Mediates Tissue Protection Research Articles

A small erythropoietin derived non-hematopoietic peptide reduces cardiac inflammation, attenuates age associated declines in heart function and prolongs healthspan.

Aging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age? We conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan-Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions. Chronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life. Administration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan.

Targeting the innate repair receptor axis via erythropoietin or pyroglutamate helix B surface peptide attenuates hemolytic-uremic syndrome in mice.

Hemolytic-uremic syndrome (HUS) can occur as a systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli and is characterized by microangiopathic hemolytic anemia and acute kidney injury. Hitherto, therapy has been limited to organ-supportive strategies. Erythropoietin (EPO) stimulates erythropoiesis and is approved for the treatment of certain forms of anemia, but not for HUS-associated hemolytic anemia. EPO and its non-hematopoietic analog pyroglutamate helix B surface peptide (pHBSP) have been shown to mediate tissue protection via an innate repair receptor (IRR) that is pharmacologically distinct from the erythropoiesis-mediating receptor (EPO-R). Here, we investigated the changes in endogenous EPO levels in patients with HUS and in piglets and mice subjected to preclinical HUS models. We found that endogenous EPO was elevated in plasma of humans, piglets, and mice with HUS, regardless of species and degree of anemia, suggesting that EPO signaling plays a role in HUS pathology. Therefore, we aimed to examine the therapeutic potential of EPO and pHBSP in mice with Stx-induced HUS. Administration of EPO or pHBSP improved 7-day survival and attenuated renal oxidative stress but did not significantly reduce renal dysfunction and injury in the employed model. pHBSP, but not EPO, attenuated renal nitrosative stress and reduced tubular dedifferentiation. In conclusion, targeting the EPO-R/IRR axis reduced mortality and renal oxidative stress in murine HUS without occurrence of thromboembolic complications or other adverse side effects. We therefore suggest that repurposing EPO for the treatment of patients with hemolytic anemia in HUS should be systematically investigated in future clinical trials.

Noninvasive Low-Intensity Focused Ultrasound Mediates Tissue Protection following Ischemic Stroke.

Objective and Impact Statement. This study examined the efficacy and safety of pulsed, low-intensity focused ultrasound (LIFU) and determined its ability to provide neuroprotection in a murine permanent middle cerebral artery occlusion (pMCAO) model. Introduction. Focused ultrasound (FUS) has emerged as a new therapeutic strategy for the treatment of ischemic stroke; however, its nonthrombolytic properties remain ill-defined. Therefore, we examined how LIFU influenced neuroprotection and vascular changes following stroke. Due to the critical role of leptomeningeal anastomoses or pial collateral vessels, in cerebral blood flow restoration and tissue protection following ischemic stroke, we also investigated their growth and remodeling. Methods. Mice were exposed to transcranial LIFU (fundamental frequency: 1.1 MHz, sonication duration: 300 ms, interstimulus interval: 3 s, pulse repetition frequency: 1 kHz, duty cycle per pulse: 50%, and peak negative pressure: -2.0 MPa) for 30 minutes following induction of pMCAO and then evaluated for infarct volume, blood-brain barrier (BBB) disruption, and pial collateral remodeling at 24 hrs post-pMCAO. Results. We found significant neuroprotection in mice exposed to LIFU compared to mock treatment. These findings correlated with a reduced area of IgG deposition in the cerebral cortex, suggesting attenuation of BBB breakdown under LIFU conditions. We also observed increased diameter of CD31-postive microvessels in the ischemic cortex. We observed no significant difference in pial collateral vessel size between FUS and mock treatment at 24 hrs post-pMCAO. Conclusion. Our data suggests that therapeutic use of LIFU may induce protection through microvascular remodeling that is not related to its thrombolytic activity.

Mesenchymal stem cells-derived extracellular vesicles as ‘natural’ drug delivery system for tissue regeneration

Mesenchymal stem cells (MSCs) have abilities to mediate tissue protection through mechanisms of anti-apoptosis, anti-oxidative stress and anti-fibrosis as well as tissue regeneration through mechanisms of cell proliferation, differentiation and angiogenesis. These effects by MSCs are mediated by a variety of factors, including growth factors, cytokines and extracellular vesicles (EVs). Among these factors, EVs, containing proteins, mRNA and microRNAs (miRNA), may carry their contents into distant tissues with high stability. Therefore, the treatment with MSC-derived EVs may be promising as ‘natural’ drug delivery systems (DDS). Especially, the treatment of MSC-derived EVs with the manipulation of specific miRNAs expression has been reported to be beneficial under a variety of diseases and tissue injuries. The overexpression of specific miRNAs in the EVs might be through pre-loading method using the gene editing system by plasmid vector or post-loading method to load miRNA mimics into EVs by electroporation or calcium chloride-mediated transfection. Despite current several challenges for clinical use, it should open the next era of regenerative medicine for a variety of diseases. In this article, we highlight the therapeutic potential of MSC-derived EVs as ‘natural’ DDS and current challenges.

Accelerated capacity of glutamate uptake via blood elements as a possible tool of rapid remote conditioning mediated tissue protection

Recently, the function of blood cells in remote ischemic conditioning (RIC) mediated neuroprotection was undoubtedly confirmed. In the present paper, we have focused on the role of blood elements in glutamate homeostasis. The blood of remote conditioned (tolerant) animals was incubated ex vivo with 100 μM glutamate, and the quantitative and qualitative changes of excitatory amino acid transporters (EAAT 1, 2, and 3) were determined. We confirmed RIC mediated accelerated sequestration of extracellular glutamate via EAATs and altered distribution of that amino acid between plasma and cell elements compared to non-tolerant counterparts. The activity of EAATs was elevated in erythrocytes and monocytes, while the density of transporters was not affected. Quantitative changes of EAAT1 density were detected solely in platelets where the forced scavenging was independent of EAATs inhibition. Surprisingly, the trafficking of immunovisualised EAAT2 and 3 raised at tolerant erythrocytes and monocytes. We have found that protein synthesis underlined this process. On the other hand, depletion of protein synthesis did not significantly affect the scavenging capacity of those cell populations. Our work has demonstrated that the elevated blood scavenging of glutamate overdose could be one of the potential mechanisms underlying RIC mediated tissue protection.

An engineered non-erythropoietic erythropoietin-derived peptide, ARA290, attenuates doxorubicin induced genotoxicity and oxidative stress

Erythropoietin (EPO) applies anti-inflammatory, anti-apoptotic, anti-oxidant and cytoprotective effects besides its hematopoietic action. A nonhematopoietic peptide engineered from EPO, ARA 290, interacts selectively with the innate repair receptor and has similar possessions. ARA290 mediates tissue protection without hematopoietic side-effects of EPO which limit its clinical application. Doxorubicin (DOX) is the broad-spectrum chemotherapeutic agent, but its use is limited by the development of nonspecific toxicity on noncancerous tissues especially in cardiac cells. Mechanisms behind the DOX-induced toxicities are enhanced level of oxidative damage, inflammation and apoptosis. In the present study, we have investigated whether ARA290 acts as a chemoprotective agent modulating the cytotoxicity, genotoxicity and oxidative stress induced in vitro by DOX. The genoprotective effect of ARA290 on DOX-induced toxicity in three cell line (HepG2, HGF & Stem cell) were assessed. Cells were treated with ARA290 (50–400 nM) and DOX (1 μM) in pretreatment condition. Cytotoxicity was evaluated using the MTT assay, genoprotective effect of ARA290 were evaluated using the micronucleus test and comet assay. AR290 significantly reduced the percentage of DNA in tail and the frequency of micronuclei induced by DOX. Besides, DOX impaired anti-oxidant defense enzyme activities and induced inflammation and apoptotic cell death. ARA290 markedly attenuated DOX induced oxidative stress and protected against DOX induced inflammation and apoptotic cell death. This result proposes that ARA290 can act as a protective agent, reducing DOX-induced DNA damage and oxidative stress, and it is possible that this protection could also extend to cardiac cells.

Hormesis mediates dose-sensitive shifts in macrophage activation patterns.

The activation or polarization of macrophages to pro- or anti-inflammatory states evolved as an adaptation to protect against a spectrum of biological threats. Such an adaptation engages pro-oxidative mechanisms and enables macrophages to neutralize and kill threatening organisms (e.g., viruses, bacteria, mold), limit cancerous growths, and enhance recovery and repair processes. The present study demonstrates that (1) many diverse pharmacological, chemical and physical agents can mediate a dose/concentration-dependent shift between pro- and anti-inflammatory activation states, and (2) these shifts in activation states display biphasic dose-response relationships that are characteristic of hormesis. This study also reveals that preconditioning-another form of hormesis-similarly mediates tissue protection by the polarization of macrophages, but in this case, towards an anti-inflammatory phenotype. This assessment supports the generalizability and significance of hormesis in biology, medicine, and public health and further extends it to encompass the hormetic activation of macrophages.

Intraperitoneal injection of MSC-derived exosomes prevent experimental bronchopulmonary dysplasia

Mesenchymal stromal cell (MSC) derived exosomes mediate tissue protection and regeneration in many injuries and diseases by modulating cell protein production, protecting from apoptosis, inhibiting inflammation, and increasing angiogenesis. In the present study, daily intraperitoneal injection of MSC-derived exosomes protected alveolarization and angiogenesis in a newborn rat model of bronchopulmonary dysplasia (BPD) induced by 14 days of neonatal hyperoxia exposure (85% O2). Exosome treatment during hyperoxia prevented disruption of alveolar growth, increased small blood vessel number, and inhibited right heart hypertrophy at P14, P21, and P56. In vitro, exosomes significantly increased tube-like network formation by HUVEC, in part through a VEGF mediated mechanism. In summary, daily intraperitoneal injection of exosomes increased blood vessel number and size in the lung through pro-angiogenic mechanisms. MSC-derived exosomes therefore have both anti-inflammatory and pro-angiogenic mechanism to protect the lung from hyperoxia induced lung and heart disease associated with BPD.

MIF-2/D-DT enhances proximal tubular cell regeneration through SLPI- and ATF4-dependent mechanisms.

Macrophage migration inhibitory factor (MIF) is a cytokine with pleiotropic actions that is produced by several organs and cell types. Depending on the target cell and the inflammatory context, MIF can engage its two component receptor complex CD74 and CD44 and the chemokine receptors CXCR2/4. MIF is constitutively expressed in renal proximal tubular cells, stored in intracellular preformed pools, and released at a low rate. Recently, a second MIF-like protein (i.e., MIF-2/D-DT) has been characterized in mammals. Our study was aimed at examining the role of MIF-2/D-DT, which mediates tissue protection in the heart, in tubular cell regeneration from ischemia-reperfusion injury. We found that Mif-/-, Mif-2-/-, and Cd74-/- mice had significantly worse tubular injury compared with wild-type (WT) control mice and that treatment with MIF-2/D-DT significantly improved recovery of injured epithelial cells. RNAseq analysis of kidney tissue from the ischemia-reperfusion injury model revealed that MIF-2/D-DT treatment stimulates secretory leukocyte proteinase inhibitor (SLPI) and cyclin D1 expression. MIF-2/D-DT additionally activates of eukaryotic initiation factor (eIF) 2α and activating transcription factor (ATF) 4, two transcription factors involved in the integrated stress response (ISR), which is a cellular stress response activated by hypoxia, nutrient deprivation, and oxygen radicals. MIF-2/D-DT also inhibited apoptosis and induced autophagy in hypoxia-treated mouse proximal tubular (MPT) cells. These results indicate that MIF-2/D-DT is an important factor in tubular cell regeneration and may be of therapeutic utility as a regenerative agent in the clinical setting of ischemic acute kidney injury.

Functionalized Biopolymer Particles Enhance Performance of a Tissue-Protective Peptide under Proteolytic and Thermal Stress.

Cutaneous burns are often exacerbated by poor perfusion and subsequent necrosis of the microvasculature surrounding the primary injury. Preservation of these vessels can reduce necrotic tissue expansion and increase success rates of skin graft procedures. Recent work has identified a peptide derived from erythropoietin, ARA290, with the ability to mediate tissue protection in a variety of cell types. Here we demonstrate the advantages of fusing ARA290 to an elastin-like polypeptide (ELP) to salvage microvascular endothelial cells in harsh proteolytic conditions following thermal shock. These fusion proteins were expressed recombinantly in bacterial hosts and rapidly purified by inverse transition cycling. They were shown to spontaneously aggregate into particles at subphysiological temperatures. The bifunctional submicron particles were resistant to digestion in enzymes upregulated after burn injury. Furthermore, the data strongly suggest these ARA290-functionalized particles were superior to treatment with the peptide alone in preventing microvascular cell death in these conditions. The results bring to light an efficient and cost-effective strategy for the delivery therapeutic peptides to proteolytically active wound sites.

Direct Angiotensin II Type 2 Receptor Stimulation Acts Anti-Inflammatory Through Epoxyeicosatrienoic Acid and Inhibition of Nuclear Factor κB

Angiotensin II type 2 (AT(2)) receptors can be regarded as an endogenous repair system, because the AT(2) receptor is upregulated in tissue damage and mediates tissue protection. A potential therapeutic use of this system has only recently come within reach through synthesis of the first selective, orally active, nonpeptide AT(2) receptor agonist, compound 21 (C21; dissociation constant for AT(2) receptor: 0.4 nM; dissociation constant for angiotensin II type 1 receptor: >10,000 nM). This study tested AT(2) receptor stimulation with C21 as a potential future therapeutic approach for the inhibition of proinflammatory cytokines and of nuclear factor kappaB. C21 dose-dependently (1 nM to 1 micromol/L) reduced tumor necrosis factor-alpha-induced interleukin 6 levels in primary human and murine dermal fibroblasts. AT(2) receptor specificity was controlled for by inhibition with the AT(2) receptor antagonist PD123319 and by the absence of effects in AT(2) receptor-deficient cells. AT(2) receptor-coupled signaling leading to reduced interleukin 6 levels involved inhibition of nuclear factor kappaB, activation of protein phosphatases, and synthesis of epoxyeicosatrienoic acid. Inhibition of interleukin 6 promoter activity by C21 was comparable in strength to inhibition by hydrocortisone. C21 also reduced monocyte chemoattractant protein 1 and tumor necrosis factor-alpha in vitro and in bleomycin-induced toxic cutaneous inflammation in vivo. This study is the first to show the anti-inflammatory effects of direct AT(2) receptor stimulation in vitro and in vivo by the orally active, nonpeptide AT(2) receptor agonist C21. These data suggest that pharmacological AT(2) receptor stimulation may be an orally applicable future therapeutic approach in pathological settings requiring the reduction of interleukin 6 or inhibition of nuclear factor kappaB.

A018 Modulation of macrophage activation state protects tissue from necrosis during critical limb ischemia in thrombospondin-1-deficient mice

Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI. Using a genetic model of tsp-1-/- mice subjected to femoral artery excision, we report that tsp-1-/- mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1-/- and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1-/- mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1-/- mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1-/- mice, thereby demonstrating that macrophages mediated tissue protection in these mice. This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

Nitric Oxide and Aspirin: A New Mediator for an Old Drug

Aspirin is known to cause a multitude of pharmacologic actions through inhibition of cyclooxygenase(s) and reduced formation of prostaglandins. Recently, however, novel cytoprotective and antioxidant mechanisms of aspirin have been identified that are independent of cyclooxygenase inhibition. It was shown that aspirin directly stimulates the activity of endothelial nitric oxide (NO) synthase without affecting the expression of endothelial NO synthase. Increased NO formation was found to underlie aspirin-induced sustained protection of endothelial cells from oxidant injury. Downstream targets of NO that mediate tissue protection include the stress proteins heme oxygenase-1 (HO-1) and ferritin. Both HO-1 and ferritin have been identified as targets of, and inducible by, aspirin and, in the case of HO-1, aspirin-triggered lipoxins. It is important to note that these effects are specific to aspirin and not induced by other nonsteroidal antiinflammatory drugs such as diclofenac, indomethacin, or salicylates or by selective cyclooxygenase-2 inhibitors. HO-1 and its antioxidant product bilirubin have been reported to be not only involved in vasoprotection, but to have a similar function in gastric tissue. Stimulation of NO formation through aspirin and ensuing HO-1 induction might therefore help to reduce gastric injury or irritation. Moreover, NO functions as a smooth muscle-relaxing agent and is thus thought to counteract the reduction in gastric blood flow caused by inhibitors of prostaglandin synthesis. It is therefore conceivable that activation of these novel antioxidant pathways contributes, at least in part, to gastric tolerability and the favorable cardiovascular safety profile of aspirin.

Modulation of Macrophage Activation State Protects Tissue from Necrosis during Critical Limb Ischemia in Thrombospondin-1-Deficient Mice

BackgroundMacrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI.Methods and FindingsUsing a genetic model of tsp-1 −/− mice subjected to femoral artery excision, we report that tsp-1 −/− mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1 −/− and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1 −/− mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1 −/− mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1 −/− mice, thereby demonstrating that macrophages mediated tissue protection in these mice.ConclusionThis study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

Cytoprotective doses of erythropoietin or carbamylated erythropoietin have markedly different procoagulant and vasoactive activities.

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However, the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g., the brain) is higher than for treatment of anemia. Notably, a dose-dependent risk of adverse effects has been associated with rhEPO administration, especially in high-risk groups, including polycythemia-hyperviscosity syndrome, hypertension, and vascular thrombosis. Of note, several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity, primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover, we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here, we demonstrate that rhEPO administration in the rat increases systemic blood pressure, reduces regional renal blood flow, and increases platelet counts and procoagulant activities. In contrast, carbamylated rhEPO, a heteromeric receptor-specific ligand that is fully tissue protective, increases renal blood flow, promotes sodium excretion, reduces injury-induced elevation in procoagulant activity, and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands, which appear to elicit fewer adverse effects, may be especially useful in clinical settings for tissue protection.

Renal ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: role of macrophages

The role of monocytes/macrophages in the pathogenesis of ischemia-reperfusion injury (IRI) is unknown. We sought to determine whether activation of macrophage adenosine 2A (A(2A)) receptors (A(2A)Rs) mediates tissue protection. We subjected C57Bl/6 mice infused with clodronate [dichloromethylene bisphosphonate (Cl(2)MBP)] to IRI (32 min of ischemia followed by 24 h of reperfusion) to deplete them of macrophages. IRI induced an elevation of plasma creatinine that was reduced with Cl(2)MBP (26% of control). Adoptive transfer of murine RAW 264.7 cells reconstituted injury, an effect blocked significantly by A(2A) agonists (27% of plasma creatinine from mice reconstituted with macrophages). Macrophages subjected to A(2A) knockout by small interfering RNA were adoptively transferred to macrophage-depleted mice and reconstituted injury (110% of control mice); however, the increase in plasma creatinine was blocked by A(2A) agonists (20% of vehicle treatment). Finally, the A(2A) agonist effect on IRI was blocked in macrophage-depleted A(2A)-knockout mice reconstituted with wild-type RAW 264.7 cells. RNase protection assays 24 h after IRI demonstrated that macrophages are required for IL-6 and TGF-beta mRNA induction. However, A(2A) agonist-mediated tissue protection is independent of IL-6 and TGF-beta mRNA. We conclude that the full extent of IRI requires macrophages and that A(2A) agonist-mediated tissue protection is independent of activation of macrophage A(2A)Rs.

Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor.

The cytokine erythropoietin (Epo) is tissue-protective in preclinical models of ischemic, traumatic, toxic, and inflammatory injuries. We have recently characterized Epo derivatives that do not bind to the Epo receptor (EpoR) yet are tissue-protective. For example, carbamylated Epo (CEpo) does not stimulate erythropoiesis, yet it prevents tissue injury in a wide variety of in vivo and in vitro models. These observations suggest that another receptor is responsible for the tissue-protective actions of Epo. Notably, prior investigation suggests that EpoR physically interacts with the common beta receptor (betacR), the signal-transducing subunit shared by the granulocyte-macrophage colony stimulating factor, and the IL-3 and IL-5 receptors. However, because betacR knockout mice exhibit normal erythrocyte maturation, betacR is not required for erythropoiesis. We hypothesized that betacR in combination with the EpoR expressed by nonhematopoietic cells constitutes a tissue-protective receptor. In support of this hypothesis, membrane proteins prepared from rat brain, heart, liver, or kidney were greatly enriched in EpoR after passage over either Epo or CEpo columns but covalently bound in a complex with betacR. Further, antibodies against EpoR coimmunoprecipitated betacR from membranes prepared from neuronal-like P-19 cells that respond to Epo-induced tissue protection. Immunocytochemical studies of spinal cord neurons and cardiomyocytes protected by Epo demonstrated cellular colocalization of Epo betacR and EpoR. Finally, as predicted by the hypothesis, neither Epo nor CEpo was active in cardiomyocyte or spinal cord injury models performed in the betacR knockout mouse. These data support the concept that EpoR and betacR comprise a tissue-protective heteroreceptor.

Heme oxygenase/carbon monoxide signaling pathways: Regulation and functional significance

Carbon monoxide (CO), a gaseous second messenger, arises in biological systems during the oxidative catabolism of heme by the heme oxygenase (HO) enzymes. HO exists as constitutive (HO-2, HO-3) and inducible isoforms (HO-1), the latter which responds to regulation by multiple stress-stimuli. HO-1 confers protection in vitro and in vivo against oxidative cellular stress. Although the redox active compounds that are generated from HO activity (i.e. iron, biliverdin-IXα, and bilirubin-IXα) potentially modulate oxidative stress resistance, increasing evidence points to cytoprotective roles for CO. Though not reactive, CO regulates vascular processes such as vessel tone, smooth muscle proliferation, and platelet aggregation, and possibly functions as a neurotransmitter. The latter effects of CO depend on the activation of guanylate cyclase activity by direct binding to the heme moiety of the enzyme, stimulating the production of cyclic 3′:5′-guanosine monophosphate. CO potentially interacts with other intracellular hemoprotein targets, though little is known about the functional significance of such interactions. Recent progress indicates that CO exerts novel anti-inflammatory and anti-apoptotic effects dependent on the modulation of the p38 mitogen activated protein kinase (MAPK)-signaling pathway. By virtue of these effects, CO confers protection in oxidative lung injury models, and likely plays a role in HO-1 mediated tissue protection.