Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds

OBJECTIVE: Erythropoietin interacts with vascular endothelial growth factor (VEGF) and stimulates endothelial cell mitosis and motility; thus it may be of importance in the complex phenomenon of wound healing. The purpose of this study was to investigate the effect of recombinant human erythropoietin (rHuEPO) on experimental burn wounds. DESIGN: Randomized experiment. SETTING: Research laboratory. SUBJECTS: C57BL/6 male mice weighing 25-30 g. INTERVENTIONS: Mice were immersed in 80 degrees C water for 10 secs to achieve a deep-dermal second degree burn. Animals were randomized to receive either rHuEPO (400 units/kg/day for 14 days in 100 microL subcutaneously) or its vehicle alone (100 microl/day distilled water for 14 days subcutaneously). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of neoangiogenesis by immunohistochemistry, and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, measurement of VEGF wound content (enzyme-linked immunosorbent assay), expression (Western blot) of endothelial and inducible nitric oxide synthases, and determination of wound nitric oxide (NO) products. MEASUREMENTS AND MAIN RESULTS: rHuEPO increased burn wound reepithelialization and reduced the time to final wound closure. These effects were completely abated by a passive immunization with specific antibodies against erythropoietin. rHuEPO improved healing of burn wound through increased epithelial proliferation, maturation of the extracellular matrix, and angiogenesis. The hematopoietic factor augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31. Furthermore, rHuEPO enhanced the wound content of VEGF caused a marked expression of endothelial and inducible nitric oxide synthases and increased wound content of nitric oxide products. CONCLUSIONS: Our study suggests that rHuEPO may be an effective therapeutic approach to improve clinical outcomes after thermal injury.     

Recombinant human erythropoietin in nephrology

rEPO therapy provides a unique opportunity to correct anemia in end-stage renal failure patients. Complete correction of the anemia, although possible, has some obvious disadvantages over a partial correction with a target hemoglobin of 10-13 g/dl or a hematocrit of 30-35%, respectively. Unresponsiveness to rEPO seems to be rare; in most cases the predicted hemoglobin increase could be seen as soon as an underlying iron deficiency was treated adequately. Blood loss and aluminum toxicity are the next most frequent reasons for an inadequate response to rEPO. Hypertension (and its complications) as well as fistula clotting are the most important side-effects which require close attention when patients at risk for these complications are treated with rEPO.     

Recombinant human erythropoietin use in intensive care

OBJECTIVE: To review the literature concerning the role of recombinant human erythropoietin (rHuEPO) in reducing the need for transfusion in critically ill patients. DATA SOURCES: Articles were obtained through searches of the MEDLINE database (from 1990 to June 2001) using the key words erythropoietin, epoetin alfa, anemia, reticulocytes, hemoglobin, critical care, intensive care, critical illness, and blood transfusion. Additional references were found in the bibliographies of the articles cited. The Cochrane library was also consulted. STUDY SELECTION AND DATA EXTRACTION: Controlled, prospective, and randomized studies on the use of rHuEPO in critically ill adults were selected. DATA SYNTHESIS: Anemia is a common complication in patients requiring intensive care. It is caused, in part, by abnormally low concentrations of endogenous erythropoietin and is mainly seen in patients with sepsis and multiple organ dysfunction syndrome, in whom inflammation mediator concentrations are often elevated. High doses of rHuEPO produce a rapid response in these patients, despite elevated cytokine concentrations. There have been 3 studies on rHuEPO administration in intensive care and 1 trial in acutely burned patients. Only 2 of these studies looked at the impact of rHuEPO administration on the need for transfusion. CONCLUSIONS: Few randomized, controlled trials explore the role of rHuEPO in critical care. Only 1 was a large, randomized clinical trial, but it presents many limitations. Future outcome and safety studies comparing rHuEPO with placebo must include clinical endpoints such as end-organ morbidity, mortality, transfusion criteria, and pharmacoeconomic analysis. rHuEPO appears to provide an erythropoietic response. Optimal dosage and the real impact of rHuEPO on the need for transfusion in intensive care remain to be determined. Currently, based on the evidence available from the literature, rHuEPO cannot be recommended to reduce the need for red blood cell transfusions in anemic, critically ill patients.     

Human saliva stimulates skin and oral wound healing in vitro

Despite continuous exposure to environmental pathogens, injured mucosa within the oral cavity heals faster and almost scar free compared with skin. Saliva is thought to be one of the main contributing factors. Saliva may possibly also stimulate skin wound healing. If so, it would provide a novel therapy for treating skin wounds, for example, burns. This study aims to investigate the therapeutic wound healing potential of human saliva in vitro. Human saliva from healthy volunteers was filter sterilized before use. Two different in vitro wound models were investigated: (a) open wounds represented by 2D skin and gingiva cultures were used to assess fibroblast and keratinocyte migration and proliferation and (b) blister wounds represented by introducing freeze blisters into organotypic reconstructed human skin and gingiva. Re‐epithelialization and differentiation (keratin K10, K13, K17 expression) under the blister and inflammatory wound healing mediator secretion was assessed. Saliva‐stimulated migration of skin and oral mucosa fibroblasts and keratinocytes, but only fibroblast proliferation. Topical saliva application to the blister wound on reconstructed skin did not stimulate re‐epithelization because the blister wound contained a dense impenetrable dead epidermal layer. Saliva did promote an innate inflammatory response (increased CCL20, IL‐6, and CXCL‐8 secretion) when applied topically to the flanking viable areas of both wounded reconstructed human skin and oral mucosa without altering the skin specific keratin differentiation profile. Our results show that human saliva can stimulate oral and skin wound closure and an inflammatory response. Saliva is therefore a potential novel therapeutic for treating open skin wounds.     

Lipid peroxidation inhibition by raxofelast improves angiogenesis and wound healing in experimental burn wounds

We investigated the effects of raxofelast, a lipid peroxidation inhibitor, in an experimental model of burn wounds. C57BL/6 male mice of 25-30 g were immersed in 80 degrees C water for 10 seconds to achieve a partial-thickness scald burn. Animals received intraperitoneally either raxofelast (20 mg/kg/day for 14 days in 100 microL) or its vehicle alone (100 microL/day for 14 days). On day 14, burn areas were used for measuring conjugated dienes, reduced glutathione levels, histological damage, neoangiogenesis by immunohistochemistry and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, VEGF wound content, endothelial and inducible nitric oxide synthase (eNOS and iNOS) expression and wound nitrite content. Raxofelast decreased tissue conjugated dienes (vehicle 6.1 +/- 1.4 DeltaABS/mg protein; raxofelast 3.7 +/- 0.8 DeltaABS/mg protein), prevented tissue glutathione consumption (vehicle 3.2 +/- 0.9 micromol/g protein; raxofelast 6.7 +/- 1.8 mumol/g protein), increased epithelial proliferation, extracellular matrix maturation, and augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31 (vehicle 9.4 +/- 1.1 integrated intensity; raxofelast 14.8 +/- 1.8 integrated intensity). Furthermore, raxofelast enhanced VEGF wound content (vehicle 1.4 +/- 0.4 pg/mg protein; raxofelast 2.4 +/- 0.6 pg/mg protein), caused a marked expression of eNOS (vehicle 16.1 +/- 3 integrated intensity; raxofelast 26.2 +/- 4 integrated intensity) and iNOS (vehicle 9.1 +/- 1.8 integrated intensity; raxofelast 16.2 +/- 3.5 integrated intensity) and increased wound nitrite content. Lipid peroxidation inhibition by raxofelast may be an effective therapeutic approach to improve clinical outcomes after thermal injury.     

Recombinant human collagen III gel for transplantation of autologous skin cells in porcine full‐thickness wounds

Complex skin wounds, such as chronic ulcers and deep burns, require lengthy treatments and cause extensive burdens on healthcare and the economy. Use of biomaterials and cell transplantation may improve traditional treatments and promote the healing of difficult‐to‐treat wounds. In this study, we investigated the use of recombinant human collagen III (rhCol‐III) gel as a delivery vehicle for cultured autologous skin cells (keratinocytes only or keratinocyte–fibroblast mixtures). We examined its effect on the healing of full‐thickness wounds in a porcine wound‐healing model. Two Landrace pigs were used for the study. Fourteen deep dermal wounds were created on the back of each pig with an 8 mm biopsy punch. Syringes containing acellular rhCol‐III gel (n = 8) or rhCol‐III gel with autologous keratinocytes (n = 8) or rhCol‐III gel with autologous keratinocytes and fibroblasts (n = 8) were applied into wounds. Untreated wounds were used as controls for the treatment groups (n = 4). We used rhCol‐III gel to manufacture a cell‐delivery syringe containing autologous skin cells. In a full‐thickness wound‐healing model, we observed that rhCol‐III gel enhances early granulation tissue formation. Interestingly, we found cell type‐dependent differences in the stability of rhCol‐III in vivo. Fibroblast‐containing gel was effectively removed from the wound, whereas gels without cells or with keratinocytes only remained intact. Our results demonstrate that the properties of rhCol‐III gel for skin cell transplantation can be significantly altered in a cell type‐dependent manner. Copyright © 2013 John Wiley & Sons, Ltd.     

重组人促红细胞生成素联合高压氧对移植大鼠背部逆行超长缺血皮瓣存活率的影响

背景:有研究表明,重组人促红细胞生成素可诱导内皮前细胞分化成新生内皮细胞,高压氧则为新生内皮细胞提供增殖所需的充足氧分,加速了新生血管的发生.目的:分析币组人红细胞生成素、高压氧及其联合应用时对大鼠背部逆行超长缺血皮瓣的影响.设计、时间及地点:随机分组,细胞形态学观察,于2008-04/09在山东大学实验动物中心进行.材料:40只Wistar大鼠,于背部制作逆行超长缺血皮瓣.方法:于大鼠背部按4:1设计逆行超长缺血随意皮瓣,皮瓣原位缝合后按照不同的干预方式均分为4组,常压氧组:给予生理盐水+常压氧:高压氧组:高压氧+生理盐水:重组人促红细胞生成素组:重组人促红细胞生成素+常压氧;综合组:重组人促红细胞生成素+常压氧.干预方法:重组人促红细胞生成素的注射量为400 IU/kg与生理盐水等量,1次/d,连用10 d.高压氧为202.65 kPa,体积分数为95%;常压氧为101 kPa;体积分数为35%,给氧均于缝合后8 h进行,1 h,次,2次/d,共10 d.主要观察指标:于缝合后10 d测量各组皮瓣成活率.以血管内皮细胞生长因子和CD34作为血管标记行免疫组化染色,计数皮瓣新生血管密度.结果:定性分析:常压氧组局部坏死,腺体萎缩.高压氧组腺体形态较好,重组人促红细胞生成素组腺体增生,而综合组增生更加明显.定量分析:与常压氧组比较,后3组的血管密度值均增高(P<0.01),且呈递增趋势,以α=0.05的检验标准,各组之间的差别有显著性意义(P<0.01),方差分析结果显示重组人促红细胞生成素和高压氧之间存在协同作用,同时使用效果更好.结论:重组人促红细胞生成素和高压氧以及二者联合应用,可以促进缺血皮瓣下的毛细血管生成,提高皮瓣存活率并扩大皮瓣长宽比例,重组人促红细胞生成素促进皮瓣成活效果好于高压氧,二者同时使用效果更好.     

早期使用促红细胞生成素对早产儿贫血防治的临床研究

目的探讨早期使用重组人类促红细胞生成素(rHuEPO)防治早产儿贫血的疗效。方法将57例早产儿按入院次序分为治疗组30例、对照组27例。治疗组出生第7天给予每周rHuEPO 750 IU/kg,隔日1次,皮下注射,用药5周;对照组未用rHuEPO。两组早产儿均每天口服铁剂,按元素铁6 mg/(kg.d)计算,口服维生素E 25 mg/d,维生素C0.2 g/d。必要时输血。共观察6周。结果①两组早产儿生后血红蛋白(Hb)均渐下降,但治疗组程度较轻,经t检验,两组之间差异有高度显著性意义(P<0.01);②对照组有5例输血,而预防组仅1例输血,经χ2检验差异有高度显著性意义(P<0.01);③治疗后治疗组网织红细胞(Ret)较对照组显著升高,两组差异有高度显著性意义(P<0.01);④血清铁蛋白水平(SF)在用药期间治疗组明显低于对照组,治疗结束后,治疗组血清SF上升,两组比较差异无显著性意义(P>0.05)。结论早期使用rHuEPO能有效防治早产儿贫血,体内充足的铁储备是确保rHuEPO疗效的重要因素。     

A process model of healing and personal transformation in persons with chronic skin wounds

Utilizing Newman's research as praxis process, this research examined the patterns of those living with chronic skin wounds. Ten men and 8 women, primarily of retirement age and living with chronic skin wounds for a year or more, reflected upon important relationships and life events during two in-depth interviews and a self-expressive drawing. Emerging from each participant's pattern was the link among human development, expanding consciousness, and processing a serious physical threat. Considering data across participants, five themes emerged from the data with evolution of a process model of wholistic healing that has implications for advanced nursing practice.     

Recombinant human erythropoietin: effects on frataxin expression in vitro

BACKGROUND: Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by decreased expression of the protein frataxin, recently described to be an iron chaperone for the assembly of iron-sulphur clusters in the mitochondria, causing iron accumulation in mitochondria, oxidative stress and cell damage. Searching for compounds that could possibly influence frataxin expression, we found that the cytokine recombinant human erythropoietin (rhuEPO) significantly increases frataxin expression by a still unknown mechanism. MATERIALS AND METHODS: Isolated lymphocytes from FRDA patients, isolated human cardiac cells (fibroblasts and myocytes) from patients undergoing heart transplantation and P19 mouse cells (neuronal typ), were incubated with different concentrations of rhuEPO, and immunoblot was carried out for the detection of frataxin. RESULTS: We show for the first time that the cytokine recombinant human erythropoietin (rhuEPO) can, additionally to its reported neuro- and cardioprotective properties, increase frataxin expression in vitro. We show that rhuEPO significantly increases frataxin expression in primary lymphocytes from patients with Friedreich's ataxia. Further we show that rhuEPO can also increase frataxin expression in many other cell types; among them the most affected cell types in FRDA such as neurones and cardiac cells. CONCLUSIONS: Our results provide a scientific basis for further studies examining the effectiveness of this agent for the treatment of FRDA patients.     

Recombinant human erythropoietin prevents lipopolysaccharide-induced vascular hyporeactivity in the rat

Erythropoietin (EPO) is a hypoxia-inducible hormone that is essential for normal erythropoiesis in the bone marrow. Administration of recombinant human-EPO is currently being used for the therapy of anemia associated with chronic renal failure and cancer. Moreover, EPO reduces organ injury in experimental hemorrhagic as well as in splanchnic artery occlusion shock and preserves cardiac function after experimental cardiac I/R. Erythropoietin receptors are widely distributed in the cardiovascular system, including endothelial, smooth muscle, cardiac, and other cell types, and nonhematopoietic effects of EPO are increasingly recognized. Thus, the vasculature may be a biological target of EPO. Therefore, the aim of our study was to investigate whether EPO exerts a protective effect in septic shock by modulating vascular dysfunction and hyporeactivity. Rats received EPO (300 U/kg, i.v.) or vehicle 30 min before and 1 and 3 h after LPS (8 x 10 U/kg, i.v.). In vivo and ex vivo (aortic rings) experiments were performed to evaluate the vascular response to contracting and vasodilating agents. The expression of iNOS, intercellular adhesion molecule 1, poly(ADP)ribose polymerase, Bcl-xl, and Bcl-2 was evaluated by Western blot analysis in the rat aorta. We demonstrate that EPO significantly prevents LPS-induced vascular hyporeactivity and endothelial dysfunction. Interestingly, EPO inhibits the increase in iNOS, poly(ADP)ribose polymerase, and intercellular adhesion molecule 1 expression in the aorta of endotoxemic rats and attenuated the decline in the expression of both Bcl-xl and Bcl-2 caused by LPS. In conclusion, our data support the view that EPO has important nonerythropoietic effects protecting organ and tissue against injury and indicate that EPO may be useful in the therapy of patients with septic shock.     

Stabilization of human urine doping control samples: III. Recombinant human erythropoietin

BackgroundThe tampering of athlete's urine samples by the addition of proteolytic enzymes during the doping control sampling procedure was reported recently. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in urine samples to chemically inactivate proteolytic enzymes and improve the electrophoteric signal of erythropoietin (EPO) in human urine.MethodsThe stabilization mixture applied was a combination of antibiotics, antimycotic substances and protease inhibitors. A series of incubation experiments were conducted under controlled conditions in the presence and absence of the stabilization mixture in urine aliquots spiked with six proteases. Two different analytical techniques were applied for the qualitative and quantitative EPO measurement: isoelectric focusing (IEF) and chemiluminescent immunoassay respectively.ResultsThe addition of the chemical stabilization mixture into urine aliquots substantially improved EPO detection in the presence of proteolytic enzymes following incubation at 37 °C or storage at ? 20 °C.ConclusionsThe results of this study indicated that the stabilization of urine prior to the sample collection procedure with the proposed chemical mixture might prove to be a useful tool for the preservation of anti-doping samples.     

Recombinant basic fibroblast growth factor stimulates wound healing in healing-impaired db/db mice

The stimulatory effect of recombinant basic fibroblast growth factor (bFGF) on wound healing was assessed using healing-impaired (db/db) mice. Full-thickness wounds were made in female diabetic C57BL/KsJ db/db mice, and their normal (db/+) littermates with a punch biopsy instrument. Recombinant bFGF was applied locally to the open wound once a day. The mice were later killed and histological sections of the wounds were prepared. The degree of wound healing was evaluated using several histological parameters such as degree of reepithelialization, granulation tissue thickness, matrix density, number of infiltrated cells, and number of capillaries. Wounds from normal mice displayed good reepithelialization rates and granulation tissue formation, while wounds from db/db mice had poor responses, especially in the dermal parameters. Although the application of bFGF to wounds in the normal (db/+) mice had little effect, application of bFGF to wounds in db/db mice induced significant responses in all of the dermal parameters compared with nontreated db/db mice (p less than 0.001). In the presence of bFGF, these parameters approximated those observed in nontreated littermates. A minimum of 0.5 microgram bFGF in either single or multiple applications was required for a significant effect. bFGF that was either boiled or pretreated with neutralizing antibody had little stimulatory effect. Time-course experiments indicated that the granulation response in bFGF-treated mice peaked between 8 and 12 d, and decreased after 12 d, while matrix density continued to increase until the 18th day (p less than 0.05). The breaking strength of healed linear wounds in db/db mice was also decreased when compared with heterozygous littermates. This parameter was also improved by the administration of bFGF to the wounds (p less than 0.05).