Elevated Erythropoietin Receptor and Transforming Growth Factor-β1 Expression in Stenotic Arteriovenous Fistulae Used for Hemodialysis

Specimens

Samples were obtained from 16 patients whose characteristics are listed in Table 1. Overall study results are given in Table 2. The 16 patients were all chronic hemodialysis patients with the exception of one, who was entering the end-stage renal disease program. Twelve of 16 patients had almost occluded or severely narrowed AV fistula segments. Of these 12 patients, occluded or severely narrowed AV fistula segments were obtained from seven hemodialysis patients undergoing surgical AV fistula repair. All seven patients had been treated with intravenous rhEPO. All AV fistulae were of the Brescica-Cimino type. Stenosis was diagnosed on the basis of insufficient blood flow at hemodialysis and was sometimes detected by angiography. Patent portions adjacent to the stenotic fistulae were obtained from eight patients at the time of fistula reconstruction. In four cases, only patent segments adjacent to the stenotic portions were obtained at the time of operation. In addition, cutaneous veins were obtained from seven patients at fistula construction and reconstruction. Clinical characteristics of the three patient groups were not statistically different with respect to age, duration of hemodialysis, duration of EPO treatment, and mean dose of weekly intravenous rhEPO (Table 1). Informed consent was obtained from all patients before inclusion in the study. Specimens were fixed overnight in 4% paraformaldehyde and paraffin-embedded.

Histochemistry

Serial sections (3 μm thick) of paraffin-embedded tissues were mounted on slides coated with aminopropyl-triethoxysilane (Matsunami Co., Osaka, Japan). The deparaffinized sections were stained with hematoxylin-eosin and elastic van Gieson.

Immunohistochemistry

Serial sections were also used for immunohistochemical analysis. The primary antibodies used were rabbit anti-mouse EPO-R (Upstate Biotechnology, Lake Placid, NY), which detects the human EPO receptor, and rabbit anti-human TGF-β1 (Santa Cruz Biotechnology, Santa Cruz, CA). Goat anti-human PAI-1 (Bio-pool, Umea, Sweden) was used to detect the proteinase inhibitor. Extracellular matrices were examined using anti-fibronectin EDA+ (a gift from Dr. L. Zardi, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy). Anti-human CD68 (Dako, Carpinteria, CA) and anti-α-smooth muscle actin (Dako) were used to identify macrophages and smooth muscle cells, respectively. Sections incubated with nonimmune sera instead of the primary antibodies were used as controls.

Staining was performed using the standard avidin-biotin-peroxidase complex method. Deparaffinized sections were treated with 3% H₂O₂ for 30 min. To block nonspecific binding of primary antibodies, sections were incubated with 3% normal donkey serum in phosphate-buffered saline (PBS) for 30 min at room temperature, followed by incubation with primary antibody for 1 h at 37°C in a humidified chamber. After three washes in PBS, biotinylated donkey anti-rabbit, -mouse, or -goat IgG (Vector Laboratories, Burlingame, CA), diluted 1:500 in PBS, was applied, and incubation was continued for another 30 min at room temperature. The sections were then rinsed in PBS and incubated with avidin-biotin-peroxidase complex (ABC Elite, Vector Laboratories) for 30 min. Finally, color was developed by treating sections with the chromogenic component True-Blue (Kirkegaard and Perry Laboratories, Gaithersburg, MD) or chromogenic component containing 0.05% 3,3′-diaminobenzidine (Sigma Chemical Co., St. Louis, MO). Tissue sections were lightly counterstained with Contrast-Red (Kirkegaard and Perry Laboratories), dehydrated with ethanol, and mounted with coverslips.

Tissue sections were examined by light microscopy at ×200 magnification equipped with an ocular grid. EPO-R- and TGF-β1-positive cells were counted separately in the intima, media, and adventitia. In each of the vessel layers, four randomly selected fields were counted and the average values of the four fields were obtained (14). Analysis was performed by two independent investigators who were blinded to the clinical status of the patient.

In Situ Hybridization

Sections of paraffin-embedded tissues (3 mm thick) were subjected to in situ hybridization to detect the expression of TGF-β1 mRNA. The plasmid pmTGF-β1-A, expressing murine TGF-β1 cDNA, was kindly provided by Dr. H. L. Moses (Vanderbilt University, Nashville, TN) (24). Antisense and sense cRNA riboprobes were generated after digestion with restriction enzymes HindIII and EcoRI (Takara Shuzo, Otsu, Japan), respectively. Riboprobe was labeled with digoxigenin using a DIG RNA labeling kit (Boehringer Mannheim Biochemica, Mannheim, Germany). Tissue sections were rehydrated, digested with proteinase K (10 μg/ml; Wako Chemical, Osaka, Japan) for 15 min at 37°C, post-fixed in 4% paraformaldehyde, treated with 0.2N HCl, and acetylated in 0.1 M triethanolamine and 0.25% acetic anhydride. A hybridization solution containing 50% deionized formamide, 0.3 M NaCl, 10 mM Tris HCl, pH 8.0, 5 mM ethylenediaminetetra-acetic acid, 10 mM NaH₂PO₄, 0.2 mg/ml salmon sperm DNA (Sigma), 0.1 mg/ml yeast transfer RNA (Takara), 1 × Denhardt's solution, and 10% dextran sulfate was added to the probe. Hybridization was performed with 10 ng/μl of the digoxigenin-labeled riboprobe added to each slide and incubated for 18 h at 42°C, as described previously (25).

In Situ Binding Analysis

Staining for biotin-EPO was performed using purified rhEPO (a kind gift from Chugai Pharmaceuticals, Tokyo, Japan) labeled with biotin, as described previously (26).

Serial sections were also used for in situ binding studies according to the method of Gabius et al. (27). Deparaffinized sections were treated with 3% H₂O₂ for 30 min. To block nonspecific binding of rhEPO, sections were incubated with 3% normal donkey serum in PBS for 30 min at room temperature. Sections were then incubated with biotin-EPO. Subsequently, sections were rinsed in PBS and incubated with avidin-biotin-peroxidase complex (ABC Elite, Vector Laboratories) for 30 min. Finally, color was developed by treating sections with a peroxidase substrate, True-Blue (Kirkegaard and Perry Laboratories). Tissue sections were lightly counterstained with Contrast-Red (Kirkegaard and Perry Laboratories), dehydrated by ethanol, and mounted with coverslips.

Statistical Analyses

Values are expressed as mean ± SD. The average value was compared among groups by one-way ANOVA. Correlations between variables were assessed by linear regression analysis. Significance was defined as P < 0.05.

Histology

Cutaneous veins used as normal controls showed no intimal thickening (Figure 1A). In contrast, marked intimal hyperplasia was evident in all stenosed fistulae (Figure 1B), some of which were almost occluded (Figure 1C). The intimal hyperplasia was irregular with marked cellular proliferation and was associated with angiogenesis in some stenosed areas. In contrast, the vascular media in stenosed lesions was uniformly thickened. Compared with stenosed fistulae, patent portions of the fistulae near stenotic lesions showed only mild intimal hyperplasia.

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Figure 1.

Histologic changes in sections. (A) Cutaneous veins were thin and showed no intimal thickening as detected by elastic van Gieson staining. Arrows show thin intima (patient 4). (B) Prominent intimal hyperplasia was evident in stenosed fistulae. Note that the magnification of this photograph is similar to that of the cutaneous vein shown in A. Arrows show intimal hyperplasia (patient 1). (C) An almost occluded fistula showing irregular intimal hyperplasia and marked cell proliferation. Arrows show areas of angiogenesis (patient 10). Magnification, ×48.

Immunohistochemical Staining for EPO-R

In cutaneous veins, positive immunoreactivity for EPO-R was noted in the thin intima, media, and adventitia (Figure 2A), but strong staining was evident in the adventitia of capillaries. Positive immunostaining was evident in the cytoplasmic and perinuclear areas of cells. Compared with cutaneous veins, immunoreactivity for EPO-R was significantly increased in areas of intimal hyperplasia in stenosed fistulae (Figure 2B). EPO-R-positive cells were positively stained for actin but negative for CD68 (data not shown). As shown in Table 3, the number of EPO-R-positive cells was significantly higher, even in patent portions of AV fistulae, compared to cutaneous veins, and was even higher in stenotic lesions. Strong staining for EPO-R was especially marked in areas of intimal hyperplastic lesions exhibiting angiogenesis (Figure 2C). The number of EPO-R-positive cells did not correlate significantly with total amount of EPO treatment (r = 0.165, P = 0.487).

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Figure 2.

Immunostaining for the erythropoietin receptor (EPO-R). (A) In cutaneous veins, positive staining for EPO-R was observed in the thin intima, media, and adventitia. Arrows show thin intima (patient 4). (B) Staining for EPO-R was significantly increased in hyperplastic intima of the stenotic fistula (patient 1). (C) Strong EPO-R staining was observed in angiogenesis of the intimal hyperplastic lesions (patient 10). Magnification, ×120.

Staining for TGF-β, PAI-1, and Fibronectin

The staining pattern of TGF-β1 was similar to that of EPO-R. In cutaneous veins, positive staining for TGF-β1 was detected in the thin intima, media, and adventitia (Figure 3A). Positive immunostaining for TGF-β1 was evident in the cytoplasmic and perinuclear areas of cells. Staining for TGF-β1 was significantly increased in areas of intimal hyperplasia in stenosed fistulae (Figure 3B). As shown in Table 3, cells positively stained for TGF-β1 were significantly more numerous in stenotic compared to patent portions of fistulae and cutaneous veins. In patent parts of fistulae, TGF-β1 immunostaining was also increased compared with cutaneous veins. TGF-β expression was significantly correlated with that of EPO-R (r = 0.928, P < 0.05) (Figure 4). Immunostaining for PAI-1 was also increased in areas of intimal hyperplasia of stenosed fistulae and the staining was essentially the same as that of TGF-β1 (Figure 3D). Fibronectin EDA+ staining was also similar to that of PAI-1 (Figure 3E). Sections treated with nonimmune rabbit serum showed no immunostaining.

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Figure 3.

Immunostaining of transforming growth factor-β1 (TGF-β1), plasminogen activator inhibitor type 1 (PAI-1), and cellular fibronectin extra domain A (EDA+) and in situ hybridization of TGF-β1. (A) Positive staining for TGF-β1 was observed in the thin intima, media, and adventitia of cutaneous veins (patient 4). (B) TGF-β1 staining was markedly increased in hyperplastic intima of stenotic fistulae (patient 1). (C) Weak expression of TGF-β1 mRNA in cutaneous veins (patient 4). (D) Increased expression of TGF-β1 mRNA in stenotic fistula (patient 1). Increased immunoreactivity for PAI-1 (E) (patient 1) and fibronectin EDA+ (F) (patient 1). Magnification, ×120.

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Figure 4.

Correlation between numbers of EPO-R- and TGF-β-positive cells. The number of EPO-R-positive cells in the intima significantly correlated with the number of TGF-β-positive cells in the intima in all samples (r = 0.928, P < 0.05).

In Situ Hybridization

Cells expressing TGF-β1 mRNA as demonstrated by in situ hybridization were identical in most respects to those that stained positively with anti-TGF-β1 antibody. In cutaneous veins, TGF-β1 mRNA was observed in the thin intima, media, and adventitia (Figure 3C). Remarkably strong expression of TGF-β1 mRNA was evident in areas of intimal hyperplasia in stenotic fistulae (Figure 3D). Marked expression of TGF-β1 mRNA was noted even in patent portions of fistulae, and this was still less than in stenotic fistulae, but much higher than in cutaneous veins.

In Situ Binding Study

EPO-R expression was determined by in situ binding of biotin-EPO in serial tissue specimens. Binding was detected in cutaneous veins and fistulae. Staining of biotin-EPO bound to tissue was essentially the same as that of immunostaining for EPO-R using anti-EPO-R antibody. Greater binding of biotin-EPO was detected in areas of intimal hyperplasia (Figure 5A). Excess nonlabeled rhEPO prevented for the most part the binding of biotin-EPO (Figure 5B).

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Figure 5.

In situ binding of biotin-EPO. Increased binding of biotin-EPO was demonstrated in hyperplastic intima in stenosed sections (A) (patient 10). Addition of excess nonlabeled rhEPO almost blocked the binding of biotin-EPO (B). Magnification, ×120.