Interplay between endothelin and erythropoietin in astroglia: the role in protection against hypoxia

Abstract

We show that, under in vitro conditions, the vulnerability of astroglia to hypoxia is reflected by alterations in endothelin (ET)-1 release and capacity of erythropoietin (EPO) to regulate ET-1 levels. Exposure of cells to 24 h hypoxia did not induce changes in ET-1 release, while 48-72 h hypoxia resulted in increase of ET-1 release from astrocytes that could be abolished by EPO. The endothelin receptor type A (ETA) antagonist BQ123 increased extracellular levels of ET-1 in human fetal astroglial cell line (SV-FHAS). The survival and proliferation of rat primary astrocytes, neural precursors, and neurons upon hypoxic conditions were increased upon administration of BQ123. Hypoxic injury and aging affected the interaction between the EPO and ET systems. Under hypoxia EPO decreased ET-1 release from astrocytes, while ETA receptor blockade enhanced the expression of EPO mRNA and EPO receptor in culture-aged rat astroglia. The blockade of ETA receptor can increase the availability of ET-1 to the ETB receptor and can potentiate the neuroprotective effects of EPO. Thus, the new therapeutic use of combined administration of EPO and ETA receptor antagonists during hypoxia-associated neurodegenerative disorders of the central nervous system (CNS) can be suggested.

Figure 1.

Expression of EPO mRNA in DIV7 (A) and DIV21 (B) neonatal rat astroglial primary culture treated with BQ123 upon normoxic (N) and hypoxic (H) conditions. The data are shown in percentage to normoxic control (N).

Figure 2.

Quantification of viable human astrocytes and their ET-1 release into the culture medium. (A) ET-1 release from human astrocytes was measured 48 h after incubation with BQ123 under normoxic (N) and hypoxic (H) culture conditions. The data are presented as ratio of ET-1 concentration in treated cells to the mean of normoxic control (N); (B) Quantification of viable cells in human astrocytes culture. Trypan blue-negative viable cells were counted after 24, 48 and 72 h exposure to hypoxia (H 24 h, H 48 h, H 72 h) and compared with the respective normoxic controls (N 24 h, N 48 h, N 72 h).

Figure 3.

Release of ET-1 from human astrocytes (A) 24 h; (B) 48 h; and (C) 72 h after incubation with EPO under normoxic (N) and hypoxic (H) culture conditions.

Figure 4.

Expression of EPOR (green fluorescence) and GFAP (red fluorescence) in DIV7 neonatal rat astroglial primary culture. Cell nuclei are stained with DAPI (blue). (A) Normoxic control; (B) Cultures treated with BQ123 during normoxic conditions; (C) Hypoxic control; and (D) Cells treated with BQ123 during hypoxia. Arrows indicate the EPOR-positive bona fide neural precursors negative for GFAP; Arrowheads demonstrate the EPOR/GFAP-positive astrocytes. Scale bar in A–D 100 μm.

Figure 5.

Expression of EPOR (green fluorescence) and GFAP (red fluorescence) in rat astroglial primary culture. Cell nuclei are stained with DAPI (blue). (A) Normoxic control (DIV21); (B) Cultures treated with BQ123 during normoxic conditions (DIV21); (C) Hypoxic control (DIV21); (D) Cells treated with BQ123 during hypoxia (DIV21). Arrows indicate the EPOR-positive bona fide neural precursors and neurons negative for GFAP. Arrowheads demonstrate either EPOR−/GFAP+ astrocytes (A,B) or those positive for both EPOR and GFAP (C,D). Scale bar in A–D 100 μm ; and (E) Quantification of EPOR-positive cells in astroglial primary cultures (DIV7 and DIV21) normalized to the mean of the total amount of cells per image (n = 3) counted by DAPI. p < 0.05 was considered as significant (* p < 0.05, ** p < 0.01).