Background The mammalian target of rapamycin (mTOR) is known to play a pivotal role in the differentiation of hematopoietic stem cells (HSCs) via control of cell cycling. In addition to this function, mTOR is known to mediate translation of constitutively expressed mRNAs. Specifically, we have demonstrated the role of mTOR in translational regulation of key inflammatory mediators. In this investigation we hypothesized that an inflammatory agonist would increase synthesis of mTOR in a pool of terminally differentiated polymorphonuclear leukocytes (PMN). Methods Human HSCs were isolated from umbilical cord blood obtained from full-term neonates. This population of HSCs underwent proliferation and differentiation into mature PMNs. These cells were purified by magnetically selecting cells expressing the CD16+ cell surface antigen. The CD16+ cells were exposed to platelet-activating factor (PAF) 10 nM or HBSS for 0, 60, or 120 minutes. The cells were subsequently fixed and examined by immunocytochemistry for their expression of mTOR. Results The population of terminally differentiated PMNs demonstrated increased expression of mTOR following stimulation with PAF compared with control. Further, this population of cells demonstrated a time-dependent increase in production of mTOR consistent with the expected rapid response of primary host defense cells. Conclusions HSC-derived, terminally differentiated PMNs demonstrate functional integrity of inflammatory mediator-induced mTOR expression. This capacity likely enables the cell to control the rapid synthesis of other mediators of inflammation. This finding will be key for further investigation using this model to perform loss of mTOR function experiments on downstream inflammatory mediators and the concomitant alteration in inflammatory processes.
Rationale: Ischemic stroke prompts an inflammatory response which is associated with worse outcomes. Classic anti-inflammatory strategies were unsuccessful in clinical trials, implying other mechanisms contribute to injurious inflammation in stroke. In response to stimuli, neutrophils can release DNA web-like structures called neutrophil extracellular traps (NETs). Recently, a role for NETs in cardiovascular disease has emerged. Here, we studied whether NETs contribute to ischemic stroke outcomes. Methods: Markers of NET formation were assessed in brain tissue and plasma from ischemic stroke patients. For murine studies, we used male and female mice that were subjected to transient middle cerebral artery occlusion. Stroke outcomes were assessed 24 hours or 7 days after stroke. Results: NETs were found in brain tissue from deceased ischemic stroke patients. Ischemic stroke patients had significantly increased plasma biomarkers of NET formation including citrullinated histone H3 (p<0.0001) and MPO-DNA complexes (p<0.001) compared to matched controls. NET biomarkers positively correlated with worse stroke outcomes at discharge (p<0.05) while they did not correlate with stroke severity at admission. To target NET formation in ischemic stroke, we investigated the therapeutic potential of a recently discovered neonatal NET inhibitory factor (nNIF). nNIF specifically blocks NET formation in human and murine neutrophils without affecting other critical neutrophil functions. Mice prophylactically treated with nNIF had significantly reduced brain and plasma NETs after stroke while cerebral neutrophil recruitment remained unaffected. The reduction in NET formation was associated with significantly reduced neuronal apoptosis and smaller brain infarcts (p<0.0001). Furthermore, nNIF treated mice had improved neurological behavior and motor function, and enhanced 7-day survival after ischemic stroke (p<0.001). Importantly, these results were confirmed in diabetic mice and >18-month-old mice and nNIF was still effective when administered therapeutically, 1 hour after stroke onset. Conclusions: Our results support a pathological role for NETs in ischemic stroke and warrant further investigation into nNIF to improve stroke outcomes.
The effects of the nitric oxide (NO) donor spermine NONOate (Sp-NO, 1.0 mM) on cross-bridge recruitment and cross-bridge cycling kinetics were studied in permeabilized rabbit psoas muscle fibers. Fibers were activated at various Ca2+ concentrations (pCa, negative logarithm of Ca2+ concentration), and the pCa at which force was maximal (pCa 4.0) and approximately 50% of maximal (pCa50 5.6) were determined. Fiber stiffness was determined using 1-kHz sinusoidal length perturbations, and the fraction of cross bridges in the force-generating state was estimated by the ratio of stiffness during maximal (pCa 4.0) and submaximal (pCa 5.6) Ca2+ activation to stiffness during rigor (at pCa 4.0). Cross-bridge cycling kinetics were evaluated by measuring the rate constant for force redevelopment after quick release (by 15% of optimal fiber length, L(o)) and restretch of the fiber to L(o). Exposing fibers to Sp-NO for 10 min reduced force and the fraction of cross bridges in the force-generating state at maximal and submaximal (pCa50) Ca2+ activation. However, the effects of Sp-NO were more pronounced during submaximal Ca2+ activation. Sp-NO also reduced the rate constant for force redevelopment but only during submaximal Ca2+ activation. We conclude that Sp-NO reduces Ca2+ sensitivity by decreasing the number of cross bridges in the strongly bound state and also impairs cross-bridge cycling kinetics during submaximal activation.
Black individuals have a higher incidence of ischemic stroke (IS) than White individuals. The platelet receptor PAR4 harbors a functional dimorphism: rs773902 A/G (encoding Thr120/Ala120, respectively), and the A allele is associated with greater ex vivo platelet aggregation. The A allele frequency is 0.6 in Black individuals and 0.2 in White subjects. However, it is unknown whether the A allele contributes to racial differences in IS. In a prospective cohort, the REGARDS study, a Black subject-only analysis of 487 IS cases and 7,133 controls was performed, examining if the A allele was a risk for incident IS and worse IS outcome. We used Cox proportional hazards models to calculate hazard ratios (HR) with 95% confidence intervals (CI) of incident IS for the rs773902 A allele. Models were adjusted for age, sex, the first 5 genetic principal components, smoking, hypertension and diabetes. To directly test the effect of this PAR4 variant on IS, we generated the first humanized PAR4 (hPAR4) mouse strains expressing either hPAR4 Thr120 or hPAR4 Ala120. In REGARDS, the A allele was a risk factor for incident IS (HR 1.24; CI 1.04-1.53, p=0.046) and worse IS outcome (HR 2.04; CI 1.10-3.79, p=0.025). In a murine stroke model, hPAR4 Thr120 mice had worse stroke outcomes compared to hPAR4 Ala120 mice (60% increase, p=0.001). This phenotype was mediated, in part, by enhanced platelet activation (p=0.002), platelet-neutrophil interactions (p=0.014), and neutrophil extracellular traps (p=0.001). Direct inhibition of PAR4 with BMS-986120 was equally effective in improving stroke outcomes in hPAR4 Ala120 and Thr120 mice. In contrast, ticagrelor treatment improved stroke outcomes in hPAR4 Ala120 mice (50% decrease, p=0.007), but was ineffective in hPAR4 Thr120 mice, even when combined with aspirin. Importantly, blocking platelet-neutrophil interactions downstream of PAR4 with a murine analog of the FDA-approved P-selectin inhibitor crizanlizumab improved stroke outcomes in hPAR4 Thr120 mice (55% decrease, p=0.004). These results support a causal role for PAR4 Thr120 in IS that may explain part of the racial disparity in stroke. Our findings further suggest that different anti-platelet therapies may be needed for patients expressing PAR4 Thr120.
In the present study, we tested the hypothesis that intrinsic differences in ATP consumption rate per cross bridge exist across rat diaphragm muscle (Dia m ) fibers expressing different myosin heavy chain (MHC) isoforms. During maximum Ca 2+ activation (pCa 4.0) of single, Triton X-permeabilized Dia m fibers, isometric ATP consumption rate was determined by using an NADH-linked fluorometric technique. The MHC concentration in single Dia m fibers was determined by densitometric analysis of SDS-PAGE gels and comparison to a standard curve of known MHC concentrations. Isometric ATP consumption rate varied across Dia m fibers expressing different MHC isoforms, being highest in fibers expressing MHC 2X (1.14 ± 0.08 nmol · mm −3 · s −1 ) and/or MHC 2B (1.33 ± 0.08 nmol · mm −3 · s −1 ), followed by fibers expressing MHC 2A (0.77 ± 0.11 nmol · mm −3 · s −1 ) and MHC Slow (0.46 ± 0.03 nmol · mm −3 · s −1 ). These differences in ATP consumption rate also persisted when it was normalized for MHC concentration in single Dia m fibers. Normalized ATP consumption rate for MHC concentration varied across Dia m fibers expressing different MHC isoforms, being highest in fibers expressing MHC 2X (2.02 ± 0.19 s −1 ) and/or MHC 2B (2.64 ± 0.15 s −1 ), followed by fibers expressing MHC 2A (1.57 ± 0.16 s −1 ) and MHC Slow (0.77 ± 0.05 s −1 ). On the basis of these results, we conclude that there are intrinsic differences in ATP consumption rate per cross bridge in Dia m fibers expressing MHC isoforms.
We hypothesize that 1) the effect of denervation (DNV) is more pronounced in fibers expressing fast myosin heavy chain (MHC) isoforms and 2) the effect of DNV on maximum specific force reflects a reduction in MHC content per half sarcomere or the number of cross bridges in parallel. Studies were performed on single Triton X-100-permeabilized fibers activated at a pCa (−log Ca 2+ concentration) of 4.0. MHC content per half sarcomere was determined by densitometric analysis of SDS-PAGE gels and comparison to a standard curve of known MHC concentrations. After 2 of wk DNV, the maximum specific force of fibers expressing MHC 2X was reduced by ∼40% (MHC 2B expression was absent), whereas the maximum specific force of fibers expressing MHC 2A and MHC slow decreased by only ∼20%. DNV also reduced the MHC content in fibers expressing MHC 2X, with no effect on fibers expressing MHC 2A and MHC slow . When normalized for MHC content per half sarcomere, force generated by DNV fibers expressing MHC 2X and MHC 2A was decreased compared with control fibers. These results suggest the force per cross bridge is also affected by DNV.
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