1 and insulin-like growth factor binding protein-3 are associated with early carotid atherosclerosis. Arterioscler Thromb Vasc Biol. 2005; 25: 617–621.

Juul A, Scheike T, Davidsen M, Gyllenborg J, Jorgensen T. Low serum insulin-like growth factor I is associated with increased risk of ischemic heart disease: a population-based case-control study. Circulation. 2002; 106: 939–944.

Vasan RS, Sullivan LM, D?Agostino RB, Roubenoff R, Harris T, Sawyer DB, Levy D, Wilson PW. Serum insulin-like growth factor I and risk for heart failure in elderly individuals without a previous myocardial infarction: the Framingham Heart Study. Ann Intern Med. 2003; 139: 642–648.


作者单位:Cardiology Section (S.S., Y.H., S.-Y.S., C.V., J.T., P.D.), Department of Medicine, Tulane University School of Medicine, New Orleans La; the School of Life Sciences (J.M.), Arizona State University, Tempe; the Laboratory of Heart Failure and Stem Cells (Y.L.), Texas Heart Institute, Houston; and th

作者: Sergiy Sukhanov; Yusuke Higashi; Shaw-Yung Shai; C
医学百科App—中西医基础知识学习工具
  • 相关内容
  • 近期更新
  • 热文榜
  • 医学百科App—健康测试工具