ABSTRACT
AG490 is a tyrphostin originally described as a Janus Activated Kinase (JAK) 2 inhibitor. AG490 also inhibits epidermal growth factor receptor (EGFR) and guanylyl cyclases (GC). More recently, AG490 was associated with oxidative stress protection in experimental acute kidney injury models. We now show that AG490 is also a strong activator of the Hypoxia Inducible Factor (HIF)-1. Under normoxic conditions HIF-1α is degraded through hydroxylation, von Hippel Lindau protein (VHL)-mediated ubiquitin tagging and proteasomal degradation. AG490 increased HIF-1α protein, but not HIF-1α mRNA levels, dose- and time-dependently in cultured endothelial, vascular smooth muscle and kidney proximal tubular epithelial cells. AG490 increased HIF-1α protein half-life, suggesting that HIF-1α protein accumulation resulted from a decreased degradation. In this regard, AG490 prevented HIF-1α hydroxylation and increased HIF-1α protein levels in human renal carcinoma cells expressing VHL, but did not further increase HIF-1α in VHL negative cells. AG490 did not prevent the proteasomal degradation of other proteins. HIF-1α was not upregulated by dominant negative JAK2constructs, tyrphostin AG9, the EGFR inhibitors erbstatin and genistein, the GC inhibitor Ly83583 or cGMP analogues. Finally, AG490 also increased HIF-1α transcriptional activity evidenced by the increased HIF-1α-dependent VEGF expression. In conclusion, AG490 is a novel HIF-1α activator that increases HIF-1α half-life and protein levels through interference with HIF-1α hydroxylation and VHL-mediated degradation. This action may contribute to the cell and tissue protective effects of AG490.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Tyrphostins/pharmacology , Animals , Cattle , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Guanylate Cyclase/antagonists & inhibitors , Guanylate Cyclase/metabolism , Humans , Hydroxylation/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/metabolism , Proteasome Endopeptidase Complex/metabolism , Swine , Transcription, Genetic/drug effects , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
UNLABELLED: The sudden interruption of recombinant human erythropoietin (rHuEPO) in end-stage renal disease (ESRD) patients leads to rapid anemization. The mechanisms of this phenomenon are, however, insufficiently understood. The present study examined the response to immediate rHuEPO withdrawal in dialysis patients. METHODS: 10 chronic hemodialysis (HD) patients regularly receiving rHuEPO were studied. rHuEPO was stopped and reinitiated after 7 days. Reticulocyte profile, haemoglobin and haematocrit were measured at 0, 7 and 15 days. As a complementary study, and with the purpose of analyzying whether uremia was a relevant factor, 10 non-uremic male Wistar rats were treated with rHuEPO. After two weeks, rHuEPO was withdrawn in 5 animals, and continued for 7 additional days in the remainder. The same variables than in the human study were determined. RESULTS: Changes in reticulocyte subtypes from baseline to day 7 were: total 18.2 +/- 0.9 vs 14.3 +/- 1.8% (p < 0.06); high-fluorescence (HFR): 2.6 +/- 0.4 vs 0.75 +/- 0.2 (p < 0.001); medium-fluorescence (MFR): 13.0 +/- 1.1 vs 6.6 +/- 0.9% (p < 0.02); and low-fluorescence (LFR): 84.2 +/- 1.4 vs 92.7 +/- 1% (p NS). The baseline pattern was recovered upon 7 days of rHuEPO reinitiation (p NS). Mean hemoglobin and hematocrit decreased by day 14 (p < 0.02) in spite of rHuEPO reinitiation at day 7. In non-uremic rats, changes were similar to that in the ESRD patients. CONCLUSION: rHuEPO induces changes in the reticulocyte pattern, consisting in a reduction of immature reticulocytes. These changes appear to be independent of the presence of uremia. Accordingly, complete rHuEPO withdrawal in HD patients will cause a rapidly-developing anaemia due to an alteration in the reticulocyte maturation series; therefore, sudden rHuEPO interruption should be avoided whenever is possible. As a collateral application, the specific changes described herein have potential use for detecting illegal administration of rHuEPO.
Subject(s)
Anemia/drug therapy , Erythropoietin/administration & dosage , Kidney Failure, Chronic/blood , Renal Dialysis , Reticulocyte Count , Aged , Anemia/etiology , Animals , Doping in Sports , Drug Administration Schedule , Epoetin Alfa , Erythropoiesis/drug effects , Erythropoietin/pharmacology , Erythropoietin/therapeutic use , Female , Hematocrit , Humans , Kidney Failure, Chronic/complications , Kidney Failure, Chronic/therapy , Male , Middle Aged , Rats , Rats, Wistar , Recombinant Proteins , Recurrence , Uremia/blood , Uremia/complications , Uremia/therapyABSTRACT
La retirada brusca del tratamiento con eritropoyetina recombinante humana (rHuEPO) en pacientes con insuficiencia renal crónica provoca la reaparición de anemia. Sin embargo, los mecanismos de este fenómeno no se conocen suficientemente. El presente estudio examina la respuesta a la retirada brusca de rHuEPO en pacientes en hemodiálisis crónica (HD).El estudio se realizó en 10 pacientes en HD que recibían habitualmente rHuEPO. La administración de rHuEPO se interrumpió durante 7 días, tras los cuales se reinició. A los 0, 7 y 15 días, se midieron hemoglobina, hematocrito y el patrón de maduración de los reticulocitos. Con el fin de determinar el papel específico de la uremia en los fenómenos observados, se realizó un estudio complementario en 10 ratas Wistar tratadas con rHuEPO. Tras dos semanas con rHuEPO, se retiró el tratamiento a la mitad de los animales mientras que se mantuvo en el resto otra semana más Se determinaron hemoglobina, hematocrito y patrón de maduración de reticulocitos. Los cambios observados en los distintos subtipos reticulocíticos entre el día 0 y el día 7 fueron los siguientes: reticulocitos totales: 18,2 ñ 0,9 vs 14,3 ñ 1,2 por ciento (p < 0,05); alta fluorescencia (HFR): 2,6 ñ 0,4 vs 0,75 ñ 0,2 por ciento (p < 0,001); fluorescencia media (MFR): 13,0 ñ 1,1 vs 6,6 ñ 0,9 por ciento (p < 0,02); y baja fluorescencia (LFR): 84,2 ñ 1,4 vs 92,7 ñ 1 por ciento (p NS). El nivel basal (día 0) se recuperó a los 7 días de comenzar de nuevo la administración de rHuEPO (p NS). Los valores de hemoglobina y hematocrito disminuyeron al final del estudio (día 14) a pesar de reiniciarse la rHuEPO el día 7 (p < 0,02). Cambios similares se observaron en las ratas no urémicas. La rHuEPO induce cambios relevantes en el patrón reticulocítico, provocando una reducción de las formas inmaduras. Este cambio es independiente del estado urémico del individuo y sugiere la conveniencia de no efectuar una retirada brusca del tratamiento crónico con rHuEPO (AU)
Subject(s)
Male , Middle Aged , Humans , Rats , Female , Animals , Aged , Renal Dialysis , Reticulocyte Count , Epoetin Alfa , Rats, Wistar , Hematocrit , Renal Insufficiency, Chronic , Anemia , Doping in Sports , Drug Administration Schedule , Erythropoiesis , Uremia , Recurrence , UremiaABSTRACT
Proangiogenic, proliferative effects of tumors have been extensively characterized in subconfluent endothelial cells (EC), but results in confluent, contact-inhibited EC are critically lacking. The present study examined the effect of tumor-conditioned medium (CM) of the malignant osteoblastic cell line MG63 on monolayer, quiescent bovine aorta EC. MG63-CM and MG63-CM + CoCl(2) significantly increased EC survival in serum-starved conditions, without inducing EC proliferation. Furthermore, MG63-CM and MG63-CM + CoCl(2), both containing high amounts of vascular endothelial growth factor (VEGF), induced relevant phenotypic changes in EC (all P < 0.01) involving increase of nucleoli/chromatin condensations, nucleus-to-cytosol ratio, capillary-like vacuolated structures, vessel-like acellular areas, migration through Matrigel, growth advantage in reseeding, and factor VIII content. All these actions were significantly inhibited by VEGF and VEGF receptor (VEGFR2) blockade. Of particular importance, a set of similar effects were detected in a human microvascular endothelial cell line (HMEC). With regard to gene expression, incubation with MG63-CM abolished endogenous VEGF mRNA and protein but induced a clear-cut increase in VEGFR2 mRNA expression in EC. In terms of mechanism, MG63-CM activates protein kinase B (PKB)/Akt, p44/p42-mitogen-activated protein kinase (MAPK)-mediated pathways, as suggested by both inhibition and phosphorylation experiments. In conclusion, tumor cells activate confluent, quiescent EC, promoting survival, phenotypic, and gene expression changes. Of importance, VEGF antagonism converts MG63-CM from protective to EC-damaging effects.
