Accelerated Arteriosclerosis of Vein Grafts in Inducible NO SynthaseeC/eC Mice Is Related to Decreased Endothelial Progenitor Cell Repair
http://www.100md.com
《循环研究杂志》
the Department of Cardiac and Vascular Sciences (U.M., Z.Z., Y.H., Q.X.), St George’s University of London, London, United Kingdom
Institute for Pathophysiology (Y.Z., H.D.), Innsbruck Medical University, Austria.
Abstract
Inducible NO synthase (iNOS) is expressed by macrophages and smooth muscle cells in atherosclerotic lesions. Previously, we have established a mouse model for vein graft arteriosclerosis by grafting autologous jugular veins or vena cava to carotid arteries. Using this model, we studied the role of iNOS in the development of vein graft arteriosclerosis in iNOSeC/eC mice. Four weeks after grafting, neointimal hyperplasia of vein grafts in iNOSeC/eC mice was increased 2-fold compared with that of wild-type controls. Neointimal lesions contained mainly MAC-1+ macrophages and -actin+ smooth muscle cells (SMCs) in both vein grafts of iNOSeC/eC and iNOS+/+ mice. Immunofluorescence analysis revealed that increased iNOS expression in neointimal macrophages and SMCs of wild-type, but not iNOSeC/eC, mice coincided with increased vascular endothelial growth factor (VEGF) expression in vein grafts. When vein grafts were performed in iNOSeC/eC/TIE2-LacZ transgenic mice expressing LacZ gene only in endothelial cells, the number of -galactosidase+ cells in iNOSeC/eC vein grafts were significantly decreased. Furthermore, treatment with the NOS inhibitor NG-nitro-L-arginine methyl ester resulted in delayed endothelial progenitor cell attachment, whereas L-arginine intake through drinking water enhanced endothelial repair. Interestingly, local application of VEGF to iNOSeC/eC vein grafts restored endothelial progenitor homing and reduced neointimal lesions, whereas the VEGF receptor inhibitor SU1498 increased the lesion formation. Additionally, iNOS-deficient SMCs showed a low level of VEGF production in response to interleukin 1 stimulation. Thus, iNOS deficiency accelerates neointima formation by abrogating VEGF production and endothelial progenitor cell attachment and differentiation.
Key Words: mouse models iNOS vein grafts progenitor cells neointimal hyperplasia
Introduction
Autologous vein grafts are a common clinical procedure for vascular reconstruction, but their patency rate is limited by obliterative stenosis because of proliferative thickening of the intima. Neointimal hyperplasia develops rapidly once veins are subject to arterial blood pressure.1 A hallmark of lesion initiation is endothelial cell damage, which occurs within the first week after surgery.2 Previously, we established a mouse model of vein graft arteriosclerosis that resembles certain characteristics of human vein graft disease.3 We demonstrated that 1 of the earliest events after grafting was vascular cell apoptosis followed by endothelial progenitor cell repair.4,5 Venous endothelial cells became apoptosis in the arterial circulation and were replaced by circulating endothelial progenitors, which reached confluence within 4 weeks and differentiated into mature endothelial cells.5 Thus, the balance between apoptosis and progenitor cell repair could be crucial in determining lesion development in vein grafts.
NO is thought to be a key regulator in the development of atherosclerosis.6eC9 Classical cardiovascular risk factors, such as hypertension, diabetes mellitus, hypercholesterolemia, and smoking all impair NO function. NO is formed from L-arginine by the enzyme NO synthase (NOS). Three distinct isoenzymes of NOS are known to exist: constitutive-type isoforms, such as neuronal NOS (nNOS) and endothelial cell NOS (eNOS), and the inducible type of the enzyme (iNOS). The latter is widely distributed in a variety of cell types, including SMCs and leukocytes, responds to a variety of stimuli, and can produce a very high output of NO.10 NO is a pleiotropic signaling molecule involved in numerous processes,11 including lipid oxidation, mononuclear cell infiltration, and SMC homeostasis.12eC16 Notably, eNOS is essential for the survival, migration, and angiogenic response of mature endothelial cells and has recently been implicated in endothelial progenitor cell mobilization.17 However, it remains to be clarified whether iNOS might be important for endothelial progenitor cell recruitment to sites of injury.
To elucidate the role of iNOS in vascular graft-induced neointima formation, we performed vein bypass grafts in iNOSeC/eC and iNOS+/+ mice and compared neointimal lesions 4 and 8 weeks after surgery. We investigated whether altered progenitor cell repair in iNOS-null mice contributed to neointima formation in vivo and studied the molecular mechanism by comparing VEGF expression in vivo and in vitro.
Materials and Methods
Mice
All animal experiments were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals. iNOS+/eC mice were purchased from The Jackson Laboratory (Bar Harbor, Maine) and used as breeders. The mice were maintained on a light/dark (12/12 hour) cycle at 22°C receiving food and water ad libitum.
Three genotypes of iNOSeC/eC, iNOS+/eC, and iNOS+/+ mice were identified using the PCR protocol of The Jackson Laboratory, with a slight modification. Briefly, genomic DNA was isolated from tail tissues by enzyme digestion and precipitation. DNA (50 ng) in reaction buffer was thermocycled with 0.4 eol/L of each primer (0IMR1216: 5'-ACA TGC AGA ATG AGT ACC GG-3'; 0IMR1217: 5'-TCA ACA TCT CCT GGT GGA AC-3'; and OIMR1218: 5'-AAT ATG CGA AGT GGA CCT CG-3') at optimal temperature and separated by electrophoresis on agarose gels producing a single 108 and 270 bp band in iNOS+/+ and iNOSeC/eC mice, respectively, whereas both bands were visible in iNOS+/eC mice.
TIE2-LacZ mice18 expressing -galactosidase (gal) under the control of the endothelial-specific protein TIE2 promoter were purchased from The Jackson Laboratory. -Gal activity of cells from mice is mainly localized in the nucleus. Three genotypes of LacZeC/eC, LacZ+/eC, and LacZ+/+ mice were identified using the PCR protocol of The Jackson Laboratory (primers: 5'-ATC CTC TGC ATG GTC AGG TC-3' and 5'-CGT GGC CTG ATT CAT TCC-3'). iNOSeC/eC mice were crossed with TIE2-LacZ mice in our laboratory, and heterozygous offspring were mated to produce iNOS-deficient mice expressing -gal in endothelial cells (TIE2-LacZ/iNOSeC/eC).
Vein Graft Procedure and Treatment
For vein grafts, animals of the same genotype as donors and recipients were used. The procedure was similar to that described previously.3 Briefly, the vena cava from 3-month-old mice was harvested. The right common carotid artery was mobilized free from the bifurcation at the distal end toward the proximal, cut in the middle, and a cuff placed at the end. The cuff was made of an autoclavable nylon tube 0.63 mm in diameter outside and 0.5 mm inside (Portex Ltd, Hythe-Kent, UK). The vein segment was grafted between the 2 ends of the carotid artery by sleeving the ends of the vein over the artery cuff and ligating them together with the 8-0 suture. Immediately after vessel grafting, SU1498 (10 eol/L; Calbiochem-EMD Biosciences) or vascular endothelial growth factor (VEGF)-165 (100 ng/mL; Sigma) was applied to the adventitia dissolved in a 20% pluronic-127 gel (pH 7.2) as described previously.19,20 On contact with the tissues, the solutions gelled immediately, generating a translucent layer that enveloped the grafted vessel segment. In addition, 2 groups of mice received either NG-nitro-L-arginine methyl ester (L-NAME) (1 g/L) or L-arginine (25 g/L; Sigma) in their drinking water, a dose previously shown to reduce lesion formation in animal models.21 Supplementation was started 3 days before the vein graft procedure and continued until mice were euthanized.
Histology and Lesion Quantification
For histological analysis, in vivo perfusion with 4% phosphate-buffered formaldehyde was performed, as described previously.22 Vein grafts were harvested by cutting the implanted segments from the native vessels at the cuff end. The grafts were dehydrated in graded ethanol baths, cleared in xylol, embedded in paraffin, and sectioned. Neointimal lesions were defined as the region between the lumen and media, which contains 2 to 3 layers of condensed cells without microvessels. For lesional area measurement, sections were reviewed using a BX60 microscope (Zeiss) equipped with a camera and television monitor. Images were first scanned, saved and then overlaid by different linings to trace the lumen and media. The lesion area was determined by subtracting the area of the lumen from the area enclosed by the line inside of the media.
Reverse-Transcription Polymerase Chain Reaction
The procedure used for RT-PCR was similar to that described elsewhere.23 Total RNA was prepared with absolutely RNA RT-PCR Miniprep Kit (STRATAGENE). The following primers were used: iNOS (5'-CGA GGA GGC TGC CTG CAG ACT TGG-3' and 5'-CTG GGA GGA GCT GAT GGA GTA GTA-3') and GAPDH (5'-CGG AGT CAA CGG ATT TGG TCG TAT-3' and 5'-AGC CTT CTC CAT GGT GGT GAA GAC).
Immunofluorescent Staining
For frozen section preparation, vein grafts were harvested without perfusion and immediately frozen in liquid nitrogen. The procedure used for immunofluorescent staining was similar to that described previously.24 Briefly, serial 5-e thick frozen sections were labeled with rat monoclonal antibodies against mouse MAC-1+ (CD11b/18) leukocytes (BD Biosciences Pharmingen), CD31 (Abcam) or a mouse monoclonal antibody against -actin conjugated with Cy3 (Sigma), and a rabbit antibody against iNOS (BD Transduction Laboratories) or VEGF (Santa Cruz Biotechnology). The sections were visualized with swine anti-rat Ig-Cy3 and anti-rabbit Ig conjugated with FITC (DakoCytomation).
Immunohistochemistry
The procedure used in the present study was similar to that described previously.3 Briefly, serial 5-e thick frozen sections were cut from cryopreserved tissue blocks. The sections were overlayered with rat monoclonal antibodies against mouse MAC-1+ leukocytes (CD11b/18) or endothelial marker CD31. After washing with PBS, sections were incubated with rabbit anti-rat Ig (DakoCytomation) for 1 hour and developed with APARP. For SMC staining, a mouse monoclonal antibody against -actin (Sigma) labeled with phosphatase was used. Semiquantitive evaluation was performed at 10x25 magnification. Positive stained cells in the intima were counted on 2 regions of each section and expressed as the percentage of total nuclei per 100 e of the vessel wall. For CD31+ cells, staining intensity was measured using a computer software AxioVision.
En Face Preparation and X-Gal Staining
Mice were anesthetized and perfused with 0.9% NaCl solution and subsequently perfusion fixed with 2% formaldehyde and 0.2% glutaraldehyde (pH 7.2) for 2 and 10 minutes, respectively. The procedure for en face preparation is similar to that described elsewhere.25,26 In short, vein segments were harvested and the samples were fixed with 2% formaldehyde and 0.2% glutaraldehyde at 4°C for 24 hours. Each vessel segment (5x5 mm2) was prepared free from the adventitia and cut open. Vessel segments were mounted with the endothelium up on a glass slide (2.6x7.5 cm). The procedure for X-gal staining was similar to that described previously.27 Briefly, vein segments were incubated at 37°C for 18 hours in PBS supplemented with 1 mg/mL X-gal (Sigma). Vessel segments were rinsed with 3% DMSO in PBS and mounted with the endothelium up on a glass slide (2.6x7.5 cm). Positive stained (blue) cells were enumerated under the microscope.
Western Blot Analysis
Preparation of extracts from whole vessel wall for Western blotting was performed as described previously.28 Briefly, 20 e of protein were separated on a 10% polyacrylamide gel, blotted, and incubated with a rabbit polyclonal antibody to VEGF (1:100; Santa Cruz Biotechnology) followed by a rabbit IgG secondary antibody. To confirm equal loading, membranes were stained with antibody against actin (Santa Cruz Biotechnology).
Cell Culture and ELISA
Vascular SMCs were isolated by collagenase digestion of the mouse thoracic aorta and cultured in DMEM with 15% FCS at 37°C in an atmosphere of 5% CO2 as described previously.19 Cells were used for experiments between passage 10 and 20. On subconfluence, SMCs were placed in DMEM medium with 0.5% FCS for 24 hours (the presence of a low concentration of FCS is necessary to stabilize VEGF released into the medium) and subsequently exposed to interleukin (IL)-1 (20 ng/mL; R&D Systems). In some experiments, exposure to IL-1 occurred in the presence of the NOS inhibitor L-NAME (5 mmol/L) or L-arginine (10 mmol/L), respectively. VEGF was measured in the conditioned medium using a commercial ELISA kit (R&D Systems).
Statistical Analysis
Statistical analyses were performed using the Student’s t test, the ManneCWhitney U test, or ANOVA, respectively. A probability value of <0.05 was considered significant.
Results
Accelerated Lesions in iNOS-Deficient Mice
Before grafting, the composition of the venous wall was similar in iNOS+/+ and iNOSeC/eC mice (Figure 1a and 1b). Four weeks after grafting, neointimal hyperplasia, ie, thickening of the venous wall to more than 10 cell layers, was observed in wild-type mice (Figure 1c). Notably, neointima formation and cell density were markedly increased in vein grafts of iNOSeC/eC mice (Figure 1d). At 8 weeks, a loss of cell density was observed in both wild-type and knockout mice (Figure 1e and 1f). Figure 1g summarizes quantitative data on neointima thickness as measured microscopically. The lesion area of vein grafts derived from iNOSeC/eC mice was approximately doubled compared with wild-type controls at 4 and 8 weeks, and total cell numbers in 4-week grafts of iNOSeC/eC mice were markedly higher than those of wild-type animals (253±23 versus 108±16, counted over 100 e in length).
Cell Composition and iNOS Expression in Vein Grafts
Intensive immunostaining for MAC-1+ macrophages was observed especially in the surface area of vein grafts from iNOS+/+ mice, whereas -actin+ SMCs were found in the deeper layers of the neointima. MAC-1+ cells were the predominant cell type in neointimal lesions at 4 weeks (52% versus 69%), whereas -actin positive SMCs constituted approximately 10% of cells in lesions (11% versus 9%). Moreover, SMCs became dominant cells in neointimal lesions of 8-week vein grafts in both knockout and wild-type mice (Table I in the online data supplement available at http://circres.ahajournals.org). No significant difference in ratios of MAC-1+ macrophages and -actin+ cells were observed between iNOS+/+ and iNOSeC/eC mice, indicating that iNOS deficiency does alter neointimal size but not cell composition.
iNOS is known to be highly expressed in atherosclerotic lesions,7 but its presence in neointimal lesions of vein grafts remains to be determined. RT-PCR data presented in Figure 2a suggest increased iNOS mRNA transcription in wild-type, but not knockout, vein grafts. Similarly, immunostaining for iNOS was enhanced in neointimal macrophages and SMCs 4 weeks after grafting (Figure 2b) compared with freshly harvested veins. Vein segments stained with normal rat serum served as a negative control (data not shown). As expected, no staining for iNOS was detectable in vein grafts of iNOSeC/eC mice (Figure 2b).
Role of NO in Vein Graft Arteriosclerosis
It is well established that the activity of all NOS isoforms can be enhanced by L-arginine supplementation in the drinking water and inhibited by administration of the nonselective NOS inhibitor L-NAME. Whereas treatment with L-NAME accelerated neointima formation in wild-type mice and resulted in increased cell density, mainly mononuclear cells and SMCs, compared with untreated controls (Figure 3a-d), L-arginine supplementation reduced neointimal lesion size and inhibited mononuclear cell infiltration (Figure 3e and 3f). The graph in Figure 3 summarizes the data derived from 8 mice per group.
NO Influences Endothelial Progenitor Cell Homing
We demonstrated previously that endothelial cells in vein grafts are replaced by circulating progenitor cells. Because NO was recently implicated in endothelial progenitor cell mobilization,17 we wanted to determine whether iNOS deficiency would interfere with progenitor cell recruitment to vein grafts. Endothelial integrity was monitored using TIE2-LacZ/iNOSeC/eC mice, which express the LacZ gene in only endothelial cells. As expected, extensive endothelial loss occurred in wild-type vein grafts (data not shown) and endothelial cells were regenerated in vein grafts 4 weeks after surgery (Figure 4a). Whereas L-arginine supplementation accelerated restoration of the endothelial monolayer after grafting, the number of newly formed endothelial cells was markedly reduced in iNOSeC/eC mice and in wild-type mice treated with L-NAME (Figure 4a and 4b). These findings suggest that iNOS influences endothelial progenitor cell homing or attachment.
iNOS Influences VEGF Production
Because VEGF is a key chemokine and growth factor for EPCs, VEGF expression in vein grafts was measured by Western blot analysis and immunostaining. VEGF expression peaked in vein grafts 3 days after surgery and was significant lower in vein grafts of iNOSeC/eC mice compared with wild-type controls (Figure 5a). Interestingly, microvessels were abundant in the adventitia of vein grafts in iNOS+/+ mice but rare in iNOSeC/eC vein grafts (Figure 5b, top). Cells within these microvessels were identified to be CD31+ (red) expressing VEGF (green) (Figure 5b, middle). Neointimal SMCs also expressed high levels of VEGF at 4 weeks (Figure 5b, bottom). This local VEGF expression is likely to contribute to the recruitment of circulating endothelial progenitors to vein graft surface. To further analyze the effects of iNOS on vasa (microvessel) formation in vein grafts, sections of control vessels and vein grafts were labeled with anti-endothelial marker CD31 and quantified for both iNOSeC/eC and iNOS+/+ mice. Data shown in Figure 6a through 6d are representative images indicating that microvessels are mainly localized in the adventitia, and much less in neointimal lesions. Figure 6e summarized data of 8 animals per group indicate the significant higher in microvessel density in vein grafts derived from wild-type mice compared with knockouts.
NO-Induced VEGF Expression Plays a Role in Lesion Formation
Because SMCs in neointimal lesions expressed high levels of VEGF (Figure 5b), we cultivated aortic SMCs from both iNOSeC/eC and iNOS+/+ mice and measured VEGF production in response to IL-1 stimulation. Figure 7a demonstrates that levels of VEGF in the conditioned medium of iNOS+/+ SMCs reached a maximum after 24 and 48 hours but were significantly lower for iNOSeC/eC SMCs. Furthermore, IL-1eCinduced VEGF production in iNOS+/+ SMCs was inhibited by preincubation with NOS inhibitor L-NAME, whereas L-arginine significantly enhanced VEGF induction in iNOS+/+, but not iNOSeC/eC, SMCs (Figure 7b). Taken together, our results indicate that NO generated by iNOS contributes to VEGF production in SMCs.
To further clarify the functional relevance of NO-induced VEGF production in vivo, vein grafts were locally treated with VEGF receptor inhibitor SU1489. As shown in Figure 8a, SU1489 treatment markedly reduced the number of endothelial cells at 4 weeks after grafting in wild-type mice, whereas local VEGF transfer improved endothelial repair in iNOSeC/eC mice. Thus, VEGF treatment restored endothelial progenitor homing to the surface of iNOSeC/eC vein grafts. Concomitantly, neointima formation was enhanced in the SU1489-treated group compared with untreated controls, whereas local VEGF treatment reduced lesion formation in iNOSeC/eC mice (Figure 8b). In addition, VEGF treatment slightly increased the density of microvessels in the adventitia (data not shown). These findings support the notion that loss of VEGF expression may, at least partially, be responsible for impaired endothelial progenitor cell recruitment and increased neointima formation in iNOSeC/eC mice.
Discussion
We reported previously that 1 of the earliest cellular events in neointima formation of vein bypass grafts is cell death,4 triggering an inflammatory response26 followed by endothelial progenitor cell repair.5 In this process, circulating progenitor cells are largely responsible for replacing the endothelium in vein grafts.5,29 In the present study, we demonstrate in our murine vein graft model that iNOS deficiency accelerates neointima lesion formation by abrogating endothelial progenitor cell repair and facilitating mononuclear cell infiltration and SMC accumulation. Thus, iNOS is a crucial enzyme that attenuates lesion development in vein grafts by stimulating endothelial progenitor cell homing and differentiation.
Accumulating evidence indicates that endothelial progenitor cells in the blood have the capacity to proliferate, migrate, and differentiate into mature endothelial cells.30eC33 Previously, our group demonstrated that the number of -gal+ cells was reduced at 3 days and disappeared completely by 4 weeks after grafting, when a vein fragment from a TIE2-LacZ transgenic mouse expressing the marker gene LacZ only in endothelial cells was isografted into the carotid artery of wild-type mice.5 Conversely, -gal+ cells were observed on the surface of vein segments donated by wild-type mice isografted into TIE2-LacZ mice at 1 week and reached confluence by 4 weeks. These data show that circulating progenitor cells cover the surface of neointimal and arteriosclerotic lesions of vein grafts.34 Furthermore, other groups have demonstrated that eNOS is essential for progenitor cell mobilization17 and that iNOS contributes to endothelial progenitor cell recruitment into ischemic tissues.35 In the present study, we found that endothelial cell numbers on the surface of vein grafts are significantly reduced in iNOSeC/eC mice and in L-NAMEeCtreated wild-type mice, suggesting that NO is needed for endothelial progenitor cell attachment and differentiation.
Another important observation is the finding that locally produced VEGF in vein grafts is significantly reduced in iNOSeC/eC mice, which is related to decreased endothelial attachment. It has been shown that VEGF production in SMCs could be attenuated by inhibition of NOS activity,36 indicating a relationship between these 2 factors. NO was also shown to be involved early in angiogenesis, where inhibition of NOS activity abolished the increase in capillary proliferation.37 NO is released from endothelium by increased shear stress38,39 and in vivo in response to increased blood flow.40 NO and VEGF are known to be synergistic and/or complementary in other angiogenic situation.17,41 Our results demonstrated that iNOS-mediated NO production is a key event for VEGF production in SMCs stimulated by IL-1. Thus, NO together with NO-induced VEGF may synergistically serve as chemokines for endothelial progenitor homing and growth factors for cell proliferation and differentiation in vein grafts.
Because local concentration of VEGF could influence both endothelial repair on the surface of vein grafts and angiogenesis or vasa formation in the vessel wall, we have therefore compared the density of microvessels in grafts, and demonstrated a lower level of microvessels in iNOSeC/eC mice (Figure 6). These results indicate iNOS could also be an important enzyme for endothelial progenitor cell functions in terms of vasa formation in the adventitia. Concomitantly, Ii et al35 demonstrated that iNOS produced from EPCs appears to play a crucial role in cardioprotection, possibly via the involvement in angiogenesis. Is there any relationship among endothelial repair, neointimal formation, and vasa function in vein grafts As mentioned above, decreased microvessel density in iNOSeC/eC vein grafts may directly relate to lower level of local VEGF production that influences endothelial repair on the surface of vein grafts. On the other hand, microvessels within the vessel wall might be involved in transporting mononuclear cells and smooth muscle progenitors from the adventitial side based on observations that there are some microvessels in the media and neointima of 4-week vein grafts. Previously, we demonstrated that mononuclear cell recruitment into neointima was significantly reduced in ICAM-1eC/eC mice,26 suggesting these cells are mainly derived from endothelial side of the vein grafts. Thus, we hypothesize that neointimal cells may be mainly recruited from endothelial side of the vessel, although vasa vasorum could be involved as well.
The role of iNOS in vascular pathology is variable, and cholesterol levels appear to be among the factors determining whether iNOS is pro- or antiatherosclerotic. Although iNOS deficiency has no effect on atherosclerosis development in apoE/iNOS-dKO mice on normal chow,42 it substantially reduced atherosclerosis in apoE-null mice on Western diet.14,15 In contrast to spontaneous atherosclerosis, iNOS plays a protective role in transplant atherosclerosis and chronic cardiac rejection is accelerated in iNOSeC/eC mice.13 Similarly, our findings demonstrate that neointima formation is enhanced in vein grafts of iNOS-deficient mice. These observations suggest that iNOS plays different role in hyperlipidemia-induced atherosclerosis and vascular graft arteriosclerosis. In hyperlipidemia-induced atherosclerosis, reduction in lesion formation was associated with a decrease in plasma lipoperoxide concentrations.21 Hyperlipidemia is known to cause endothelial dysfunction, but, unlike the vein graft model, no acute endothelial damage occurs. The extensive loss of endothelial cells after grafting veins to arteries4 is followed by rapid progenitor cell repair.5 Therefore, different strategies for modulating iNOS activity would be needed to treat native atherosclerosis and vein graft stenosis.
In summary, we demonstrated that iNOS deficiency results in exacerbated arteriosclerosis in vein grafts in a murine model. The mechanisms of iNOS-enhanced lesion formation involve delayed endothelial repair, ie, progenitor homing and differentiation, in which NO-mediated VEGF production by vascular cells is crucial. Our findings provide basic information for developing a new strategy for treatment of vein graft disease.
Acknowledgments
This work was supported by grants from the British Heart Foundation (RG/04/008) and Oak Foundation.
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Chiu JJ, Wung BS, Hsieh HJ, Lo LW, Wang DL. Nitric oxide regulates shear stress-induced early growth response-1. Expression via the extracellular signal-regulated kinase pathway in endothelial cells. Circ Res. 1999; 85: 238eC246.
Davis ME, Cai H, Drummond GR, Harrison DG. Shear stress regulates endothelial nitric oxide synthase expression through c-Src by divergent signaling pathways. Circ Res. 2001; 89: 1073eC1080.
Johnson DL, McAllister TN, Frangos JA. Fluid flow stimulates rapid and continuous release of nitric oxide in osteoblasts. Am J Physiol. 1996; 271: E205eCE208.
Parenti A, Morbidelli L, Cui XL, Douglas JG, Hood JD, Granger HJ, Ledda F, Ziche M. Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal-regulated kinase1/2 activation in postcapillary endothelium. J Biol Chem. 1998; 273: 4220eC4226.
Knowles JW, Reddick RL, Jennette JC, Shesely EG, Smithies O, Maeda N. Enhanced atherosclerosis and kidney dysfunction in eNOS(-/-)Apoe(-/-) mice are ameliorated by enalapril treatment. J Clin Invest. 2000; 105: 451eC458.(Ursula Mayr, Yiping Zou, )
Institute for Pathophysiology (Y.Z., H.D.), Innsbruck Medical University, Austria.
Abstract
Inducible NO synthase (iNOS) is expressed by macrophages and smooth muscle cells in atherosclerotic lesions. Previously, we have established a mouse model for vein graft arteriosclerosis by grafting autologous jugular veins or vena cava to carotid arteries. Using this model, we studied the role of iNOS in the development of vein graft arteriosclerosis in iNOSeC/eC mice. Four weeks after grafting, neointimal hyperplasia of vein grafts in iNOSeC/eC mice was increased 2-fold compared with that of wild-type controls. Neointimal lesions contained mainly MAC-1+ macrophages and -actin+ smooth muscle cells (SMCs) in both vein grafts of iNOSeC/eC and iNOS+/+ mice. Immunofluorescence analysis revealed that increased iNOS expression in neointimal macrophages and SMCs of wild-type, but not iNOSeC/eC, mice coincided with increased vascular endothelial growth factor (VEGF) expression in vein grafts. When vein grafts were performed in iNOSeC/eC/TIE2-LacZ transgenic mice expressing LacZ gene only in endothelial cells, the number of -galactosidase+ cells in iNOSeC/eC vein grafts were significantly decreased. Furthermore, treatment with the NOS inhibitor NG-nitro-L-arginine methyl ester resulted in delayed endothelial progenitor cell attachment, whereas L-arginine intake through drinking water enhanced endothelial repair. Interestingly, local application of VEGF to iNOSeC/eC vein grafts restored endothelial progenitor homing and reduced neointimal lesions, whereas the VEGF receptor inhibitor SU1498 increased the lesion formation. Additionally, iNOS-deficient SMCs showed a low level of VEGF production in response to interleukin 1 stimulation. Thus, iNOS deficiency accelerates neointima formation by abrogating VEGF production and endothelial progenitor cell attachment and differentiation.
Key Words: mouse models iNOS vein grafts progenitor cells neointimal hyperplasia
Introduction
Autologous vein grafts are a common clinical procedure for vascular reconstruction, but their patency rate is limited by obliterative stenosis because of proliferative thickening of the intima. Neointimal hyperplasia develops rapidly once veins are subject to arterial blood pressure.1 A hallmark of lesion initiation is endothelial cell damage, which occurs within the first week after surgery.2 Previously, we established a mouse model of vein graft arteriosclerosis that resembles certain characteristics of human vein graft disease.3 We demonstrated that 1 of the earliest events after grafting was vascular cell apoptosis followed by endothelial progenitor cell repair.4,5 Venous endothelial cells became apoptosis in the arterial circulation and were replaced by circulating endothelial progenitors, which reached confluence within 4 weeks and differentiated into mature endothelial cells.5 Thus, the balance between apoptosis and progenitor cell repair could be crucial in determining lesion development in vein grafts.
NO is thought to be a key regulator in the development of atherosclerosis.6eC9 Classical cardiovascular risk factors, such as hypertension, diabetes mellitus, hypercholesterolemia, and smoking all impair NO function. NO is formed from L-arginine by the enzyme NO synthase (NOS). Three distinct isoenzymes of NOS are known to exist: constitutive-type isoforms, such as neuronal NOS (nNOS) and endothelial cell NOS (eNOS), and the inducible type of the enzyme (iNOS). The latter is widely distributed in a variety of cell types, including SMCs and leukocytes, responds to a variety of stimuli, and can produce a very high output of NO.10 NO is a pleiotropic signaling molecule involved in numerous processes,11 including lipid oxidation, mononuclear cell infiltration, and SMC homeostasis.12eC16 Notably, eNOS is essential for the survival, migration, and angiogenic response of mature endothelial cells and has recently been implicated in endothelial progenitor cell mobilization.17 However, it remains to be clarified whether iNOS might be important for endothelial progenitor cell recruitment to sites of injury.
To elucidate the role of iNOS in vascular graft-induced neointima formation, we performed vein bypass grafts in iNOSeC/eC and iNOS+/+ mice and compared neointimal lesions 4 and 8 weeks after surgery. We investigated whether altered progenitor cell repair in iNOS-null mice contributed to neointima formation in vivo and studied the molecular mechanism by comparing VEGF expression in vivo and in vitro.
Materials and Methods
Mice
All animal experiments were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals. iNOS+/eC mice were purchased from The Jackson Laboratory (Bar Harbor, Maine) and used as breeders. The mice were maintained on a light/dark (12/12 hour) cycle at 22°C receiving food and water ad libitum.
Three genotypes of iNOSeC/eC, iNOS+/eC, and iNOS+/+ mice were identified using the PCR protocol of The Jackson Laboratory, with a slight modification. Briefly, genomic DNA was isolated from tail tissues by enzyme digestion and precipitation. DNA (50 ng) in reaction buffer was thermocycled with 0.4 eol/L of each primer (0IMR1216: 5'-ACA TGC AGA ATG AGT ACC GG-3'; 0IMR1217: 5'-TCA ACA TCT CCT GGT GGA AC-3'; and OIMR1218: 5'-AAT ATG CGA AGT GGA CCT CG-3') at optimal temperature and separated by electrophoresis on agarose gels producing a single 108 and 270 bp band in iNOS+/+ and iNOSeC/eC mice, respectively, whereas both bands were visible in iNOS+/eC mice.
TIE2-LacZ mice18 expressing -galactosidase (gal) under the control of the endothelial-specific protein TIE2 promoter were purchased from The Jackson Laboratory. -Gal activity of cells from mice is mainly localized in the nucleus. Three genotypes of LacZeC/eC, LacZ+/eC, and LacZ+/+ mice were identified using the PCR protocol of The Jackson Laboratory (primers: 5'-ATC CTC TGC ATG GTC AGG TC-3' and 5'-CGT GGC CTG ATT CAT TCC-3'). iNOSeC/eC mice were crossed with TIE2-LacZ mice in our laboratory, and heterozygous offspring were mated to produce iNOS-deficient mice expressing -gal in endothelial cells (TIE2-LacZ/iNOSeC/eC).
Vein Graft Procedure and Treatment
For vein grafts, animals of the same genotype as donors and recipients were used. The procedure was similar to that described previously.3 Briefly, the vena cava from 3-month-old mice was harvested. The right common carotid artery was mobilized free from the bifurcation at the distal end toward the proximal, cut in the middle, and a cuff placed at the end. The cuff was made of an autoclavable nylon tube 0.63 mm in diameter outside and 0.5 mm inside (Portex Ltd, Hythe-Kent, UK). The vein segment was grafted between the 2 ends of the carotid artery by sleeving the ends of the vein over the artery cuff and ligating them together with the 8-0 suture. Immediately after vessel grafting, SU1498 (10 eol/L; Calbiochem-EMD Biosciences) or vascular endothelial growth factor (VEGF)-165 (100 ng/mL; Sigma) was applied to the adventitia dissolved in a 20% pluronic-127 gel (pH 7.2) as described previously.19,20 On contact with the tissues, the solutions gelled immediately, generating a translucent layer that enveloped the grafted vessel segment. In addition, 2 groups of mice received either NG-nitro-L-arginine methyl ester (L-NAME) (1 g/L) or L-arginine (25 g/L; Sigma) in their drinking water, a dose previously shown to reduce lesion formation in animal models.21 Supplementation was started 3 days before the vein graft procedure and continued until mice were euthanized.
Histology and Lesion Quantification
For histological analysis, in vivo perfusion with 4% phosphate-buffered formaldehyde was performed, as described previously.22 Vein grafts were harvested by cutting the implanted segments from the native vessels at the cuff end. The grafts were dehydrated in graded ethanol baths, cleared in xylol, embedded in paraffin, and sectioned. Neointimal lesions were defined as the region between the lumen and media, which contains 2 to 3 layers of condensed cells without microvessels. For lesional area measurement, sections were reviewed using a BX60 microscope (Zeiss) equipped with a camera and television monitor. Images were first scanned, saved and then overlaid by different linings to trace the lumen and media. The lesion area was determined by subtracting the area of the lumen from the area enclosed by the line inside of the media.
Reverse-Transcription Polymerase Chain Reaction
The procedure used for RT-PCR was similar to that described elsewhere.23 Total RNA was prepared with absolutely RNA RT-PCR Miniprep Kit (STRATAGENE). The following primers were used: iNOS (5'-CGA GGA GGC TGC CTG CAG ACT TGG-3' and 5'-CTG GGA GGA GCT GAT GGA GTA GTA-3') and GAPDH (5'-CGG AGT CAA CGG ATT TGG TCG TAT-3' and 5'-AGC CTT CTC CAT GGT GGT GAA GAC).
Immunofluorescent Staining
For frozen section preparation, vein grafts were harvested without perfusion and immediately frozen in liquid nitrogen. The procedure used for immunofluorescent staining was similar to that described previously.24 Briefly, serial 5-e thick frozen sections were labeled with rat monoclonal antibodies against mouse MAC-1+ (CD11b/18) leukocytes (BD Biosciences Pharmingen), CD31 (Abcam) or a mouse monoclonal antibody against -actin conjugated with Cy3 (Sigma), and a rabbit antibody against iNOS (BD Transduction Laboratories) or VEGF (Santa Cruz Biotechnology). The sections were visualized with swine anti-rat Ig-Cy3 and anti-rabbit Ig conjugated with FITC (DakoCytomation).
Immunohistochemistry
The procedure used in the present study was similar to that described previously.3 Briefly, serial 5-e thick frozen sections were cut from cryopreserved tissue blocks. The sections were overlayered with rat monoclonal antibodies against mouse MAC-1+ leukocytes (CD11b/18) or endothelial marker CD31. After washing with PBS, sections were incubated with rabbit anti-rat Ig (DakoCytomation) for 1 hour and developed with APARP. For SMC staining, a mouse monoclonal antibody against -actin (Sigma) labeled with phosphatase was used. Semiquantitive evaluation was performed at 10x25 magnification. Positive stained cells in the intima were counted on 2 regions of each section and expressed as the percentage of total nuclei per 100 e of the vessel wall. For CD31+ cells, staining intensity was measured using a computer software AxioVision.
En Face Preparation and X-Gal Staining
Mice were anesthetized and perfused with 0.9% NaCl solution and subsequently perfusion fixed with 2% formaldehyde and 0.2% glutaraldehyde (pH 7.2) for 2 and 10 minutes, respectively. The procedure for en face preparation is similar to that described elsewhere.25,26 In short, vein segments were harvested and the samples were fixed with 2% formaldehyde and 0.2% glutaraldehyde at 4°C for 24 hours. Each vessel segment (5x5 mm2) was prepared free from the adventitia and cut open. Vessel segments were mounted with the endothelium up on a glass slide (2.6x7.5 cm). The procedure for X-gal staining was similar to that described previously.27 Briefly, vein segments were incubated at 37°C for 18 hours in PBS supplemented with 1 mg/mL X-gal (Sigma). Vessel segments were rinsed with 3% DMSO in PBS and mounted with the endothelium up on a glass slide (2.6x7.5 cm). Positive stained (blue) cells were enumerated under the microscope.
Western Blot Analysis
Preparation of extracts from whole vessel wall for Western blotting was performed as described previously.28 Briefly, 20 e of protein were separated on a 10% polyacrylamide gel, blotted, and incubated with a rabbit polyclonal antibody to VEGF (1:100; Santa Cruz Biotechnology) followed by a rabbit IgG secondary antibody. To confirm equal loading, membranes were stained with antibody against actin (Santa Cruz Biotechnology).
Cell Culture and ELISA
Vascular SMCs were isolated by collagenase digestion of the mouse thoracic aorta and cultured in DMEM with 15% FCS at 37°C in an atmosphere of 5% CO2 as described previously.19 Cells were used for experiments between passage 10 and 20. On subconfluence, SMCs were placed in DMEM medium with 0.5% FCS for 24 hours (the presence of a low concentration of FCS is necessary to stabilize VEGF released into the medium) and subsequently exposed to interleukin (IL)-1 (20 ng/mL; R&D Systems). In some experiments, exposure to IL-1 occurred in the presence of the NOS inhibitor L-NAME (5 mmol/L) or L-arginine (10 mmol/L), respectively. VEGF was measured in the conditioned medium using a commercial ELISA kit (R&D Systems).
Statistical Analysis
Statistical analyses were performed using the Student’s t test, the ManneCWhitney U test, or ANOVA, respectively. A probability value of <0.05 was considered significant.
Results
Accelerated Lesions in iNOS-Deficient Mice
Before grafting, the composition of the venous wall was similar in iNOS+/+ and iNOSeC/eC mice (Figure 1a and 1b). Four weeks after grafting, neointimal hyperplasia, ie, thickening of the venous wall to more than 10 cell layers, was observed in wild-type mice (Figure 1c). Notably, neointima formation and cell density were markedly increased in vein grafts of iNOSeC/eC mice (Figure 1d). At 8 weeks, a loss of cell density was observed in both wild-type and knockout mice (Figure 1e and 1f). Figure 1g summarizes quantitative data on neointima thickness as measured microscopically. The lesion area of vein grafts derived from iNOSeC/eC mice was approximately doubled compared with wild-type controls at 4 and 8 weeks, and total cell numbers in 4-week grafts of iNOSeC/eC mice were markedly higher than those of wild-type animals (253±23 versus 108±16, counted over 100 e in length).
Cell Composition and iNOS Expression in Vein Grafts
Intensive immunostaining for MAC-1+ macrophages was observed especially in the surface area of vein grafts from iNOS+/+ mice, whereas -actin+ SMCs were found in the deeper layers of the neointima. MAC-1+ cells were the predominant cell type in neointimal lesions at 4 weeks (52% versus 69%), whereas -actin positive SMCs constituted approximately 10% of cells in lesions (11% versus 9%). Moreover, SMCs became dominant cells in neointimal lesions of 8-week vein grafts in both knockout and wild-type mice (Table I in the online data supplement available at http://circres.ahajournals.org). No significant difference in ratios of MAC-1+ macrophages and -actin+ cells were observed between iNOS+/+ and iNOSeC/eC mice, indicating that iNOS deficiency does alter neointimal size but not cell composition.
iNOS is known to be highly expressed in atherosclerotic lesions,7 but its presence in neointimal lesions of vein grafts remains to be determined. RT-PCR data presented in Figure 2a suggest increased iNOS mRNA transcription in wild-type, but not knockout, vein grafts. Similarly, immunostaining for iNOS was enhanced in neointimal macrophages and SMCs 4 weeks after grafting (Figure 2b) compared with freshly harvested veins. Vein segments stained with normal rat serum served as a negative control (data not shown). As expected, no staining for iNOS was detectable in vein grafts of iNOSeC/eC mice (Figure 2b).
Role of NO in Vein Graft Arteriosclerosis
It is well established that the activity of all NOS isoforms can be enhanced by L-arginine supplementation in the drinking water and inhibited by administration of the nonselective NOS inhibitor L-NAME. Whereas treatment with L-NAME accelerated neointima formation in wild-type mice and resulted in increased cell density, mainly mononuclear cells and SMCs, compared with untreated controls (Figure 3a-d), L-arginine supplementation reduced neointimal lesion size and inhibited mononuclear cell infiltration (Figure 3e and 3f). The graph in Figure 3 summarizes the data derived from 8 mice per group.
NO Influences Endothelial Progenitor Cell Homing
We demonstrated previously that endothelial cells in vein grafts are replaced by circulating progenitor cells. Because NO was recently implicated in endothelial progenitor cell mobilization,17 we wanted to determine whether iNOS deficiency would interfere with progenitor cell recruitment to vein grafts. Endothelial integrity was monitored using TIE2-LacZ/iNOSeC/eC mice, which express the LacZ gene in only endothelial cells. As expected, extensive endothelial loss occurred in wild-type vein grafts (data not shown) and endothelial cells were regenerated in vein grafts 4 weeks after surgery (Figure 4a). Whereas L-arginine supplementation accelerated restoration of the endothelial monolayer after grafting, the number of newly formed endothelial cells was markedly reduced in iNOSeC/eC mice and in wild-type mice treated with L-NAME (Figure 4a and 4b). These findings suggest that iNOS influences endothelial progenitor cell homing or attachment.
iNOS Influences VEGF Production
Because VEGF is a key chemokine and growth factor for EPCs, VEGF expression in vein grafts was measured by Western blot analysis and immunostaining. VEGF expression peaked in vein grafts 3 days after surgery and was significant lower in vein grafts of iNOSeC/eC mice compared with wild-type controls (Figure 5a). Interestingly, microvessels were abundant in the adventitia of vein grafts in iNOS+/+ mice but rare in iNOSeC/eC vein grafts (Figure 5b, top). Cells within these microvessels were identified to be CD31+ (red) expressing VEGF (green) (Figure 5b, middle). Neointimal SMCs also expressed high levels of VEGF at 4 weeks (Figure 5b, bottom). This local VEGF expression is likely to contribute to the recruitment of circulating endothelial progenitors to vein graft surface. To further analyze the effects of iNOS on vasa (microvessel) formation in vein grafts, sections of control vessels and vein grafts were labeled with anti-endothelial marker CD31 and quantified for both iNOSeC/eC and iNOS+/+ mice. Data shown in Figure 6a through 6d are representative images indicating that microvessels are mainly localized in the adventitia, and much less in neointimal lesions. Figure 6e summarized data of 8 animals per group indicate the significant higher in microvessel density in vein grafts derived from wild-type mice compared with knockouts.
NO-Induced VEGF Expression Plays a Role in Lesion Formation
Because SMCs in neointimal lesions expressed high levels of VEGF (Figure 5b), we cultivated aortic SMCs from both iNOSeC/eC and iNOS+/+ mice and measured VEGF production in response to IL-1 stimulation. Figure 7a demonstrates that levels of VEGF in the conditioned medium of iNOS+/+ SMCs reached a maximum after 24 and 48 hours but were significantly lower for iNOSeC/eC SMCs. Furthermore, IL-1eCinduced VEGF production in iNOS+/+ SMCs was inhibited by preincubation with NOS inhibitor L-NAME, whereas L-arginine significantly enhanced VEGF induction in iNOS+/+, but not iNOSeC/eC, SMCs (Figure 7b). Taken together, our results indicate that NO generated by iNOS contributes to VEGF production in SMCs.
To further clarify the functional relevance of NO-induced VEGF production in vivo, vein grafts were locally treated with VEGF receptor inhibitor SU1489. As shown in Figure 8a, SU1489 treatment markedly reduced the number of endothelial cells at 4 weeks after grafting in wild-type mice, whereas local VEGF transfer improved endothelial repair in iNOSeC/eC mice. Thus, VEGF treatment restored endothelial progenitor homing to the surface of iNOSeC/eC vein grafts. Concomitantly, neointima formation was enhanced in the SU1489-treated group compared with untreated controls, whereas local VEGF treatment reduced lesion formation in iNOSeC/eC mice (Figure 8b). In addition, VEGF treatment slightly increased the density of microvessels in the adventitia (data not shown). These findings support the notion that loss of VEGF expression may, at least partially, be responsible for impaired endothelial progenitor cell recruitment and increased neointima formation in iNOSeC/eC mice.
Discussion
We reported previously that 1 of the earliest cellular events in neointima formation of vein bypass grafts is cell death,4 triggering an inflammatory response26 followed by endothelial progenitor cell repair.5 In this process, circulating progenitor cells are largely responsible for replacing the endothelium in vein grafts.5,29 In the present study, we demonstrate in our murine vein graft model that iNOS deficiency accelerates neointima lesion formation by abrogating endothelial progenitor cell repair and facilitating mononuclear cell infiltration and SMC accumulation. Thus, iNOS is a crucial enzyme that attenuates lesion development in vein grafts by stimulating endothelial progenitor cell homing and differentiation.
Accumulating evidence indicates that endothelial progenitor cells in the blood have the capacity to proliferate, migrate, and differentiate into mature endothelial cells.30eC33 Previously, our group demonstrated that the number of -gal+ cells was reduced at 3 days and disappeared completely by 4 weeks after grafting, when a vein fragment from a TIE2-LacZ transgenic mouse expressing the marker gene LacZ only in endothelial cells was isografted into the carotid artery of wild-type mice.5 Conversely, -gal+ cells were observed on the surface of vein segments donated by wild-type mice isografted into TIE2-LacZ mice at 1 week and reached confluence by 4 weeks. These data show that circulating progenitor cells cover the surface of neointimal and arteriosclerotic lesions of vein grafts.34 Furthermore, other groups have demonstrated that eNOS is essential for progenitor cell mobilization17 and that iNOS contributes to endothelial progenitor cell recruitment into ischemic tissues.35 In the present study, we found that endothelial cell numbers on the surface of vein grafts are significantly reduced in iNOSeC/eC mice and in L-NAMEeCtreated wild-type mice, suggesting that NO is needed for endothelial progenitor cell attachment and differentiation.
Another important observation is the finding that locally produced VEGF in vein grafts is significantly reduced in iNOSeC/eC mice, which is related to decreased endothelial attachment. It has been shown that VEGF production in SMCs could be attenuated by inhibition of NOS activity,36 indicating a relationship between these 2 factors. NO was also shown to be involved early in angiogenesis, where inhibition of NOS activity abolished the increase in capillary proliferation.37 NO is released from endothelium by increased shear stress38,39 and in vivo in response to increased blood flow.40 NO and VEGF are known to be synergistic and/or complementary in other angiogenic situation.17,41 Our results demonstrated that iNOS-mediated NO production is a key event for VEGF production in SMCs stimulated by IL-1. Thus, NO together with NO-induced VEGF may synergistically serve as chemokines for endothelial progenitor homing and growth factors for cell proliferation and differentiation in vein grafts.
Because local concentration of VEGF could influence both endothelial repair on the surface of vein grafts and angiogenesis or vasa formation in the vessel wall, we have therefore compared the density of microvessels in grafts, and demonstrated a lower level of microvessels in iNOSeC/eC mice (Figure 6). These results indicate iNOS could also be an important enzyme for endothelial progenitor cell functions in terms of vasa formation in the adventitia. Concomitantly, Ii et al35 demonstrated that iNOS produced from EPCs appears to play a crucial role in cardioprotection, possibly via the involvement in angiogenesis. Is there any relationship among endothelial repair, neointimal formation, and vasa function in vein grafts As mentioned above, decreased microvessel density in iNOSeC/eC vein grafts may directly relate to lower level of local VEGF production that influences endothelial repair on the surface of vein grafts. On the other hand, microvessels within the vessel wall might be involved in transporting mononuclear cells and smooth muscle progenitors from the adventitial side based on observations that there are some microvessels in the media and neointima of 4-week vein grafts. Previously, we demonstrated that mononuclear cell recruitment into neointima was significantly reduced in ICAM-1eC/eC mice,26 suggesting these cells are mainly derived from endothelial side of the vein grafts. Thus, we hypothesize that neointimal cells may be mainly recruited from endothelial side of the vessel, although vasa vasorum could be involved as well.
The role of iNOS in vascular pathology is variable, and cholesterol levels appear to be among the factors determining whether iNOS is pro- or antiatherosclerotic. Although iNOS deficiency has no effect on atherosclerosis development in apoE/iNOS-dKO mice on normal chow,42 it substantially reduced atherosclerosis in apoE-null mice on Western diet.14,15 In contrast to spontaneous atherosclerosis, iNOS plays a protective role in transplant atherosclerosis and chronic cardiac rejection is accelerated in iNOSeC/eC mice.13 Similarly, our findings demonstrate that neointima formation is enhanced in vein grafts of iNOS-deficient mice. These observations suggest that iNOS plays different role in hyperlipidemia-induced atherosclerosis and vascular graft arteriosclerosis. In hyperlipidemia-induced atherosclerosis, reduction in lesion formation was associated with a decrease in plasma lipoperoxide concentrations.21 Hyperlipidemia is known to cause endothelial dysfunction, but, unlike the vein graft model, no acute endothelial damage occurs. The extensive loss of endothelial cells after grafting veins to arteries4 is followed by rapid progenitor cell repair.5 Therefore, different strategies for modulating iNOS activity would be needed to treat native atherosclerosis and vein graft stenosis.
In summary, we demonstrated that iNOS deficiency results in exacerbated arteriosclerosis in vein grafts in a murine model. The mechanisms of iNOS-enhanced lesion formation involve delayed endothelial repair, ie, progenitor homing and differentiation, in which NO-mediated VEGF production by vascular cells is crucial. Our findings provide basic information for developing a new strategy for treatment of vein graft disease.
Acknowledgments
This work was supported by grants from the British Heart Foundation (RG/04/008) and Oak Foundation.
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