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From Allen Bradley Medical Sciences Laboratory, Medical College of Wisconsin, Milwaukee, WI.
ABSTRACT
Objective— Previous studies have suggested that neointimal formation, a central cause of vein graft stenosis, has several potential cell sources. It was hypothesized that neointimal cells arise primarily from the cells of the vein graft.
Methods and Results— This study investigated vein graft neointimal cell origins using a model of vein-to-artery cross-transplantation between transgenic Rosa26 mice (constitutive expression of bacterial ?-galactosidase marker gene) and wild-type mice. Vein-originating cells survived and make a major contribution to neointimal formation within the vein graft, mostly adjacent to the lumen/endothelium, suggesting an intimate association with endothelial cells. Cross-transplantation of veins from thrombomodulin promoter-driven ?-galactosidase reporter transgenic mice to wild-type arteries demonstrated survival of vein graft endothelial cells. Neointimal thickening was greater at the proximal and, to a lesser extent, distal ends, in comparison to the middle of the graft. By contrast, arterial grafts had almost no neointimal formation throughout the graft. The relative neointimal wall thickness is much greater in this model compared with other murine and larger-species vein graft models, even showing near-occlusive stenosis of the perianastomotic region.
Conclusions— Vein graft neointimal cells arise predominantly from vein-derived cells, suggesting clinical relevance of stenosis-inhibiting therapies directed at the vein graft.
Vein grafting between Rosa26 (constitutive marker gene expression) and wild-type mice demonstrated that neointimal cells originate predominantly from the vein graft. Neointimal proliferation appeared early and adjacent to the graft-preserved endothelium. Neointimal thickening was progressive with time and was greater at the proximal and distal ends of the graft.
Key Words: bypass graft ? neointima ? Rosa26 ? thrombomodulin ? stenosis
Introduction
Vascular bypass surgery using vein grafts has become an established procedure for treating many conditions, most notably myocardial infarction (eg, coronary bypass) and limb claudication (eg, femoral-popliteal bypass grafting).1,2 Despite high early success rates of these procedures, it is estimated that 20% to 50% of cases undergo stenotic occlusion of the graft within 5 years,3–5 caused by an inward growth of neointimal cells.6 Experimental models that mimic clinical vein grafting show a characteristic burst of neointimal cell proliferation early after grafting (3 to 7 days), which leads to a thickened intimal layer by 1 month.7,8 This neointimal growth appears to stabilize beyond 1 to 2 months in animal models9,10 and does not create significant graft stenosis at any time, thus differing from the clinical progression of this complication.
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The source of neointimal cells remains controversial. Studies using arterial injury models (eg, balloon angioplasty) have presented evidence for medial smooth muscle cells, adventitial (myo)fibroblasts, and blood-borne stem cells as major contributors to the neointima.11–15 Studies using vein graft models have supported adjacent arterial media as a major source for neointimal cells.7,16 Arterial and organ allotransplantation models have found that circulating progenitor cells and adjacent (recipient) arterial media are the major contributors to transplant-associated neointimal formation.15,17–19 A recent study using a murine vein graft model demonstrated that the neointimal cells within the graft arise from both the vein graft and the recipient artery.20 The following study confirms this result using a new murine model that has direct analogy to clinical vein grafting and stenosis. Furthermore, the findings show that neointima cells from the vein graft arise from and proliferate predominantly at the innermost (luminal) aspect, suggesting involvement of vein-derived endothelial cells.
Methods
Vein Graft Model
A new model of vein graft transplantation in mice was developed by the author and is similar to recently described murine vein graft models20–22 but uses a smaller-diameter graft, a branch of the jugular vein, interpositioned to the femoral artery. This model has more clinical analogy in terms of graft-to-artery diameter match and it places the graft into a similar anatomic location as in clinical femoral–popliteal bypass grafting, with graft reconstruction of a primarily muscular (nonelastic) artery. Grafts were transplanted between C57Bl/6J wild-type mice and Rosa26 mice on the C57Bl/6J background (Jackson Labs, Bar Harbor, Me; a transgenic line that constitutively expresses the ?-galactosidase reporter gene in all cells23) or TM-LacZ heterozygous mice (?-galactosidase reporter gene under the thrombomodulin promoter, expressed only in endothelial cells; kindly provided by Hartmut Weiler) on the C57Bl/6J background.24
Animal protocols were approved by the Institutional Animal Care and Use Committee and were in accordance with NIH and AALAC guidelines. After intraperitoneal pentobarbital induction of anesthesia (60 mg/kg body weight), a branch of the external jugular vein (2 mm in length) was dissected and transplanted (in reverse orientation) from one mouse into the femoral artery of another mouse, using 2 end-to-end microvascular anastomoses to achieve graft placement. A series of arterial grafts was also done, transplanting a femoral arterial graft (2 mm in length) from one mouse into the femoral artery of another mouse. The microsurgical procedure was performed at 60x magnification with a Wild 651 operating microscope, using 6 to 10 interrupted stitches of 11-0 or 12- 0 nylon suture per end-to-end anastomosis. A single intravenous bolus of heparin (5 U in 100 μL) was given immediately before reflow of the femoral artery. Patency of the grafted vessel was evaluated by mechanically emptying and observing refill downstream from the graft25 immediately after repair and again at 1 and 5, 10, or 30 days after grafting.
Experimental Design
Grafts harvested from wild-type mice were transplanted into Rosa26 mice. Grafts harvested from Rosa26 mice or TM-LacZ mice were transplanted into wild-type mice.
Evaluation
At 5, 10, or 30 days after grafting, grafted vessels were doubly ligated outside the graft margins, immediately removed, and immersion-fixed in 4% formaldehyde/0.5% glutaraldehyde in phosphate-buffered saline (PBS) for 10 minutes. After washing in PBS, samples were immersed overnight in 5-bromo-4-chloro-3-indolyl-?-D-galactoside (X-gal) stain (specific histochemical stain for ?-galactosidase presence).23 Most samples were then processed through ascending ethanol and xylene, paraffin-embedded, and cross-sectioned at 6 μm. Several samples were placed in 30% sucrose after initial X-gal staining, then frozen for cryostat sectioning at 12 μm thickness. Deparaffinized slides were brought back through xylene and ethanol to PBS, with further staining in X-gal. Photomicrographs were taken through a Westover light microscope with a Polaroid DMC-1 camera and images were captured as computer files. Slides were further stained with hematoxylin and eosin (H&E), Verhoeff elastin, Masson trichrome stain, or Nuclear Fast Red counterstain (Sigma).
Immunohistochemical staining was performed with primary antibodies to -smooth-muscle actin (1:200 dilution; Sigma) or CD-31 (anti-PECAM, 1:100 dilution; Pharmingen), followed by biotin-linked secondary antibodies and avidin-HRP, and final staining with diaminobenzidine.
Histomorphometry was accomplished on digital images using NIH ImageTool 3.0 software. Slides displaying evident sutures were used to identify the vascular repair sites. Representative slides from the proximal, middle, and distal portions of the graft and adjacent recipient artery portions underwent analysis. Neointimal and medial thicknesses and areas were measured: Tissue layer thickness measurements were made at 8 points 45° apart around the vessel circumference and averaged. Vessel diameters were determined by measuring the inner (luminal) circumference and dividing by to get the diameter of a circle based on the given perimeter; this data manipulation gave a more accurate measure of the lumen diameter because tissue sections were often collapsed and noncircular. Intimal and medial areas were determined by subtracting the total inner luminal area from the total outer area for each layer. The data for each parameter were statistically compared among groups with ANOVA and between groups with Student-Newman-Kuehls posthoc test, using P<0.05 to assign statistical significance.
Results
Gross Evaluation
A total of 51 vein grafts and 16 arterial grafts were attempted. Seven of the vein grafts and 2 of the arterial grafts were not completed because of technical errors. Eight mice failed to recover from anesthesia (7 vein grafts and 1 arterial graft), possibly caused by hemorrhagic losses. Of the 37 vein grafts completed, 30 (81%) were patent at harvest; the failed grafts were thrombosed at 1 day postoperatively and at harvest. The 13 completed arterial grafts had a 92% success rate (12/13), with the 1 failure caused by thrombosis at 1 day postoperatively. All grafts that were thrombosed at 1 day postoperatively had resorbing or obliterated grafts at postoperative days 5, 10, or 30. Patent grafts had clearly evident pulsatile flow within the graft and distal artery at every observation time.
Rosa26 grafts placed into wild-type mice showed a large amount of blue-stained tissue after X-gal staining and before sectioning; this blue region did not extend substantially into the recipient artery on either side of the graft. Conversely, wild-type grafts placed into Rosa26 mice had extensive blue staining of the recipient artery, with a more lightly blue-stained tissue overlying the graft (data not shown). Very little blue-stained tissue was grossly evident in TM-LacZ grafts placed into wild-type arteries.
Histology/Histochemistry/Immunohistochemistry
Sections from Rosa26 veins grafted into wild-type arteries showed distinct blue (X-gal) staining of the inner neointimal cells, with progressively less stain toward the media at all time points (Figure 1a). Arterial grafts had little neointima formation that stained blue along with the medial and inner adventitial layers (Figure 1b). Vein graft neointima was highly pronounced at 30 days, with little cellularity or thickness of the medial layer (Figure 1c). Arterial graft media was atrophic and had lost thickness (Figure 1d) relative to the adjacent artery.
Figure 1. Cross-sections of X-gal–stained Rosa26 vein graft (A) and arterial graft (B) harvested at 30 days, showing heavy blue staining primarily in the luminal neointima (A) and arterial media (B). Masson trichrome stain of Rosa26 vein graft (C) and arterial graft (D) at 30 days, showing thick proximal vein graft neointima (C) and relatively little arterial graft neointima (D, within black-stained internal elastic lamina). X-gal-stained TM-LacZ-to-wild-type vein graft at 5 days (E); Rosa26-to-wild–type vein graft at 5 (F) and 30 (G) days; wild–type-to-Rosa26 vein graft at 30 days (H and I are counterstained with Nuclear Fast Red; blue arrows in I show cell sites of recipient-originating neointimal cells). Bars in A–D=60 μm; bars in E–I=15 μm; lum=vessel lumen. Black arrows indicate adventitial-medial boundary (green stain in C and D).
Endothelium was preserved and showed clear X-gal staining in TM-LacZ vein grafts (Figure 1e). In Rosa26 vein grafts, the progression of neointimal growth with time appeared to be initiated and maintained near the endothelial layer, from 5 days (Figure 1f) to 30 days (Figure 1g). In contrast, wild-type vein grafts placed in Rosa26 arteries showed little X-gal staining near the lumen, with most recipient-derived (X-gal-positive) cells appearing in the adventitia and at later times in the outer media (Figure 1h). Some of these grafts showed regions of blue-stained neointima on a portion of the cross-sectioned graft, suggesting a more pronounced influx of recipient cells of unknown origin in these regions (Figure 1i).
Several vein grafts showed marked stenosis at the vascular repair site (Figure Ia, available online at http://atvb.ahajournals. org), even causing near-occlusion of the lumen (Figure Ib), in comparison to the adjacent arterial lumen diameter (Figure Ic). Adjacent sections and double-stained sections (X-gal staining, followed by H&E) showed predominantly graft-originating cells in the stenotic, neointima-filled repair zone.
The neointima of both vein and artery grafts demonstrated only partial staining for -smooth-muscle actin, which was found more densely in the inner-most layer and most prominently at the 30-day evaluation time (Figure IIa, available online at http://atvb.ahajournals.org). The residual medial layer of both vein and artery grafts had little cellularity and associated poor staining of -smooth-muscle actin. The endothelial cell marker, PECAM, was evident as a single cell layer in all grafts evaluated, at all time points (Figure IIb). Together with the positive X-gal staining in TM-LacZ grafts, this indicated maintenance of the endothelium in this murine vascular graft model.
Histomorphometry
The neointimal growth at 30 days was thickest in the proximal (upstream) portion of vein grafts (103±31 μm; mean±SD) in comparison to the central (49±27 μm) and distal (58±23 μm) regions and the adjacent recipient artery (5±2 μm) (P<0.01). Although the distal region showed greater neointimal thickness than the central region, this did not achieve statistical significance (P>0.05). All regions of the vein graft had dramatically thicker neointima in comparison to that of arterial grafts (Figure 2a; P<0.01). Arterial graft medial thickness decreased slightly (mean=19 μm) but not significantly from that of the adjacent artery (Figure 2a). In contrast, vein graft media was substantially reduced in comparison to the artery; however, the range of thickness (10 to 20 μm) was greater than that of control jugular vein media (range: 4 to 5 μm). Neointimal and medial areas reflected the wall thickness data, with significantly greater neointimal areas throughout the vein graft in comparison to the arterial grafts and adjacent recipient artery (P<0.01; Figure IIIa, available online at http://atvb.ahajournals.org). Total medial area apparently rose slightly (but did not achieve significance) within the vein graft; medial and intimal areas of arterial grafts were relatively constant and comparable to the adjacent artery (Figure IIIa). Lumen areas of vein grafts at all points after 30 days were much greater than those of the adjacent arterial regions of the vessel (P<0.01; Figure 2b); the central and distal regions of the vein graft also had significantly greater lumen areas in comparison to the proximal region (P<0.01). Arterial graft luminal areas increased slightly but insignificantly from those of the adjacent artery.
Figure 2. A, Neointimal and medial wall thicknesses (in microns) for vein grafts (n=8; black circles=neointima, black squares=media) and arterial grafts (n=7; white circles=neointima, white squares=media) at 30 days throughout the graft. "Proximal artery" and "distal artery" were measured from sections of the recipient artery on the respective sides of the graft. Data are means±SD. B, Vessel lumen areas (in square microns) for vein grafts (closed circles) and arterial grafts (white circles) at 30 days throughout the graft; lumen areas were calculated from circles that matched the measured luminal circumferences of the cross sections.
The time course of vein graft neointimal development showed progressive increases from 5 to 10 days and from 10 to 30 days at all points along the graft. The proximal neointima thickness increased from 14±11 μm at 5 days to 30±14 μm at 10 days, both significantly less than the thickness of 103±31 μm at 30 days (P<0.01; Figure 3a). Similar time-course changes were seen at the middle and distal portions of the graft. Relatedly, the neointimal area at all points in the graft increased with time, whereas the lumen area was found to progressively decrease from 5 to 30 days (Figure 3b).
Figure 3. Bar graphs of mean histomorphometric data for vein grafts at 5 days (n=5), 10 days (n=7), and 30 days (n=8). A, Neointimal wall thicknesses (in microns). B, Vessel lumen areas (in square microns); lumen areas were calculated from circles that matched the measured luminal circumferences of the cross-sections. Data are means±SD.
Discussion
The ability to interpose a vein graft in a murine artery with a consistent and relatively high amount of neointimal formation demonstrates the potential for this model to evaluate vein graft stenosis and methods to inhibit neointimal growth. Whereas the relative contribution of neointimal growth to wall thickness is small for rat, rabbit, and larger animal models,7–11,16 the proximal portion of the vein graft in the current model had a stenotic area that greatly reduced the luminal area (Figure 2b), even to apparent occlusion at the anastomotic site (Figure IIb). This finding of severe luminal stenosis at 1 month differs from the milder degrees of neointimal formation found in other vein graft models and is directly analogous to clinical graft stenotic occlusion. Furthermore, the low level of arterial graft neointima/stenosis is also consistent with clinical outcomes of low stenotic risk with arterial anastomosis or grafting.3–6
The findings of this study contrast with those of Redwood and Tennant16 in which a lack of evidence for rat vein graft cell survival was found. These investigators used a similar cross-transplantation model, using male grafts placed into female recipients, with polymerase chain reaction analysis of DNA isolated from grafts to identify an SRY (Y chromosome) target to determine male cell presence. Their inability to confirm donor graft cell presence may be model- or species-dependent and warrants further study. The clear evidence of vein-derived neointimal cells by the present study, supported by the findings of Hu et al,20 indicates that in murine systems a significant proportion of neointima is derived from cells of the vein graft. Whether this is also true in humans remains to be determined.
The findings of this study conflict with those of a recent report by Xu et al,26 a group that has an established model of murine vein grafting.20,21 Using bone marrow transplantation from Rosa26 or TIE-LacZ (endothelial cell-specific ?-gal–expressing) mice into lethally irradiated wild-type mice, which subsequently received wild-type vein grafts, it was shown that endothelial cells on the graft surface were absent initially (<3 days after grafting) and were progressively re-established by circulating progenitor cells.26 This finding is in marked contrast to that of the vein-derived endothelial phenotype found on the luminal surface at 5, 10, and 30 days in the present report (Figures 1e and IIb). This disparity may be caused by the difference in graft models. The use of a vein graft with larger diameter by Xu et al, together with a nonuniform artery-to-vein anastomosing technique (ligating the vein to the artery over a plastic ring) and graft placement in a higher-flow (carotid) artery may have caused greater shear stress and/or turbulence across the lumen surface leading to endothelial sloughing and subsequent "hematopoietic stem cell" replacement. In comparison, small vein grafts (ie, a branch of, not the main, external jugular vein) inserted by direct suture (uniform anastomosis) into the femoral artery (lower flow/shear) may have allowed preservation of the endothelium in the present report. A recent study27 on different models of artery injury, using marker gene marrow transplants, found little or no endothelial replacement by circulating cells when the injury was less traumatic to the vessel (ligation or cuff injury) in comparison with a denuding (intraluminal wire) injury model. In a rabbit vein graft study, Ehsan et al28 documented an immediate decrease in endothelial cell density on the graft surface that was associated with arterial pressure-induced stretch. The endothelial cells displayed rapid proliferation in the first 48 hours, resulting in re-establishment of baseline endothelial cell density three days after graft placement. This contrasts with the time course of Xu et al26 in which the circulation-derived endothelium was sparse at 3 days and was not re-established until 1 month. In further support of the findings herein of endothelial preservation on arterialized veins, Kwei et al29 used a murine artery-to-vein shunt model to demonstrate that the endothelial cells are maintained in the first 7 days, with smooth muscle cell (neointimal) formation within the vein wall. Although a shunt model is not directly analogous to vein grafting, it suggests that the conditions under which veins are manipulated can affect preservation of the endothelium. It remains to be determined which murine vein graft model is more analogous to the clinical condition of vein graft stenosis. Taken together, these studies suggest that >1 mechanism may need to be considered in the quest to control clinical vein graft neointimal development.
The medial layer of murine veins is very thin, generally only 1 cell layer in thickness. After graft placement cells in this layer have minimal survival with no apparent proliferation. In contrast, smooth muscle-like cells (ie, -smooth muscle actin-positive) are found most prominently on the lumen-ward side of the neointima, often with a nonsmooth-muscle zone adjacent to the residual media (Figure IIa). This is indirect evidence suggesting that neointimal cells do not arise from medial or adventitial layers. Furthermore, at least in this model, endothelial cells from the vein graft survive (Figure 1e) and the neointimal proliferative burst early after graft placement is seen predominantly in direct juxtaposition to the endothelial cells (Figure 1f). This suggests a possible endothelial origin for neointimal cells. In fact, others have found that intimal mesenchymal cells can arise from endothelial cells during development,30 and of more relevance, adult endothelial cells can be induced to undergo transdifferentiation into a smooth muscle phenotype in vitro,31,32 sometimes expressing simultaneous markers for both smooth muscle and endothelial cells.33 This presumptive pluripotent transdifferentiation of endothelial cells has even been extended to cardiac muscle.34 Vein graft neointimal cells not directly on the lumen surface did not display phenotypic traits of endothelial cells (PECAM expression, Figure IIb; thrombomodulin promoter-driven ?-gal expression, Figure 1e). If these cells arise from the endothelium, they apparently undergo a complete (and rapid) transdifferentiation to a smooth muscle cell type. Alternatively, these cells may have arisen from medial smooth muscle cells that maintained a close association with the endothelium, leaving their normal medial-layer region (ie, rapidly crossing or breaking down the internal elastic lamina). The proximity to the endothelium may indicate a paracrine influence on neointimal growth from endothelium-derived factors or from circulating growth factors that diffuse through a porous graft endothelial boundary. Experimental approaches to distinguish these different possibilities will need to be developed.
The TM-LacZ heterozygous transgenic mouse expresses lower levels of endothelial thrombomodulin than do wild-type mice.24 Thrombomodulin expression is downregulated in endothelial cells subjected to arterial shear stress in vitro35 and in vein grafts placed under arterial flow conditions.36–38 Despite presumably lower thrombomodulin expression levels, the TM-LacZ vein grafts herein had clear evidence of thrombomodulin activity as indicated by ?-gal marker expression (Figure 1e). Thus, the antithrombotic activity of thrombomodulin is apparently maintained in this murine model of vein grafting. Whether this activity has a major influence in vein graft thrombosis and/or neointimal formation is not known.
More extensive research has been done on neointimal progression occurring after balloon angioplasty. Animal models either replicate the procedure or use an endothelial denuding technique to cause arterial injury. Recent studies have indicated that the main sources of neointimal cells in these models are the arterial medial smooth muscle, periadventitial fibroblasts, and/or blood-borne (hematogenous) cells.11–15 Allotransplant-associated vascular neointima has also been indicated to arise from the recipient.15,17–19 These findings are in marked contrast to the data presented herein, and particularly by Hu et al20 in which wild-type vein grafts were placed in wild-type mice that had previously undergone irradiation and bone marrow replacement with Rosa26 (or SM-22 promoter-driven ?-gal expressing) bone marrow. These investigators found no evidence of bone marrow-derived cells in the graft neointima. The paucity of Rosa26 cells in the inner layers of the neointima after wild-type to Rosa26 grafting in the present study (Figure 1h and 1i) further negates a hematogenous origin for a major source of neointimal cells in vein grafts. These contrasting findings suggest a profound difference in vein graft neointimal formation relative to its development after angioplasty and allotransplantation, indicating that separate therapeutic interventions may be needed for each procedure. Neointimal formation originating from cells of the vein graft suggests that therapeutic avenues could be targeted at the vein graft, perhaps applied selectively during ex vivo transfer from vein harvest to surgical graft placement.
Acknowledgments
Supported by a Medical College of Wisconsin New Investigator grant.
Acknowledgments
The author is grateful to Chao-Ying Chen for histologic preparations and to Bern Teitz (Micrins Inc, Lake Forest, Ill) for providing 11-0 and 12-0 microsutures for this study.
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