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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1614-1621

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1614-1621.)
© 1997 American Heart Association, Inc.

Articles

Monocyte Chemotactic Protein-1 Expression Is Associated With the Development of Vein Graft Intimal Hyperplasia

V.K. Stark; J.R. Hoch; T.F. Warner; ; D.A. Hullett

From the Departments of Surgery and Pathology (T.F.W.), University of Wisconsin, Madison.


*    Abstract
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Abstract Infiltration of immunologically active cells into vein grafts is concomitant with the development of intimal hyperplasia (IH) and often leads to obliterative stenosis and graft failure. Previous work has demonstrated the prolonged presence of monocytes and macrophages in vein grafts. The stimuli attracting these macrophages remain unidentified. Monocyte chemotactic protein-1 (MCP-1), a potent and specific chemokine for monocytes/macrophages, is secreted by smooth muscle cells, endothelial cells, fibroblasts, and leukocytes, all of which are present in grafted veins. In this study, we examined the temporal profile of MCP-1 gene expression in rat vein grafts by using reverse transcription–polymerase chain reaction (PCR) and immunohistochemistry. Epigastric vein–to–femoral artery bypass grafts were microsurgically placed and harvested at various time points after grafting. Histological analysis confirmed the consistent development of IH. PCR was performed and relative levels of MCP-1 quantified by autoradiography. Our results show that MCP-1 mRNA levels increased 28-fold by 4 hours after grafting and up to 117-fold by 1 week. After this time MCP-1 mRNA levels decreased; nonetheless, even at 8 weeks after grafting, message levels remained elevated 7-fold above baseline. Immunoreactive MCP-1 protein and ED1+ macrophages were detected at all time points; the degree of immunostaining correlated with MCP-1 mRNA levels. Our results support the hypothesis that upregulation of MCP-1 gene expression in vein grafts results in the recruitment of monocytes and tissue macrophages to the vein wall, which leads to IH. The correlation between monocyte/macrophage infiltration and IH suggests a critical role for these cells in IH development.


Key Words: vein graft • intimal hyperplasia • cytokines • macrophages • MCP-1


*    Introduction
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 *Introduction
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Autologous vein grafts remain the only surgical alternative for many types of vascular reconstruction; unfortunately, the failure rate of these grafts after 1 year approaches 20%.1 The principal cause of graft failure is the development of IH, a fibroproliferative thickening of the neointima often leading to obliterative stenosis. Although the hallmarks of IH—the migration/proliferation of smooth muscle–like cells and the deposition of extracellular matrix—are well described,2 the etiology of this process remains poorly understood, and no successful clinical interventions have been identified.

IH develops after diverse interventions such as vein and prosthetic arterial bypass grafts, creation of arteriovenous fistulas, and percutaneous transluminal balloon angioplasty, all of which cause substantial injury to the involved vessels. Thus, the resulting IH can be considered an overexuberant vascular wound-healing response. Central to wound healing and inflammation are activated monocytes and macrophages. In addition to their phagocytic functions, monocytes and macrophages are secretory cells, producing mitogenic, fibrogenic, and angiogenic factors that can influence tissue remodeling3 4 and thus may play a role in IH development.

In a Lewis rat model of vein graft IH, we have demonstrated sustained macrophage infiltration into the vein wall and development of IH after acute inflammation has subsided.5 We have also detected mRNA transcripts of macrophage-associated cytokines (IL-1, PDGF, and TGF-ß) in vein graft tissue at several postoperative time points.6 Our observations and the work of others point to continuous involvement of macrophages within vein bypass grafts, possibly modulating the events of IH via their cytokines.

The mechanism by which monocytes/macrophages are continuously recruited to healing vein grafts is unknown. The most potent and specific chemotactic and activating factor described for these cells is MCP-1.7 It is secreted by various cells, including endothelial cells, smooth muscle cells, and fibroblasts, all of which are present in vein grafts. In previous work, we detected the presence of MCP-1 mRNA at several time points after vein graft implantation. In the present study, we demonstrate semiquantitatively the bimodal upregulation of MCP-1 mRNA expression in rat vein grafts, followed closely by a marked increase in MCP-1 immunoreactive protein, monocyte/macrophage infiltration, and IH development.


*    Methods
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Microsurgical Vein Grafting and Harvest
Epigastric vein–to–common femoral artery interposition grafts were placed in 82 male Lewis rats (350 to 450 g) via aseptic microsurgical techniques as previously described.5 In brief, each animal was anesthetized intraperitoneally with ketamine and xylazine, and the epigastric vein and common femoral artery were dissected. An 8-mm segment of relaxed epigastric vein was excised, reversed, and interposed into a 3-mm arterial defect, rendering a 1-cm completed graft. Each anastomosis was completed with 8 to 10 interrupted sutures of 10-0 nylon. After a 45-minute total ischemic time hemostasis was restored, patency was confirmed, and the wound was irrigated and closed. The animal was closely observed until recovery. Animal care complied with the Principles of Laboratory Animal Care (formulated by the National Society for Medical Research) and the Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, revised 1985). Harvest time points in this study included 1 and 4 hours; 1 and 4 days; and 1, 2, 4, and 8 weeks. All grafts were compared with nongrafted contralateral epigastric vein controls.

Histology and Immunohistochemistry
One set of randomly chosen vein grafts (5 grafts per time point) was used for these experiments. Transverse 5-µm sections of formalin-fixed, paraffin-embedded control and graft tissues were cut at 500-µm intervals and placed on coated slides. Serial sections from the proximal region (displaying the earliest IH) were chosen for staining. Specimens were deparaffinized extensively and rehydrated in graded ethanol solutions. Sections were stained with hematoxylin/eosin and Verhoeff–van Gieson's elastin stains for histological and morphometric analyses. For immunostaining, nonspecific binding was blocked with 2% BSA in PBS. Slides were incubated with polyclonal rabbit antibody to rat MCP-1 (a generous gift of Dr Jeffrey Warren, University of Michigan, Ann Arbor; 10 µg/mL) at 4°C overnight. Quenching of endogenous peroxidase with 1% H2O2 in PBS was followed by incubation with biotinylated anti-rabbit secondary antibody (Dako) at room temperature for 1 hour. Serial sections were stained with monoclonal antibodies ED1 (5 µg/mL, Harlan Bioproducts for Science), which recognizes >95% of all monocytes and macrophages, and ED2 (10 µg/mL, Harlan Bioproducts for Science, which recognizes resident macrophages only, followed by biotinylated anti-mouse secondary antibody. Other antibodies included asm-1 (anti–{alpha}-actin, 20 µg/mL; Boehringer Mannheim) and polyclonal rabbit anti–factor VIII (Dako). Incubation with streptavidin-peroxidase was followed by the addition of the substrate 3'3'-diaminobenzidine (Vector Labs). Some specimens were counterstained lightly with hematoxylin. All washes were done in PBS at pH 7.5. To ensure specificity of the antibody, parallel staining with either mouse IgG1, mouse IgG2A, or rabbit IgG at the same concentration as the test antibody was performed. Tissues from rat spleen and cervical lymph nodes were used as positive controls.

TEM
A separate set of grafts at 1 day and 1, 2, and 8 weeks (n=2 per time point) was used for TEM. Two nongrafted epigastric veins were also analyzed. Tissue was fixed in 2.5% glutaraldehyde buffered in 0.1 mol/L sodium cacodylate, pH 7.2, and postfixed in 1% OsO4. After dehydration in graded alcohol solutions, the specimens were embedded in Epon-Araldite. Thick (1 µm) sections were cut and stained with toluidine blue. Thin (90 nm) sections were cut, placed on copper grids, and stained with uranyl acetate and lead citrate. Three separate sections from each segment were examined by one of the authors (T.W.) using a Hitachi H500 TEM at various magnifications.

RNA Extraction, Reverse Transcription, Semiquantitative PCR, and Southern Blot Hybridization
A separate set of vein grafts (4 grafts per time point) was randomly chosen for PCR analysis. Tissue was processed according to previously published methods with modifications.5 6 In brief, each patent graft was excised, placed into Trizol denaturing solution (GIBCO BRL), and homogenized. Chloroform extractions, washes, and precipitations were performed. Purity and yields were determined by spectrophotometry. RNA was reverse transcribed using Superscript II (GIBCO BRL) according to the manufacturer's instructions. Controls included transcription of a known amount of spleen RNA and omission of the enzyme. DNA template (100 ng of input reverse-transcribed RNA) was combined with sense and antisense primers (Table 1Down) specific for MCP-1 or G3PDH, [{alpha}-32P]dCTP (5 µCi per reaction), dNTPs, buffer, and Taq polymerase (Pharmacia). To ensure that amplification was performed in the linear range, optimal cycle numbers and annealing temperatures were determined (data not shown). Samples were subjected to 28 (G3PDH) or 30 (MCP-1) cycles of amplification in a thermal cycler (Stratagene). One cycle was defined as 94°C for 1 minute, 66°C for 1 minute, and 72°C for 2 minutes. Rat spleen cDNA served as the positive control. Negative controls included omission of the template and enzyme. PCR fragments were run on 1.8% agarose gels, which were stained with ethidium bromide, photographed, and subjected to confirmatory Southern hybridization. The DNA was denatured, transferred, and immobilized on a Zeta-Probe blotting membrane (Bio-Rad). Hybridization proceeded according to the manufacturer's instructions. The DNA was hybridized to a 32P 5' end–labeled specific probe at 55°C overnight. Oligonucleotide probes internal to the original PCR primers were used (Table 1Down). After extensive washing, the filter was dried and autoradiography performed. In addition, the PCR fragments were electrophoresed on 7% polyacrylamide gels, after which autoradiography and scintillation counting of relevant bands were performed. Confirmatory densitometry was done using computerized image analysis. Primers and probes were either derived from the published cDNA sequences of rat MCP-18 and G3PDH9 or purchased from Clontech.


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  		Table 1. Oligonucleotide Sequences for Reverse Transcription PCR and Southern Hybridization

Image and Statistical Analyses
Image analysis to quantify IH was performed as previously described5 with modifications. All tissue sections were imaged using a videomicroscope and analyzed using Image-1 image analysis software (Fryer Co). Analysis was done in a blinded fashion. Total neointimal areas were calculated by using the internal elastic lamina as the line of demarcation between the intima and the media-adventitia. A mean area for each section was derived from three measurements; all the means per time point were averaged to derive an overall mean. Total and neointimal cells as well as the number of ED1+ and ED2+ cells per cross section were counted. Results were expressed as mean±SEM. To determine whether values represented significant differences from nonsurgical controls, a one-way ANOVA was performed followed by Fisher's protected least significant difference procedure to compare the means of the treatments. The relative abundance of MCP-1 protein as detected by immunostaining was scored subjectively on a graded scale in a blinded fashion.


*    Results
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IH Is Induced in Grafts
Animal survival and graft patency rates were 100%. Predictably, IH was induced in all grafts, with neointimal areas increasing progressively throughout all time points (Fig 1ADown). Total cell numbers increased until 4 weeks and then began to decline (Fig 1BDown). In contrast, the numbers of cells in the neointima continued to increase. Morphometric and histological changes observed in the vein wall were consistent with our previously reported results.5 6 Early endothelial denudation was accompanied by platelet and leukocyte adherence to the subendothelial space. The degeneration of medial cells and marked mononuclear cell infiltration were detected by 1 day. Transmural mononuclear cells were visible by 1 week (Fig 2ADown). By 2 weeks, a regenerated endothelium and an eccentric neointima were clearly discernible in all grafts (Fig 2BDown). Neointimal areas and graft cellularity had increased significantly by 4 weeks (Fig 2CDown).



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  		Figure 1. IH is induced in rat vein grafts. A, Bar chart shows mean neointimal areas per proximal cross section (µm2; n=5 per time point); error bars denote SEM. The neointimal areas of 1-, 2-, 4-, and 8-week vein grafts were statistically different from that of nongrafted epigastric vein intima (P=.0001). After 1 day, there was always a significant increase in area between consecutive time points (P<.05). B, Bar chart shows the results of cell counting. Total and neointimal hematoxylin-stained nuclei per proximal cross section were counted manually. Values were derived from 5 grafts per time point; error bars represent SEM.



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  		Figure 2. Photomicrographs show development of intimal hyperplasia in healing vein grafts. Nongrafted contralateral epigastric veins did not develop intimal hyperplasia. A, Marked cellular infiltration, luminal fibrin, and focal aggregates of mononuclear leukocytes (arrows) were seen in a 1-week graft. B, Eccentric developing neointima and continued cellular infiltration in a 2-week graft. C, A 4-week graft displays florid intimal hyperplasia. The media is markedly acellular, and some infiltrating cells are shown within the adventitia. L indicates lumen; N, neointima; M, media; and A, adventitia. Hematoxylin/eosin staining (original magnification x80).

TEM confirmed and extended all histological findings. Medial smooth muscle cell degenerative changes and extensive cell death were notable. Infiltrating mononuclear leukocytes were present luminal and abluminal to the internal elastic membrane and were surrounded by cell debris (Fig 3ADown); this zone of necrosis persisted throughout all later time points. At 2 weeks, the majority of neointimal cells were identified as mononuclear leukocytes (Fig 3BDown). Also present were smooth muscle–like cells with ultrastructural characteristics consistent with those of myofibroblasts: irregular outlines, disorganized microfilaments, and abundant, rough endoplasmic reticulum. These cells were the major component of the neointima at 4 and 8 weeks.



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  		Figure 3. TEM photographs display mononuclear cells in rat vein grafts. A, In a 1-week graft, a mononuclear cell (arrow) is shown penetrating the endothelium; other mononuclear cells are visible in the neointima (arrowheads). Original magnification x6900. B, Neointima of a 2-week graft is mainly composed of mononuclear cells (arrowheads), fibrin, and cell debris. Morphology of the cells is consistent with that of monocytes late in their transformation to macrophages. The area above the internal elastic lamina (EL) is the neointima. Original magnification x6100. L indicates lumen; M, media; and F, fibrin.

ED1+ and ED2+ Macrophages Infiltrate and Persist in Healing Vein Grafts
Grafts were further analyzed immunohistochemically for the presence of monocytes and macrophages. Immunostaining was performed using the monoclonal antibodies ED1, which recognizes >95% of all rat monocytes and macrophages, and ED2, which recognizes resident macrophages only. Nongrafted epigastric veins contained no ED1+ cells. ED1+ monocytes were detected at the luminal surface by 1 day after grafting and were seen transmurally by 1 week (Fig 4ADown). ED1+ cell infiltration was maximal at 2 weeks. These cells were detected within the developing neointima, at the shoulders of developing lesions, and in the medial-adventitial layer. After 4 weeks, ED1+ macrophages were seen near the internal elastic lamina at the base of the neointima (Fig 4BDown). ED2+ resident macrophages were detected in small numbers in the adventitia of the normal vein. They were no longer detectable immediately after grafting but were detected by 1 week in the adventitia and increased steadily until 4 weeks (Fig 4CDown), after which their numbers remained significantly higher than in the normal epigastric vein. They were rarely seen outside the adventitia. The results of ED1+ and ED2+ cell counting are presented in Fig 5Down.



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  		Figure 4. Immunoperoxidase staining demonstrates the presence of monocytes/macrophages within healing vein grafts. Normal nonsurgical epigastric veins contained few macrophages. All graft sections stained in parallel with isotype-matched control antibodies were negative. A, In a 1-week graft, ED1+ monocytes and macrophages (arrows) are visible both at the luminal surface and scattered throughout the vessel wall. Original magnification x130. B, In a 4-week graft, granular ED1+ staining (arrows) is detectable in the interface between the developing neointima and the media/adventitia and within the adventitia. Original magnification x130. C, ED2+ resident macrophages (solid arrows) are clearly visible in the adventitia of an 8-week graft. The neointima contains no ED2+ macrophages. The internal elastic lamina (open arrow) separates the neointima from the media/adventitia. L indicates lumen; N, neointima; M, media; and A, adventitia. Original magnification x80.



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  		Figure 5. ED1+ cells are greatly increased after vein graft implantation. Bar chart illustrates the total numbers of ED1+ and ED2+ cells infiltrating the vein graft wall. After 1 day, the number of ED1+ cells in vein grafts differ significantly from those in nongrafted control veins (P=.0001). The appearance of ED2+ resident macrophages in the wall lags behind that of ED1+ monocytes/macrophages by {approx}1 week and is restricted to the adventitia. At 4-8 weeks, ED2+ cell counts significantly exceed those of nonsurgical control veins (P<.05). Values represent means derived from 5 grafts per time point; error bars denote SEM.

ED1+ Cells Predominate in the Developing Neointima
As shown in Table 2Down, ED1+ monocytes/macrophages predominated in the neointima at 1 day and 2 weeks after grafting. By 2 weeks, the neointima had enlarged and become morphologically well distinguished from the other wall layers. As the neointima continued to develop, monocytes and macrophages became progressively less numerous as other cell types, such as myofibroblasts, smooth muscle cells, and endothelial cells, populated the neointima. Nevertheless, the number of ED1+ cells remained significantly higher than in control veins.


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  		Table 2. Predominance of ED1+ Cells During Neointimal Development

MCP-1 mRNA Is Bimodally Upregulated in Vein Bypass Grafts
To investigate the most likely chemoattractant for graft-infiltrating monocytes and macrophages, semiquantitative reverse transcription-PCR was used to assess relative MCP-1 mRNA levels in vein grafts. MCP-1 mRNA levels were normalized against those of G3PDH, a constitutively expressed gene that is present in all cells. In normal epigastric veins, there was a low level of MCP-1 expression (Fig 6Down). After grafting, levels were increased variably by 1 hour and consistently by 4 hours, at 28-fold above controls. There was another increase at 1 week (117-fold above baseline) prior to the peak in macrophage number. Levels decreased substantially by 4 and 8 weeks but remained elevated 7-fold over control levels. The results of densitometry confirmed scintillation counting (data not shown).



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  		Figure 6. MCP-1 mRNA levels are augmented after vein graft implantation. A, Bar chart demonstrates the bimodal upregulation of MCP-1 mRNA in healing vein grafts, as quantified by PCR amplification. Four grafts at each time point were subjected to PCR, and each reaction was quantified by scintillation counting of relevant bands excised from two separate polyacrylamide gels. Bars represent mean values of MCP-1 counts normalized to those of G3PDH; error bars denote SEM. After grafting, there is an upregulation of MCP-1 mRNA, which persists throughout all assessed time points (overall P=.0001). The increase in mRNA at 4 hours differs significantly from that at 1 hour (P=.03) and at 4 days (P=.05), thus suggesting a true peak followed by a decline in message. The increase at 1 week differs significantly from that at both 4 days (P=.0001) and 2 weeks (P=.02), indicating a true second peak of upregulation. B, Representative inverse scan of an ethidium bromide–stained agarose gel demonstrates reverse transcription PCR amplification products of G3PDH (upper 408-bp band) and MCP-1 (lower 354-bp band) mRNA in vein grafts harvested at various time points. The intensity of MCP-1 bands varies, whereas that of G3PDH bands remains relatively constant. LN indicates lymph node; S, spleen; and EV, nongrafted epigastric vein.

Immunoreactive MCP-1 Is Increased After Vein Grafting
To detect MCP-1 protein in vein grafts, immunostaining with a specific antibody was performed on paraffin sections. Stained sections were scored in a blinded fashion; the temporal profile of MCP-1 localization is presented in Table 3Down. The pattern of protein expression agreed with reverse transcription-PCR results. In the normal epigastric vein, weak cytoplasmic and extracellular staining was seen in the intimal endothelium, with some diffuse staining throughout the medial and adventitial layers. Throughout all time points after grafting, marked staining was seen in the adventitia. At 1 hour after grafting, some focal luminal and medial staining associated with degenerating endothelial cells and smooth muscle cells remained but was absent by 4 hours. By 1 to 4 days, patchy luminal staining was detectable near adherent mononuclear cells and the remaining endothelium. Adventitial staining surrounding resident fibroblasts and infiltrating cells had increased, but medial staining was conspicuously absent. We did not detect any MCP-1–positive polymorphonuclear leukocytes. At 1 to 2 weeks, immunoreactive MCP-1 was seen in the adventitia, at the luminal edge, and in the developing neointima, often within the fibrin matrix and surrounding the infiltrating mononuclear cells. After 4 weeks, staining was confined mainly to the area around the internal elastic lamina at the base of the neointima, coincident with ED1+ monocytes/macrophages (Fig 7Down), as well as the luminal surface. Staining in the adventitia, though slightly weaker than at earlier time points overall, was also seen occasionally in the vasa vasorum.


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  		Table 3. Qualitative Comparison of Immunoreactive MCP-1 Protein Localization in Vein Graft Cross Sections



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  		Figure 7. Immunoperoxidase staining demonstrates colocalization of MCP-1 and ED1+ cells in the vein graft wall. A, Photomicrograph of an 8-week graft cross section stained with a rabbit polyclonal antibody against rat MCP-1 reveals marked MCP-1 deposition at the base of the neointima (large arrow), as well as some staining both at the endothelial surface (small arrow) and in the adventitia. Serial section stained with rabbit IgG was negative (data not shown). No counterstain (original magnification x130). B, Section stained with ED1 antibody against rat monocytes/macrophages reveals positive cells residing mainly at the base of the neointima (arrow), with a few cells in the adventitia. The staining is more granular, and the distribution appears both cytoplasmic and extracellular, suggesting the possibility of antigen shedding. Serial section stained with mouse IgG1 was negative (not shown). Differential interference contrast photography, no counterstain (original magnification x105). L indicates lumen, N, neointima; M, media; and A, adventitia.


*    Discussion
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 *Discussion
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IH in vein bypass grafts, a condition often leading to obliterate stenosis and subsequent graft failure, is characterized by a thickened neointima composed of mesenchymal cells and extracellular matrix. It is thought that IH begins as a wound healing response to vascular injury that evolves into a chronic condition of unchecked proliferation.2 The development of IH in vein grafts may be regulated by mediators of wound healing and inflammation, such as activated monocytes and macrophages.10 These mononuclear leukocytes possess multiple functions, including phagocytosis; antigen presentation; and secretion of mitogenic, fibrogenic, angiogenic, and chemotactic factors.3 4 11 12 Their importance in atherosclerosis is well documented,13 and their presence has been demonstrated in human vein grafts and in animal models of vein graft IH.5 14 15 16 17 18

The factors that recruit monocytes and macrophages to vein grafts have not yet been identified. One likely chemokine is MCP-1, a potent monocyte chemotactic and activating factor belonging to the C-C family of intercrine cytokines.7 MCP-1 is synthesized by endothelial cells19 20 21 , smooth muscle cells,22 fibroblasts,19 23 24 25 and monocytes/macrophages,24 26 27 28 all of which are present in vein grafts. MCP-1 is constitutively expressed in vascular tissues21 ; this expression can be further stimulated by various factors. MCP-1 has been localized within macrophage-rich atherosclerotic lesions and abdominal aortic aneurysms.29 30 31 In a rabbit arterial injury model, antibodies against MCP-1 have been shown to decrease IH,32 lending further credence to our hypothesis. Upregulation of this factor has been demonstrated in other disease processes involving macrophages, eg, rheumatoid arthritis,33 34 pulmonary disorders,35 36 hepatitis,37 chronic tissue rejection,38 and tumor development.39 40 In addition, MCP-1 upregulation has been correlated with macrophage infiltration in normal dermal wound repair.41 These observations strongly suggest a role for MCP-1 in IH development. In an effort to elucidate monocyte/macrophage recruitment in healing vein grafts, we examined MCP-1 gene expression in rat vein bypass grafts. This semiquantitative study reports biphasic MCP-1 upregulation at both the mRNA and protein levels in vein grafts that precedes maximal macrophage infiltration and IH development.

In the normal rat epigastric vein, there is weakly detectable MCP-1 mRNA expression, consistent with the low basal level of MCP-1 mRNA found in normal vessels.21 There are macrophages, albeit in low numbers, detectable in the adventitia. Early after surgery, MCP-1 gene expression increases in vein grafts. Various factors are candidates for affecting MCP-1 mRNA expression in these grafts. Thrombin, present as part of the coagulation cascade, is known to induce MCP-1 expression.42 The proinflammatory cytokines IL-1{alpha} and TNF-{alpha}, expressed early in healing vein grafts5 (J.R.H., unpublished observations, 19{bullet}{bullet}), have been shown in vitro to increase MCP-1 gene expression.19 20 TGF-ß, a fibrogenic cytokine with pleiotropic effects, and PDGF, a smooth muscle cell mitogen, also influence MCP-1 expression33 43 ; both are increased in hyperplastic vein grafts and restenotic arteries.6 44 45 46 Other possible mediators of MCP-1 upregulation in healing vein grafts include MCSF47 and IFN-{gamma}.28 It is known that transcription of MCP-1, as an early-response gene, is induced by fluid shear stress via a shear-inducible element in the promoter48 49 ; thus, it is conceivable that other hemodynamic factors also affecting vein grafts, such as compliance mismatch and pulsatile flow, may influence MCP-1 gene expression in vascular cells. Many of these proteins and hemodynamic factors have been associated with IH development.2 5 6 44 45 46 50 51

In our model, the levels of MCP-1 mRNA fluctuated in a biphasic manner, with peaks at 4 hours and 1 week. These peaks were followed by detectable MCP-1 immunoreactivity and immediately preceded maximal monocyte/macrophage infiltration peaks at 1 day and 2 weeks. It is notable that MCP-1 transcript levels did not return to baseline after acute inflammation had subsided ({approx}4 weeks). Instead, MCP-1 mRNA levels remained elevated 7-fold above control levels and macrophages persisted, suggesting a state of chronic inflammation. This finding contrasts with the disappearance of MCP-1 mRNA expression and macrophage infiltration at 2 weeks in healing dermal wounds41 and perhaps reflects the pathological consequences of vein grafting. Our findings suggest that this chemokine continuously recruits monocytes and macrophages to the healing grafts during the development of IH.

There is very weak MCP-1 protein expression as detected by immunohistochemical staining in the normal epigastric vein. Coincidentally, few macrophages are seen. Early after grafting, staining is reduced, corresponding to the partial loss of endothelial cells. Medial staining is attenuated, possibly as a result of a decrease in resident medial cells. Ultrastructural examination of vein graft tissue has shown that the majority of medial smooth muscle cells degenerate and die within hours after grafting (V.K.S., unpublished data, 19{bullet}{bullet}). However, MCP-1 protein remains detectable in the fibroblast-rich adventitia. MCP-1 immunoreactivity reappears at the luminal surface, concomitant with infiltrating cells. One to 2 weeks after grafting, immunostaining is augmented on the luminal surface, in the developing neointima, and in the adventitia. Possible sources of MCP-1 protein in vein grafts are regenerated endothelial cells, lymphocytes, and adventitial myofibroblasts. Medial immunostaining is conspicuously absent, reflecting the paucity of cells in this layer. At later time points, MCP-1 staining is seen occasionally at the luminal surface in the endothelium and most frequently in the necrotic zone surrounding the internal elastic lamina, an area where macrophages reside in the vein graft wall. Resident macrophages, which do not stain strongly for MCP-1, appear to be a population normally present in vessels and thus, may not be important in IH development.

MCP-1 may not be the sole chemoattractant for monocytes and macrophages in vein grafts; in light of the complex cytokine milieu of vein grafts, the potential for additive or synergistic effects of MCP-1 and other factors is great. In addition, MCP-1 may be recruiting other leukocytes such as T cells,52 which are present in hyperplastic rat vein grafts.5 Our results show that elevated levels of MCP-1 mRNA and protein correlate with transmural macrophage infiltration during early neointimal development. Overall, the evidence suggests an integral role for monocytes/macrophages in the development of IH in healing vein grafts. The persistence of both MCP-1 expression and macrophage infiltration in grafted veins further supports the idea that IH is a prolonged healing response. The relationship between MCP-1 expression and neointimal development merits further investigation, possibly highlighting clinical therapeutic strategies for the prevention of vein graft IH.


*    Selected Abbreviations and Acronyms
 
G3PDH = glyceraldehyde 3-phosphate dehydrogenase
IH = intimal hyperplasia
IL = interleukin
MCP-1 = monocyte chemotactic protein 1
PCR = polymerase chain reaction
PDGF = platelet-derived growth factor
TEM = transmission electron microscopy
TGF = transforming growth factor


*    Acknowledgments
 
This work was supported in part by grant No. 94-Gb-42 of the American Heart Association.


*    Footnotes
 
Reprint requests to John R. Hoch, MD, Department of Surgery—H4/736 CSC, University of Wisconsin, 600 Highland Ave, Madison, WI 53792.

Received August 26, 1996; accepted October 28, 1996.


*    References
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 *References
 

  1. Dalman RL, Taylor LM. Infrainguinal revascularization procedures. In: Porter JM, Taylor LM, eds. Basic Data Underlying Clinical Decision Making in Vascular Surgery. St Louis, Mo: Quality Medical Publishers; 1994:141-143.
  2. Davies MG, Hagen P-O. Pathobiology of intimal hyperplasia. Br J Surg.. 1994;81:1254-1269.[Medline] [Order article via Infotrieve]
  3. Kovacs EJ, DiPietro LA. Fibrogenic cytokines and connective tissue production. FASEB J.. 1994;8:854-861.[Abstract]
  4. Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leukoc Biol.. 1994;55:410-422.[Abstract]
  5. Hoch JR, Stark VK, Hullett DA, Turnipseed WD. Vein graft intimal hyperplasia: leukocytes and cytokine gene expression. Surgery.. 1994;116:463-471.[Medline] [Order article via Infotrieve]
  6. Hoch JR, Stark VK, Turnipseed WD. The temporal relationship between the development of vein graft intimal hyperplasia and growth factor gene expression. J Vasc Surg.. 1995;22:51-58.[Medline] [Order article via Infotrieve]
  7. Rollins BJ. JE/MCP-1: An early-response gene encodes a monocyte-specific cytokine. Cancer Cells.. 1991;3:517-524.[Medline] [Order article via Infotrieve]
  8. Yoshimura T, Takeya M, Takahashi K. Molecular cloning of rat monocyte chemoattractant protein-1 (MCP-1) and its expression in rat spleen cells and tumor cell lines. Biochem Biophys Res Commun.. 1991;174:504-509.[Medline] [Order article via Infotrieve]
  9. Tso JY, Sun X-H, Kao T-H, Reece KS, Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res.. 1985;13:2485-2502.[Abstract/Free Full Text]
  10. Adams DO, Hamilton TA. The cell biology of macrophage activation. Annu Rev Immunol.. 1984;2:283-318.[Medline] [Order article via Infotrieve]
  11. Rappolee DA, Mark D, Banda MJ, Werb Z. Wound macrophages express TGF{alpha} and other growth factors in vivo: analysis by mRNA phenotyping. Science.. 1988;241:708-712.[Abstract/Free Full Text]
  12. Nathan CF. Secretory products of macrophages. J Clin Invest.. 1987;79:319-326.
  13. Hansson GK. Immune and inflammatory mechanisms in the development of atherosclerosis. Br Heart J. 1993;69(suppl):S38-S41.
  14. Kockx MM, Cambier BA, Bortier HE, De Meyer GR, Van Cauwelaert PA. The modulation of smooth muscle cell phenotype is an early event in human aorto-coronary saphenous vein grafts. Virchows Archiv A Pathol Anat.. 1992;420:155-162.
  15. Kockx MM, Cambier BA, Bortier HE, De Meyer GR, Declercq SC, Van Cauwelaert PA, Bultinck J. Foam cell replication and smooth muscle cell apoptosis in human saphenous vein grafts. Histopathology.. 1994;25:365-371.[Medline] [Order article via Infotrieve]
  16. Amano J, Suzuki A, Sunamori M, Tsukada T, Numano F. Cytokinetic study of aortocoronary bypass vein grafts in place for less than six months. Am J Cardiol.. 1991;67:1234-1236.[Medline] [Order article via Infotrieve]
  17. Adcock GD, Adcock OT, Wheeler JR, Gregory RT, Snyder SO, Gayle RG, Trivedi AN. Arterialization of reversed autogenous vein grafts: quantitative light and electron microscopy of canine jugular vein grafts harvested and implanted by standard or improved techniques. J Vasc Surg.. 1993;6:283-295.
  18. Zwolak RM, Kirkman TR, Clowes AW. Atherosclerosis in rabbit vein grafts. Arteriosclerosis.. 1989;9:374-378.[Abstract/Free Full Text]
  19. Strieter RM, Wiggins R, Phan SH, Wharram BL, Showell HJ, Remick DG, Chensue SW, Kunkel SL. Monocyte chemotactic protein gene expression by cytokine-treated human fibroblasts and endothelial cells. Biochem Biophys Res Commun.. 1989;162:694-700.[Medline] [Order article via Infotrieve]
  20. Rollins BJ, Yoshimura T, Leonard EJ, Pober JS. Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE. Am J Pathol.. 1990;136:1229-1233.[Abstract]
  21. Li YS, Shyy YJ, Wright JG, Valente AJ, Cornhill JF, Kolattukudy PE. The expression of monocyte chemotactic protein (MCP-1) in human vascular endothelium in vitro and in vivo. Mol Cell Biochem.. 1993;126:61-68.[Medline] [Order article via Infotrieve]
  22. Graves DT, Jiang YL, Williamson MJ, Valente AJ. Identification of monocyte chemotactic activity produced by malignant cells. Science.. 1989;245:1490-1493.[Abstract/Free Full Text]
  23. Rollins BJ, Morrison ED, Stiles CD. Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. Proc Natl Acad Sci U S A.. 1988;85:3738-3742.[Abstract/Free Full Text]
  24. Rollins BJ, Stier P, Ernst T, Wong GG. The human homolog of the JE gene encodes a monocyte secretory protein. Mol Cell Biol.. 1989;9:4687-4695.[Abstract/Free Full Text]
  25. Lukacs NW, Chensue SW, Smith RE, Strieter RM, Warmington K, Wilke C, Kunkel SL. Production of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1{alpha} by inflammatory granuloma fibroblasts. Am J Pathol.. 1994;144:711-718.[Abstract]
  26. Matsushima K, Larsen CG, DuBois GC, Oppenheim JJ. Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line. J Exp Med.. 1989;169:1485-1490.[Abstract/Free Full Text]
  27. Cushing SD, Fogelman AM. Monocytes may amplify their recruitment into inflammatory lesions by inducing monocyte chemotactic protein. Arterioscler Thromb.. 1992;12:78-82.[Abstract/Free Full Text]
  28. Brieland JK, Flory CM, Jones ML, Miller GR, Remick DG, Warren JS, Fantone JC. Regulation of monocyte chemoattractant protein-1 gene expression and secretion in rat pulmonary alveolar macrophages by lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1beta. Am J Respir Cell Mol Biol.. 1995;12:104-109.[Abstract]
  29. Nelken NA, Coughlin SR, Gordon D, Wilcox JN. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest.. 1991;88:1121-1127.
  30. Yla-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshimura T, Leonard EJ, Witztum JL, Steinberg D. Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci U S A.. 1991;88:5252-5256.[Abstract/Free Full Text]
  31. Koch AE, Kunkel SL, Pearce WH, Shah MR, Parikh D, Evanoff HL, Haines GK, Burdick MD, Strieter RM. Enhanced production of the chemotactic cytokines interleukin-8 and monocyte chemoattractant protein-1 in human abdominal aortic aneurysms. Am J Pathol.. 1993;142:1423-1431.[Abstract]
  32. Guzman LA, Whitlow PL, Beall CJ, Kolattakudy P. Monocyte chemotactic protein antibody inhibits restenosis in the rabbit atherosclerotic model. Circulation. 1993;88(pt 2).
  33. Villiger PM, Terkeltaub R, Lotz M. Production of monocyte chemoattractant protein-1 by inflamed synovial tissue and cultured synoviocytes. J Immunol.. 1992;149:722-727.[Abstract]
  34. Akahoshi T, Wada C, Endo H, Hirota K, Hosaka S, Takagishi K, Kondo H, Kashiwazaki S, Matsushima K. Expression of monocyte chemotactic and activating factor in rheumatoid arthritis: regulation of its production in synovial cells by interleukin-1 and tumor necrosis factor. Arthritis Rheum.. 1993;36:762-771.[Medline] [Order article via Infotrieve]
  35. Jones ML, Warren JS. Monocyte chemoattractant protein-1 in a rat model of pulmonary granulomatosis. Lab Invest.. 1992;66:498-503.[Medline] [Order article via Infotrieve]
  36. Jones ML, Mulligan MS, Flory CM, Ward PA, Warren JS. Potential role of monocyte chemoattractant protein 1/JE in monocyte/macrophage-dependent IgA immune complex alveolitis in the rat. J Immunol.. 1992;149:2147-2154.[Abstract]
  37. Marra F, Valente AJ, Pinzani M, Abboud HE. Cultured human liver fat-storing cells produce monocyte chemotactic protein-1: regulation by proinflammatory cytokines. J Clin Invest.. 1993;92:1674-1680.
  38. Russell ME, Adams DH, Wyner LR, Yamashita Y, Halnon NJ, Karnovsky MJ. Early and persistent induction of monocyte chemoattractant protein-1 in rat cardiac allografts. Proc Natl Acad Sci U S A.. 1993;90:6086-6090.[Abstract/Free Full Text]
  39. Negus RPM, Stamp GWH, Relf MG, Burke F, Malik STA, Bernasconi S, Allavena P, Sozzani S, Mantovani A, Balkwill FR. The detection and localization of monocyte chemoattractant protein-1 (MCP-1) in human ovarian cancer. J Clin Invest.. 1995;95:2391-2396.
  40. Sato K, Kuratsu J-I, Takeshima H, Yoshimura T, Ushio Y. Expression of monocyte chemoattractant protein-1 in meningioma. J Neurosurg.. 1995;82:874-878.[Medline] [Order article via Infotrieve]
  41. DiPietro LA, Polverini PJ, Rahbe SM, Kovacs EJ. Modulation of JE/MCP-1 expression in dermal wound repair. Am J Pathol.. 1995;146:868-875.[Abstract]
  42. Colotta F, Sciacca FL, Sironi M, Luini W, Rabiet MJ, Mantovani A. Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin. Am J Pathol.. 1994;144:975-985.[Abstract]
  43. Cochran BH, Reffel AC, Stiles CD. Molecular cloning of gene sequences regulated by platelet-derived growth factor. Cell.. 1983;33:939-947.[Medline] [Order article via Infotrieve]
  44. Majesky MW, Reidy MA, Bowen-Pope DF, Hart CE, Wilcox JN, Schwartz SM. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol.. 1990;111:2149-2156.[Abstract/Free Full Text]
  45. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor ß1 during repair of arterial injury. J Clin Invest.. 1991;88:904-910.
  46. Nikol S, Isner JM, Pickering JG, Kearney M, Leclerc G, Weir L. Expression of transforming growth factor-ß1 is increased in human vascular restenosis lesions. J Clin Invest.. 1992;90:1582-1592.
  47. Shyy Y-J, Wickham LL, Hagan JP, Hsieh H-J, Hu Y-L, Telian SH, Valente AJ, Sung K-L, Chien S. Human monocyte colony-stimulating factor stimulates the gene expression of monocyte chemotactic protein-1 and increases the adhesion of monocytes to endothelial monolayers. J Clin Invest.. 1993;92:1745-1751.
  48. Shyy Y-J, Hsieh H-J, Hu Y-L, Usami S, Chien S. Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium. Proc Natl Acad Sci U S A.. 1994;91:4678-4682.[Abstract/Free Full Text]
  49. Shyy Y-J, Lin M-C, Han J, Lu Y, Petrime M, Chien S. The cis-acting phorbol ester "12-O-tetradecanoylphorbol 13-acetate"-responsive element is involved in shear stress-induced monocyte chemotactic protein 1 gene expression. Proc Natl Acad Sci U S A.. 1995;92:8069-8073.[Abstract/Free Full Text]
  50. Agnelli G. Thrombin plays a pivotal role in vascular re-occlusion after PTCA and coronary thrombolysis. Cardiovasc Res.. 1996;31:232-234.[Medline] [Order article via Infotrieve]
  51. Dobrin PB, Littooy FN, Endean ED. Mechanical factors predisposing to intimal hyperplasia and medial thickening in autogenous vein grafts. Surgery.. 1989;105:393-400.[Medline] [Order article via Infotrieve]
  52. Carr MW, Roth SJ, Luther E, Rose SS, Springer TA. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci U S A.. 1994;91:3652-3656.[Abstract/Free Full Text]



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