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From the Surgical Research Laboratories,* Children’s Hospital, Boston; the Department of Pathology, Brigham and Women’s Hospital, Boston; the Department of Radiology, Beth Israel Deaconess Medical Center, Boston; the Department of Radiology, Children’s Hospital, Boston; and Harvard Medical School,¶ Boston, Massachusetts
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Abstract |
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Exposure to the new biomechanical environment of the arterial circulation is thought to be an important stimulus for the vascular remodeling of a venous bypass graft.3 Several anatomical and cellular changes in grafted veins have been documented. For example, in a model of canine femoral vein grafts, arterialized veins appear thicker than normal saphenous veins, with infiltration of polymorphonuclear leukocytes into the medial layer 2 days after surgery.4 Moreover, a role for the extracellular matrix and its regulators (matrix metalloproteinases, MMPs) has been established. Thus, when human saphenous vein segments were perfused ex vivo under arterial versus venous conditions, a significant increase in the production of MMP-9 and MMP-2 under arterial flow was observed, together with increased secretion of MMP-9 and higher retention of MMP-2 in the extracellular space.5,6 In humans, tenascin-C expression has been documented in the media and adventitia of patent saphenous vein grafts but not in occluded grafts or normal arteries and veins.7
Furthermore, several large animal models of vein bypass grafts were developed to characterize in detail venous arterialization in vivo.8,9,10,11 In a well-characterized rabbit model of jugular vein interposition into the carotid artery, arterialized veins demonstrated altered sensitivity to bradykinin,12 norepinephrine, histamine, serotonin,13 adenosine,14 and dopamine.15 Tissue factor expression was increased in the vessel intima 3 days after grafting, but was not detected 14 or 28 days after grafting.16 There was also differential expression of collagen III and IV over time in the arterialized bypass grafts.17 In addition, changes in gene expression have been characterized, such as down-regulation of endothelin B receptors18 and an initial reduction in thrombomodulin expression by the luminal endothelium.19 Vascular endothelial growth factor mRNA expression was increased in vein grafts of dogs20 and rats.21
While arterialization of venous bypass grafts involves numerous remodeling processes, the initial adaptation of the vein segment may subsequently lead to significant vascular pathologies. Several animal models have been designed to resemble the pathological consequences of venous arterialization. For example, in mice, arteriosclerotic and neointimal hyperplastic lesions were induced in arterialized veins using vascular cuffs or an end-to-side surgical anastomosis.22,23 Using the model originally described by Zou et al,22 the role of specific genes in the development of accelerated atherosclerotic lesions was assessed.24,25,26 Although these murine models allow for the study of venous graft pathology, the normal early physiological adaptive response of the venous vessel to the arterial circulation remains elusive. Here, we developed a novel model of venous arterialization in mice by creating an arterio-venous connection between the common carotid artery and the external jugular vein in situ, and described the early morphological and functional changes of a vein exposed to the arterial circulation.
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Materials and Methods |
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C57BL/6J male mice, 8 to 10 weeks old, were anesthetized by intraperitoneal injection of Avertin (tribromoethanol, Fisher Scientific, Pittsburgh, PA) 125 to 240 mg/kg. Analgesia was administered pre-operatively as a subcutaneous injection of Buprenex (buprenorphine) 0.05 to 0.1 mg/kg. After adequate shaving and preparation of the neck skin, a 1-cm right paramidline vertical incision was made through skin and fascia. The cervical fat was dissected and excised to expose the external jugular vein (JV). All branches of the JV were ligated using 10–0 ethilon (Ethicon, Somerville, NJ) and divided. The JV was clamped distally and ligated proximally with 8–0 silk (Ethicon) and divided. The common carotid artery (CCA) was clamped proximally and ligated just below the carotid bifurcation with 8–0 silk. A perfluorocarbon biocompatible microvascular catheter (Fine Science Tools, San Francisco, CA), measuring 400 µm in outer diameter and 200 µm in inner diameter, was cut into 2-mm segments and soaked in heparin (100 units/ml) before the surgical intervention. A carotid arteriotomy was made between the proximal clamp and distal ligation, and the catheter was inserted into the CCA proximally and the JV distally. The catheter was secured to both vessels by circumferential ligation using 8–0 silk. The CCA was divided between the distal ligation and catheter. Upon removal of the microvascular clamps, pulsatile flow was visualized in the JV. Sham surgery was performed in which cervical dissection was completed, and all branches of the JV were ligated and divided using 10–0 ethilon. A microvascular catheter was placed adjacent to the JV. In all cases, the skin was closed using 4–0 vicryl in a continuous fashion. Operative time averaged 40 to 60 minutes. All animal procedures were performed with approval from the Institutional Animal Care and Use Committee.
Imaging and Hemodynamic Analysis
Magnetic resonance angiography (MRA) was performed with an 8.5 Tesla micro imaging system, operating at 360 MHz proton frequency (DRX-360, Burker BioSpin MRI, Inc, Karlsruhe, Germany). Mice were anesthetized by inhalation of 1 to 2% isofluorane and placed in a radio frequency coil (I.D. 20 mm). After localizer images, a two-dimensional phase contrast (PC) sequence was conducted over the entire neck region. The imaging parameters were as follows: repetition time/echo time, 20 to 25 msec/5.5 msec; field of view, 16 x 16 mm2; slice thickness, 1 mm; number of slices, 16 to 18; matrix size 128 x 128; number of excitations, 4; flip angle, 60 degrees; and the velocity-encoding coefficient, 10 cm/sec. The maximum intensity projection (MIP) as post-processing technique was applied to all PC MR images in each animal. The total imaging time was 9 to 11 minutes. Mice were imaged 1 day and 7 days after surgery.
Duplex Doppler ultrasound was performed on the right, surgical arterialized JV and the left, non-surgical CCA and JV. Mice were anesthetized by intraperitoneal injection of Avertin (tribromoethanol) 125 to 240 mg/kg. Imaging was performed using a high-resolution linear transducer operating at a scanning frequency of 15 MHz. The field of view was limited to the most superficial (5 mm) structures of the neck. Scale, wall filter, and gain settings were optimized for both color and pulsed Doppler studies. Each vessel was identified by color Doppler, and lumenal diameter of the vessel was obtained by placing measurement calipers on the frozen video display of a representative color image of each vessel. A sample volume measuring <2 mm was placed over each vessel for hemodynamic sampling during real-time scanning. Multiple sampling of flow was obtained for at least 5 consecutive seconds on each vessel of interest. Mice were imaged 1 day and 7 days after surgery.
Tissue Isolation
Vessels were harvested at 1 day, 3 days, and 7 days after surgery. At the time of harvest, the animals were anesthetized under inhaled isofluorane, and the arterialized vein was exposed through the previous incision. Graft patency was confirmed by the visual appearance of pulsatile arterial blood flow in the external jugular vein. After confirmation of patency, the abdominal and thoracic cavities were opened by a midline incision. The animal was euthanised under anesthesia by cardiac puncture and incision of the left renal vein. The animal was perfusion-fixed (110 mm Hg) using phosphate-buffered saline (PBS) followed by 2% paraformaldehyde through the left ventricle. A 4-mm segment of the arterialized vein was isolated distal to the microvascular catheter anastomosis, and the contralateral JV was harvested as a control at all times studied. All vessels were dissected from the surrounding tissue and fat. The vessels were immediately oriented and snap-frozen in OCT for further analysis.
Immunohistochemical Analysis
Sequential 5-µm sections of the arterialized vein, beginning 1 mm distal to the anastomosis of the microvascular catheter, and the contralateral JV control vessel were processed for histology and immunohistochemistry. Hematoxylin and eosin staining was performed for gross morphological examination. All sections were mounted using Permount (Fisher, Medford, MA).
Frozen sections were fixed with acetone, blocked with normal serum, avidin, and biotin solutions, and then incubated with primary antibody for 1 hour at room temperature under moist conditions. Sections were sequentially treated with goat anti-rat biotin at 1:200 dilution (Jackson Immunochemicals, West Grove, PA), blocked for endogenous peroxidase using 0.3% hydrogen peroxide for 20 minutes, and then treated with the ABC-Elite peroxidase kit (Vector Laboratory, Burlingame, CA). Finally, sections were developed using AEC chromagen and counterstained with Gill’s hematoxylin No. 2. Negative controls were performed for all antibodies by substituting an isotype-match antibody.
Serial sections were stained with anti-
-smooth muscle actin (1:150) conjugated to alkaline phosphatase (Sigma, St. Louis, MO) and developed with Fast Red (Vector Laboratory) for 30 minutes at room temperature. Vessels were also labeled with a mouse monoclonal antibody against proliferating cell nuclear antigen (PCNA, BD Biosciences, San Diego, CA), following incubation with rat anti-mouse CD16/CD32 to block the mouse Fc receptors. Sections were also analyzed for the presence of vascular endothelium (CD31, BD Biosciences), E-selectin (BD Biosciences), vascular cell adhesion molecule (VCAM-1, BD Biosciences), neutrophils (Gr-1, BD Biosciences), and monocytes and macrophages (Mac-1/CD11b, Biodesign, Saco, ME). The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay (TUNEL, Intergen Company, Purchase, NY) was performed for in situ detection of apoptotic bodies.
Permeability Assay
Under general anesthesia, mice from all time points were injected with Evans Blue dye (60 mg/kg) via the left retro-orbital plexus, 30 minutes before euthanasia, followed by harvest of the arterialized vein and the contralateral JV and CCA. All vessels were rinsed in PBS. Another group of 7-day post-surgery mice were given recombinant human vascular endothelial growth factor (rhVEGF, 300 ng) by right retro-orbital injection, 10 minutes after receiving Evans Blue dye, and 30 minutes before vessel harvest.
Statistics
Data are expressed as mean ± SD. Comparisons were made using a two-tailed Student’s t-test. Differences were considered to be significant at P < 0.01.
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Results |
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We performed this surgery in a total of 17 mice, and at the time of vessel harvest, 11 vessels were found to be patent (65% overall success rate). Thus, a minimum of three mice was studied at each time point for changes in vessel morphology and immunohistochemical analyses. The major cause of failure was thrombosis, therefore, venous segments that appeared thrombosed were excluded from analysis. Vascular patency was confirmed before vessel perfusion and harvest by the gross appearance of pulsatile arterial blood flow in the external jugular vein.
Non-Invasive Imaging and Hemodynamic Characteristics
To assess vessel patency and the overall anatomical features of the surgical model we performed magnetic resonance angiography (MRA) in the head and neck region of mice 1 day and 7 days after surgery. Blood flow was visualized in the non-surgical JV and CCA, and the surgical JV exposed to the carotid arterial circulation (Figure 1)
. As is typical for MRA imaging, stationary tissues were not visualized secondary to post-processing suppression techniques. Interestingly, the JV exposed to arterial flow demonstrated an increased diameter distally in comparison to the distal contralateral JV.
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demonstrated a pattern of normal venous flow with respiratory variation and retrograde cardiac pulsations of variable amplitude. The JV exposed to arterial flow (Figure 2, C and F)
showed little respiratory variation and a pattern of pulsatility that was directly related to the cardiac cycle; it was of continuous but lower amplitude compared to normal arterial pulsatility in the contralateral CCA (Figure 2, B and E)
. In addition, flow during diastole was higher than that observed in the contralateral CCA.
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To determine the early morphological changes in the venous vascular wall in response to the arterial circulation, an immunohistochemical study was performed at 1, 3, and 7 days after surgery. All sections were derived from the JV exposed to the arterial circulation 1 mm distal to the microvascular catheter anastomosis. As controls we used the contralateral JV for each time point. No differences were observed among time 0 controls and 1, 3, and 7 days post-surgery JV contralateral controls. Thus, all figures show one representative control. Hematoxylin and eosin-stained sections demonstrated alterations in the venous vascular wall at the earliest time point studied, 24 hours after surgery. On postoperative day 1, the presence of an acellular band surrounding the vessel lumen was noticed (Figure 3B)
with a concomitant increase in the number of cells present in the vascular wall (Figure 3F)
when compared to controls (Figure 3E)
. After 3 days, the acellular layer was replaced by cellular components, which appeared disorganized (Figure 3G)
and non-uniform around the vessel lumen (Figure 3C)
. At 7 days, however, the cellular layers around the vascular wall appeared in organized layers (Figure 3, D and H)
. Circumferential media thickening was quantitatively assessed at this time point (control, 9.78 µm ± 0.77, n = 3; and 7-days post-surgery 44.40 µm ± 5.84*, n = 3; asterisk indicate that this value is significantly different from control value; P < 0.01). As arterialization of the external jugular vein progressed, the vessel also acquired a more elastic quality, as seen by the wide lumen of the arterialized vein after 7 days (Figure 3D)
in comparison to the typical collapsed lumen of the control vein (Figure 3A)
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. Furthermore, after 7 days there was evidence of neovascularization in the vascular wall by the appearance of microvessels (Figure 4D)
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. After 24 hours of arterial circulation, the subendothelial cell layer showed smooth muscle actin expression (Figure 5B)
. Three days after surgery, smooth muscle actin expression was found in several cells of the remodeling media (Figure 5C)
. After 7 days, smooth muscle actin expression appeared in well-organized concentric layers of the vascular wall (Figure 5D)
.
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, however, within 24 hours of arterialization, there were positively stained cells in the endothelial layer and other regions of the vascular wall (arrows, Figure 5F
). After 3 days, proliferating cells were more prevalent in the vascular wall in comparison to the endothelium (arrow, Figure 5G
), and by 7 days, the proliferative activity was infrequent in the vascular wall, consistent with the location of smooth muscle actin expression (Figure 5H)
. Thus, proliferation of smooth muscle cells may have contributed to the increase in vascular wall thickness and mass after 7 days of exposure to the arterial circulation.
To assess for the incidence of apoptosis during vascular remodeling in this model, we performed the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Using this analysis, we found little evidence of apoptosis. Only a few TUNEL-positive cells were located in the vascular wall at 1 day (Figure 5J)
, with the highest number of apoptotic cells observed at 3 days (Figure 5K)
. There was no apoptotic activity after 7 days of arterialization (Figure 5L)
, and control vessels demonstrated no evidence of apoptosis at all time points examined (Figure 5I)
.
Expression of Adhesion Molecules and Presence of Inflammatory Cells
To assess the inflammatory response during arterialization in this model, we stained vessels for E-selectin and VCAM-1, inducible endothelial-expressed adhesion molecules. The presence of inflammatory cells was also studied using Gr-1, a marker specific for neutrophils, and Mac-1, a marker for monocytes and macrophages. Twenty-four hours after surgery, E-selectin was sporadically expressed in a few endothelial cells (Figure 6B)
, and there was little expression of VCAM-1 at this time point (Figure 6F)
. Neutrophils appeared in the vascular wall after 1 day of exposure to the arterial circulation (Figure 6J)
. Furthermore, some monocytes and macrophages were present along the lumen and vascular wall after 1 day (Figure 6N)
. At 3 days, there was an increase in E-selectin (Figure 6C)
and VCAM-1 (Figure 6G)
expression in the endothelium with a concomitant influx of neutrophils and macrophages appearing in all layers of the vascular wall (Figure 6, K and O)
. It is possible that some of these inflammatory cells were recruited from the circulation in response to the increased expression of E-selectin and VCAM-1 on endothelial cells. Interestingly, after 7 days of arterialization, the inflammatory response appeared resolved with no expression of E-selectin (Figure 6D)
or VCAM-1 (Figure 6H)
, and no evidence of neutrophils or macrophages in the vascular wall (Figure 6, L and P)
. At all time points studied, control vessels demonstrated little evidence of inflammation with no E-selectin expression (Figure 6A)
, minimal VCAM-1 expression (Figure 6E)
, and no neutrophils or macrophages were present in the vascular wall (Figure 6I
, 6 mol/L). Non-surgical control vessels in Figure 6
were taken from mice 3 days after surgery, and days 1 and 7 control vessels are not shown. Sham surgery vessels at 1 day and 3 days demonstrated minimal expression of E-selectin and VCAM-1 (data not shown).
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To determine whether the arterialized vein exhibits functional changes in comparison to the native jugular vein, we tested for differences in vascular permeability. After intravenous Evans Blue dye injection, the contralateral control JV demonstrated extravasation of Albumin-Evans Blue in the vascular wall, and minimal extravasation was seen in the control CCA. Under these conditions, the aorta remained predominantly unstained, with some Evans Blue dye extravasation at the aortic arch (data not shown). The arterialized vein, 7 days after surgery, showed extravasation of dye at the region of the microvascular catheter anastomosis, but remained impermeable 4 mm distally (Figure 7A)
. Similar results were obtained using vessels that had been exposed to the arterial circulation for 1 and 3 days (data not shown). Nevertheless, addition of rhVEGF 10 minutes after Evans Blue dye injection resulted in extravasation of dye in the arterialized vein with a concomitant increase in the control CCA, 7 days after surgery (Figure 7B)
.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Previous studies suggest that positive remodeling of an arteriole to an artery is influenced by modulators of inflammation. For example, in a rabbit model of collateral circulation arteriogenesis, the expression of ICAM-1 and VCAM-1 preceded the appearance of monocytes.30 Furthermore, leukocyte adherence has been reported in areas of endothelial injury in canine bypass grafts.8 We also observed the expression of adhesion molecules and inflammatory cells in this model at 1 and 3 days after surgery. Interestingly, Zou et al31 showed in their mouse model of neointima formation in venous bypass grafts that the absence of intercellular adhesion molecule-1 expression resulted in diminished intimal lesion formation, reduced leukocyte adhesion, and lower monocyte/macrophage accumulation in neointimal lesions. This fundamental connection between inflammatory mediators and vascular remodeling underscores the influence of cellular interactions in affecting phenotypic modulations. Thus, the temporal coordination of VCAM-1 and E-selectin expression together with the presence of neutrophils, monocytes/macrophages in this model may contribute to the structural changes observed.
To begin to characterize the functional adaptation of the arterialized vessel in this model, we examined whether the arterialization process could modulate vascular permeability. Previous studies have shown that venules are more permeable than arterioles and capillaries, demonstrating an arteriovenous gradient of permeability in normal vessels.32,33 Regional differences in vascular permeability have also been characterized in vivo. In rabbits, permeability is enhanced in the aortic arch and aortic ostia,34 which is consistent with areas of low-density lipoprotein (LDL) retention and hemodynamicvariation.35 To demonstate that the decrease in permeability is endothelium-mediated we intravenously injected mice with VEGF. The addition of VEGF caused an increase in permeability with extravasation of Albumin-Evans Blue Dye in all vessels. In rats, VEGF was shown to increase capillary and venular permeability by opening endothelial intercellular junctions and inducing fenestrae in the endothelium.36 In this model, the decrease in permeability of the arterialized vein in comparison to the normal external jugular vein reflects functional changes in the endothelial phenotype and/or alterations of the normal endothelial-intimal architecture.
In summary, we have developed a novel mouse model of venous arterialization that defines early cellular and molecular adaptations of a venous vessel in response to a new local arterial biomechanical environment. The use of this model in the context of the genetic and genomic tools available in mice should help in the identification and functional characterization of genes implicated in venous arterialization.
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Acknowledgements |
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Footnotes |
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Supported by grants from National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH) (P50-HL56985 to M.A.G.), NIH (R01 CA64481–08 to J.F.), The Society of University Surgeons (to S.K.), The Abbott Trust at Children’s Hospital (to J.F. and G.G.-C.), and The Leet Patterson Trust (to G.G.-C. and S.K.).
Accepted for publication September 17, 2003.
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