Promises and pitfalls in erythopoietin-mediated tissue protection: are nonerythropoietic derivatives a way forward?

Abstract

The essential biological role of erythropoietin (EPO) in maintaining erythrocyte mass has been well understood for many years. Although EPO is required for the maturation of red cells, it also has strong procoagulant effects on the vascular endothelium and platelets, which limit erythrocyte losses after hemorrhage. Like other members of the type 1 cytokine superfamily, EPO has multiple biological activities. For the past 10 years, multiple investigators have shown that EPO acts as a locally produced antagonist of proinflammatory cytokines that are generated by the innate immune response in response to infection, trauma, or metabolic stress. Specifically, EPO inhibits apoptosis of cells surrounding a locus of injury, reduces the influx of inflammatory cells, and recruits tissue-specific stem cells and endothelial progenitor cells. Available evidence suggests that these multiple, nonerythropoietic effects of EPO are mediated by a tissue protective receptor (TPR) that is distinct from the homodimeric receptor responsible for erythropoiesis. Notably, activation of the TPR requires a higher concentration of EPO than is needed for maximal erythropoiesis. Unfortunately, these higher concentrations of EPO also stimulate hematopoietic and procoagulant pathways, which can cause adverse effects and, therefore, potentially limit the clinical use of EPO for tissue protection. To circumvent these problems, the EPO molecule has been successfully modified in a variety of ways to interact only with the TPR. Early clinical experience has shown that these compounds appear to be safe, and proof of concept trials are ready to begin.

FIGURE 1

Erythropoietin signals via 2 distinct receptor isoforms. The hematopoietic receptor is a high affinity homodimer that mediates hematological and vascular effects. In contrast, current evidence suggests that the TPR is a lower affinity heteromer composed of EPOR and βCR (CD131).

FIGURE 2

Erythropoietin binds to (EPOR)₂ by bridging across an assembled homodimer. Helices A–D of EPO associate via hydrophobic interactions to form a compact, globular configuration. Sites 1 and 2 (indicated by dashed boxes) within the topography of the EPO molecule bind with high affinity to localized regions on each EPOR monomer. The aqueous face of the helix B is oriented away from the interior of the receptor, indicated by the dashed ellipse. Reprinted with permission from Proc Natl Acad Sci U S A.

FIGURE 3

The nonhematopoietic derivative carbamoylated EPO protects cardiomyocytes from staurosporine-induced apoptosis in vitro. Mean percentage of myocytes having undergone apoptosis after isolation from rat or mouse hearts. Shown are data for rat control (n = 9), S (Staurosporine, n = 10), rat S plus C (S plus CEPO, n = 10), mouse S plus C (n = 4), and mouse S plus E (S plus EPO; n = 4). *, P < 0.05; **, P < 0.01 versus staurosporine. Reprinted with permission from Proc Natl Acad Sci U S A.

FIGURE 4

βCR knockout mice subjected to spinal cord compressive injury do not respond to either rhEPO or CEPO. Normal or CD131 knockout male mice of 8 to 16 weeks of age received a moderate compressive lesion of the spinal cord, followed immediately by a single intraperitoneal dose of rhEPO or CEPO (10 μg/kg of body weight: the equivalent of 1000 IU of EPO) and were subsequently evaluated for motor function for 6 weeks. Mortality was similar between groups (≈10%–20%). Wild-type mice responded to CEPO with a complete recovery within 4 weeks. In contrast, βcR knockout animals exhibited little recovery in motor function among the CEPO, rhEPO, or saline groups after 6 weeks. Reprinted with permission from Proc Natl Acad Sci U S A.

FIGURE 5

ARA 290 was synthesized based on the linear sequence of helix B indicated by the dashed ellipse in Figure 2 (boxed region; single letter amino acid code; U: pyroglutamate). Circled residues show those amino acid residues on the aqueous face of helix B and a linear peptide comprising only these residues was manufactured. Reprinted with permission from Proc Natl Acad Sci U S A.

FIGURE 6

ARA 290 improves renal ischemia-reperfusion injury. Renal function was assessed by measuring plasma creatinine in mice (n = 12 each group) 24 hours after being subjected to sham operation or renal ischemia–reperfusion injury (bilateral renal pedicle occlusion for 30 min). Phosphate-buffered saline (PBS) or ARA 290 (8.0 nmol/kg of body weight) was administered intraperitoneally 1 minute, 6 hours, and 12 hours into reperfusion. Data represent mean and SEM; ***, P < 0.001 versus PBS. Reprinted with permission from Proc Natl Acad Sci U S A.