There is a growing appreciation that our microbial environment in the gut plays a critical role in the maintenance of health and the pathogenesis of disease. Probiotic, beneficial gut microbes, administration can directly attenuate cardiac injury and post-myocardial infarction (MI) remodelling, yet the mechanisms of cardioprotection are unknown. We hypothesised that administration of Bifidobacterium animalis subsp. lactis 420 (B420), a probiotic with known anti-inflammatory properties, to mice will mitigate the pathological impact of MI, and that anti-inflammatory T regulatory (Treg) immune cells are necessary to impart protection against MI as a result of B420 administration. Wild-type male mice were administered B420, saline or Lactobacillus salivarius 33 (Ls-33) by gavage daily for 14 or 35 days, and underwent ischemia/reperfusion (I/R). Pretreatment with B420 for 10 or 28 days attenuated cardiac injury from I/R and reduced levels of inflammatory markers. Depletion of Treg cells by administration of anti-CD25 monoclonal antibodies eliminated B420-mediated cardio-protection. Further cytokine analysis revealed a shift from a pro-inflammatory to an anti-inflammatory environment in the probiotic treated post-MI hearts compared to controls. To summarise, B420 administration mitigates the pathological impact of MI. Next, we show that Treg immune cells are necessary to mediate B420-mediated protection against MI. Finally, we identify putative cellular, epigenetic and/or post-translational mechanisms of B420-mediated protection against MI.
Osteopontin (OPN) is a secreted extracellular phosphoprotein involved in diverse biologic functions, including inflammation, cell migration, and antiapoptotic processes. Here we investigate the neuroprotective potential of OPN to reduce cell death using both in vitro and in vivo models of ischemia. We show that incubation of cortical neuron cultures with OPN protects against cell death from oxygen and glucose deprivation. The effect of OPN depends on the Arg-Gly-Asp (RGD)-containing motif as the protective effect of OPN in vitro was blocked by an RGD-containing hexapeptide, which prevents integrin receptors binding to their ligands. Osteopontin treatment of cortical neuron cultures caused an increase in Akt and p42/p44 MAPK phosphorylation, which is consistent with OPN-inducing neuroprotection via the activation of these protein kinases. Indeed, the protective effect of OPN was reduced by inhibiting the activation of Akt and p42/p44 MAPK using LY294002 and U0126, respectively. The protective effect of OPN was also blocked by the protein synthesis inhibitor cycloheximide, suggesting that the neuroprotective effect of OPN required new protein synthesis. Finally, intracerebral ventricular administration of OPN caused a marked reduction in infarct size after transient middle cerebral artery occlusion in a murine stroke model. These data suggest that OPN is a potent neuroprotectant against ischemic injury.
Osteopontin (OPN), a large secreted glycoprotein with an arginine, glycine, aspartate (RGD) motif, can bind and signal through cellular integrin receptors. We have shown previously that OPN enhances neuronal survival in the setting of ischemia. Here, we sought to increase the neuroprotective potency of OPN and improve the method of delivery with the goal of identifying a treatment for stroke in humans. We show that thrombin cleavage of OPN improves its ability to ligate integrin receptors and its neuroprotective capacity in models of ischemia. Thrombin-cleaved OPN is a twofold more effective neuroprotectant than the untreated molecule. We also tested whether OPN could be administered intranasally and found that it is efficiently targeted to the brain via intranasal delivery. Furthermore, intranasal administration of thrombin-treated OPN confers protection against ischemic brain injury. Osteopontin mimetics based on the peptide sequences located either N or C terminal to the thrombin cleavage site were generated and tested in models of ischemia. Treatment with successively shorter N-terminal peptides and a phosphorylated C-terminal peptide provided significant neuroprotection against ischemic injury. These findings show that OPN mimetics offer promise for development into new drugs for the treatment of stroke.