The prolyl 4-hydroxylase inhibitor GSK360A decreases post-stroke brain injury and sensory, motor, and cognitive behavioral deficits - Fig 4

<p>(<b>A)</b> The blood plasma levels of EPO 5 hours and 24 hours after 30 mg/kg, p.o. GSK360A administration is presented. The GSK360A treated group had significantly higher levels of EPO compared to vehicle group even at 24 hours after administration (e.g., >80-fold over the vehicle group for both time points). (<b>B)</b> The blood plasma levels of VEGF 5 hours and 24 hours after GSK360A administration is presented. GSK360A treated group had significantly higher levels of VEGF at both time points compared to vehicle group. N = 5 rats per group. Two-way ANOVA test followed by post hoc analysis using the Bonferroni procedure for multiple comparisons; *p < 0.05 and **p < 0.001 when compared with vehicle group.</p

Mechano Growth Factor peptide (MGF), the COOH terminus of unprocessed Insulin-like growth factor 1 (IGF-1), has no apparent effect on muscle myoblasts or primary muscle stem cells

A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24 amino acid COOH-terminal peptide which is cleaved from the NH3 terminal 70 amino acid mature IGF-1 protein. This COOH-terminal peptide was termed “mechano-growth factor” (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus at our two pharmaceutical companies we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This too failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of pERK, but not pAkt in cardiac myocytes. While a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF

Body weight was measured over the course of the study.

<p>Thus, 24 hours prior to tMCAO Stroke body weight was measured and represents the “Pre” measurement. This was followed by the oral (gavage) administration of 30mg/kg GSK360A at 18 and 5 hours prior to Stroke that was produced in rats at time 0. Then body weight was measured at 5 hours, 1 day, 1 week and 3 weeks after stroke. No significant differences in body weight were observed between the vehicle and the GSK360A treated groups over the course of study. N = 8 rats per group. Two-way ANOVA test followed by post hoc analysis using the Bonferroni procedure for multiple comparisons. There was no difference in body weight over time (p = 0.7064) between vehicle and GSK360A groups.</p

Effect of GSK360A on APA learning performance 3 weeks after tMCAO stroke.

<p><b>(A)</b> The Two-Way ANOVA indicated that the group and group x trial interaction effects were significant (p < 0.01). Thus, looking at the interaction of groups over trials, the GSK360A treated group received significantly less shocks (i.e., made less errors in avoiding the shock quadrant) over trials (p < 0.01). Specifically, the GSK360A treated group received significantly less shocks in trial 5 and trial 7. <b>(B)</b> The effect of GSK360A was not due to differences in rat movements during the trials, as there was no significant difference in distance traveled per trial between the two groups. N = 8 rats per group. Two-Way ANOVA, followed by post hoc analysis using the Bonferroni procedure for multiple comparisons; *p < 0.05 and **p < 0.01 when compared with vehicle groups.</p

Toward improved myocardial maturity in an organ-on-chip platform with immature cardiac myocytes.

In&nbsp;vitro studies of cardiac physiology and drug response have traditionally been performed on individual isolated cardiomyocytes or isotropic monolayers of cells that may not mimic desired physiological traits of the laminar adult myocardium. Recent studies have reported a number of advances to Heart-on-a-Chip platforms for the fabrication of more sophisticated engineered myocardium, but cardiomyocyte immaturity remains a challenge. In the anisotropic musculature of the heart, interactions between cardiac myocytes, the extracellular matrix (ECM), and neighboring cells give rise to changes in cell shape and tissue architecture that have been implicated in both development and disease. We hypothesized that engineered myocardium fabricated from cardiac myocytes cultured in&nbsp;vitro could mimic the physiological characteristics and gene expression profile of adult heart muscle. To test this hypothesis, we fabricated engineered myocardium comprised of neonatal rat ventricular myocytes with laminar architectures reminiscent of that observed in the mature heart and compared their sarcomere organization, contractile performance characteristics, and cardiac gene expression profile to that of isolated adult rat ventricular muscle strips. We found that anisotropic engineered myocardium demonstrated a similar degree of global sarcomere alignment, contractile stress output, and inotropic concentration-response to the β-adrenergic agonist isoproterenol. Moreover, the anisotropic engineered myocardium exhibited comparable myofibril related gene expression to muscle strips isolated from adult rat ventricular tissue. These results suggest that tissue architecture serves an important developmental cue for building in&nbsp;vitro model systems of the myocardium that could potentially recapitulate the physiological characteristics of the adult heart. Impact statement With the recent focus on developing in&nbsp;vitro Organ-on-Chip platforms that recapitulate tissue and organ-level physiology using immature cells derived from stem cell sources, there is a strong need to assess the ability of these engineered tissues to adopt a mature phenotype. In the present study, we compared and contrasted engineered tissues fabricated from neonatal rat ventricular myocytes in a Heart-on-a-Chip platform to ventricular muscle strips isolated from adult rats. The results of this study support the notion that engineered tissues fabricated from immature cells have the potential to mimic mature tissues in an Organ-on-Chip platform

Overall schematic of basic experimental studies and designs.

<p>Two-pretreatments of GSK360A (30mg/kg) at 18 hours and 1 hour prior the experiment was administrated orally. (A) Pilot pharmacokinetic and pharmacodynamic tests including plasma, kidney and brain GSK360A levels and blood EPO and VEGF levels were measured 5 and 24 hours after and data were presented in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184049#pone.0184049.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184049#pone.0184049.g004" target="_blank">4</a>. (B) Ischemic stroke was induced by tMCAO and GSK360A at 18 and 5 hours prior to Stroke that was produced in rats at time 0. The effects of GSK360A on body weight, sensory-motor neurological deficits, cognitive function, biochemical changes on HIF related protein/molecular and brain infarcts were evaluated (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184049#pone.0184049.g005" target="_blank">5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184049#pone.0184049.g010" target="_blank">10</a>).</p

GSK360A increased kidney EPO and brain VEGF mRNA after stroke.

<p>(A) EPO mRNA in the kidney was significantly increased by GSK360A at 5 hours post-stroke compared to vehicle group. Levels of EPO mRNA returned to vehicle treated levels by 24 hours post-stroke.(B) VEGF mRNA in the brain ischemic hemisphere (above) and in the non-ischemic hemisphere (not shown) was increased by GSK360A at 5 hours and 24 hours post-stroke compared to vehicle group (p = 0.002). N = 5 rats per group. Two-Way ANOVA test, followed by post hoc analyses using the Bonferroni procedure for multiple comparisons; **p < 0.01 when compared with vehicle groups.</p