The ergogenic effect of recombinant human erythropoietin on VO2max depends on the severity of arterial hypoxemia

Treatment with recombinant human erythropoietin (rhEpo) induces a rise in blood oxygen-carrying capacity (CaO(2)) that unequivocally enhances maximal oxygen uptake (VO(2)max) during exercise in normoxia, but not when exercise is carried out in severe acute hypoxia. This implies that there should be a threshold altitude at which VO(2)max is less dependent on CaO(2). To ascertain which are the mechanisms explaining the interactions between hypoxia, CaO(2) and VO(2)max we measured systemic and leg O(2) transport and utilization during incremental exercise to exhaustion in normoxia and with different degrees of acute hypoxia in eight rhEpo-treated subjects. Following prolonged rhEpo treatment, the gain in systemic VO(2)max observed in normoxia (6-7%) persisted during mild hypoxia (8% at inspired O(2) fraction (F(I)O(2)) of 0.173) and was even larger during moderate hypoxia (14-17% at F(I)O(2) = 0.153-0.134). When hypoxia was further augmented to F(I)O(2) = 0.115, there was no rhEpo-induced enhancement of systemic VO(2)max or peak leg VO(2). The mechanism highlighted by our data is that besides its strong influence on CaO(2), rhEpo was found to enhance leg VO(2)max in normoxia through a preferential redistribution of cardiac output toward the exercising legs, whereas this advantageous effect disappeared during severe hypoxia, leaving augmented CaO(2) alone insufficient for improving peak leg O(2) delivery and VO(2). Finally, that VO(2)max was largely dependent on CaO(2) during moderate hypoxia but became abruptly CaO(2)-independent by slightly increasing the severity of hypoxia could be an indirect evidence of the appearance of central fatigue.

Impaired renal endothelial nitric oxide synthase and reticulocyte production as modulators of hypertension induced by recombinant human erythropoietin in the rat

INTRODUCTION AND AIMS: Hypertension is a common side effect of recombinant human erythropoietin (rHuEPO) therapy; however, the exact pathways remain to be elucidated. The discovery of non-hematopoietic actions of rHuEPO increased the number of patients that could putatively benefit from this therapy; however, to achieve those effects higher doses are usually needed, which increase the risk and incidence of adverse events. Our aim was to study the effect of a broad range of rHuEPO doses on hematological and biochemical parameters, blood pressure and renal function and damage in the rat, focusing on endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factors (HIFs). METHODS: Male Wistar rats were divided in 5 groups receiving different doses of rHuEPO (100, 200, 400 and 600 IU/kg body weight (BW)/week) and saline solution (control), during 3 weeks. Blood and 24h urine were collected to perform hematological and biochemical analysis. Blood pressure (BP) was measured by the tail-cuff method. The kidney tissue was collected to mRNA and protein expression assays and to characterize renal lesions. RESULTS: A dose-dependent increase in red blood cells count, hematocrit and hemoglobin levels was found with rHuEPO therapy, in rHuEPO200, rHuEPO400 and rHuEPO600 groups. Increased reticulocyte count was found in the rHuEPO400 and rHuEPO600 groups. BP raised in all groups receiving rHuEPO. The rHuEPO200 and rHuEPO600 groups presented increased kidney protein levels of HIF2α and a reduction in kidney protein levels of eNOS, along with the highest grade of vascular and tubular renal lesions. CONCLUSIONS: Our study showed that rHuEPO-induced hypertension might involve indirect (hematological) and direct (renal) effects which varies according to the dose used. Thus, rHuEPO therapy should be performed rationally and under adequate surveillance, as hypertension develops even with lower doses. Especial caution with higher doses should be taken, as rHuEPO-induced hypertension leads to early renal damage without alterations in traditional markers of renal function, thus masking the serious adverse effects and risks.

Effect of recombinant human erythropoietin expressions of apoptosis genes in rats following traumatic brain injury

Purpose: To explore the effect of recombinant human erythropoietin (r-HuEPO) on apoptosis in rats after traumatic brain injury. Methods: A total of 48 traumatic brain-injured Sprague Dawley (SD) rats were obtained by improved Feeney’s traumatic brain injury model, and were randomly divided into four groups: normal salinetreated rats (control) and rats treated with r-HuEPO at doses of 1000 U/kg, 3000 U/kg and 5000 U/kg. Brain tissues were collected on the 7th day after trauma surgery. Apoptotic cells, and NF-kappa B (NFĸB)-, c-myc-, and Fas/Fasl-positive cells were identified in brain tissues by immunohistochemical assay. Results: After treatment with r-HuEPO (3000 and 5000 U/kg), expression of NF-κB and Fas/Fasl were significantly decreased (p < 0.05) compared to control rats, especially at the 5000 U/kg dose (p < 0.01). However, for c-myc, no significant difference was observed between r-HuEPO treatment and control groups (p > 0.05). Compared to the 1000 U/kg r-HuEPO group, Fas/Fasl expression levels were significantly lower in the 3000 and 5000 U/kg r-HuEPO groups (p < 0.05). Additionally, expression of NF-κB and Fasl in the 5000 U/kg r-HuEPO group was significantly lower than that in the 3000 U/kg r- HuEPO group (p < 0.05). Moreover, the number of apoptotic cells in the r-HuEPO group (5000 U/kg) was significantly lower than in the control group (p < 0.05). Conclusion: Thus, r-HuEPO may be beneficial for treating traumatic brain injury via inhibition of NFkappa B and Fas/Fasl expressions.

Does recombinant human Epo increase exercise capacity by means other than augmenting oxygen transport?

[EN] This study was performed to test the hypothesis that administration of recombinant human erythropoietin (rHuEpo) in humans increases maximal oxygen consumption by augmenting the maximal oxygen carrying capacity of blood. Systemic and leg oxygen delivery and oxygen uptake were studied during exercise in eight subjects before and after 13 wk of rHuEpo treatment and after isovolemic hemodilution to the same hemoglobin concentration observed before the start of rHuEpo administration. At peak exercise, leg oxygen delivery was increased from 1,777.0+/-102.0 ml/min before rHuEpo treatment to 2,079.8+/-120.7 ml/min after treatment. After hemodilution, oxygen delivery was decreased to the pretreatment value (1,710.3+/-138.1 ml/min). Fractional leg arterial oxygen extraction was unaffected at maximal exercise; hence, maximal leg oxygen uptake increased from 1,511.0+/-130.1 ml/min before treatment to 1,793.0+/-148.7 ml/min with rHuEpo and decreased after hemodilution to 1,428.0+/-111.6 ml/min. Pulmonary oxygen uptake at peak exercise increased from 3,950.0+/-160.7 before administration to 4,254.5+/-178.4 ml/min with rHuEpo and decreased to 4,059.0+/-161.1 ml/min with hemodilution (P=0.22, compared with values before rHuEpo treatment). Blood buffer capacity remained unaffected by rHuEpo treatment and hemodilution. The augmented hematocrit did not compromise peak cardiac output. In summary, in healthy humans, rHuEpo increases maximal oxygen consumption due to augmented systemic and muscular peak oxygen delivery.

Selective in vitro glycosylation of recombinant proteins:semi-synthesis of novel homogeneous glycoforms of human erythropoietin

Background: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure–function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins

Polycaprolactone-based scaffold plus recombinant human bone morphogenetic protein (rhBMP-2) in an ovine model of anterior spinal fusion

Synthetic scaffolds combined with growth factors have the potential to replace allograft or autograft as a graft material for spinal interbody fusion. Such tissue engineering approaches may be useful in Adolescent Idiopathic Scoliosis (AIS) surgery, however there are no studies to date examining the use of such biodegradable implants in combination with biologics in a thoracic spine model. This in vivo study examines the use of biodegradable polycaprolactone (PCL) based scaffolds with rhBMP-2 as a bone graft substitute in a sheep thoracic fusion model, where an anterior approach is used to simulate minimally invasive surgical deformity correction in the setting of AIS.

Polycaprolactone-based scaffold plus recombinant human bone morphogenic protein rhBMP-2) in a sheep thoracic spine fusion model

Adolescent idiopathic scoliosis is a complex three dimensional deformity affecting 2-3% of the general population. The resulting spinal deformity consists of coronal curvature, hypokyphosis of the thoracic spine and vertebral rotation in the axial plane with posterior elements turned into the curve concavity. The potential for curve progression is heightened during the adolescent growth spurt. Success of scoliosis deformity correction depends on solid bony fusion between adjacent vertebrae after the intervertebral (IV) discs have been surgically cleared and the disc spaces filled with graft material. Recently a bioactive and resorbable scaffold fabricated from medical grade polycaprolactone has been developed for bone regeneration at load bearing sites. Combined with rhBMP-2, this has been shown to be successful in acting as a bone graft substitute in a porcine lumbar interbody fusion model when compared to autologous bone graft alone. The study aimed to establish a large animal thoracic spine interbody fusion model, develop spine biodegradable scaffolds (PCL) in combination with biologics (rhBMP-2) and to establish a platform for research into spine tissue engineering constructs. Preliminary results demonstrate higher grades of radiologically evident bony fusion across all levels when comparing fusion scores between the 3 and 6 month postop groups at the PCL CaP coated scaffold level, which is observed to be a similar grade to autograft, while no fusion is seen at the scaffold only level. Results to date suggest that the combination of rhBMP-2 and scaffold engineering actively promotes bone formation, laying the basis of a viable tissue engineered constructs.

Biological performance of a polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in an ovine thoracic interbody fusion model

Introduction. We develop a sheep thoracic spine interbody fusion model to study the suitability of polycaprolactone-based scaffold and recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within the thoracic spine. The surgical approach is a mini- open thoracotomy with relevance to minimally invasive deformity correction surgery for adolescent idiopathic scoliosis. To date there are no studies examining the use of this biodegradable implant in combination with biologics in a sheep thoracic spine model. Methods. In the present study, six sheep underwent a 3-level (T6/7, T8/9 and T10/11) discectomy with randomly allocated implantation of a different graft substitute at each of the three levels; (i) calcium phosphate (CaP) coated polycaprolactone based scaffold plus 0.54µg rhBMP-2, (ii) CaP coated PCL- based scaffold alone or (iii) autograft (mulched rib head). Fusion was assessed at six months post-surgery. Results. Computed Tomographic scanning demonstrated higher fusion grades in the rhBMP-2 plus PCL- based scaffold group in comparison to either PCL-based scaffold alone or autograft. These results were supported by histological evaluations of the respective groups. Biomechanical testing revealed significantly higher stiffness for the rhBMP-2 plus PCL- based scaffold group in all loading directions in comparison to the other two groups. Conclusions. The results of this study demonstrate that rhBMP-2 plus PCL-based scaffold is a viable bone graft substitute, providing an optimal environment for thoracic interbody spinal fusion in a large animal model.

Characterisation of Polycaprolatone-Based Scaffold Plus Recombinant Human Morphogenetic Protein - 2 (RHBMP-2) in an Ovine Thoracic Spine Model

This thesis represents a step forward in the development of a pre-clinical model investigating a suitable substitute for host bone for use in human spinal fusion. By way of an animal model, it examines the biological performance of a novel bone graft substitute comprised of a combination of a custom-designed biodegradable material and biologics.

Ability of recombinant human bone morphogenetic protein 2 to enhance bone healing in the presence of tobramycin : evaluation in a rat segmental defect model

Objective To determine whether locally applied tobramycin influences the ability of recombinant human bone morphogenetic protein 2 (rhBMP-2) to heal a segmental defect in the rat femur. Methods The influence of tobramycin on the osteogenic differentiation of mesenchymal stem cells was first evaluated in vitro. For the subsequent, in vivo experiments, a 5-mm segmental defect was created in the right femur of each of 25 Sprague-Dawley rats and stabilized with an external fixator and four Kirschner wires. Rats were divided in four groups: empty control, tobramycin (11 mg)/absorbable collagen sponge, rhBMP-2 (11 μg)/absorbable collagen sponge, and rhBMP-2/absorbable collagen sponge with tobramycin. Bone healing was monitored by radiography at 3 and 8 weeks. Animals were euthanized at 8 weeks and the properties of the defect were compared with the intact contralateral femur. Bone formation in the defect region was assessed by dual-energy x-ray absorptiometry, microcomputed tomography, histology, and mechanical testing. Results Tobramycin exerted a dose-dependent inhibition of alkaline phosphatase induction and calcium deposition by mesenchymal stem cells cultured under osteogenic conditions. The inhibition was reversed in the presence of 500 ng/mL of rhBMP-2. Segmental defects in the rat femora failed to heal in the absence of rhBMP-2. Tobramycin exerted no inhibitory effects on the ability of rhBMP-2 to heal these defects and increased the bone area of the defects treated with rhBMP-2. Data obtained from all other parameters of healing, including dual-energy x-ray absorptiometry, microcomputed tomography, histology, and mechanical testing, were unaffected by tobramycin. Conclusions Although our in vitro results suggested that tobramycin inhibits the osteogenic differentiation of mesenchymal stem cells, this could be overcome by rhBMP-2. Tobramycin did not impair the ability of rhBMP-2 to heal critical-sized femoral defects in rats. Indeed, bone area was increased by nearly 20% in the rhBMP-2 group treated with tobramycin. This study shows that locally applied tobramycin can be used in conjunction with rhBMP-2 to enhance bone formation at fracture sites.