(1/3892) Human, rat, and mouse kidney cells express functional erythropoietin receptors.

BACKGROUND: Erythropoietin (EPO), secreted by fibroblast-like cells in the renal interstitium, controls erythropoiesis by regulating the survival, proliferation, and differentiation of erythroid progenitor cells. We examined whether renal cells that are exposed to EPO express EPO receptors (EPO-R) through which analogous cytokine responses might be elicited. METHODS: Normal human and rat kidney tissue and defined cell lines of human, rat, and mouse kidney were screened, using reverse transcription-polymerase chain reaction, nucleotide sequencing, ligand binding, and Western blotting, for the expression of EPO-R. EPO's effects on DNA synthesis and cell proliferation were also examined. RESULTS: EPO-R transcripts were readily detected in cortex, medulla, and papilla of human and rat kidney, in mesangial (human, rat), proximal tubular (human, mouse), and medullary collecting duct cells (human). Nucleotide sequences of EPO-R cDNAs from renal cells were identical to those of erythroid precursor cells. Specific 125I-EPO binding revealed a single class of high- to intermediate-affinity EPO-Rs in each tested cell line (kD 96 pm to 1. 4 nm; Bmax 0.3 to 7.0 fmol/mg protein). Western blots of murine proximal tubular cell membranes revealed an EPO-R protein of approximately 68 kDa. EPO stimulated DNA synthesis and cell proliferation dose dependently. CONCLUSION: This is the first direct demonstration, to our knowledge, that renal cells possess EPO-Rs through which EPO stimulates mitogenesis. This suggests currently unrecognized cytokine functions for EPO in the kidney, which may prove beneficial in the repair of an injured kidney while being potentially detrimental in renal malignancies.  (+info)

(2/3892) Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: functional analysis of signaling molecules by retrovirus-mediated expression.

Erythropoietin (EPO) and its cell surface receptor (EPOR) play a central role in proliferation, differentiation, and survival of erythroid progenitors. Signals induced by EPO have been studied extensively by using erythroid as well as nonerythroid cell lines, and various controversial results have been reported as to the role of signaling molecules in erythroid differentiation. Here we describe a novel approach to analyze the EPO signaling by using primary mouse fetal liver hematopoietic cells to avoid possible artifacts due to established cell lines. Our strategy is based on high-titer retrovirus vectors with a bicistronic expression system consisting of an internal ribosome entry site (IRES) and green fluorescent protein (GFP). By placing the cDNA for a signaling molecule in front of IRES-GFP, virus-infected cells can be viably sorted by fluorescence-activated cell sorter, and the effect of expression of the signaling molecule can be assessed. By using this system, expression of cell-survival genes such as Bcl-2 and Bcl-XL was found to enhance erythroid colony formation from colony-forming unit-erythroid (CFU-E) in response to EPO. However, their expression was not sufficient for erythroid colony formation from CFU-E alone, indicating that EPO induces signals for erythroid differentiation. To examine the role of EPOR tyrosine residues in erythroid differentiation, we introduced a chimeric EGFR-EPOR receptor, which has the extracellular domain of the EGF receptor and the intracellular domain of the EPOR, as well as a mutant EGFR-EPOR in which all the cytoplasmic tyrosine residues are replaced with phenylalanine, and found that tyrosine residues of EPOR are essential for erythroid colony formation from CFU-E. We further analyzed the function of the downstream signaling molecules by expressing modified signaling molecules and found that both JAK2/STAT5 and Ras, two major signaling pathways activated by EPOR, are involved in full erythroid differentiation.  (+info)

(3/3892) Iron depletion by phlebotomy with recombinant erythropoietin prior to allogeneic transplantation to prevent liver toxicity.

Iron overload may induce liver toxicity after hematopoietic stem cell transplantation (HSCT), but it is not known if iron depletion prior to HSCT can reduce the risk of severe toxicity in this setting. We used subcutaneous recombinant erythropoietin (EPO) (25 UI/kg) three times a week and phlebotomy once a week, to prevent liver toxicity in a patient with advanced acute leukemia and liver disease due to severe iron overload, previous drug toxicity and hepatitis C viral infection. Over the 9 months prior to allogeneic HSCT, 34 phlebotomies were carried out. Serum ferritin dropped from 2964 to 239 microg/l and the ALT dropped to near normal values. At allogeneic HSCT no liver toxicity was observed, suggesting that iron depletion in the pretransplant period may contribute to reducing transplant-related toxicity in selected cases.  (+info)

(4/3892) Association of plasma fibrinogen concentration with vascular access failure in hemodialysis patients.

BACKGROUND: Elevated plasma fibrinogen is an important risk factor for coronary artery disease in the general population and patients with chronic renal failure. High plasma fibrinogen may trigger thrombus formation in arteriovenous fistulas. We performed a prospective, cohort study to evaluate the association of plasma fibrinogen concentration with vascular access failure in patients undergoing long-term haemodialysis. METHODS: Between September 1989 and October 1995, 144 patients underwent a vascular access operation. In March 1997, 102 patients (56 M, 46 F) who had been followed up for more than 18 months (median; 37 months, range; 18-102 months) were included in the study. The median age of the patients was 52 years (range; 19-78 years). In 35 patients, renal disease was secondary to diabetes mellitus. The type of vascular access was a polytetrafluoroethylene (PTFE) graft in 17 patients. Seventy-seven patients received recombinant human erythropoietin (r-HuEPO) therapy during the follow-up period. Plasma fibrinogen, albumin, total cholesterol, hematocrit, platelets and creatinine were measured at the time of operation. Vascular access failure was defined as the occurrence of complications requiring transluminal angioplasty, thrombolytic therapy or surgical repair. RESULTS: Thirty-eight patients had at least one vascular access failure and the incidence was 0.3 (range; 0-2.4) episodes per patient-year. The survival rate of vascular access was 78% (native fistula; 80%, PTFE graft; 71%) after 12 months and 70% (native fistula; 73%, PTFE graft; 51%) after 24 months. Older age, a PTFE graft, r-HuEPO therapy, higher hematocrit, lower albumin and higher fibrinogen levels were significantly associated with vascular access failure, whereas gender, diabetes mellitus, total cholesterol and platelet count were not. Plasma fibrinogen was inversely correlated with albumin (r=-0.38, P=0.001). The cumulative vascular access survival was significantly lower in patients with high plasma fibrinogen levels (> or = 460 mg/dl) compared with patients with low levels (< 460 mg/dl) (P=0.007). Independent risk factors for vascular access failure analysed by Cox's proportional hazards model were older age (RR; 1.36 by 10-year increment), higher fibrinogen level (RR; 1.20 by 100 mg/dl increment), PTFE graft (RR; 2.28) and r-HuEPO therapy (RR; 3.79). CONCLUSION: High plasma fibrinogen level is an independent risk factor for vascular access failure in haemodialysis patients.  (+info)

(5/3892) Advances in the biological therapy and gene therapy of malignant disease.

Biological and gene therapy of cancer have become important components of clinical cancer research. Advances in this area are based on evidence for the presence of tumor antigens, antitumor immune responses, evasion of host control by tumors, and the recognition of host defense failure in cancer patients. These mechanisms are being corrected or exploited in the development of biological and gene therapy. Over the last decade, 9 biological therapies have received Food and Drug Administration approval, and another 12 appear promising and will likely be approved in the next few years. Our approach to gene therapy has been to allogenize tumors by the direct intratumoral injection of HLA-B7/beta2-microglobulin genes as plasmid DNA in a cationic lipid into patients with malignant melanoma. In four Phase I studies, we found a 36% response by the local injected tumor and a 19% systemic antitumor response. In other cancers, gene transfer, expression, and an intratumoral T-cell response were seen, but no clinical response was seen. A variety of follow-up studies with HLA-B7 and other genes are planned. Evasion of host control is now a major target of gene therapy. Strategies to overcome this include up-regulation of MHC and introduction of cell adhesion molecules into tumor cells, suppression of transforming growth factor and interleukin 10 production by tumor cells, and blockade of the fas ligand-fas interaction between tumor cells and attacking lymphocytes. With these approaches, it seems likely that gene therapy may become the fifth major modality of cancer treatment in the next decade.  (+info)

(6/3892) Identification of the poly(C) binding protein in the complex associated with the 3' untranslated region of erythropoietin messenger RNA.

Hypoxia regulates expression of erythropoietin (EPO), a glycoprotein that stimulates erythrocytosis, at the level of transcription and also possibly at the level of messenger RNA (mRNA) stability. A pyrimidine-rich region within the EPO mRNA 3' untranslated region was implicated in regulation of EPO mRNA stability element and shown to bind protein factors. In the present study we wished to identify the protein factor binding to the pyrimidine-rich sequence in the EPO mRNA stability element. Using mobility shift assays, ultraviolet light cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroelution of protein factors from the gel slices corresponding to the ribonucleoprotein complexes, we found that two isoforms of a 40 kD poly(C) binding protein (PCBP, also known as alphaCP or hnRNPE), PCBP1, and PCBP2 are present in that complex. In Hep3B or HepG2 cells hypoxia induces neither expression of PCBP nor formation of the ribonucleoprotein complex associated with EPO mRNA that involves PCBP.  (+info)

(7/3892) Erythropoietin depresses nitric oxide synthase expression by human endothelial cells.

We have recently shown that erythropoietin (EPO)-induced hypertension is unrelated to the rise in hematocrit and is marked by elevated cytosolic [Ca+2] and nitric oxide (NO) resistance. The present study was done to determine the effect of EPO on NO production and endothelial NO synthase (eNOS) expression by endothelial cells. Human coronary artery endothelial cells were cultured to subconfluence and then were incubated for 24 hours in the presence of either EPO (0, 5, and 20 U/mL) alone or EPO plus the calcium channel blocker felodipine. The experiments were carried out with quiescent (0.5% FCS) and proliferating (5% FCS) cells. Total nitrate and nitrite, eNOS protein, DNA synthesis (thymidine incorporation), and cell proliferation (cell count) were determined. In addition, NO production in response to acetylcholine stimulation was tested. EPO resulted in a dose-dependent inhibition of basal and acetylcholine-stimulated NO production and eNOS protein expression and also led to a significant dose-dependent stimulation of DNA synthesis in endothelial cells. The inhibitory effects of EPO on NO production and eNOS expression were reversed by felodipine. Thus, EPO downregulates basal and acetylcholine-stimulated NO production, depresses eNOS expression, and stimulates proliferation in isolated human endothelial cells. The suppressive effects of EPO on NO production and on eNOS expression are reversed by calcium channel blockade.  (+info)

(8/3892) Expression of the erythropoietin receptor by trophoblast cellsin the human placenta.

Nonclassical sites of erythropoietin (EPO) and erythropoietin receptor (EPO-R) expression have been described that suggest new physiological roles for this hormone unrelated to erythropoiesis. The recent finding of EPO expression by trophoblast cells in the human placenta prompted us to consider whether these cells also express EPO-R. With use of immunocytochemistry, EPO-R was identified in villous and extravillous cytotrophoblast cells, as well as in the syncytiotrophoblast at all gestational ages. EPO-R was also expressed by cells within the villous core, including endothelial cells of fetoplacental blood vessels. Placental tissues and isolated and immunopurified trophoblast cells, as well as trophoblast-derived choriocarcinoma Jar cells, expressed immunoreactive EPO-R on Western blot. EPO-R mRNA was also detected in the same placental tissues and trophoblast cells by nested-primer reverse transcription-polymerase chain reaction. Finally, EPO-R was functional insofar as the receptor was phosphorylated on tyrosine residues in response to exogenous EPO treatment of cultured trophoblast or Jar cells. Thus, the present findings support the hypothesis that trophoblast cells of the human placenta express EPO-R. In view of these results, taken together with previous work demonstrating EPO expression by the same cells, an autocrine role for this hormone in the survival, proliferation, or differentiation of placental trophoblast cells is proposed.  (+info)