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Am J Physiol Heart Circ Physiol 294: H2219-H2230, 2008. First published March 7, 2008; doi:10.1152/ajpheart.00650.2007
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Increased shear stress with upregulation of VEGF-A and its receptors and MMP-2, MMP-9, and TIMP-1 in venous stenosis of hemodialysis grafts

Sanjay Misra,1 Alex A. Fu,1 Alessandra Puggioni,2 Kamran M. Karimi,2 Jaywant N. Mandrekar,3 James F. Glockner,1 Luis A. Juncos,4 Bilal Anwer,5 Antonio M. McGuire,6 and Debabrata Mukhopadhyay7

1Department of Radiology, 2Division of Vascular Surgery, 3Division of Biostatistics, 4Division of Hypertension and Nephrology, 5University of Minnesota Medical School, 6Mayo Medical School, and 7Department of Biochemistry and Molecular Biology, Minneapolis; and Mayo Clinic College of Medicine, Rochester, Minnesota

Submitted 5 June 2007 ; accepted in final form 28 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Venous injury and subsequent venous stenosis formation are responsible for hemodialysis graft failure. Our hypothesis is that these pathological changes are in part related to changes in wall shear stress (WSS) that results in the activation of matrix regulatory proteins causing subsequent venous stenosis formation. In the present study, we examined the serial changes in WSS, blood flow, and luminal vessel area that occur subsequent to the placement of a hemodialysis graft in a porcine model of chronic renal insufficiency. We then determined the corresponding histological, morphometric, and kinetic changes of several matrix regulatory proteins including VEGF-A, its receptors, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and TIMP-2. WSS was estimated by obtaining blood flow and luminal vessel area by performing phase-contrast MRI with magnetic resonance angiography in 21 animals at 1 day after graft placement and prior to death on day 3 (n = 7), day 7 (n = 7), and day 14 (n = 7). At all time points, the mean WSS at the vein-to-graft anastomosis was significantly higher than that at the control vein (P < 0.05). WSS had a bimodal distribution with peaks on days 1 and 7 followed by a significant reduction in WSS by day 14 (P < 0.05 compared with day 7) and a decrease in luminal vessel area compared with control vessels. By day 3, there was a significant increase in VEGF-A and pro-MMP-9 followed by, on day 7, increased pro-MMP-2, active MMP-2, and VEGF receptor (VEGFR)-2 (P < 0.05) and, by day 14, increased VEGFR-1 and TIMP-1 (P < 0.05) at the vein-to-graft anastomosis compared with control vessels. Over time, the neointima thickened and was composed primarily of {alpha}-smooth muscle actin-positive cells with increased cellular proliferation. Our data suggest that hemodialysis graft placement leads to early increases in WSS, VEGF-A, and pro-MMP-9 followed by subsequent increases in pro-MMP-2, active MMP-2, VEGFR-1, VEGFR-2, and TIMP-1, which may contribute to the development of venous stenosis.

wall shear stress; matrix metalloproteinases; vascular endothelial growth factor-A; hemodialysis grafts; vascular biology; tissue inhibitors of matrix metalloproteinase

MAINTENANCE OF VASCULAR ACCESS PATENCY is essential to secure optimal delivery of hemodialysis to patients with end-stage renal disease. While the arteriovenous fistula is the preferred vascular access, over 50% of end-stage renal disease patients undergoing hemodialysis in the United States require polytetrafluoroethylene (PTFE) grafts for access (7). Unfortunately, the patency of PTFE grafts is only 50% at 1 yr and 25% at 2 yr (52). This high failure rate results primarily from venous stenosis, which is caused by intimal hyperplasia with the subsequent impairment of blood flow and thrombosis at the vein-to-graft anastomosis (47). Consequently, understanding the mechanisms that lead to the formation of intimal hyperplasia is of critical importance in developing strategies that delay or prevent graft failure.

Intimal hyperplasia is produced by the proliferation of vascular smooth muscle cells together with matrix deposition (31, 47, 51, 59). While various cytokines have been implicated in this process, one that may be of particular importance is VEGF, specifically VEGF-A. This is because VEGF-A is important in vascular remodeling and restenosis, and its expression is increased in patients with failed grafts (51). However, because these observations are from clinical specimens that are at the end stage of the venous stenotic and thrombotic process, it is not clear whether the increased VEGF contributed to the development of intimal hyperplasia or was a result of the advanced process. In fact, to date, no studies have examined the temporal expression of VEGF-A during graft failure. Consequently, the role of VEGF-A and its receptors in hemodialysis graft failure remains incompletely understood (55).

Another group of cytokines that are thought to play a key role in hemodialysis graft failure and remodeling of vein grafts used as arterial conduits are matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs) (6, 35, 36, 38, 49). This contention is based on the observations that an imbalance of MMP activity over TIMPs promotes the migration and proliferation of smooth muscle cells (2, 21) and that certain MMPs increase the bioavailability of VEGF-A, potentially accelerating the development of intimal hyperplasia (30). Previous work from our laboratory also supports a prominent role for MMPs in hemodialysis graft failure; we reported that PTFE grafts in pigs exhibited early upregulation of MMP-2 associated with increased cell migration from the adventitia to the media and intima and the subsequent formation of venous stenosis (35). Furthermore, nonspecific MMP-2 and MMP-9 inhibitors reduced the formation of neointima in the same model (49). Finally, a recent study from our laboratory showed in human hemodialysis graft samples that there was significantly increased expression of pro-MMP-2, pro-MMP-9, and TIMP-1 compared with control vessels (38). Thus, these studies have suggested that MMPs (specifically MMP-2 and MMP-9) may play a prominent role in the pathogenesis of intimal hyperplasia of hemodialysis grafts. However, these studies focused on MMP activity without examining the temporal expression of TIMPs in hemodialysis graft failure.

The mechanisms that can potentially contribute to early venous injury with subsequent initiation of the signaling pathways that promote intimal hyperplasia are multifactorial and include hemodynamic factors such as high and low wall shear stress (WSS) (10, 11, 15–17, 26, 29, 34, 57), shear stress gradients (57), turbulent flow (17, 18, 32), eddy currents (32), local vessel hypoxia (37, 38), and others (32). Our laboratory has been especially interested in the effect of WSS (the dragging force of the blood on the endothelium) because we and others have shown that it is increased at the vein-to-graft anastomosis of hemodialysis grafts (11, 40) and because it regulates the expression of many proteins including MMP-2, MMP-9, VEGF-A, and VEGF receptor (VEGFR)-2 (1, 53, 58). Recently, there have been several reports describing the use of phase-contrast magnetic resonance angiography with MRI (PC MRA/MRI) to determine blood flow and luminal vessel area to estimate WSS (3, 33, 40, 44). Thus, our overall hypothesis is that the formation of intimal hyperplasia resulting in venous stenosis is in part related to changes in WSS that results in the activation of matrix regulatory proteins causing subsequent venous stenosis formation. In the present study, we examined the serial changes in WSS, blood flow, and luminal vessel area that occur subsequent to the placement of a hemodialysis graft in a porcine model of chronic renal insufficiency. The aim of this study was to determine temporal changes in WSS, blood flow, and luminal vessel area (using PC MRA/MRI) and the expression of matrix regulatory proteins [VEGF-A and its receptors, MMP-2, and MMP-9, and their inhibitors (TIMP-1 and TIMP-2)] after PTFE graft placement in a porcine model of early venous stenosis formation. Because hemodialysis grafts are used in patients with advanced renal failure and the regulation of the targeted cytokines may be markedly different in the uremic milieu, we performed these experiments using a porcine model of renal insufficiency (37, 39).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Institutional Animal Care and Use Committee approval was obtained prior to the performance of any procedures. Housing and handling of the animals was performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals revised in 2000.

Overview of study design. Experiments were performed in 21 castrated juvenile male domestic pigs (40–50 kg, Larson Products, Sargent, MN) with chronic renal insufficiency induced by subtotal renal infarction (37, 39). Twenty-eight days after renal infarction, arteriovenous PTFE grafts were placed from the carotid artery to the ipsilateral jugular vein (Fig. 1A). Animals were killed on day 3 (n = 7), day 7 (n = 7), and day 14 (n = 7) following graft placement. Histological and morphometric analysis of the vein-to-graft anastomosis and control vessels were determined. Temporal expression of matrix regulatory proteins (VEGF-A, VEGFR-1, VEGFR-2, MMP-2, MMP-9, TIMP-1, and TIMP-2) were determined from tissue samples removed from the vein-to-graft anastomosis and control vessels harvested at the time of death. Luminal vessel area, blood flow, and graft patency at the vein-to-graft anastomosis and control vessels were determined serially in all animals on day 1 after graft placement and prior to death using PC MRA/MRI (Fig. 1B). All MRI evaluations were performed at the vein-to-graft anastomosis and contralateral control vessels.


Figure 1
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  		Fig. 1. Placement of the polytetrafluoroethylene (PTFE) hemodialysis graft and representative MRI and phase-contrast magnetic resonance angiography (PC MRA) of the vein-to-graft anastomosis. A: placement of the PTFE hemodialysis graft. CA, control carotid artery; CV, control jugular vein; VS, venous stenosis, IA, inflow artery. B: MRI with PC MRA performed on day 14 that shows high grade VS formation. RSA, right subclavian artery; LSA, left subclavian artery. C: PC MRA showing the direction of blood vessels in the opposite direction as depicted by black in the VS and white in the IA. D: magnitude of the blood flow in the same vessels. E: a hematoma surrounding the PTFE graft. T, trachea.

 
Anesthesia. Prior to all procedures including the MRI procedure, animals were fasted for 12 h. They were initially anesthetized with a combination of 5 mg/kg tiletamine hydrochloride (50 mg/ml) and zolazepam hydrochloride (50 mg/ml), 2 mg/kg xylazine (Bayer, Shawnee Mission, KS), and 0.06 mg/kg glycopyrrolate given intramuscularly. To induce additional anesthesia, an intravenous fluid line was placed in the ear vein for the delivery of zolazepam hydrochloride (5 mg/kg) as needed. During the procedure, animals were intubated and placed on a positive-pressure ventilator delivering oxygen (3–5 ml/kg) and isoflurane (1–3%). The end-tidal CO2 volume, oxygen saturation, heart rate, electrocardiogram, and blood pressure were monitored throughout the surgical procedures.

Creation of chronic renal insufficiency by renal artery embolization. Renal insufficiency was induced by subjecting the pigs to total embolization of the left kidney and partial embolization of the right kidney, as previously described (37, 39). Briefly, 6-Fr sheaths were placed in the right femoral artery, and the left renal artery was selected using a 5-Fr tapered angled glide catheter (Boston Scientific, Natick, MA). Through this catheter, 150- to 250-µm polyvinyl acrylide particles (PVA Contour, Boston Scientific, Boston, MA) were mixed with iodinated contrast and normal saline in a 1:1 concentration and infused until the left renal artery was completely occluded. Next, either the right upper or lower pole artery was selected and embolized completely in a similar fashion. The sheath was removed, and hemostasis was obtained by manual compression. The pig was treated for 5 days with antibiotics to prevent infection. Blood urea nitrogen (BUN) and creatinine were determined prior to embolization, at the time of graft placement, and at time of death.

PTFE graft placement. PTFE grafts were placed 28 days after renal artery embolization as previously described with the following modifications (35). An arteriovenous PTFE graft (4 mm diameter by 7 cm long, Gore, Flagstaff, AZ) was placed from either the right or left carotid artery to the ipsilateral jugular vein (Fig. 1A) and the contralateral vessels were isolated to serve as controls. Animals were killed on day 3 (n = 7), day 7 (n = 7), and day 14 (n = 7) after graft placement, and vein-to-graft anastomosis with contralateral vessels were used for Western blot, zymography, and histological analyses. Plavix (75 mg by mouth, Bristol-Myers Squibb/Sanofi Pharmaceuticals Partnership, Bridgewater, NJ) was started the night before and given daily until the animal was killed.

Cine PC MRI and contrast-enhanced MRA. To assess hemodynamic changes and vascular remodeling of the vein-to-graft anastomosis and control vessels, MRI was performed as previously described (35, 40). Animals were anesthetized for the procedure as previously described. MRI was performed serially in all animals 1 day after graft placement and prior to death on days 3, 7, and 14 following graft placement (Fig. 1B). This was repeated three times for each anatomic location (vein-to-graft anastomosis and contralateral vessels), and the average value was used. Luminal vessel area, blood flow, and graft patency was determined using MRI. Cine PC MRI and contrast-enhanced MRA was conducted 24 h after graft placement and prior to the animal being killed. Flow, velocity, and area measurements were obtained on the grafted vessels and on the contralateral nongrafted vessels at 20 different phases of the cardiac cycle. Using the cine PC MRI and contrast-enhanced MRA data, the following parameters were calculated: WSS, Reynolds number (Re), and luminal vessel area. All magnetic resonance examinations were performed using a Signa CVi 1.5 Tesla system (GE Medical Systems, Milwaukee, WI) with a torso-phased array coil positioned over the upper chest. After an initial three-plane localizing scan, a test bolus of gadopentetate dimeglumine (1–2 ml) was given (Magnevist, Berlex Laboratories, Wayne, NJ) followed by a 20-ml saline flush injected at 3 ml/s. A single slice overlying the thoracic aorta was scanned repeatedly about once per second, and the time delay was noted between the injection of the contrast medium and its arrival in the aorta.

Gadolinium-enhanced three-dimensional (3-D) MRA was performed using the following scan parameters: repetition time 5 ms, echo time 1.7 ms, flip angle 35°, 0.75 excitations, 0.75 phase field of views, 62.5-kHz receiver bandwidth, elliptic-centric phase encoding, 256 x 192 scan matrix, and 20 x 15-cm field of view, giving an in-plane resolution of 0.78 x 0.78 mm. Thirty to forty sections (1.2–1.4 mm) were obtained with 50% overlapping reconstruction in the z-direction. The scan time was 20–25 s. Contrast medium (30 ml) was injected at 3 ml/s followed by a 20-ml saline flush at the same rate. An appropriate scan delay derived from the test bolus sequence was chosen to ensure that the acquisition of the central portion of k-space corresponded with peak arterial enhancement.

The two-dimensional cine PC MRI sequences were conducted immediately after the 3-D MRA (Fig. 1B). Acquisitions were positioned perpendicular to the appropriate vessels at locations selected from maximum intensity projection images and reformatted images from the 3-D MRA as follows: 1) the jugular vein 2 cm distal to the vein-to-graft anastomosis and 2) the contralateral control jugular vein. Scan variables were as follows: repetition time 13.2 ms, echo time 4.9 ms, flip angle 30°, 1 excitation, receiver bandwidth 15.6 kHz, 256 x 224 matrix, and 14-cm field of view, for an in-plane resolution of 0.55 x 0.62 mm. Slice thickness was 5 mm. The velocity-encoding gradient was set to 100 cm/s unless aliasing was identified on initial acquisitions. Electrocardiogram triggering was provided by a peripheral pulse oximeter. Acquisition times were generally 20–30 s. Imaging planes (slices) were placed perpendicular to the artery or vein, with positioning guided by MRA images. Using retrospective gating and view sharing, the images were reconstructed to 20 evenly spaced time points in the cardiac cycle. Segmented k-space acquisition produced 8 views/segment. Quantitative flow information was obtained only in the direction perpendicular to the slice (Fig. 1, C and D). All flow calculations were made on an Advantix Windows workstation (Cardiac and Flow Analysis Tools, AW Release 4.0, GE Medical Systems). This was performed by a dedicated radiologist that was fellowship trained in MRI (J. F. Glockner). Flow measurements were repeated three times within 5 mm of each other at the vein-to-graft anastomosis, and averaged values from these three measurements were used. Previous work from our institution has shown the accuracy of blood flow measurements in 5-mm-diameter vessels to be 0.6–4.4% for blood flow rates of 315 and 540 ml/min (27). The intrareviewer coefficient of variability for blood flow was low between three different radiologists when interpreting the images, and, therefore, only one radiologist interpreted the blood flow images for the present study (27).

Vessel harvesting. To harvest the vein-to-graft anastomosis, both carotid and jugular vessels were dissected free of the surrounding soft tissue, and a heparin bolus of 250 U/kg was given intravenously. The vein-to-graft anastomosis ~2 cm toward the heart (venous stenosis; Fig. 1A) was removed as previously described (35, 37). These specimens were snap frozen in liquid nitrogen and stored at –80°C for subsequent Western blot analysis and zymography. In three animals from each time point, samples from the vein-to-graft anastomosis and control vessels were removed and embedded in paraffin for immunohistochemical analysis (35).

Histological and morphometric analysis. Hematoxylin and eosin (H&E), Verhoeff's van Gieson (VVG), PCNA, Masson's trichrome, VEGF-A, VEGFR-1, and {alpha}-smooth muscle actin staining were performed on the vein-to-graft anastomosis and control vein removed from animals on day 3 (n = 3), day 7 (n = 3), and day 14 (n = 3) as previously described (35, 54). The thickness of the intima, media, and adventitia of the vein-to-graft anastomosis and control vein was determined from sections stained with VVG stain and pooled together for each time point as previously described (35).

Re. At every point, to determine whether the blood flow was laminar rather than turbulent, the Re was calculated as follows: Re = (r x Formula x d)/m, where r is the vessel diameter, Formula is the average velocity of blood, d is the density of blood, and m is viscosity. The viscosity and density of blood were estimated as 0.00345 N·s·m–2 and 1,000 kg/l, respectively. A Re of <2,000 generally indicates the lack of turbulence (11, 62), and at every point measured within the venous stenosis, the Re was <2,000. Thus laminar flow was assumed to exist for all WSS calculations.

Calculation of WSS. The average WSS throughout the cardiac cycle was determined using Poiseuille's law. For steady laminar flow in a tube, the WSS (in N/m2) may be calculated directly from Formula (in cm/s) with the following formula: WSS = 8Formulaa/A, where a is viscosity (0.00345 N·s·m–2) and A is the tube diameter (in cm) (40).

Western blot analysis and SDS-PAGE zymography. Matrix regulatory protein expression was assessed with Western blot analysis and zymography on whole tissue lysate using 100 µg protein (tissue). Immunoreactive signals were detected by ECL (Amersham Pharmacia Biotech, Piscataway, NJ) following the manufacturer's instructions. The antibodies and antisera used included VEGF-A (Santa Cruz Biotechnology, SC-7269, mouse anti-human), VEGFR-1 [Santa Cruz Biotechnology, SC-504, rabbit anti-human (C-1158), lot G1505], VEGFR-2 [Santa Cruz Biotechnology, SC-316, lot F2204, rabbit anti-human (C-17)], TIMP-1 (R&D Systems, clone no. 63515, Mouse IgG2B), and TIMP-2 (R&D Systems, clone no. 127711, mouse IgG1). The proteins of interest exhibit a high degree of homology between pigs and human; these antibodies have been used in pigs as previously described (4, 23, 28, 43). Zymography was performed on whole tissue lysate as previously described (35–37).

Statistical methods. The WSS was calculated from the PC MRA/MRI data and averaged for each time point for the same group of animals (i.e., day 3, day 7, and day 14) for the vein-to-graft anastomosis. Values are expressed as means ± SD. ANOVA was used first to compare the means across the three groups of animals (i.e., day 3, day 7, and day 14). If the ANOVA F-test P value was statistically significant (P < 0.05) or showed a trend toward significance, pairwise t-tests were performed to compare each group to every other group. Paired t-tests were used to compare vessels (venous stenosis and control vein) within animals for each group (day 3, day 7, or day 14 death). A P value of 0.05 or less was considered statistically significant. SAS version 9 (SAS Institute, Cary, NC) was used for statistical analyses.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Surgical outcomes. There were no complications as a result of the embolization procedure other than the expected induction of renal insufficiency. BUN and creatinine prior to the induction of renal insufficiency were 9.11 ± 2.66 mg/dl and 1.28 ± 0.19 mg/dl, respectively. BUN and creatinine at the time of graft placement were 22.2 ± 10.6 mg/dl (P < 0.05 compared with before graft placement) and 2.47 ± 0.61 mg/dl (P < 0.05 compared with before graft placement), respectively. Twenty-one pigs underwent the placement of 21 (4 x 7 cm) PTFE grafts: 2 grafts on the left and 19 grafts on the right, with the contralateral vessels serving as controls (Fig. 1). It was common to have a postoperative hematoma after the graft placement on day 1 (Fig. 1E).

Histological and morphometric analysis. H&E, VVG, {alpha}-smooth muscle actin, PCNA , and Masson's trichrome staining were performed at the vein-to-graft anastomosis and control vessels on days 3, 7, and 14, and representative sections from day 14 are shown in Fig. 2. This revealed a thickened neointima composed primarily of {alpha}-smooth muscle actin-positive cells with increased cell proliferation in the intima and media compared with controls (Fig. 2). In addition, there was increased matrix accumulation within the media and neointima. The thickness of the intima, media, and adventitia of the vein-to-graft anastomosis and control vessels was determined on days 3, 7, and 14 (Fig. 3). By days 7–14, the average thickness of the intima of the vein-to-graft anastomosis was significantly higher than the average thickness of the intima of control vessels (P < 0.05). The mean thickness of the media of the vein-to-graft anastomosis was greater than the mean thickness of the media of control vessels on days 3 and 7 (P < 0.05). The mean thickness of the adventitia of the vein-to-graft anastomosis was significantly greater than the mean thickness of the adventitia of control vessels at all time points (P < 0.05). These results indicate that by day 14, there is a thickened neointima composed primarily of {alpha}-smooth muscle cell actin-positive cells with increased proliferation within the intima and media compared with control vessels.


Figure 2
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  		Fig. 2. Morphological changes in the venous wall and the development of neointimal hyperplasia. Animals were killed at the three various time points, and representative sections from a day 14 animal are shown. The explanted vein-to-graft anastomosis and contralateral jugular vein were stained using hematoxyln and eosin (H&E), Verhoeff's van Gieson (VVG) stain, {alpha}-smooth muscle (SM) actin, PCNA, and Masson's trichrome. The results show that neointimal hyperplasia had developed by day 14 compared with the control vein. An analysis of the expression of {alpha}-SM actin at the vein-to-graft anastomosis and normal vein was performed. The control external jugular vein showed well-organized layers of SM cells expressing {alpha}-SM actin appearing red-brown. On day 14 after the graft placement, the neointima and media at the venous anastomosis stained more intensely than the control for {alpha}-SM actin. Cells positive for PCNA are brown. There was an increased number of cells staining brown in the neointima of the venous anastomosis by day 14 compared with the control vein. The control external jugular vein showed the most extracellular matrix (appearing blue by trichrome staining) in the media and adventitia. By day 14, more cells (appearing pink) accumulated in the media and neointima accompanied by prominent extracellular matrix deposition in the media and inner neointima.

 

Figure 3
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  		Fig. 3. Thickness of the nointima, media, and adventitia at the vein-to-graft anastomosis (VS) and control veins (CV) over time. Specimens from explanted vein-to-graft anastomosis and control vessels were obtained from pigs killed on days 3, 7, and 14. The thickness of the neointima, media, and adventitia from the vein-to-graft anastomosis and control vessels were individually quantified using computer-assisted planimetry on VVG-stained slides and was pooled for the vein-to-graft anastomosis and control veins on days 3, 7, and 14. The average thickness of the intima from the vein-to-graft anastomosis increased over time compared with control vessels. By days 7 and 14, it was significantly increased compared with control vessels (P < 0.05). The mean thickness of the media and adventitial of the vein-to-graft anastomosis was higher than that of the control vein at all time points. By days 3 and 7, the average thickness of the media was significantly higher than the control veins (P < 0.05). At all time points, the average thickness of the adventitia of the vein-to-graft anastomosis was significantly higher than the control vein (P < 0.05). Data are means ± SD. *P < 0.05.

 
Luminal vessel area, blood flow, and WSS measurements by MRI at the vein-to-graft anastomosis. MRI was performed to determine the luminal vessel area and blood flow of the vessels over time in the animals. The Re at all time points was <1,000, and, therefore, we assumed that the blood flow was not turbulent. At all time points, the WSS of the vein-to-graft anastomosis was significantly higher than that of the control vein (0.6 ± 0.22 N/m2, P < 0.05). The mean WSS at the vein-to-graft anastomosis had a bimodal peak. The mean WSS was 7.3 ± 2.1 N/m2 by day 1, decreased to 5.1 ± 1.8 N/m2 by day 3, subsequently peaked at 8.8 ± 1.6 N/m2 by day 7 (P < 0.05, compared with days 3 and 14), and was 4.3 ± 0.5 N/m2 by day 14 (Fig. 4). At all time points, the luminal vessel area of the vein-to-graft anastomosis was significantly less than that of the control vein (17.3 ± 4.6 mm2). The luminal vessel area followed a similar pattern and was 12.6 ± 3.3 mm2 by day 1, increased slightly to 13.2 ± 3.6 mm2 by day 3, subsequently decreased to 10.6 ± 2.3 mm2 by day 7, and was 17.1 ± 3.8 mm2 by day 14. Similarly, at all time points, the blood flow through the vein-to-graft anastomosis was significantly higher than that of the control vein (95 ± 45 ml/min, P < 0.05). The blood flow was 727 ± 392 ml/min by day 1, decreased to 619 ± 327 ml/min by day 3, increased to 719 ± 170 ml/min by day 7, and was 724 ± 321 ml/min by day 14. Overall, these results indicate that the WSS has a bimodal peak with an early increase by day 1 and a subsequent decrease and second peak by day 7, which was significantly higher than on days 3 and 14.


Figure 4
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  		Fig. 4. MRI measurements of the vein-to-graft anastomosis on days 1, 3, 7, and 14. The wall shear stress (WSS) was calculated from the MRI data. At all time points, the mean WSS of the VS was significantly higher (P < 0.05) than the control vein. By day 7, the WSS was significantly higher than on days 3 and 14 (P < 0.05). Data are means ± SD. *P < 0.05.

 
Protein expression of VEGF-A, VEGFR-1, and VEGFR-2 at the vein-to-graft anastomosis on days 3, 7, and 14 after graft placement. Protein expression was determined by Western blot analysis on both the vein-to-graft anastomosis and control vessels to determine the temporal relationship of VEGF-A, VEGFR-1 (Flt-1), and VEGFR-2 (KDR) on days 3, 7, and 14. Scanning densitometry values from immunoblots obtained from protein samples of the stenotic vein were divided by the control vein for each time point (Figs. 5Go–7). By day 3, we observed a significantly higher expression of VEGF-A compared with the control vein (P < 0.05; Fig. 5), which was localized to the intima by immunohistochemistry (Fig. 5D). By day 7, we observed a significantly higher expression of VEGFR-2 at the vein-to-graft anastomosis compared with the control vein (P < 0.05; Fig. 6). By day 14, there was a significant elevation in VEGFR-1 observed compared with control vessels, which was localized to the neointima by immunohistochemistry (P < 0.05; Fig. 7). Overall, these results indicate an early expression of VEGF-A followed by VEGFR-2 and VEGFR-1 at the vein-to-graft anastomosis compared with control vessels.


Figure 5
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  		Fig. 5. A: graph representing the appropriate protein band of VEGF-A from Western blot analysis. B: graph representing the appropriate band for β-actin from Western blot analysis for protein loading. C: semiquantitative analysis for VEGF-A performed on days 3, 7, and 14. The normalized density of VEGF-A was significantly higher at the vein-to-graft anastomosis compared with the control vein by day 3. *Significantly higher value (P < 0.05). Data are means ± SD. D: immunohistochemistry for VEGF-A for localization of expression on day 3. By immunohistochemistry, brown staining cells are positive for VEGF-A. Representative sections from a day 3 animal are shown. The left and middle left columns are from the vein-to-graft anastomosis, and middle right and right columns are from the control vein. The left and middle right columns are x10 magnification, and the middle left and right columns are x40 magnification. There were more cells staining brown located in the intima and media at the vein-to-graft anastomosis compared with controls.

 

Figure 6
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  		Fig. 6. A: graph representing the appropriate protein band of VEGF receptor (VEGFR)-2 from Western blot analysis. B: graph representing the appropriate band for β-actin from Western blot analysis for protein loading. C: semiquantitative analysis performed on days 3, 7, and 14. The normalized density of VEGFR-2 was significantly higher on day 7 compared with day 3. *Significantly higher value (P < 0.05). Data are means ± SD.

 

Figure 7
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  		Fig. 7. A: graph representing the appropriate protein band of VEGFR-1 from Western blot analysis. B: graph representing the appropriate band for β-actin from Western blot analysis for protein loading. C: semiquantitative analysis performed on days 3, 7, and 14. The normalized density of VEGFR-1 was significantly higher on day 14 compared with the control vein. *Significantly higher value (P < 0.05). Data are means ± SD. D: immunohistochemistry for VEGFR-1 for localization of expression on day 14. Representative sections from a day 14 animal are shown. The left and middle left columns are from the vein-to-graft anastomosis, and the middle right and right columns are from the control vein. The left and middle right columns are x10 magnification, and the middle left and right columns are x40 magnification. By immunohistochemistry, brown staining cells are positive for VEGF-A and VEGFR-1. There were more cells staining brown located in the intima and media at the vein-to-graft anastomosis compared with controls.

 
Protein expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 at the vein-to-graft anastomosis on days 3, 7, and 14 after graft placement. Protein expression was determined by zymography and Western blot analysis on both the vein-to-graft anastomosis and control vessels to determine the temporal relationship of MMP-2, MMP-9, TIMP-1, and TIMP-2 on days 3, 7, and 14. Scanning densitometry values from immunoblots or zymography obtained from protein samples of the stenotic vein were divided by the control vein for each time point (Fig. 8). By days 7 and 14, there was a significant increase in both pro-MMP-2 and active MMP-2 at the vein-to-graft anastomosis compared with control veins (P < 0.05; Fig. 8B). In addition, pro-MMP-9 was significantly elevated at the vein-to-graft anastomosis compared with control veins at all time points (P < 0.05; Fig. 8D). No active MMP-9 was observed. By day 14, TIMP-1 was significantly higher (2.8-fold, P < 0.05) at the vein-to-graft anastomosis compared with the control vein (Fig. 8G). There were no differences in TIMP-2 at any of the time points compared with the control vein. Overall, these results show that there is an early activation of pro-MMP-9 by day 3 followed by increased expression of both pro-MMP-2 and active MMP-2 on days 7–14 with a significant increase in TIMP-1 by day 14.


Figure 8
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  		Fig. 8. A: graph representing the appropriate protein bands of pro-matrix metalloproteinase (MMP)-2 and MMP-2 by zymography. STD, standard. B: semiquantitative analysis performed on days 3, 7, and 14. The normalized density of pro-MMP-2 and active MMP-2 at the vein-to-graft anastomosis was significantly higher than the control veins by days 7 and 14. *Significantly higher value (P < 0.05). Data are means ± SD. C: graph representing the appropriate protein band of pro-MMP-9 by zymography. D: semiquantitative analysis performed on days 3, 7, and 14. The normalized density of pro-MMP-9 was significantly higher (*) at all time points compared with the control vein. E: graph representing the appropriate protein band of tissue inhibitor of matrix metalloproteinase (TIMP)-1 by Western blot analysis. F: graph representing the appropriate band for β-actin from Western blot analysis for protein loading. G: semiquantitative analysis performed on days 3, 7, and 14. The normalized density of TIMP-1 was significantly higher on day 14 compared with the control vein. *Significantly higher value (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The mechanisms underlying the formation of venous stenosis in hemodialysis vascular grafts have not been well defined. It has been hypothesized that hemodynamic factors such as changes in WSS (10, 11, 15–17, 26, 29, 34, 56, 57), turbulent flow (17, 18, 32), eddy currents (32), local vessel hypoxia (37, 38), and others (32) contribute to venous stenosis formation. Subsequently, these changes are felt to result in increased smooth muscle cell proliferation and migration with the elaboration of matrix regulatory proteins implicated in stenosis formation. In the present study, we employed a porcine model of chronic renal insufficiency in which we placed arteriovenous PTFE grafts. WSS was significantly higher at the vein-to-graft anastomosis compared with the control vein at all time points. It had a bimodal peak with significant elevation by day 7 compared with days 3 and 14. This was associated with significantly higher amounts of VEGF-A and pro-MMP-9 by day 3 followed by significantly higher expression of pro-MMP-2, active MMP-2, and VEGFR-2 by day 7 and significantly higher expression of pro-MMP-2, active MMP-2, VEGFR-1, and TIMP-1 by day 14. By immunohistochemitry, VEGF-A expression was localized to the intima and media by day 3, and, by day 14, VEGFR-1 expression was localized to the neointima. By day 14, there was significant neointimal thickening, composed primarily of cells staining for {alpha}-smooth muscle cell actin with a thickened media and adventitia at the vein-to-graft anastomosis.

Because this is a new experimental animal model employing chronic renal insufficiency in which we placed arteriovenous PTFE grafts, we first confirmed that the histological and morphometric changes that occurred at the vein-to-graft anastomosis were similar to those observed in other experimental animal models. The hallmark of venous stenosis in hemodialysis graft failure is neointimal thickening composed primarily of cells staining positive for {alpha}-smooth muscle actin accompanied with a thickened media. In addition, increased cellular proliferation and migration have been observed in the media and intima. In the present study, histological and morphometric evaluation of the vein-to-graft anastomosis confirmed this pathological process. We observed that the neointima thickened over time and that the thickness was significantly higher than that in the control vein by days 7–14 with a thickened media. Finally, increased cellular proliferation was observed in the intima and media. All these findings are consistent with previous observations in other experimental animal models and clinical specimens (6, 31, 47, 51, 59).

WSS, which represents the dragging force exerted by flow on the endothelium, regulates the expression of many proteins, including MMP-2, MMP-9, VEGF-A, and VEGFR-2 (1, 53, 58, 61), raising the possibility that it may be a key factor in the initiation of venous stenosis. Consequently, it is important to know the temporal changes that occur in WSS after the establishment of an arteriovenous graft and how these relate to venous stenosis. Previous studies have estimated WSS by directly measuring blood flow and velocity using ultrasound (11, 61) or MRI (3, 40, 44). In these studies, the WSS at the venous anastomosis of arteriovenous PTFE grafts ranged between 2 and 5 N/m2 (11, 15). Furthermore, computational fluid dynamic modeling has been performed at the vein-to-graft anastomosis, which has shown similar findings (26, 29, 57). In the present study, we used PC MRA/MRI to noninvasively measure the temporal changes in hemodynamic parameters that occur early in the development of venous stenosis. We found that the WSS at the vein-to-graft anastomosis was significantly higher than that at the control vein and exhibited a bimodal distribution on days 1 and 7. It peaked on day 7, which was significantly higher than days 3 and 14 after hemodialysis graft placement. The increased WSS at the vein-to-graft anastomosis at day 1 after graft placement likely reflects changes related to surgical trauma caused by the placement of the grafts. The changes in WSS follow the histological and morphometric changes that are occurring. By day 14, the neointima and media have thickened and a venous stenosis has formed, which decreases the blood flow and subsequent WSS. In our previous study in animals with 4-mm-diameter by 2-cm-long (straight) PTFE grafts placed from the iliac artery to iliac vein, the mean WSS was lower on days 3 and 7 (2–3 N/m2) after graft placement and was 2 N/m2 by day 14 The luminal vessel area and blood flow followed a similar pattern to the WSS. Taken collectively, there were early increases in WSS, which peaks by day 7 and decreases by day 14, and as the vein-to-graft anastomosis remodels and venous stenosis forms, the luminal vessel area decreases and, subsequently, the WSS decreases.

WSS regulates the expression of many proteins, including VEGF-A, VEGFR-2, MMP-2, and MMP-9 (1, 53, 58). VEGF is a multifunctional cytokine, and its function includes the regulation of cellular proliferation and migration. By day 3, we observed significantly increased expression of VEGF-A at the vein-to-graft anastomosis, which was localized to the intima (endothelial cells) by immunohistochemistry. It has been shown in animal models of atherosclerosis and restenosis after angioplasty that inhibition of VEGF-A will decrease restenosis (5, 8, 12, 42). VEGF-A has the ability to stimulate endothelial cell migration directly or indirectly by binding to its two tyrosine receptor kinases, VEGFR-1 and VEGFR-2 (9). In our previous work in a porcine model of hemodialysis grafts, increased cell migration was observed by day 7 after graft placement (35). VEGFR-2 is expressed by endothelial cells, and, in the present study, by day 7, the expression of VEGFR-2 was significantly higher at the vein-to-graft anastomosis than control veins, which corresponded with the significantly higher WSS. These findings are consistent with data from cultured endothelial cells exposed to shear stress, which showed that VEGFR-2 has a shear stress-responsive region (53, 58). By day 14, there was significantly higher expression of VEGFR-1, which was localized to the neointima by immunohistochemistry. Smooth muscle cells are known to express VEGFR-1 (20), and the increased amount of VEGFR-1 likely reflects an increase in total smooth muscle cell mass. Finally, the increase in VEGFR-1 is in agreement with previous studies in animal models of coronary stenosis and atherosclerosis in which elevated levels of VEGFR-1 were observed (19, 41, 54). The exact role of VEGFR-1 remains controversial; however, it has been shown to be involved in the induction of MMP-9 and release of vascular-specific growth factors.

Because certain MMPs can increase the bioavailability of VEGF-A, potentially accelerating the formation of venous stenotis, we examined the expression of MMPs and their inhibitiors in the venous stenosis (6, 30, 35, 36, 38, 49). In our previous study in a porcine model, MMP-2 activity peaked at the vein-to-graft anastomosis by day 7 (35). In the present study, we observed similar findings with significantly increased expression of pro-MMP-2 and active MMP-2 by days 7 and 14 at the vein-to-graft anastomosis compared with control veins. In addition, significantly increased expression of pro-MMP-9 was observed by day 3, which declined over the course of the study but remained higher than that of the control vein. The increased expression of pro-MMP-2, active MMP-2, and pro-MMP-9 is associated with increased cellular proliferation and migration, which occurs by days 3–7 (2, 14, 21).

Imbalance of MMP activity over TIMP activity has been shown to promote cell migration and proliferation. Because of this observation, we investigated the role of TIMP-1 and TIMP-2 in early venous stenosis formation. We observed significantly higher levels of TIMP-1 (2.8-fold) by day 14. TIMP-1 is a natural inhibitor of MMP-2, which has been shown to be involved in cellular migration. Overexpression of TIMP-1 has been shown to decrease stenosis formation in an animal model of arterial pathology; however, to date, this has not been studied in hemodialysis graft failure (2, 13, 21). Finally, recent work from our laboratory has shown that there is increased expression of pro-MMP-2, pro-MMP-9, and TIMP-1 in clinical specimens removed from patients with PTFE hemodialysis grafts (38). Taken collectively, at early time points (days 3 and 7), there is an elaboration of pro-MMP-2, active MMP-2, and pro-MMP-9, which favors cellular proliferation and migration. By day 14, this has started to decrease, and there is an increased expression of TIMP-1, favoring a decrease in cellular migration and proliferation. This corresponds with the histological and morphometric analysis, which shows that venous stenosis has formed, primarily composed of vascular smooth muscle cells ({alpha}-smooth muscle cell actin-positive cells) with increased cellular proliferation.

One important aspect of our present study is that the experiments were conducted in pigs with renal insufficiency, thus modeling the clinical scenario in a more relevant fashion. Previous experimental studies that have examined mechanisms of arteriovenous graft stenosis have predominantly been performed in animals with normal kidney function (6, 22, 24, 25, 31, 35, 36, 40, 48–51, 59). While these studies are very useful in examining the diverse mechanisms by which venous stenosis may develop, they may not accurately depict the prevalent mechanisms in humans because hemodialysis grafts are used in patients with advanced renal failure in which the uremic milieu may markedly alter the regulation of the targeted cytokines. For instance, renal insufficiency is associated with increased oxidative stress, which has been associated with increased levels of VEGF-A and other proteins (45, 46, 60), raising the possibility that WSS may increase VEGF-A levels to even a greater degree in uremic patients. In the present study, we created renal insufficiency with subtotal renal infarction causing chronic kidney disease prior to the placement of the grafts (37, 39). In these animals, there were significantly increased BUN and creatinine prior to graft placement compared with baseline values, and the characteristics of their arteriovenous grafts are consistent with those observed in clinical specimens.

In conclusion, the findings from the present study add to the current knowledge of the hemodynamic parameters and vascular remodeling changes that occur in early venous stenosis formation in hemodialysis grafts. After the placement of a PTFE graft, there is a significantly higher WSS at the vein-to-graft anastomosis (Fig. 9). We hypothesize that the early increases (day 3) in WSS results in an increased expression of VEGF-A and pro-MMP-9 at the vein-to-graft anastomosis, followed by an increased expression of VEGFR-2, pro-MMP-2, and active MMP-2 by day 7. The increased expression of VEGF-A, VEGFR-2, pro-MMP-2, active MMP-2, and pro-MMP-9 reflects changes manifested by increased cellular migration and proliferation leading to venous stenosis formation. By day 14, there is a higher amount of VEGFR-1 and TIMP-1; the increase in VEGFR-1 reflects the increased smooth muscle mass, whereas the increased TIMP-1 activity represents a shift toward a decrease in cell migration and proliferation and further venous stenosis formation. Understanding the role of these proteins in venous stenosis formation in hemodialysis grafts will help improve outcomes in patients.


Figure 9
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  		Fig. 9. Schematic depicting potential pathways from the present study.

 

   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants CA-78383, HL-072178, and HL-70567 and by a grant from the American Cancer Society (to D. Mukhopadhyay). This work was also supported in part by a research grant from the Mayo Clinic and the Gonda Vascular Center. In addition, unrestricted research grants were provided by the Cook Group, Incorporated; MedComp; and Atrium Medical.


   ACKNOWLEDGMENTS
 
The authors thank Jill L. Anderson and Steve Krage for the help with the animal experiments, Tanya Hoskin for the help with the statistical analysis, and Dr. Kallmes for the use of the laboratory.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Misra, Dept. of Radiology, Mayo Clinic College of Medicine, 200 First St. SW, Alfred 6460, Rochester, MN 55905 (e-mail: misra.sanjay{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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