A single topical dose of erythropoietin applied on a collagen carrier enhances calvarial bone healing in pigs

Background and purpose

The osteogenic potency of erythropoietin (EPO) has been documented. However, its efficacy in a large-animal model has not yet been investigated; nor has a clinically safe dosage. The purpose of this study was to overcome such limitations of previous studies and thereby pave the way for possible clinical application. Our hypothesis was that EPO increases calvarial bone healing compared to a saline control in the same subject.

Methods

We used a porcine calvarial defect model. In each of 18 pigs, 6 cylindrical defects (diameter: 1 cm; height: 1 cm) were drilled, allowing 3 pairwise comparisons. Treatment consisted of either 900 IU/mL EPO or an equal volume of saline in combination with either autograft, a collagen carrier, or a polycaprolactone (PCL) scaffold. After an observation time of 5 weeks, the primary outcome (bone volume fraction (BV/TV)) was assessed with high-resolution quantitative computed tomography. Secondary outcome measures were histomorphometry and blood samples.

Results

The median BV/TV ratio of the EPO-treated collagen group was 1.06 (CI: 1.02–1.11) relative to the saline-treated collagen group. Histomorphometry showed a similar median effect size, but it did not reach statistical significance. Autograft treatment had excellent healing potential and was able to completely regenerate the bone defect independently of EPO treatment. Bony ingrowth into the PCL scaffold was sparse, both with and without EPO. Neither a substantial systemic effect nor adverse events were observed. The number of blood vessels was similar in EPO-treated defects and saline-treated defects.

Interpretation

Topical administration of EPO on a collagen carrier moderately increased bone healing. The dosing regime was safe, and could have possible application in the clinical setting. However, in order to increase the clinical relevance, a more potent but still clinically safe dose should be investigated.Erythropoietin (EPO) is a hematopoietic growth factor that stimulates the formation of red blood cells. In recent years, the non-hematopoietic effects of EPO have been investigated. Of interest for skeletal tissue engineering, the pleiotropic capabilities of EPO include osteogenic and angiogenic potencies (Rölfing et al. 2012). Subcutaneous injections of 250 IU/kg EPO were found to enhance bone formation 6 weeks after operation in a spinal fusion model in rabbits (Rölfing et al. 2012). The validity of the methodology of this study was confirmed in a recently published meta-analysis (Riordan et al. 2013, Rölfing and Bünger 2013). Other independent research groups have reported increased bone formation in mice and rats after daily treatment with 200–6,000 IU/kg EPO (Bozlar et al. 2006, Holstein et al. 2007, 2011, Shiozawa et al. 2010, Garcia et al. 2011, Kim et al. 2012). Furthermore, vascularization of 3-dimensional scaffolds for bone tissue regeneration remains a challenge. The described pleiotropic functions of EPO may overcome this limitation of skeletal tissue engineering in the future. EPO could possibly facilitate angiogenesis directed into the core of the scaffold, thereby facilitating bony ingrowth. Moreover, EPO promotes a direct and indirect osteogenic stimulation of mesenchymal stromal cells (Shiozawa et al. 2010, Rölfing et al. 2013).Translation of these promising in vitro and in vivo data into clinical trials requires a physiological dosage of EPO in order to avoid its known complications, such as thromboembolism (Ehrenreich et al. 2009, Shiozawa et al. 2010, Kim et al. 2012, Rölfing et al. 2012). Notably, we observed an extremely high hematocrit level after 250 IU/kg EPO for 20 days in a rabbit model (Rölfing et al. 2012). In other in vivo studies, repetitive EPO injections ranging from 500 to 6,000 IU/kg were administered. These treatment regimes have a systemic effect, and thus hold the risk of adverse events. Testing of the efficacy of a clinically safe dose of EPO is therefore necessary before clinical trials can be considered. Aiming for clinical progress and feasibility, the present study was carried out with a view to evaluating the efficacy of a single, low-dose EPO to stimulate bone healing in a large-animal study. The dose of EPO was chosen based on the following considerations. The translation of the minimally effective dose in cell studies into large-animal models is difficult (Rölfing et al. 2013). The rather low dosage of 2,700 IU/animal, equivalent to 18.5 ± 2.0 IU/kg, was chosen in order to minimize the systemic effect of EPO due to safety concerns and in order to minimize the potential effect on the within-subject controls. In anemic patients, 20–240 IU/kg are injected subcutaneously or intravenously 3 times a week. The hypothesis was that 900 IU site-specifically applied EPO would increase bony ingrowth compared to a saline-treated control in a porcine calvarial defect model.     

Astrocyte glycogenolysis is triggered by store‐operated calcium entry and provides metabolic energy for cellular calcium homeostasis

Astrocytic glycogen, the only storage form of glucose in the brain, has been shown to play a fundamental role in supporting learning and memory, an effect achieved by providing metabolic support for neurons. We have examined the interplay between glycogenolysis and the bioenergetics of astrocytic Ca²⁺ homeostasis, by analyzing interdependency of glycogen and store‐operated Ca²⁺ entry (SOCE), a mechanism in cellular signaling that maintains high endoplasmatic reticulum (ER) Ca²⁺ concentration and thus provides the basis for store‐dependent Ca²⁺ signaling. We stimulated SOCE in primary cultures of murine cerebellar and cortical astrocytes, and determined glycogen content to investigate the effects of SOCE on glycogen metabolism. By blocking glycogenolysis, we tested energetic dependency of SOCE‐related Ca²⁺ dynamics on glycogenolytic ATP. Our results show that SOCE triggers astrocytic glycogenolysis. Upon inhibition of adenylate cyclase with 2',5'‐dideoxyadenosine, glycogen content was no longer significantly different from that in unstimulated control cells, indicating that SOCE triggers astrocytic glycogenolysis in a cAMP‐dependent manner. When glycogenolysis was inhibited in cortical astrocytes by 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol, the amount of Ca²⁺ loaded into ER via sarco/endoplasmic reticulum Ca²‐ATPase (SERCA) was reduced, which suggests that SERCA pumps preferentially metabolize glycogenolytic ATP. Our study demonstrates SOCE as a novel pathway in stimulating astrocytic glycogenolysis. We also provide first evidence for a new functional role of brain glycogen, in providing local ATP to SERCA, thus establishing the bioenergetic basis for astrocytic Ca²⁺ signaling. This mechanism could offer a novel explanation for the impact of glycogen on learning and memory. GLIA 2014;62:526–534     

The Osteogenic Effect of Erythropoietin on Human Mesenchymal Stromal Cells is Dose-Dependent and Involves Non-Hematopoietic Receptors and Multiple Intracellular Signaling Pathways

Erythropoietin (EPO) is a pleiotropic growth factor. Of interest for skeletal tissue engineering, the non-hematopoietic capabilities of EPO include its osteogenic and angiogenic potencies. The main aim of this study was to investigate the dose–response relationship and determine the lowest effective dose of EPO that reliably increases the osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Additional aims were to elucidate the surface receptors and to investigate the role of the intracellular signaling pathways by blocking the mammalian target of rapamycin (mTOR), Jak-2 protein tyrosine kinase (JAK2), and phosphoinositide 3-kinases (PI3K). The primary outcome measures were two mineralization assays, Arsenazo III and alizarin red, applied after 10, 14, and 21 days. Moreover, alkaline phosphatase activity, cell number, and cell viability were determined after 2 and 7 days. A proportional dose–response relationship was observed. In vivo, the lowest effective dose of 20 IU/ml should be used for further research to accommodate safety concerns about adverse effects. Ex vivo, the most effective dose of 100 IU/ml could facilitate vascularization and bone ingrowth in cell-based scaffolds. The expression of non-hematopoietic receptors EPOR and CD131 was documented, and EPO triggered all three examined intracellular pathways. Future studies of the efficacy of EPO in cell-based tissue engineering can benefit from our findings.     

Activity of Galidesivir in a Hamster Model of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) has claimed the lives of millions of people worldwide since it first emerged. The impact of the COVID-19 pandemic on public health and the global economy has highlighted the medical need for the development of broadly acting interventions against emerging viral threats. Galidesivir is a broad-spectrum antiviral compound with demonstrated in vitro and in vivo efficacy against several RNA viruses of public health concern, including those causing yellow fever, Ebola, Marburg, and Rift Valley fever. In vitro studies have shown that the antiviral activity of galidesivir also extends to coronaviruses. Herein, we describe the efficacy of galidesivir in the Syrian golden hamster model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Treatment with galidesivir reduced lung pathology in infected animals compared with untreated controls when treatment was initiated 24 h prior to infection. These results add to the evidence of the applicability of galidesivir as a potential medical intervention for a range of acute viral illnesses, including coronaviruses.     

Loss of function,missense, and intronic variants in NOTCH1 confer different risks for left ventricular outflow tract obstructive heart defects in two European cohorts

Loss of function variants in NOTCH1 cause left ventricular outflow tract obstructive defects (LVOTO). However, the risk conferred by rare and noncoding variants in NOTCH1 for LVOTO remains largely uncharacterized. In a cohort of 49 families affected by hypoplastic left heart syndrome, a severe form of LVOTO, we discovered predicted loss of function NOTCH1 variants in 6% of individuals. Rare or low-frequency missense variants were found in 16% of families. To make a quantitative estimate of the genetic risk posed by variants in NOTCH1 for LVOTO, we studied associations of 400 coding and noncoding variants in NOTCH1 in 1,085 cases and 332,788 controls from the UK Biobank. Two rare intronic variants in strong linkage disequilibrium displayed significant association with risk for LVOTO amongst European-ancestry individuals. This result was replicated in an independent analysis of 210 cases and 68,762 controls of non-European and mixed ancestry. In conclusion, carrying rare predicted loss of function variants in NOTCH1 confer significant risk for LVOTO. In addition, the two intronic variants seem to be associated with an increased risk for these defects. Our approach demonstrates the utility of population-based data sets in quantifying the specific risk of individual variants for disease-related phenotypes.     

Health-related quality of life at hospital discharge as a predictor for 6-month unplanned readmission and all-cause mortality of acutely admitted older medical patients

Quality of Life Research - To assess whether health-related quality of life (HRQOL) status, using the European Quality of life-5 dimensions (EQ5D), in acutely admitted older medical patients was...