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Nephrol Dial Transplant (2003) 18: 814-818
© 2003 European Renal Association-European Dialysis and Transplant Association

Brief Report

Detection of cell-cycle regulators in failed arteriovenous fistulas for haemodialysis

Rick de Graaf1, Ruben Dammers1, Tryfon Vainas1, Arnold P. G. Hoeks2 and Jan H. M. Tordoir1,

1 Department of Surgery, University Hospital Maastricht and 2 Department of Biophysics, University Maastricht, Maastricht, The Netherlands



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Chronic renal failure (CRF) patients rely on arteriovenous fistulas (AVFs) for haemodialysis vascular access. Intimal hyperplastic stenoses result in failure of AVFs and frequent intervention is required to maintain vascular access. The extent of intimal hyperplasia depends on the interplay between cyclins and cyclin-dependent kinases (e.g. cdk2), positively regulating cell-cycle progression. cdk activity is negatively modulated by the interaction with cdk inhibitory proteins, such as p21Waf1 and p27Kip1. Little is known about the expression of these proteins in the development of intimal hyperplasia in AVFs.

Methods. p21Waf1, p27Kip1, cdk2 and Proliferating Cell Nuclear Antigen immunoreactivity was determined in 18 failed AVFs from 16 CRF patients and 10 non-diseased vessels (five arteries and five veins).

Results. The percentage of p21Waf1-positive cells was significantly lower in AVFs (3±1%), compared with normal veins and arteries (62±4 and 63±4%, respectively; P<0.001). cdk2-positive cells were significantly higher in AVFs (40.7±3.7%) than in normal veins and arteries (2±1 and 0±0%, respectively; P<0.001). Although no difference in p27Kip1 immunoreactivity was found between AVFs (37±17%) and veins (23±8%; P=0.208), it was lower in healthy arteries (17±11%; P=0.037).

Conclusions. The data suggest that in failed AVFs, p21Waf1, but not p27Kip1, is related to intimal hyperplasia. This is the first report to show involvement of cell-cycle regulators in AVF-related human intimal hyperplasia.

Keywords: cell-cycle; fistula; hyperplasia; intimal; stenosis



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
Intimal hyperplastic stenoses in surgically created arteriovenous fistulas (AVFs) for haemodialysis remain a serious problem in maintaining vascular access. One-year primary patency rates vary from 53 to 88% for autogenous radio-cephalic wrist fistulas to 37–70% when prosthetic grafts are used [1]. Pathological proliferation and migration of vascular smooth muscle cells (VSMC) resulting in intimal thickening is the main cause of graft stenosis and occlusion within the first year after AVF construction [2,3]. In normal vascular tissue, quiescent VSMCs are found in the G0 phase of the cell cycle and VSMCs proliferate at a low rate. However, on stimulation VSMCs can exit their quiescent state and progress through the G1 and G1/S transition of the cell cycle [4,5].

Cell-cycle progression is positively regulated by the formation of complexes comprised of cyclin-dependent kinases (cdk) and their activating subunits, the cyclins. Cdk activity is, in turn, negatively modulated by the interaction with cdk inhibitory proteins (CKIs), resulting in cell-cycle arrest [6]. Progression through the first gap (G1) phase requires both cyclin D-cdk4/6 (early G1) and cyclin E-cdk2 (late G1) [5]. Specific CKIs (p21Waf1 and p27Kip1) are capable of blocking cell-cycle progression by binding to and inhibiting the activation of cyclin E-associated–cdk complexes [7]. Both p21Waf1 and p27Kip1 inhibit VSMC proliferation by reducing cdk2 activity [8].

Expression of these CDKs and CKIs in arterial balloon injury models and the role of these cell-cycle proteins in VSMC proliferation have been investigated in cultured bovine, porcine and human endothelial and smooth muscle cells as well as in human atherosclerotic lesions and normal porcine arteries [9,10]. However, it is not known how the expression of these cell-cycle regulators relate to venous intimal hyperplasia as a result of haemodynamic forces which are relevant in AVFs. Therefore, we investigated the expression of p21Waf1, p27Kip1, cdk2 and Proliferating Cell Nuclear Antigen (PCNA) in AVFs and non-diseased vascular tissue.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Tissue specimens
Eighteen specimens of stenotic lesions of failing AVFs were obtained from 16 patients (eight men and eight women; age 59.8±3.4 years) undergoing AVF revision at the surgical department of the University Hospital Maastricht. Two patients underwent a second revision during which primarily stenosed tissue was collected. Hypertension was diagnosed in 12 patients (75%) and only one patient presented with non-insulin-dependent diabetes. The diagnosis of stenosis was based on generally accepted Duplex ultrasound criteria and confirmed by angiography [11]. The study was approved by the joint Medical Ethical Committee of the Maastricht University and the University Hospital Maastricht and the patients were included after written informed consent.

In 14 of 18 AVFs vascular access was created through a prosthetic brachial-anticubital forearm loop access, while in the four other cases, an autogenous radial-cephalic direct wrist fistula was constructed. AVF revision was performed 27 months (range 1–80 months) after primary access creation. Surgical repair was performed by placement of an interposition graft, allowing collection of the stenotic process and adjacent vessel segments. In all patients, the stenotic lesion was focal and narrowing or occluding the lumen. All 18 specimens of stenotic lesions were taken from the direct vicinity of the venous anastomic site distal to the AVF. During revision, the stenotic lesions were obtained and fixed in 10% formalin for 4 h, followed by 18 h in 70% ethanol, and paraffin-embedded for immunocytochemistry. Sections (4-mm thick) were stained with haematoxylin and eosin, and immunostained with p21Waf1, p27Kip1, cdk2 and PCNA antibodies.

Five normal arteries were obtained during obduction from patients who died of non-cardiovascular cause. Remnants of vein grafts used for arterial revascularization were obtained (n=5). Warm ischaemia time was <5 min in all veins. Both non-diseased arteries and veins were formalin fixed, paraffin-embedded and stained with aforementioned antibodies.

Immunocytochemistry
Serial sections of the tissues were incubated with the following primary antibodies: a mouse monoclonal anti-human p21Waf1 antibody, 1:50 dilution (NeoMarkers, Inc., Fremont, CA, USA); a mouse monoclonal anti-human p27Kip1 antibody, 1:50 dilution (NeoMarkers); a mouse monoclonal anti-human cdk2 antibody, 1:50 dilution (NeoMarkers); and a mouse monoclonal anti-human PCNA antibody, 1:30 dilution (NeoMarkers). Sections (4-mm thick) were deparaffinized in three changes of xylene, and rehydrated in 90, 70 and 50% ethanol. Slides were boiled in citrate buffer for 20 min, and incubated in 2% BSA/PBS/Tween for 20 min to block aspecific binding activity. Primary antibodies were diluted in TBS and applied to the slides for 60 min at room temperature. After three washes with TBS for 5 min, a biotinylated horse anti-mouse secondary antibody (1:400 dilution, Dako) was applied for 30 min at room temperature, followed by a steptavidin amplification reagent (Dako) for 30 min at room temperature. Finally, Fast Red was used for 10 min at room temperature, yielding a red reaction product. Visualization of all nuclei in the tissue sections was realized with a haematoxylin counterstain.

Total nuclei, and positive nuclei were counted in the (neo-)intima of the different vascular segments by means of a microscope-based video image analysing system (Analysis®, Soft Imaging System, Münster, Germany). Four random high-power fields of each specimen were analysed.

Statistical analysis
Data management and statistical analysis were performed with the SPSS 10.0 package for Windows (SPSS, Chicago, IL, USA). Data are presented as mean values and standard errors of the mean (SEM). Mean percentages in the patient group and control tissues were compared by means of ANOVA with Bonferroni correction. An analysis of covariance was performed to correlate patient characteristics (such as sex, age and present cardiovascular risk factors) and graft survival to cell-cycle regulating proteins.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Cell-cycle regulators in AVFs and non-diseased vascular tissue
All vascular segments were stained with p21Waf1, p27Kip1, cdk2 and PCNA antibodies and total nucleic, and immunoreactive nucleic count was determined in four random high-power fields. The average number of nuclei counted was 4416 in AVFs, 1363 in non-diseased veins and 1262 in non-diseased arteries.

Whereas p21Waf1 was clearly expressed to a greater extent in non-diseased veins and arteries, cdk2- and PCNA-positive cells were more frequently detected in failed AVFs (Figure 1Go). p27Kip1 immunoreactivity was observed both in non-diseased and intimal hyperplastic tissue (Figure 1Go). Figure 2Go shows the percentage of positively stained cells for p21Waf1, p27Kip1, cdk2 and PCNA, respectively. The percentage p21Waf1-positive cells was significantly lower in AVFs (3±1%) compared with non-diseased veins and arteries (62±9% and 63±8%, respectively; P<0.001). In contrast, cdk2- and PCNA-positive cells were abundantly present in AVFs (41±16 and 37±15%, respectively) whereas immunopositive cells were almost absent in non-diseased veins (0±0% and 0±0%, respectively; P<0.001) and arteries (2±2 and 1±0.4%, respectively; P<0.001). p27Kip1 immunoreactivity was lower in arteries compared with failed AVFs (17±11 and 37±14%, respectively; P=0.037). However, between veins and AVFs no difference was found in p27Kip1 immunoreactivity (23±8%; P=0.208). No variations were observed between autogenous and prosthetic accesses concerning any cell-cycle regulating protein. Therefore, we decided not to report on these two types of access separately. Correction for the variables age, sex and cardiovascular risk factors had no influence on the level of significance. No relation was found between the different cell-cycle regulating proteins and patient history of former AVFs or the number of revisions. Furthermore, no relation was found between age, sex, cardiovascular risk factors and cell-cycle regulating proteins.



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  		Fig. 1.  Representative immunocytochemical findings of cell-cycle regulators in normal arterial and venous tissue, and neointima of failed AVFs. Fast Red-stained nuclei are shown in normal veins: (A) p21Waf1, (B) p27Kip1, (C) cdk2 and (D) PCNA; normal arteries: (E) p21Waf1, (F) p27Kip1, (G) cdk2 and (H) PCNA; AVFs: (I) p21Waf1, (J) p27Kip1, (K) cdk2 and (L) PCNA expression. p21Waf1 and p27Kip1 immunoreactivity is higher in normal arteries and veins, compared with AVFs (A and B, E and F, I and J) versus higher cdk2 and PCNA immunoreactivity in AVFs compared with arteries and veins (C and D, G and H, K and L). Bar, 100 µm.

 


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  		Fig. 2.  Expression of p21Waf1, p27Kip1, cdk2 and PCNA in AVFs for haemodialysis, normal veins and arteries. Data are presented as mean percentages±standard error of the means. *P<0.001 compared with expression in AVFs; {dagger}P<0.05 compared with expression in AVFs.

 



   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
Although cell-cycle regulator expression in normal, balloon-injured and atherosclerotic vascular tissue has been described [9,10], this has not yet been reported in human intimal hyperplastic lesions.

Normal human arteries and veins are mainly characterized by the presence of quiescent VSMCs in the media. This is supported by our finding that p21Waf1 and p27Kip1 are abundantly expressed in non-diseased human arteries and veins. In contrast, cdk2 expression was absent in veins and minimally expressed in arteries. Considering the aforementioned characteristics of the cell-cycle machinery, these results agree with earlier studies, showing a correlation between CKI (p21Waf1, p27Kip1) expression and VSMC quiescence [8,9,12]. We did not obtain any non-stenosed venous material from the direct vicinity of the stenosed lesion. This tissue would probably be more suitable as a control specimen, as high-flow conditions in this part of the fistula might also initiate changes in the expression of cell-cycle proteins. However, ethical constraints would arise from clinical unnecessary excision of tissue indispensable for the patient.

Intimal hyperplasia, ensuing stenosis is characterized by an increased proliferation and migration of VSMCs, which is the main cell type present in the neointima of AVFs [13]. p27Kip1 and p21Waf1 expression might be expected to be reduced in intimal hyperplasia. However, in the present study p27Kip1 does not seem to play a significant role. In contrast, p21Waf1 expression was considerably lower in stenotic regions of failing AVFs. These findings support the notion that p21Waf1 is a negative regulator of VSMC proliferation and thus intimal hyperplasia in AVFs [8,9,12]. The prevailing theory of p21Waf1 negatively modulating cdk2 expression is reflected by our finding that cdk2 is profusely expressed in intimal hyperplastic lesions.

Wei et al. [14] have shown a temporally and spatially coordinated expression of cdk2, cyclins E and A, and PCNA in a rat balloon-injury model. Unfortunately, we were not able to examine cell-cycle protein expression through time due to the retrospective nature of the study. Further investigations in time-dependent cell-cycle protein expression are needed to elucidate cell-cycle protein function in intimal hyperplasia and eventually providing a site for intervention.

Recently, there has been a growing interest in the role of therapeutical means in preventing restenosis after vascular reconstruction. For example, rapamycin has been shown to inhibit VSMC proliferation and migration in vitro and arterial intimal thickening after balloon injury in vivo by increasing p27Kip1 levels, and inhibiting pRb phosphorylation within the vessel wall [15]. The exact mechanism of action of rapamycin on p27Kip1 levels is unclear. Our results show that p27Kip1 is not suppressed in AVFs. As rapamycin induces p27Kip1 expression, this prospective therapeutical agent might not be the ideal choice for the prevention of stenoses in AVFs. Another possible agent, tranilast, has been found to inhibit proliferation and migration of VSMCs and collagen synthesis [16] by induction of p21Waf1 and p53, preventing restenosis after PTCA [17]. As a result of the induction of p21Waf1 levels, and subsequent decrease of cdk2 activity, tranilast blocks cell-cycle progression in the G1 phase. In the present paper, we demonstrated dramatic reduction of p21Waf1 levels in occluded AVFs. It might be anticipated that an imposed stabilization or even up-regulation of p21Waf1 might inhibit VSMC proliferation, thereby preventing AVF stenosis.

The altered expression of several cell-cycle proteins in failed AVFs contributes to intimal hyperplasia. As the cell-cycle machinery is accessible for therapeutic intervention, it is our opinion that it might be an attractive target to prevent intimal hyperplasia in AVFs. Large randomized placebo-controlled intervention studies are required to prove noteworthy reduction in intimal hyperplasia after AVF construction.



   Acknowledgments
 
The authors would like to thank Dr Frank Stassen for critically reading the manuscript and providing valuable advice.



   Notes
 
Correspondence and offprint requests to: Jan H. M. Tordoir, MD, PhD, Department of Surgery, University Hospital Maastricht, P. Debyelaan 25, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Email: j.tordoir{at}surgery.azm.nl Back



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Burger H, Kluchert BA, Kootstra G et al. Survival of arteriovenous fistulas and shunts for haemodialysis. Eur J Surg 1995; 161:327–334[Medline]
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Received for publication: 12. 8.02
Accepted in revised form: 27.11.02


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