Hormones – Cytokines – Signaling

Kidney International (2000) 57, 1895–1904; doi:10.1046/j.1523-1755.2000.00039.x

Exposure of endothelial cells to recombinant human erythropoietin induces nitric oxide synthase activity

Debendranath Banerjee, Marilis Rodriguez, Mihir Nag and John W Adamson

The Lindsley F. Kimball Research Institute of The New York Blood Center, New York, New York, USA

Correspondence: Debendranath Banerjee, Ph.D., The Lindsley F. Kimball Research Institute of The New York Blood Center, New York, New York 10021, USA. E-mail: dbanerje@nybc.org

Received 5 August 1999; Revised 20 October 1999; Accepted 2 December 1999.

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Abstract

Exposure of endothelial cells to recombinant human erythropoietin induces nitric oxide synthase activity.

Background

 

Anemic patients with chronic renal failure receiving recombinant human erythropoietin (rHuEPO) therapy frequently develop hypertension through an unknown mechanism. We hypothesize that EPO receptors (EPORs) on endothelial cells (ECs) in various sites of vasculature may mediate the activities of nitric oxide synthase (NOS) and/or the release of endothelin-1 (ET-1), contributing to blood pressure changes. We tested this hypothesis using primary cultures of ECs obtained from human coronary artery (HCAEC), pulmonary artery (HPAEC), dermis (HDEC), and umbilical vein (HUVEC).

Methods

 

EPORs were measured by 125I-EPO binding. The effect of EPO on EPOR, ET-1, and NOS mRNA levels was assessed by quantitative reverse transcription-polymerase chain reaction. Cellular NOS activity and ET-1 release into the medium was measured by the NOSdetect assay and by radioimmunoassay kits.

Results

 

Short-term (4 h) treatment with EPO (4 U/mL) did not change the number or affinity of EPOR per cell. Neither were there any changes in the amount of EPOR, ET-1, and NOS transcripts (cDNA/mug of mRNA) nor in ET-1 release and NOS activity. In HUVEC only, 24-hour exposure to EPO caused a threefold increase in NOS transcript. In other cells, EPO treatment for six days increased NOS activity by twofold to fourfold.

Conclusions

 

We show that upon extended exposure, EPO induces NOS activity but does not affect ET-1 release. These findings indicate that the hypertensive effect of EPO is not likely to be caused by a direct effect on ECs.

Keywords:

erythropoietin, receptor, endothelin, nitric oxide, anemia, chronic renal failure

Erythropoietin (EPO) is a glycoprotein hormone that is the primary regulator of erythropoiesis1. EPO is produced by the kidney in adults and by the liver in fetal life1,2. EPO functions through its interaction with a single chain cell surface receptor of the cytokine receptor superfamily3,4. The EPO receptor (EPOR) on erythroid progenitor cells is the primary target for EPO binding5,6.

In hematopoietic cells, EPOR mRNA is expressed at moderate levels7. EPOR or EPOR mRNA are also expressed on nonhematopoietic cells, including human umbilical vein endothelial cell (HUVEC)8,9, rat brain capillary endothelial cells (ECs)10, murine hippocampal and cerobrocortical areas11, and primary cultured hippocampal and cortical neurons12,13. The EPORs are functional in HUVEC and mouse brain.

The angiogenic effect of EPO has been studied in ovariectomized mice in which an injection of EPO into the uterine cavity promoted blood vessel formation in the endometrium14. EPO stimulates proliferation and migration of human and bovine EC and also angiogenesis of the rat thoracic aorta8,15.

Many anemic patients with chronic renal failure receive treatment with recombinant human erythropoietin (rHuEPO) with beneficial results. The major side-effect of this treatment is the development of clinically significant hypertension16,17,18. Several factors have been implicated in rHuEPO-associated hypertension: an increase in hematocrit and red blood cell mass resulting in increased blood viscosity19, a loss of hypoxic vasodilation20, a direct vasoconstrictor effect21, and an increase in calcium uptake by vascular smooth muscle cells22,23. Nevertheless, the principal cause of EPO-induced hypertension is unknown. The angiogenic nature of EPO and the occurrence of EPOR on ECs suggest that EPO may directly stimulate ECs.

In humans, clinical events such as hypertension and thrombosis are often localized in specific vessels. This pattern, in part, may be due to the heterogeneity of EC themselves [reviewed in24,25. Since the discovery of EC-dependent vasodilation by Furchgott and Zawadzki, ECs have been recognized as an important functional unit. Upon induction by vasoactive agents, such as acetylcholine and bradykinin, ECs secrete short-lived relaxing factor(s) causing relaxation of underlying smooth muscle cells [reviewed in26,27. One endothelium-derived relaxing factor is nitric oxide (NO)28. EC-dependent vasoconstriction has also been observed in response to various physiological stimuli such as thrombin29, hypoxia30, and mechanical stretch31. Endothelin-1 (ET-1) is the most potent vasoconstrictor peptide produced by ECs32,33. Vascular smooth muscle cells and glomerular mesangial cells respond to ET-1 by cellular contraction and proliferation34,35. To date, very little information is available on the effect of EPO on endothelium from different vascular sites.

In this study, we obtained primary cultures of EC prepared from various sites of human vasculature and examined the levels of EPO binding to the cells and the effect of EPO on EPOR, ET-1, and NO synthase (NOS) mRNA expression by quantitative reverse transcription-polymerase chain reaction (RT-PCR). We also measured the effect of EPO on ET-1 release into the culture medium and endothelial NOS enzyme activities. We found that EPORs are present on all ECs tested and that EPOR, ET-1, and NOS mRNA levels are unaffected by treatment with EPO. However, the extended exposure of vascular ECs to EPO at super-physiological concentrations induced NOS activity, which may contribute to the regulation of blood pressure.

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METHODS

Cells and cell culture

Primary cultures of human ECs, prepared from umbilical vein (HUVEC), coronary artery (HCAEC), pulmonary artery (HPAEC), and dermis (HDEC) were purchased from Clonetics (San Diego, CA, USA). Cells were cultured at 37°C in modified MCDB 131 medium (Clonetics) supplemented with 5% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA) in an air-5% CO2 atmosphere at constant humidity. Cells were maintained in continuous culture and used within three to five passages.

125I-EPO binding reactions

For binding studies (3-[125I]iodotyrosyl)rHuEPO (Amersham Corp., Arlington Heights, IL, USA) was used. The experimental procedures for the binding of 125I-EPO to ECs have been reported elsewhere8. Briefly, confluent monolayers of ECs (1 times 106 cells/well) were washed with binding medium (0.05 mol/L phosphate buffer, pH 7.4, containing 150 mmol/L NaCl, 68 mmol/L CaCl2, 50 mmol/L MgCl2, and 1 mg/mL human serum albumin) and incubated at 22°C with 1.0 mL binding buffer containing 60,000 counts per min (cpm) of 125I-EPO(Sp. Act. 300 to 900 Ci/mmol). At the end of specified time of incubation, wells were gently washed three times with prewarmed phosphate-buffered saline (PBS) containing 1% FBS, and the cells were then solubilized in lysis buffer [20 mmol/L HEPES, pH 7.4, 1% Triton X-100, 10% (vol/vol) glycerol, and 0.1 mg/mL of bovine serum albumin]. The radioactivity of the lysates was measured in a gamma counter. Nonspecific binding was assessed in the presence of a 100-fold molar excess of unlabeled EPO added at the start of incubation and subtracted from the total to calculate net specific binding.

Preparation of RNA

Approximately 1 to 5 times 107 cells, either treated with rHuEPO (4 U/mL) for four hours or untreated, were washed twice with PBS, and RNA was extracted with TRIzol reagent as recommended by the supplier (Life Technologies, Grand Island, NY, USA). mRNA was adsorbed onto oligo(dT)-cellulose columns (Qiagen, Valencia, CA, USA). The total amount and concentration of mRNA were determined spectrophotometrically.

Reverse transcription of RNA and synthesis of cDNA

An aliquot of mRNA (0.5 to 1 mug) was incubated at 42°C for one hour in 20 muL of 10 mmol/L Tris-HCl, pH 8.8, containing 50 mmol/L KCl, 0.1% Triton X-100, 5 mmol/L MgCl2, 1 mmol/L each dNTPs, 20 units of RNAsin, and 0.5 mug oligo dT15 primer. The cDNA was synthesized by primer extension using 15 units of avian myeloblastosis virus (AMV) reverse transcriptase per mug of RNA (Promega, Madison, WI, USA).

Preparation of internal standard DNA and amplification of cDNA

An internal standard was constructed by PCR amplification of genomic DNA using the following primer sets: (1) 5'-GCACCGAGTGTGTGCTGAGC-3' and 5'-GGTCAGCAGCACCAGGATGA-3' for hEPOR36, (2) 5'-CCGTATGGACTTGGAAGCCC-3' and 5'-CTGGTTTGTCTTAGGTGTTCC-3' for ET-137, and (3) 5'-CCTCCCCCCGGCTGGGTGCG-3' and 5'-GCACCTCCAGAAGCGTGGG-3' for NOS38. The same primer sets were used for cDNA amplification.

Quantitative polymerase chain reaction

To quantitate gene-specific mRNA, multiple reaction mixtures were prepared with known amounts of cDNA. Serial dilutions of internal standard cDNA and [alpha-32P]CTPwere added to each tube and coamplified. The products were analyzed by gel electrophoresis. The bands representing gene-specific cDNA and the standard DNA fragment were recovered and counted, and the results were plotted as cpm versus the concentration of external standard.

Endothelin-1 assay

The medium from cultured ECs, either exposed for four hours to rHuEPO (4 U/mL) or untreated, was harvested and centrifuged at 500 times g at 4°C for 10 minutes. The recovered supernatants were then stored at -70°C. ET-1 determinations were performed using an ET-1 radioimmunoassay kit (Peninsula Lab., Belmont, CA, USA).

Nitric oxide synthase assay

Confluent monolayers of ECs, either treated with rHuEPO (4 U/mL) daily for six days or untreated, were washed twice with PBS, harvested, and homogenized. The homogenate was centrifuged, and the supernatant protein concentration was adjusted to 1 mg/mL. The NOS activity was measured using a NOSdetect assay kit according to the supplier's specification (Stratagene, La Jolla, CA, USA).

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RESULTS

125I-EPO binding study

To compare binding of 125I-EPO to EC from various vascular sites, cells were exposed to 125I-EPO with or without a 100-fold excess of unlabeled EPO for increasing time intervals. The time dependence of binding of 125I-rHuEPO to ECs from various sources is shown in Figure 1. All ECs readily bound 125I-EPO, and the specific binding increased with time, reaching a plateau in about four hours for HUVEC and HCAEC. The plateauing time was slightly longer (approximately 6 h) for HDEC and HPAEC. The fluctuation of increase binding in various ECs may correspond to the differences in the internalization and degradation of EPO as the binding assays were performed at 22°C. The concentration dependence of EPO binding was measured over the range from 1 to 18 nmol/L Figure 2. Specific binding was found to be saturable. The mean number of EPOR per cell was calculated by Scatchard plot analysis Figure 3. The calculated number of binding sites was 44,668 for HUVEC, 49,845 for HCAEC, 56,106 for HDEC, and 58,093 for HPAEC, while the dissociation constants (Kd) were 5.6 times 10-9 mol/L for HUVEC, 7.7 times 10-9 mol/L for HCAEC, 1.10 times 10-8 mol/L for HDEC, and 1.50 times 10-8 mol/L for HPAEC. Exposure of cells for two days to EPO resulted in no significant change in the number of receptor sites (data not shown).

Figure 1.
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Kinetics of 125I-recombinant human erythropoietin (rHuEPO) binding to endothelial cells. Monolayers of human endotheial cells prepared from (A) umbilical vein (HUVEC), (B) coronary artery (HCAEC), (C) dermis (HDEC), and (D) pulmonary artery (HPAEC) were incubated with 60,000 cpm of 125I-EPO (Sp. act. 166 Ci/mmol) in 1.0 mL phosphate buffered saline (PBS) containing CaCl2 and MgCl2 for increasing time intervals at 22°C. At the end of each time point, cells were washed, and the specific radioactivity associated with each well was determined as described in the Methods section. The results from three independent experiments are shown. Error bars represent standard errors (N = 3).

Full figure and legend (25K)

Figure 2.
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Dose response of 125I-EPO binding to endothelial cells. Confluent monolayers of (A) HUVEC, (B) HCAEC, (C) HDEC, and (D) HPAEC were washed and incubated with increasing amounts of 125I-EPO (1 to 18 pmol) for four hours at 22°C. Monolayers were washed, and the specific radioactivity associated with each well was determined as described in the Methods section. The results from three independent experiments are shown. Error bars represent standard errors (N = 3).

Full figure and legend (27K)

Figure 3.
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Scatchard analysis of 125I-EPO binding to endothelial cells. Plots represent Scatchard analyses of binding data shown in Figure 2. The data suggest the presence of a single class receptor with different affinities in different cell types. For (A) HUVEC, Kd = 5.6 times 10-9 mol/L and 44,668 receptor sites per cell; (B) HCAEC, Kd = 7.7 times 10-9 mol/L and 49,845 receptor sites per cell; (C) HDEC, Kd = 1.10 times 10-8 mol/L and 56,106 receptor sites per cell; (D) HPAEC, Kd = 1.5 times 10-8 mol/L and 58,093 receptor sites per cell.

Full figure and legend (28K)

Quantitation of mRNA expression

To confirm that EPO binding correlated with EPOR gene expression, ECs were cultured with or without EPO for four hours, and EPOR mRNA was measured by quantitative RT-PCR. The results are shown in Figure 4. The expected 197 bp cDNA fragment and 285 bp internal standard DNA were amplified from ECs from all sources. Further cloning and sequencing confirmed that the 197 bp fragment represented hEPOR mRNA. The amount of hEPOR-specific cDNA/mug of mRNA determined in duplicate by quantitative PCR was 5.1 times 10-4 ng for HUVEC, 11.0 times 10-4 ng for HPAEC, 4.0 times 10-4 ng for HCAEC, and 64.0 times 10-4 for HDEC. With the exception of HCAEC, treatment of these cells with EPO for up to four hours had little or no effect on EPOR mRNA. In contrast, a twofold increase of EPOR mRNA was measured in EPO-treated HCAEC culture Table 1. Treatment of HUVEC cells with EPO for 1, 2, 4, 6, and 24 hours had little or no effect on EPOR mRNA (data not shown).

Figure 4.
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Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Polymerase chain reaction (PCR) analysis of human erythropoietin receptor (hEPOR) transcripts in control and EPO-treated HUVEC, HPAEC, HDEC, and HCAEC. Monolayers of HUVEC, HCAEC, HDEC, and HPAEC were incubated with EPO (4 U/mL) for four hours or were untreated, and mRNA was prepared as described in the Methods section. cDNA was reverse transcribed from mRNA isolated from respective endothelial cells. Multiple reactions for hEPOR-specific PCR amplification of cDNA were carried out with hEPOR primers. Prior to amplification, increasing amounts of standard DNA and [alpha-32P]dCTP were added to each reaction. (A) The amplification products were resolved by agarose gel electrophoresis. Left panel show control gels and right panels show EPO-treated gels (upper band 285 bp standard DNA and lower band 197 bp hEPOR cDNA; bp, size markers). (B) For quantitation, the hEPOR and the standard DNA bands were cut, the radioactivity present in each band was determined and plotted against the amount of standard added to each reaction mixture. Symbols are: (filled circle) standard DNA; (filled square) hEPOR-specific PCR product. The amount of standard corresponding to the point at which the two PCR products are equal is an indication of the amount of hEPOR cDNA present.

Full figure and legend (48K)


Effects of rHuEPO on ET-1 gene expression and ET-1 secretion

A direct link between ET-1 and EPO has been established from the findings that HUVECs have EPOR8,9 and that rHuEPO increases ET-1 release by bovine pulmonary artery ECs39. We therefore examined the effect of EPO on ET-1 expression by cultured HUVEC, HCAEC, HPAEC, and HDEC. As shown in Figure 5, a 290 bp ET-1–specific partial cDNA fragment and a 450 bp internal standard DNA were amplified by ET-1 primer sets and RT-PCR. The 290 bp fragment was cloned, and its identity confirmed by sequencing. The quantitative values are shown in Table 2. The amount of ET-1–specific cDNA/mug of mRNA in duplicate quantitative PCR was 2.8 times 10-2 ng for HUVEC, 3.8 times 10-2 ng for HPAEC, 2.6 times 10-2 ng for HCAEC, and 0.34 times 10-2 ng for HDEC. These values were unchanged in HCAEC, HPAEC, and HDEC cultures exposed to rHuEPO. However, the level of ET-1 transcript rose twofold in EPO-treated HUVEC cultures. Treatment of HUVEC cells with EPO for 1, 2, 4, 6, and 24 hours had no significant effect on ET-1 mRNA (data not shown).

Figure 5.
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Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Polymerase chain reaction (PCR) analysis of endothelin-1 (ET-1) transcripts in untreated and EPO-treated HUVEC, HPAEC, HDEC, and HCAEC. Monolayers of HUVEC, HCAEC, HDEC, and HPAEC cells were either incubated with EPO (4 U/mL) for four hours or were untreated, and mRNA was prepared as described in the Methods section. Reverse transcription of total RNA isolated from respective endothelial cells with ET-1–specific primers and multiple reactions for PCR amplification of cDNA were carried out with ET-1 primers and standard DNA as described in Figure 4. (A) The amplification products were resolved by agarose gel electrophoresis. Left panels show controls and right panels show EPO treated (upper band 450 bp standard DNA and lower band 290 bp ET-1 cDNA; bp, size markers). (B) Quantitation was achieved as described in Figure 4. ET-1 cDNA and the standard DNA bands were cut, the radioactivity present in each band was determined and plotted against the amount of standard added to each reaction mixture. Symbols are: (filled circle) standard DNA; (filled square) ET-1-specific PCR product to each reaction mixture.

Full figure and legend (49K)


Table 3 shows ET-1 release by cultured HUVEC, HCAEC, HPAEC, and HDEC during a four-hour treatment with 4 U/mL rHuEPO. All cells released ET-1 into the media. About 35 to 48 pg of ET-1 were released by 2 times 105 cells. These values also did not change with EPO treatment, consistent with the results shown in Table 2.


Effect of rHuEPO on NOS activity

Nitric oxide is thought to play a central role in vascular homeostsis. NO, a heterodiatomic free radical product, is generated through the oxidation of L-arginine to L-citrulline by NOS. NO has been demonstrated to inhibit thrombosis, cytokine-induced vascular cell adhesion molecule-1 (VCAM) expression, leukocyte adhesion to endothelium, and smooth muscle proliferation and migration40,41,42. To investigate the effect of EPO on NOS activity, we measured NOS mRNA levels by RT-PCR. The results are shown in Figure 6. Partial cDNA fragments (approximately 230 bp) and standard DNA (approximately 500 bp) were isolated, cloned, and characterized by sequencing. The quantitative values are shown in Table 4. The NOS-specific mRNA in ECs varied from 1.0 times 10-3 to 1.8 times 10-3 ng/mug of mRNA. Exposure of the cells to rHuEPO had little effect. However, the presence of EPO for 24 hours induced a threefold increase in NOS-specific mRNA (5.4 times 10-3 ng cDNA/mug of mRNA) compared with control cultures of HUVEC (1.8 times 10-3 ng cDNA/mug of mRNA). The increase values were maintained for up to six days upon daily removal and replenishment with fresh EPO-supplemented medium. Short-term exposure for one, two, four, and six hours to EPO, HUVEC exhibited no effect on NOS-specific mRNA (data not shown).

Figure 6.
Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author
Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Polymerase chain reaction (PCR) analysis of nitric oxide synthase (NOS) transcripts in EPO-treated and nontreated HUVEC, HPAEC, HDEC, and HCAEC. Monolayers of HUVEC, HCAEC, HDEC, and HPAEC were either incubated with EPO (4 U/mL) for four hours or were untreated, and mRNA was prepared as described in the Methods section. Reverse transcription of total RNA isolated from respective endothelial cells with NOS-specific primers and multiple reactions for PCR amplification of cDNA were carried out with NOS primers and standard DNA, as described in Figure 5. (A) The amplification products were analyzed by agarose gel electrophoresis. Left panels show controls and right panels show EPO treated (upper 500 bp band standard DNA and lower 200 bp NOS cDNA band; bp, size markers). (B) For quantitation, the NOS and the standard bands were cut, the radioactivity present in each band was determined and plotted against the amount of standard added to each reaction mixture. Symbols are: (filled circle) standard DNA; (filled square) NOS-specific PCR product.

Full figure and legend (61K)


To confirm that the increased NOS-specific mRNA resulted in increased NOS activity, we determined the effect of EPO on NOS activity. As shown in Table 5, long-term EPO treatment resulted in a twofold to fourfold increase in NOS activity in all ECs.


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DISCUSSION

The present study demonstrates that ECs prepared from various regions of the vascular tree bind EPO in a dose-dependent manner. The scattering of data points at a low ligand concentration in Scatchard analysis indicates the possibility of more than one binding site. Our finding of a large number of relatively low-affinity binding sites on all EC types tested Figure 3 is consistent with the findings of Anagnostou et al8. This is in contrast with earlier studies that measured the binding of EPO to its receptors and showed that erythroid progenitor cells have only a small number of cell surface receptors43,44,45. The low number of receptors per cell for growth factors is common in hematopoietic cells46.

The ligand affinity of EPOR expressed on EC is much lower than that on erythroid cells; the Kd for EPO binding to EPOR on EC varies from approximately 5 to 15 nmol/L8,10, while the Kd for the high-affinity receptor on erythroid cells is approximately 50 pmol/L47. The reason for the expression of only the low-affinity receptor on ECs is unclear. Expression of only the low-affinity receptor might be crucial to prevent unregulated angiogenesis by endogenous EPO. The presence of EPOR on ECs may originate from the close relationship between vasculogenesis and hematopoiesis in early development48 and between angiogenesis and hematopoiesis later in development49.

Compared with OCI-M1 or other human erythroid cell lines, HUVECs have many more EPOR and a lower expression of EPOR mRNA9. In our study, the mean number of EPOR per cell varied from 44,668 to 58,093, but the quantity of EPOR mRNA was less than the amount reported for OCI-M1 cells. The significance of this discrepancy is not clear. In erythroid cells, less than 5% of the newly synthesized EPORs are found on the cell surface, and the rest are degraded in the endoplasmic reticulum50,51.

Quantitative determination of mRNA expression failed to detect any increase in EPOR gene expression in EPO-treated HUVEC, HPAEC, and HDEC cultures. In contrast, a twofold increase of EPOR mRNA was found in EPO-treated HCAEC culture Table 1. Similar determinations of ET-1 transcripts revealed no increase in ET-1 mRNA in EPO-treated HPAEC, HCAEC, and HDEC cultures, whereas a twofold increase of ET-1 mRNA was found in EPO-treated HUVEC Table 2. The reasons for these differences are not clear. It is possible that the untreated HCAEC and HUVEC cultures contained fewer ECs, and thus, the technique can be limited by its inability to distinguish heterogeneous cell populations.

Normally, ECs contribute to the regulation of blood pressure and blood flow by releasing vasodilators such as NO and prostacyclin (PGI2), as well as vasoconstrictors including ET-1 and platelet activating factor (PAF) [reviewed in52. ECs synthesize ET-153. ET-1 is not stored in granules but is transcribed after stimulation by hypoxia, shear stress, or ischemia54. rHuEPO has been shown to have a direct stimulatory effect on ET-1 release from cultured bovine PAEC39. This stimulatory effect was time dependent, peaked at 4 hours, and reached a plateau at 12 hours. In contrast, we saw no effect on ET-1 release in response to EPO in any of the four human EC types in culture. This may be due to differences in culture conditions.

Endothelial cells produce NO, a free radical generated through the oxidation of L-arginine to L-citrulline by NO synthase55. One isoform, eNOS, is constitutively active in EC and can be further stimulated by thrombin, bradykinin, or shear stress56. Our study demonstrates that short-term treatment of ECs in culture has little or no effect on NOS transcript. However, EC cultures exposed to EPO for several days increased their production of NO. Although we did not measure NO release into the medium and it is impossible to directly extrapolate these results to the in vivo situation, the increased production of NO may contribute to the regulation of blood pressure.

Heidenreich, Rahn, and Zidek21 and Carlini et al39 have shown that rHuEPO has a direct vasoconstrictive effect. In addition, in vivo studies of Buemi, Allegra, and Frisina57 and the results of the present studies Table 5 are consistent with a vasodilatation effects of EPO. EPO may act by modifying the balance of vasoconstrictive and vasodilatory effects to favor vasoconstriction in the setting of renal failure. Why hypertension is only seen in this patient group remains unresolved.

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References

  1. Krantz, SB: Erythropoietin. Blood 1991 77: 419–434,  | PubMed | ISI | ChemPort |
  2. Jelkmann, W: Erythropoietin: Structure, control of production, and function. Physiol Rev 1992 72: 449–489,  | PubMed | ISI | ChemPort |
  3. Bazan, JF: Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci USA 1990 87: 6934–6938,  | PubMed | ChemPort |
  4. D'Andrea, AD, Lodish, HF, Wong, GG: Expression cloning of the murine eythropoietin receptor. Cell 1989 57: 277–285,  | Article | PubMed | ChemPort |
  5. Wu, H, Liu, X, Jaenisch, R, Lodish, HF: Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell 1995 83: 59–67,  | Article | PubMed | ISI | ChemPort |
  6. Lin, CS, Lim, SK, D'Agati, V, Costantini, F: Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis. Genes Dev 1996 10: 154–164,  | PubMed | ISI | ChemPort |
  7. Orlic, D, Anderson, S, Biesecker, LG, Sorrentino, BP, Bodine, DM: Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45, NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7. Proc Natl Acad Sci USA 1995 92: 4601–4605,  | PubMed | ChemPort |
  8. Anagnostou, A, Lee, ES, Kessimian, N, Levinson, R, Steiner, M: Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells. Proc Natl Acad Sci USA 1990 87: 5978–5982,  | PubMed | ChemPort |
  9. Anagnostou, A, Liu, Z, Steiner, M, Chin, K, Lee, ES, Kessimian, N, Noguchi, CT: Erythropoietin receptor mRNA expression in human endothelial cells. Proc Natl Acad Sci USA 1994 91: 3974–3978,  | PubMed | ChemPort |
  10. Yamaji, R, Okada, T, Moriya, M, Naito, M, Tsuruo, T, Miyatake, K, Nakano, Y: Brain capillary endothelial cells express two forms of erythropoietin receptor mRNA. Eur J Biochem 1996 239: 494–500,  | Article | PubMed | ISI | ChemPort |
  11. Morishita, E, Masuda, M, Nagao, M, Yasuda, Y, Sasaki, R: Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience 1997 76: 105–116,  | Article | PubMed | ISI | ChemPort |
  12. Morishita, E, Narita, H, Nishida, M, Kawashima, N, Yamagishi, K, Masuda, S, Nagao, M, Hatta, H, Sasaki, R: Anti-erythropoietin receptor monoclonal antibody: Epitope mapping, quantification of the soluble receptor and detection of the solubilized transmembrane receptor and the receptor expressing cells. Blood 1996 88: 465–471,  | PubMed | ISI | ChemPort |
  13. Digicaylioglu, M, Bichet, S, Marti, HH, Wenger, RH, Rivas, LA, Bauer, C, Gassmann, M: Localization of specific erythropoietin binding sites in defined areas of the mouse brain. Proc Natl Acad Sci USA 1995 92: 3717–3720,  | PubMed | ChemPort |
  14. Yasuda, Y, Masuda, S, Chikuma, M, Inoue, K, Nagao, M, Sasaki, R: Estrogen-dependent production of erythropoietin in uterus and its implication in uterine angiogenesis. J Biol Chem 1998 273: 25381–25387,  | Article | PubMed | ISI | ChemPort |
  15. Carlini, RG, Reyes, AA, Rothstein, M: Recombinant human erythropoietin stimulates angiogenesis in vitro. Kidney Int 1995 47: 740–745,  | PubMed | ISI | ChemPort |
  16. Bode-Boger, SM, Bodger, RH, Kuhn, M, Radermacher, J, Frolich, JC: Endothelin release and shift in prostaglandin balance are involved in the modulation of vascular tone by recombinant erythropoietin. J Cardiovasc Pharmacol 1992 20: 25–28,  | PubMed |
  17. Caravaca, F, Pizarro, JL, Arrobas, M, Cubero, JJ, Garcia, MC, Perez-Miranda, M: Antiplatelet therapy and development of hypertension induced by recombinant human erythropoietin in uremic patients. Kidney Int 1994 45: 845–851,  | PubMed | ISI | ChemPort |
  18. Raine, A, Roger, S: Effects of erythropoietin on blood pressure. Am J Kidney Dis 1991 18: 76–83,  | PubMed | ISI | ChemPort |
  19. Steffen, HM, Brunner, R, Muller, R, Degenhardt, S, Pollok, M, Lang, R, Baldamus, CA: Peripheral hemodynamics, blood viscosity and the renin-angiotensin system in hemodialysis patients under therapy with recombinant human erythropoietin. Contrib Nephrol 1989 76: 292–298,  | PubMed | ChemPort |
  20. Duke, M, Abelman, W: The hemodynamic response to chronic anemia. Circulation 1969 39: 503–515,  | PubMed | ISI | ChemPort |
  21. Heidenreich, S, Rahn, KH, Zidek, W: Direct vasopressor effect of recombinant human erythropoietin on renal resistance vessels. Kidney Int 1991 39: 259–265,  | PubMed | ISI | ChemPort |
  22. Tepel, M, Wischniowski, H, Zidek, W: Erythropoietin increases cytosolic free calcium concentration and thrombin induced changes in cytosolic free calcium in platelets from spontaneously hypertensive rats. Biochem Biophys Res Commun 1991 177: 991–997,  | Article | PubMed | ISI | ChemPort |
  23. Miller, BA, Bell, LL, Lynch, CJ, Cheung, JY: Erythropoietin modulation of intracellular calcium: A role for tyrosine phosphorylation. Cell Calcium 1994 16: 481–490,  | Article | PubMed | ISI | ChemPort |
  24. Augustin, HG, Kozian, DH, Johnson, RC: Differentiation of endothelial cells: Analysis of the constitutive and activated endothelial cell phenotypes. Bioessays 1994 16: 901–906,  | Article | PubMed | ISI | ChemPort |
  25. Dejana, E: Endothelial adherens junctions: Implications in the control of vascular permeability and angiogenesis. J Clin Invest 1996 98: 1949–1953,  | PubMed | ISI | ChemPort |
  26. Furchgott, RF: Role of endothelium in responses of vascular smooth muscle. Circ Res 1983 53: 557–573,  | PubMed | ISI | ChemPort |
  27. Furchgott, RF: The role of endothelium in responses of vascular smooth muscle to drugs. Annu Rev Pharmacol Toxicol 1984 24: 175–197,  | Article | PubMed | ISI | ChemPort |
  28. Palmer, RM, Ferrige, AG, Moncada, S: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987 327: 524–526,  | Article | PubMed | ISI | ChemPort |
  29. De Mey, JG, Vanhoutte, PM: Heterogeneous behavior of the canine arterial and venous wall. Importance of the endothelium. Circ Res 1982 51: 439–447,  | PubMed | ChemPort |
  30. Harder, DR: Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact endothelium. Circ Res 1987 60: 102–107,  | PubMed | ISI | ChemPort |
  31. Katusic, ZS, Shepherd, JT, Vanhoutte, PM: Endothelium-dependent contraction to stretch in canine basilar arteries. Am J Physiol 1987 252: H671–H673,  | PubMed | ISI | ChemPort |
  32. Yanagisawa, M, Kurihara, H, Kimura, S, Tomobe, Y, Kobayashi, M, Mitsui, Y, Yazaki, Y, Goto, K, Masaki, T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988 332: 411–415,  | Article | PubMed | ISI | ChemPort |
  33. Clozel, M, Fischli, W: Human cultured endothelial cells do secrete endothelin-1. J Cardiovasc Pharmacol 1989 13(Suppl 5):S229–S231,  | PubMed | ISI | ChemPort |
  34. Marsden, PA, Danthuluri, NR, Brenner, BM, Ballerman, BJ, Brock, TA: Endothelin action on vascular smooth muscle involves inositol triphosphate and calcium mobilization. Biochem Biophys Res Commun 1989 158: 86–93,  | Article | PubMed | ISI | ChemPort |
  35. Highsmith, RF, Pang, DC, Rapoport, RM: Endothelial cell derived vasoconstrictors: Mechanism of action in vascular smooth muscle. J Cardiovasc Pharmacol 1989 13(Suppl 5):S36–S44,  | PubMed | ISI | ChemPort |
  36. Noguchi, CT, Bae, KS, Chin, K, Wada, Y, Schechter, AN, Hankins, WD: Cloning of the human erythropoietin. Blood 1991 78: 2548–2556,  | PubMed | ISI | ChemPort |
  37. Bloch, KD, Friedrich, SP, Lee, MN, Eddy, RL, Shows, TB, Quertermous, T: Structural organization and chromosomal assignment of the gene encoding endothelin. J Biol Chem 1989 264: 10851–10857,  | PubMed | ISI | ChemPort |
  38. Marsden, PA, Heng, HHQ, Scherer, SW, Stewart, RJ, Hall, AV, Shi, XM, Tsui, LC, Schappert, KT: Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem 1993 268: 17478–17488,  | PubMed | ISI | ChemPort |
  39. Carlini, RG, Dusso, AS, Obialo, CI, Alvarez, UM, Rothstein, M: Recombinant human erythropoietin (rHuEPO) increases endothelin-1 release by endothelial cells. Kidney Int 1993 43: 1010–1014,  | PubMed | ISI | ChemPort |
  40. Dusting, GJ: Nitric oxide in cardiovascular disorders. J Vasc Res 1995 32: 143–161,  | PubMed | ISI | ChemPort |
  41. Wennmalm, A: Endothelial nitric oxide and cardiovascular disease. J Intern Med 1994 235: 317–327,  | PubMed | ISI | ChemPort |
  42. Decaterina, R, Libby, P, Thannickal, VJ, Peng, HB, Rajavashisth, TB, Gimbrone, MA Jr, Shin, WS, Liao, JK: Nitric oxide decreases cytokine-induced endothelial activation: Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest 1995 96: 60–68,  | PubMed | ISI | ChemPort |
  43. Broudy, VC, Lin, N, Egrie, J, De Huen, C, Weiss, T, Papayannopoulou, T, Adamson, JW: Identification of the receptor for erythropoietin on human and murine erythroleukemia cells and modulation by phorbol ester and dimethyl sulfoxide. Proc Natl Acad Sci USA 1988 85: 6513–6517,  | PubMed | ChemPort |
  44. Todokoro, K, Kanazawa, S, Amanuma, H, Ikawa, Y: Specific binding of erythropoietin to its receptor on responsive mouse erythroleukemia cells. Proc Natl Acad Sci USA 1987 84: 4126–4130,  | PubMed | ChemPort |
  45. Fraser, JK, Nicholls, J, Coffey, C, Lin, FK, Berridge, MV: Down-modulation of high-affinity receptors for erythropoietin on murine erythroblasts by interleukin 3. Exp Hematol 1988 16: 769–773,  | PubMed | ISI | ChemPort |
  46. Krantz, SB, Sawyer, ST, Sawada, KI: Purification of erythroid progenitor cells and characterization of erythropoietin receptors. Br J Cancer 1988 58(Suppl 9):31–35,  | ISI |
  47. Nagao, M, Matsumoto, S, Masuda, S, Sasaki, R: Effect of tunicamycin treatment on ligand binding to the erythropoietin receptor: Conversion from two classes of binding sites to a single class. Blood 1993 81: 2503–2510,  | PubMed | ISI | ChemPort |
  48. Flamme, I, Risau, W: Introduction of vasculogenesis and hematopoiesis in vitro. Development 1992 116: 435–439,  | PubMed | ISI | ChemPort |
  49. Asahara, T, Murohara, T, Sullivan, A, Silver, M, van der Zee, R, Li, T, Witzenbichler, B, Schatteman, G, Isner, JM: Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997 275: 964–967,  | Article | PubMed | ISI | ChemPort |
  50. Youssoufian, H, Longmore, G, Neumann, D, Yoshimura, A, Lodish, HF: Structure, function and activation of the erythropoietin receptor. Blood 1993 81: 2223–2236,  | PubMed | ISI | ChemPort |
  51. Neumann, D, Wikstrom, L, Watowich, SC, Lodish, HF: Intermediates in degradation of the erythropoietin receptor accumulate and are degraded in lysosomes. J Biol Chem 1993 268: 13639–13649,  | PubMed | ISI | ChemPort |
  52. Cines, DB, Pollak, ES, Buck, CA, Loscalzo, J, Zimmerman, GA, McEver, RP, Pober, JS, Wick, TM, Konkle, BA, Schwartz, BS, Barnathan, ES, McCrae, KR, Hug, BA, Schmidt, AM, Stern, DM: Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 1998 91: 3527–3561,  | PubMed | ISI | ChemPort |
  53. Levin, ER: Endothelins. N Engl J Med 1996 333: 356–363,  | ISI |
  54. Nakamura, S, Naruse, M, Maruse, K, Demura, H, Uemura, H: Immunochemical localization of endothelin in cultured bovine endothelial cell. Histochemistry 1990 94: 475–477,  | Article | PubMed | ISI | ChemPort |
  55. Stamler, JS, Singel, DJ, Loscalzo, J: Biochemistry of nitric oxide and its redox-activated forms. Science 1992 258: 1898–1902,  | PubMed | ISI | ChemPort |
  56. Topper, JN, Cai, J, Falb, D, Gimbrone, MA Jr: Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: Cyclooxygenase-3, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress. Proc Natl Acad Sci USA 1996 93: 10417–10422,  | Article | PubMed | ChemPort |
  57. Buemi, M, Allegra, A, Frisina, N: Eritropoietina: Non solo anemia renale. G Ital Nefrol 1995 12: 9–15,
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Acknowledgments

We wish to thank Mr. Tellervo Huima and Ms. Yelena Oksov for the preparation of the illustrations.

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