Fibroblast Growth Factor 23 and Exercise Capacity in Heart Failure with Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is characterized by left ventricular hypertrophy and decreased exercise capacity. Fibroblast growth factor 23 (FGF23), a hormone involved in phosphate, vitamin D, and iron homeostasis, is linked to left ventricular hypertrophy and HF. We measured c-terminal FGF23 (cFGF23) and intact FGF23 (iFGF23) levels and examined their associations with exercise capacity in patients with HFpEF. METHODS AND RESULTS: Using multivariable linear regression and linear mixed models, we studied the associations of cFGF23 and iFGF23 with baseline and mean weekly change over 24 weeks in peak oxygen consumption and 6-minute walk distance in individuals enrolled in the Phosphodiesterase-5 Inhibition to Improve Clinical Status and Exercise Capacity in HFpEF trial. Our study population included 172 individuals with available plasma for cFGF23 and iFGF23 measurements. Median (25th-75th percentile) baseline cFGF23 and iFGF23 levels were 208.7 RU/mL (132.1-379.5 RU/mL) and 90.3 pg/mL (68.6-128.5 pg/mL), respectively. After adjustment for cardiovascular disease and hematologic and kidney parameters, higher cFGF23 was independently associated with a lower peak oxygen consumption at baseline. Higher iFGF23 was independently associated with shorter 6-minute walk distance at baseline. No significant associations were appreciated with the longitudinal outcomes. CONCLUSIONS: In patients with HFpEF, higher FGF23 levels are independently associated with decreased exercise capacity at baseline.

Plate reader-based cell viability assays for glioprotection using primary rat optic nerve head astrocytes.

Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute critically to extracellular matrix synthesis during development and throughout life. In glaucoma, and in related disorders affecting the optic nerve and the optic nerve head, pathological changes include altered astrocyte gene and protein expression resulting in their activation and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA in order to slow glaucoma and related pathologies are of high clinical relevance. Herein, we describe the development of standardized methods that will allow for the systematic advancement of such glioprotective strategies. These include isolation, purification and culture of primary adult rat ONHAs, optimized immunocytochemical protocols for cell type validation, as well as plate reader-based assays determining cellular viability, proliferation and the intracellular redox state. We validated and standardized our protocols by performing a glioprotection study using primary ONHAs. Specifically, we measured protection against exogenously-applied oxidative stress using tert-butylhydroperoxide (tBHP) as a model of disease-mediated oxidative stress in the retina and optic nerve head by the prototypic antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Levels of oxidative stress were increased in the response to exogenously applied tBHP and were assessed by 6-carboxy-2', 7' dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Normalized DCFDA fluorescence showed a maximal 5.1-fold increase; the half-maximal effect (EC50) for tBHP was 212 ± 25 µM. This was paralleled very effectively in the assays measuring cell death and cell viability with half-maximal effects of 241 ± 20 µM and 194 ± 5 µM for tBHP in the lactate dehydrogenase (LDH) release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays, respectively. Pre-treatment with 100 µM Trolox decreased the sensitivity of ONHAs to tBHP. Half-maximal effects increased to 396 ± 12 µM tBHP in the LDH release assay and to 383 ± 3 µM tBHP in the MTT assay. Vehicle treatment (0.1% v/v ethanol) did not significantly affect cellular responses to tBHP. Antioxidant treatment increases ONHA viability and reduces the deleterious effects of oxidative stress. Our experiments provide important feasibility data for utilizing primary rat ONHAs in plate reader-based assays assessing novel therapeutics for glioprotection of the optic nerve and the optic nerve head in glaucoma and related disorders. Furthermore, our novel, standardized protocols have the potential to be readily adapted to high-throughput and high-content testing strategies.

An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience.

INTRODUCTION: Quantification of lactate dehydrogenase (LDH) release is a widely accepted assay for the quantitative determination of cell viability and late-stage apoptosis. Major disadvantages of commercially available LDH assay kits include proprietary formulations, limited options for optimization and high cost, all resulting in limited reproducibility in research applications. Here, we describe a novel, custom LDH assay suitable in the context of plate reader-based screening of drug candidates for glioprotection, but with wide applicability to other cell types and experimental paradigms. METHODS: We developed a novel and highly reproducible LDH release assay that is more cost-effective than commercially available assays with comparable performance. The assay was validated by assessing 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid antioxidant protection against tert-butylhydroperoxide-induced oxidative stress in C6 astroglioma cells. Assay performance was validated by direct comparison and compatible with other methods of measuring cellular viability, namely 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 6-carboxy-2', 7' dichlorodihydrofluorescein diacetate assays. RESULTS: There was no statistically significant difference between results obtained with the novel custom assay and a commercially available assay CytoTox96® (Promega, Madison, WI). DISCUSSION: The novel custom LDH release assay allows the reproducible quantification of cell viability and is highly cost-effective when compared to commercially available assays (approximately 25 times cheaper). In addition and in contrast to commercially available assays, the identification and detailed description of all assay components and procedures provide greater control over experimental conditions and design. We provide a detailed standard operating procedure permitting our novel assay to be readily adapted depending on experimental requirements.