Erythropoietin is involved in angiogenesis in human primary melanoma

In this study, the extent of angiogenesis, evaluated as microvascular volume density, immunoreactivity of tumour cells to erythropoietin (Epo) and of endothelial cells to Epo receptor (EpoR) have been correlated in human primary melanoma specimens. Results showed that Epo/EpoR expression correlate with angiogenesis and tumour thickness. These findings suggest that Epo is secreted by tumour cells and it affects vascular endothelial cells via its receptor and promotes angiogenesis in a paracrine manner, playing an important role in melanoma angiogenesis.     

The role of erythropoietin and erythropoietin receptor in malignant laryngeal tumors

Erythropoietin (Epo) is a glycoprotein hormone responsible for erythropoiesis. Its effect is realized by binding erythropoietin receptor (EpoR) expressed on erythroid progenitor cells. Hypoxia is the main stimulus for the secretion of erythropoietin. Anemia is an independent negative prognostic factor for survival in patients with malignant diseases. Synthetic forms of erythropoietin are used in clinical oncology practice to increase the level of hemoglobin. As well as endogenous they can bind to EpoR. Considering the fact that most effects of synthetic Epo are negative, the role of endogenous Epo/EpoR has become an extremely important issue. The authors do not agree on most items related to the effects of exogenous Epo and EpoR in patients with head and neck carcinomas. We are investigating the expression of Epo/EpoR in the tissue of malignant laryngeal carcinoma. Our hypothesis is that less differentiated laryngeal carcinomas will have a higher level of endogenous Epo/EpoR expression. Therefore, in patients with positive Epo/EpoR we expect shorter survival and poorer locoregional disease control. We anticipate that our hypothesis may help to provide the role of endogenous Epo/EpoR in patients with malignant tumors of the larynx. If the assumptions of this study are confirmed, the patients with laryngeal carcinomas whose tumor cells express Epo/EpoR should not be considered for the treatment of anemia with recombinant erythropoietin in any case. We also point out that our research will expand the knowledge of the biology of laryngeal tumor cells and that the results could be utilized as basic knowledge in development of future therapeutic strategies.     

Soluble erythropoietin receptor is present in the mouse brain and is required for the ventilatory acclimatization to hypoxia

While erythropoietin (Epo) and its receptor (EpoR) have been widely investigated in brain, the expression and function of the soluble Epo receptor (sEpoR) remain unknown. Here we demonstrate that sEpoR, a negative regulator of Epo's binding to the EpoR, is present in the mouse brain and is down-regulated by 62% after exposure to normobaric chronic hypoxia (10% O₂ for 3 days). Furthermore, while normoxic minute ventilation increased by 58% in control mice following hypoxic acclimatization, sEpoR infusion in brain during the hypoxic challenge efficiently reduced brain Epo concentration and abolished the ventilatory acclimatization to hypoxia (VAH). These observations imply that hypoxic downregulation of sEpoR is required for adequate ventilatory acclimatization to hypoxia, thereby underlying the function of Epo as a key factor regulating oxygen delivery not only by its classical activity on red blood cell production, but also by regulating ventilation.     

Erythropoietin and erythropoietin receptor in hepatocellular carcinoma: correlation with vasculogenic mimicry and poor prognosis

To evaluate erythropoietin (Epo) and erythropoietin receptor (EpoR) expression, its relationship with vasculogenic mimicry (VM) and its prognostic value in human hepatocellular carcinoma (HCC), we examined Epo/EpoR expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-Schiff) double staining on 92 HCC specimens. The correlation between Epo/EpoR expression and VM formation was analyzed using two-tailed Chi-square test and Spearman correlation analysis. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between Epo/EpoR expression and VM formation (P < 0.05). Patients with Epo or EpoR expression exhibited poorer overall survival (OS) than Epo-negative or EpoR-negative patients (P < 0.05). Epo-positive/VM-positive and EpoR-positive/VM-positive patients had the worst OS (P < 0.05). In multivariate survival analysis, age, Epo and EpoR were independent prognostic factors related to OS. These results will provide evidence for further research on HCC microcirculation patterns and also will provide new possible targets for HCC diagnosis and treatment.     

Erythropoietin regulates hypoxic ventilation in mice by interacting with brainstem and carotid bodies

Apart from its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions in brain, retina and heart upon ischaemic injury. However, the physiological non-erythroid functions of Epo remain unclear. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate Epo's impact on ventilation upon hypoxic exposure. Tg21 mice showed improved ventilatory response to severe acute hypoxia and moreover improved ventilatory acclimatization to chronic hypoxic exposure. Furthermore, following bilateral transection of carotid sinus nerves that uncouples the brain from the carotid body, Tg21 mice adapted their ventilation to acute severe hypoxia while chemodenervated wild-type (WT) animals developed a life-threatening apnoea. These results imply that Epo in brain modulates ventilation. Additional analysis revealed that the Epo receptor (EpoR) is expressed in the main brainstem respiratory centres and suggested that Epo stimulates breathing control by alteration of catecholaminergic metabolism in brainstem. The modulation of hypoxic pattern of ventilation after i.v. injection of recombinant human Epo in WT mice and the dense EpoR immunosignal observed in carotid bodies showed that these chemoreceptors are sensitive to plasma levels of Epo. In summary, our results suggest that Epo controls ventilation at the central (brainstem) and peripheral (carotid body) levels. These novel findings are relevant to understanding better respiratory disorders including those occurring at high altitude.     

Erythropoietin/erythropoietin-receptor system is involved in angiogenesis in human hepatocellular carcinoma

AIMS: To correlate microvascular density and erythropoietin (Epo)/Epo-receptor (EpoR) expression in endothelial and tumour cells with histopathological type in hepatocellular carcinoma (HCC). Methods AND RESULTS: Specimens of primary HCC obtained from 50 patients who had undergone curative hepatectomy were investigated immunohistochemically by using anti-CD31, anti-Epo and anti-EpoR antibodies. Poorly differentiated HCC had a higher degree of vascularization than other stages and Epo/EpoR expression in both tumour and endothelial cells increased in parallel with grade of malignancy and was highly correlated with the extent of angiogenesis. CONCLUSIONS: Epo/EpoR levels correlate with angiogenesis and progression of patients with HCC and these findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by hepatic tumour cells and it affects vascular endothelial cells via its receptors and promotes angiogenesis in a paracrine manner. It is thus suggested that Epo is an important factor in hepatic tumour angiogenesis. Understanding the mechanisms of HCC angiogenesis provides a basis for a rational approach to the development of antiangiogenic therapy in patients with hepatic cancer.     

Erythropoietin/erythropoietin receptor system is involved in angiogenesis in human neuroblastoma

AIMS: Previous studies have shown that increased vascularity is associated with tumour progression in human neuroblastoma (NB). The involvement of erythropoietin (Epo) in tumour angiogenesis has also been reported. The aim of this study was to correlate microvascular density and Epo/Epo-receptor (EpoR) expression in endothelial and tumour cells to the clinical stage of NB. METHODS AND RESULTS: Specimens of NB obtained from 20 patients were investigated immunohistochemically by using anti-CD31, anti-Epo and anti-EpoR antibodies. The extent of angiogenesis was found to be up-regulated in advanced disease. In keeping with this observation, Epo/EpoR expression in tumour and endothelial cells, respectively, was also highly correlated with the extent of angiogenesis and higher clinical stage. CONCLUSIONS: The correlation of Epo/EpoR expression with angiogenesis and tumour progression suggests the presence of a loop in the Epo-EpoR system. Epo is secreted by tumour cells and affects vascular endothelial cells via its receptor, promoting tumour angiogenesis in a paracrine manner. Data suggest that Epo represents an important mediator in NB angiogenesis. Understanding the mechanisms of NB angiogenesis provides the basis for a rational approach to the development of antiangiogenic therapy in patients affected by NB.     

促红细胞生成素在斑马鱼胚胎发育中抑制肾脏细胞凋亡

目的:研究促红细胞生成素(Epo)在斑马鱼早期胚胎肾脏细胞发育中的凋亡和坏死方面起到的作用。方法:使用吗啉反义寡核苷酸技术通过胚胎注射使Epo及其受体(EpoR)的表达下调,并用real-time PCR及血红蛋白染色进行基因下调的有效性验证。在胚胎受精48 h观察肾脏形态并进行TUNEL凋亡染色,使用流式细胞术分析Annexin V凋亡染色,并用Western blot进行Akt磷酸化验证。结果:Epo及EpoR基因表达下调的斑马鱼胚胎在受精48 h可见明显肾小球囊样改变,且肾小管颈部缩短消失。TUNEL及Annexin V染色结果提示受精48 h后,Epo及EpoR基因表达下调的斑马鱼胚胎中肾脏凋亡细胞增多,但EpoR基因下调的斑马鱼胚胎内主要表现为晚期凋亡细胞及坏死细胞增多。Western blot实验结果提示Epo及EpoR基因表达下调的斑马鱼胚胎较对照组Akt磷酸化减少(P0.05)。结论:促红细胞生成素可引起Akt磷酸化。促红细胞生成素通过保护肾脏细胞免于凋亡和坏死而在斑马鱼胚胎肾脏发育中发挥重要作用。     

人参总皂苷诱导红系血细胞增殖的信号转导研究

目的研究人参总皂苷(TSPG)刺激红系血细胞增殖有关的细胞内信号转导途径。方法用集落形成实验检测TSPG刺激红系造血祖细胞(BFU-E、CFU-E)增殖的能力;噻唑蓝法(MTT)检测红细胞生成素(Epo)对脐血细胞增殖的影响;Western blotting法检测TSPG刺激脐血细胞24 h后EpoR表达的变化;免疫沉淀法检测TSPG对EpoR介导的蛋白酪氨酸激酶JAK2和信号传导及转录激活因子5(STAT5)的酪氨酸磷酸化的影响。结果1.TSPG10~75 mg/L均能提高脐血细胞形成BFU-E、CFU-E的集落产率,以25 mg/L提高幅度最明显;2.以MTT法测定得出Epo 2×103~5×104IU/L均能促进脐血细胞的增殖,以5×103IU/L促进增殖效果最明显;3.在不同剂量的TSPG刺激下,造血细胞EpoR的表达变化不明显;4.TSPG作用造血细胞24 h后,加入Epo继续刺激0、2、5、30 min,JAK2和STAT5的酪氨酸磷酸化均增强。结论TSPG在促进造血细胞增殖的过程中,对造血细胞EpoR表达的影响不显著,但可能在EpoR介导的信号转导过程中增强JAK2和STAT5的酪氨酸磷酸化发挥重要作用。     

Increased expression of erythropoietin receptor in human pterygial tissues

Erythropoietin (Epo) induces physiological activities such as cell proliferation, migration, and angiogenesis in Epo receptor (EpoR)-expressing vascular endothelial and tumor cells. Recently, it has been demonstrated that growth factor-independent proliferation is frequently observed during the cell transformation process. Pterygium is a fibrovascular proliferating tissue that includes transformed cells. The aim of this study was to examine the localization of Epo and EpoR proteins in human pterygial tissues. Eleven samples including nine pterygia and two normal bulbar conjunctivas, which were surgically excised, were studied. Formalin-fixed, paraffin-embedded tissue sections were constructed and then were examined by immunohistochemistry with anti-Epo and EpoR antibodies. Cytoplasmic immunoreactivity for EpoR was heterogeneously detected in basal and suprabasal cells of the pterygium epithelium. In the pterygium stroma, a variety of endothelial cells forming vascular cavities showed cytolasmic immunoreactivity for EpoR. In normal conjunctival epithelium, a few basal cells showed a weak homogeneous immunoreactivity for EpoR in the cytoplasm. The number of EpoR-expressing epithelial cells was much higher in the pterygium compared to the normal conjunctiva. EpoR expression was marginally detected in stromal microvessels of the normal conjunctiva. Immunoreactivity for Epo was not noted in pterygium epithelium and stroma, and in normal conjunctiva. These results suggest that the Epo-independent EpoR-signaling pathway plays a potential role in cell proliferation and angiogenesis in human pterygium.     

Identification of a sensitive anti-erythropoietin receptor monoclonal antibody allows detection of low levels of EpoR in cells

Erythropoietin (Epo) binds and activates the Epo receptor (EpoR) on the surface of erythroid progenitor cells resulting in formation of erythrocytes. Recently, EpoR was reported to be expressed on non-erythroid cells suggesting a role for Epo outside of erythropoiesis. However those studies employed antibodies with questionable specificity and the significance of the observations are controversial. In order to accurately determine the expression of EpoR proteins in cells, we have generated a panel of novel anti-human EpoR monoclonal antibodies. One of these antibodies (A82) was particularly sensitive and it detected the EpoR protein on intact cells by flow cytometry and by western blot analysis with cell lysates. Both methods were optimized and using them, EpoR protein was detected by western immunoblotting with lysates from fewer than 200 EpoR positive control cells and the positive signals were proportional to EpoR protein expression level with a minimal signal in EpoR negative cells. The proteins detected by western blot analysis using A82 included full-length EpoR (～ 59 kDa) as well as smaller EpoR fragments derived from the EPOR gene. These results indicate that A82 can be used to examine low level EpoR expression in cells by western and flow cytometry allowing an improved understanding of EpoR expression and metabolism.     

Permanent focal cerebral ischemia activates erythropoietin receptor in the neonatal rat brain

Erythropoietin (Epo) has been shown to act as a neurotrophic and neuroprotective factor via binding to its receptor (EpoR) which is activated in adult brains following hypoxia and ischemia. However, no evidence suggests that cerebral ischemia can activate EpoR in the neonatal brain. In the present study, the changes in EpoR expression were investigated using a modified model of permanent focal cerebral ischemia (FCI) in 7-day-old rat pups. Western blot analysis with an anti-rabbit EpoR antibody revealed a significant increase in the EpoR protein in the ischemic areas, starting from 6 to 12 h after FCI. Moreover, many EpoR-positive cells were detected in the ischemic areas from 12 h after FCI, and the positive cells were identified as neurons and microglia/macrophage but not astrocytes 24 h after FCI. Additionally, double staining with a red in situ apoptosis detection kit and the EpoR antibody indicated that EpoR-positive cells were in apoptotic cell death in the ischemic area. Therefore, these results suggest that EpoR is activated in the ischemic areas of neonatal rats and plays an important role in brain injury during development.     

Snail1 mediates hypoxia-induced melanoma progression

Tumor hypoxia is a known adverse prognostic factor, and the hypoxic dermal microenvironment participates in melanomagenesis. High levels of hypoxia inducible factor (HIF) expression in melanoma cells, particularly HIF-2α, is associated with poor prognosis. The mechanism underlying the effect of hypoxia on melanoma progression, however, is not fully understood. We report evidence that the effects of hypoxia on melanoma cells are mediated through activation of Snail1. Hypoxia increased melanoma cell migration and drug resistance, and these changes were accompanied by increased Snail1 and decreased E-cadherin expression. Snail1 expression was regulated by HIF-2α in melanoma. Snail1 overexpression led to more aggressive tumor phenotypes and features associated with stem-like melanoma cells in vitro and increased metastatic capacity in vivo. In addition, we found that knockdown of endogenous Snail1 reduced melanoma proliferation and migratory capacity. Snail1 knockdown also prevented melanoma metastasis in vivo. In summary, hypoxia up-regulates Snail1 expression and leads to increased metastatic capacity and drug resistance in melanoma cells. Our findings support that the effects of hypoxia on melanoma are mediated through Snail1 gene activation and suggest that Snail1 is a potential therapeutic target for the treatment of melanoma.