Inhibition of TIM‐4 protects against cerebral ischaemia‐reperfusion injury

TIM‐4 plays an important role in ischaemia‐reperfusion injury of liver and kidney; however, the effects of TIM‐4 on cerebral ischaemia‐reperfusion injury (IRI) are unknown. The purpose of the present study was to investigate the potential role of TIM‐4 in experimental brain ischaemia‐reperfusion injury. In this study, cerebral ischaemia reperfusion was induced by transient middle cerebral artery occlusion (MCAO) for 1 hour in C57/BL6 mice. The TIM‐4 expression was detected in vivo or vitro by real‐time quantitative polymerase chain reaction, Western blot and flow cytometric analysis. In vivo, the administration of anti‐TIM‐4 antibodies significantly suppressed apoptosis, inhibited inflammatory cells and enhanced anti‐inflammatory responses. In vitro, activated microglia exhibited reduced cellular proliferation and induced IRI injury when co‐cultured with neurons; these effects were inhibited by anti‐TIM‐4 antibody treatment. Similarly, microglia transfected with TIM‐4 siRNA and stimulated by LPS + IFN‐γ alleviated the TIM‐4‐mediated damage to neurons. Collectively, our data indicate that the inhibition of TIM‐4 can improve the inflammatory response and exerts a protective effect in cerebral ischaemia‐reperfusion injury.     

D1 receptor‐mediated endogenous tPA upregulation contributes to blood‐brain barrier injury after acute ischaemic stroke

Blood‐brain barrier (BBB) integrity injury within the thrombolytic time window is becoming a critical target to reduce haemorrhage transformation (HT). We have previously reported that BBB damage was initially damaged in non‐infarcted striatum after acute ischaemia stroke. However, the underlying mechanism is not clear. Since acute ischaemic stroke could induce a significant increase of dopamine release in striatum, in current study, our aim is to investigate the role of dopamine receptor signal pathway in BBB integrity injury after acute ischaemia using rat middle cerebral artery occlusion model. Our data showed that 2‐h ischaemia induced a significant increase of endogenous tissue plasminogen activator (tPA) in BBB injury area and intra‐striatum infusion of tPA inhibitor neuroserpin, significantly alleviated 2‐h ischaemia‐induced BBB injury. In addition, intra‐striatum infusion of D1 receptor antagonist SCH23390 significantly decreased ischaemia‐induced upregulation of endogenous tPA, accompanied by decrease of BBB injury and occludin degradation. More important, inhibition of hypoxia‐inducible factor‐1 alpha with inhibitor YC‐1 significantly decreased 2‐h ischaemia‐induced endogenous tPA upregulation and BBB injury. Taken together, our data demonstrate that acute ischaemia disrupted BBB through activation of endogenous tPA via HIF‐1α upregulation, thus representing a new therapeutic target for protecting BBB after acute ischaemic stroke.     

MiR‐21‐5p/dual‐specificity phosphatase 8 signalling mediates the anti‐inflammatory effect of haem oxygenase‐1 in aged intracerebral haemorrhage rats

Intracerebral haemorrhage (ICH) is a severe neurological disorder caused by bleeding within the brain tissue. Inflammation has been implicated in ICH pathogenesis and is a potential therapeutic target for ICH. Haemin, an activator of haem oxygenase‐1 (HO‐1), rapidly increases HO‐1 protein expression and activity and has been shown to distinctly affect anti‐inflammatory functions after central nervous system (CNS) injury. However, less is known about the mechanisms that underlie the anti‐inflammatory effects of haemin in aged rats post‐ICH. Here, we performed microarray analysis to identify miRNAs that respond strongly to HO‐1 regulation in ICH rats and found that miR‐21‐5p induced the most significant change. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and Gene Ontology (GO) analysis, we focused on dual‐specificity phosphatase 8 (DUSP8) from the predicted miR‐21‐5p targets. Luciferase reporter assays confirmed that miR‐21‐5p bound directly to DUSP8. MiR‐21‐5p upregulation in vitro downregulated DUSP8 expression. Importantly, intracerebroventricularly injecting antagomir for miR‐21‐5p (A‐miR‐21‐5p), which was used to inhibit miR‐21‐5p in aged ICH rats, significantly reduced the neurological defects, repaired cognitive impairment, alleviated blood–brain barrier (BBB) permeability, inhibited neuronal apoptosis posthaemorrhage and accelerated haematoma absorption. In addition, serum miR‐21‐5p levels were notably elevated in patients relative to healthy individuals and were correlated with National Institutes of Health Stroke Scale (NIHSS) scores and clinical outcomes. In summary, A‐miR‐21‐5p increased HO‐1 expression in cerebral haematomas, thus eliciting the DUSP8‐modulated perifocal neuroprotective effect of haemin. MiR‐21‐5p with haemin therapy may be a potential therapy post‐ICH.     

The role and therapeutic potential of heat shock proteins in haemorrhagic stroke

Heat shock proteins (HSPs) are induced after haemorrhagic stroke, which includes subarachnoid haemorrhage (SAH) and intracerebral haemorrhage (ICH). Most of these proteins function as neuroprotective molecules to protect cerebral neurons from haemorrhagic stroke and as markers to indicate cellular stress or damage. The most widely studied HSPs in SAH are HSP70, haeme oxygenase‐1 (HO‐1), HSP20 and HSP27. The subsequent pathophysiological changes following SAH can be divided into two stages: early brain injury and delayed cerebral ischaemia, both of which determine the outcome for patients. Because the mechanisms of HSPs in SAH are being revealed and experimental models in animals are continually maturing, new agents targeting HSPs with limited side effects have been suggested to provide therapeutic potential. For instance, some pharmaceutical agents can block neuronal apoptosis signals or dilate cerebral vessels by modulating HSPs. HO‐1 and HSP70 are also critical topics for ICH research, which can be attributed to their involvement in pathophysiological mechanisms and therapeutic potential. However, the process of HO‐1 metabolism can be toxic owing to iron overload and the activation of succedent pathways, for example, the Fenton reaction and oxidative damage; the overall effect of HO‐1 in SAH and ICH tends to be protective and harmful, respectively, given the different pathophysiological changes in these two types of haemorrhagic stroke. In the present study, we focus on the current understanding of the role and therapeutic potential of HSPs involved in haemorrhagic stroke. Therefore, HSPs may be potential therapeutic targets, and new agents targeting HSPs are warranted.     

The vascular protective role of oestradiol: a focus on postmenopausal oestradiol deficiency and aneurysmal subarachnoid haemorrhage

The steroid hormone, oestradiol, has pleiotropic functions. The protective effects of oestradiol are attributed to its anti‐inflammatory, antioxidant, anti‐atherogenic, anti‐apoptotic, vasodilatory activities and regulation of micro RNA. Oestradiol upregulates endothelial nitric oxide synthase gene expression and increases the production of nitric oxide, an important vasodilator. It suppresses the renin–angiotensin system and monitors haemodynamic stress. The hormone maintains the integrity of blood vessels by reducing oxidative stress while upregulating the expression of antioxidant enzymes and prevents vascular inflammation by regulating pro‐ and anti‐inflammatory cytokines. Aneurysmal subarachnoid haemorrhage (aSAH) occurring as a consequence of the rupture of an intracranial aneurysm is a devastating cerebrovascular event, representing 5–7% of all strokes. Postmenopausal women are more susceptible to aSAH compared to men in the same age group. This gender disparity has been attributed to reduced levels of the vascular protective hormone oestradiol following menopause. This review is focused on the protective role of oestradiol on vasculature and how the drop in oestradiol levels after menopause dramatically increases the incidence of aSAH in women. During menopause, oestradiol deficiency may affect vascular integrity causing dysregulation of vascular homeostasis by affecting the renin–angiotensin–aldosterone system (RAAS) and inflammatory and apoptotic cascades, resulting in the weakening of the cerebral arterial wall and potentially to development of an aneurysm and its rupture. In view of the role of oestradiol in maintaining vascular integrity, treatments involving hormone replacement could be a promising approach in postmenopausal women who are at risk of developing or rupturing an intracranial aneurysm.     

Ischaemia alters the effects of cardiomyocyte‐derived extracellular vesicles on macrophage activation

Myocardial ischaemia is associated with an exacerbated inflammatory response, as well as with a deregulation of intercellular communication systems. Macrophages have been implicated in the maintenance of heart homeostasis and in the progression and resolution of the ischaemic injury. Nevertheless, the mechanisms underlying the crosstalk between cardiomyocytes and macrophages remain largely underexplored. Extracellular vesicles (EVs) have emerged as key players of cell‐cell communication in cardiac health and disease. Hence, the main objective of this study was to characterize the impact of cardiomyocyte‐derived EVs upon macrophage activation. Results obtained demonstrate that EVs released by H9c2 cells induced a pro‐inflammatory profile in macrophages, via p38MAPK activation and increased expression of iNOS, IL‐1β and IL‐6, being these effects less pronounced with ischaemic EVs. EVs derived from neonatal cardiomyocytes, maintained either in control or ischaemia, induced a similar pattern of p38MAPK activation, expression of iNOS, IL‐1β, IL‐6, IL‐10 and TNFα. Importantly, adhesion of macrophages to fibronectin was enhanced by EVs released by cardiomyocytes under ischaemia, whereas phagocytic capacity and adhesion to cardiomyocytes were higher in macrophages incubated with control EVs. Additionally, serum‐circulating EVs isolated from human controls or acute myocardial infarction patients induce macrophage activation. According to our model, in basal conditions, cardiomyocyte‐derived EVs maintain a macrophage profile that ensure heart homeostasis, whereas during ischaemia, this crosstalk is affected, likely impacting healing and post‐infarction remodelling.     

Haematoma scavenging in intracerebral haemorrhage: from mechanisms to the clinic

The products of erythrocyte lyses, haemoglobin (Hb) and haem, are recognized as neurotoxins and the main contributors to delayed cerebral oedema and tissue damage after intracerebral haemorrhage (ICH). Finding a means to efficiently promote absorption of the haemolytic products (Hb and haem) around the bleeding area in the brain through stimulating the function of the body's own garbage cleaning system is a novel clinical challenge and critical for functional recovery after ICH. In this review, available information of the brain injury mechanisms underlying ICH and endogenous haematoma scavenging system is provided. Meanwhile, potential intervention strategies are discussed. Intracerebral blood itself has ‘toxic’ effects beyond its volume effect after ICH. Haptoglobin–Hb–CD163 as well as haemopexin–haem–LRP1 is believed to be the most important endogenous scavenging pathway which participates in blood components resolution following ICH. PPARγ–Nrf2 activates the aforementioned clearance pathway and then accelerates haematoma clearance. Meanwhile, the scavenger receptors as novel targets for therapeutic interventions to treat ICH are also highlighted.     

Cerebral inflammation contributes to encephalopathy and brain edema in acute liver failure: protective effect of minocycline

Encephalopathy and brain edema are serious complications of acute liver failure (ALF). The precise pathophysiologic mechanisms responsible have not been fully elucidated but it has been recently proposed that microglia‐derived proinflammatory cytokines are involved. In the present study we evaluated the role of microglial activation and the protective effect of the anti‐inflammatory drug minocycline in the pathogenesis of hepatic encephalopathy and brain edema in rats with ALF resulting from hepatic devascularisation. ALF rats were killed 6 h after hepatic artery ligation before the onset of neurological symptoms and at coma stages of encephalopathy along with their appropriate sham‐operated controls and in parallel with minocycline‐treated ALF rats. Increased OX‐42 and OX‐6 immunoreactivities confirming microglial activation were accompanied by increased expression of interleukins (IL‐1β, IL‐6) and tumor necrosis factor‐alpha (TNF‐α) in the frontal cortex at coma stage of encephalopathy in ALF rats compared with sham‐operated controls. Minocycline treatment prevented both microglial activation as well as the up‐regulation of IL‐1β, ΙL‐6 and TNF‐α mRNA and protein expression with a concomitant attenuation of the progression of encephalopathy and brain edema. These results offer the first direct evidence for central proinflammatory mechanisms in the pathogenesis of brain edema and its complications in ALF and suggest that anti‐inflammatory agents may be beneficial in these patients.     

Anti‐inflammatory effects of doxepin hydrochloride against LPS‐induced C6‐glioma cell inflammatory reaction by PI3K‐mediated Akt signaling

Recent studies have shown that tricyclic antidepressants (TCAs) may have anti‐inflammatory and anticonvulsant effects in addition to its antidepressant effects. So far, the nonantidepressant effects of TCAs and their molecular pharmacological mechanisms remain completely unclear. Chronic inflammation in the brain parenchyma may be related to the pathogenesis and progression of various neurodegenerative diseases. As a common antidepressant and anti‐insomnia drug, doxepin also may be a potential anti‐inflammatory and anticonvulsant drug, so the study on the anti‐inflammatory protective effect of doxepin and its molecular mechanism has become a very important issue in pharmacology and clinical medicine. Further elucidating the anti‐inflammatory and neuroprotective effects of doxepin and its molecular mechanism may provide the important theoretical and clinical basis for the prevention and treatment of neurodegenerative disease. This study was designed to understand the glio‐protective mechanism of doxepin against the inflammatory damage induced by lipopolysaccharide (LPS) exposure in C6‐glioma cells. We found the treatment of C6‐glioma cells with LPS results in deleterious effects, including the augmentation of inflammatory cytokine levels (tumor necrosis factor‐α, interleukin‐1β), and suppresses the Akt phosphorylation. Furthermore, our outcomes demonstrated that doxepin was able to suppress these effects induced by LPS, through activation of the phosphatidylinositol‐3‐kinase‐mediated protein kinase B (Akt) pathway. To sum up, these results highlight the potential role of doxepin against neuroinflammatory‐related disease in the brain.     

Netrin‐1 alleviates subarachnoid haemorrhage‐induced brain injury via the PPARγ/NF‐KB signalling pathway

Netrin‐1 (NTN‐1) is a novel drug to alleviate early brain injury following subarachnoid haemorrhage (SAH). However the molecular mechanism of NTN‐1‐mediated protection against early brain injury following SAH remains largely elusive. This study aims to evaluate the effects and mechanisms of NTN‐1 in protecting SAH‐induced early brain injury. The endovascular perforation SAH model was constructed using male C57BL/6J mice, and recombinant NTN‐1 was administrated intravenously. Mortality rates, SAH grade, brain water content, neurological score and neuronal apoptosis were evaluated. The expression of PPARγ, Bcl‐2, Bax and nuclear factor‐kappa B (NF‐κB) were detected by Western blot. Small interfering RNA specific to NTN‐1 receptor, UNC5B, and a selective PPARγ antagonist, bisphenol A diglycidyl ether (BADGE), were applied in combination with NTN‐1. The results suggested that NTN‐1 improved the neurological deficits, reduced the brain water content and alleviated neuronal apoptosis. In addition, NTN‐1 enhanced PPARγ and Bcl‐2 expression and decreased the levels of Bax and NF‐κB. However, the neuroprotection of NTN‐1 was abolished by UNC5B and BADGE. In conclusion, our results demonstrated that NTN‐1 attenuates early brain injury following SAH via the UNC5B PPARγ/NF‐κB signalling pathway.     

Prophylactic effects of ellagic acid and rosmarinic acid on doxorubicin‐induced neurotoxicity in rats

Doxorubicin (DOX) is a chemotherapeutic agent widely used in human malignancies. Its long‐term use cause neurobiological side effects. The aim of the present study was to investigate the prophylactic effect exerted by daily administration of ellagic acid (EA) and rosmarinic acid (RA) on DOX‐induced neurotoxicity in rats. Our data showed that DOX‐induced significant elevation of brain malondialdehyde, tumor necrosis factor‐alpha (TNF‐α), inducible nitric oxide synthase (iNOS), caspase‐3, and cholinesterase associated with significant reduction in reduced glutathione, monoamines namely serotonin, dopamine, as well as norepinephrine. Concomitant administration of EA (10 mg/kg/day, p.o. for 14 days) and/or RA (75 mg/kg/day, p.o. for 14 days) with DOX significantly mitigated the neural changes induced by DOX. Meanwhile, treatment ameliorated pro‐inflammatory cytokines as TNF‐α, iNOS, and attenuated oxidative stress biomarkers as well as brain monoamines. In conclusion, EA and RA can effectively protect against DOX‐induced neurotoxicity, and the mechanisms underlying the neuroprotective effect are potentially associated with its antioxidant, anti‐inflammatory, and antiapoptotic properties.     

Macrophage polarization: a key event in the secondary phase of acute spinal cord injury

Acute spinal cord injury (SCI) has become epidemic in modern society. Despite advances made in the understanding of the pathogenesis and improvements in early recognition and treatment, it remains a devastating event, often producing severe and permanent disability. SCI has two phases: acute and secondary. Although the acute phase is marked by severe local and systemic events such as tissue contusion, ischaemia, haemorrhage and vascular damage, the outcome of SCI are mainly influenced by the secondary phase. SCI causes inflammatory responses through the activation of innate immune responses that contribute to secondary injury, in which polarization‐based macrophage activation is a hallmarker. Macrophages accumulated within the epicentre and the haematoma of the injured spinal cord play a significant role in this inflammation. Depending on their phenotype and activation status, macrophages may initiate secondary injury mechanisms and/or promote CNS regeneration and repair. When it comes to therapies for SCI, very few can be performed in the acute phase. However, as macrophage activation and polarization switch are exquisitely sensitive to changes in microenvironment, some trials have been conducted to modulate macrophage polarization towards benefiting the recovery of SCI. Given this, it is important to understand how macrophages and SCI interrelate and interact on a molecular pathophysiological level. This review provides a comprehensive overview of the immuno‐pathophysiological features of acute SCI mainly from the following perspectives: (i) the overview of the pathophysiology of acute SCI, (ii) the roles of macrophage, especially its polarization switch in acute SCI, and (iii) newly developed neuroprotective therapies modulating macrophage polarization in acute SCI.     

Human adipose stromal cell therapy improves survival and reduces renal inflammation and capillary rarefaction in acute kidney injury

Damage to endothelial cells contributes to acute kidney injury (AKI) by causing impaired perfusion, while the permanent loss of the capillary network following AKI has been suggested to promote chronic kidney disease. Therefore, strategies to protect renal vasculature may impact both short‐term recovery and long‐term functional preservation post‐AKI. Human adipose stromal cells (hASCs) possess pro‐angiogenic and anti‐inflammatory properties and therefore have been tested as a therapeutic agent to treat ischaemic conditions. This study evaluated hASC potential to facilitate recovery from AKI with specific attention to capillary preservation and inflammation. Male Sprague Dawley rats were subjected to bilateral ischaemia/reperfusion and allowed to recover for either two or seven days. At the time of reperfusion, hASCs or vehicle was injected into the suprarenal abdominal aorta. hASC‐treated rats had significantly greater survival compared to vehicle‐treated rats (88.7% versus 69.3%). hASC treatment showed hastened recovery as demonstrated by lower creatinine levels at 48 hrs, while tubular damage was significantly reduced at 48 hrs. hASC treatment resulted in a significant decrease in total T cell and Th17 cell infiltration into injured kidneys at 2 days post‐AKI, but an increase in accumulation of regulatory T cells. By day 7, hASC‐treated rats showed significantly attenuated capillary rarefaction in the cortex (15% versus 5%) and outer medulla (36% versus 18%) compared to vehicle‐treated rats as well as reduced accumulation of interstitial alpha‐smooth muscle actin‐positive myofibroblasts. These results suggest for the first time that hASCs improve recovery from I/R‐induced injury by mechanisms that contribute to decrease in inflammation and preservation of peritubular capillaries.     

The autophagy–lysosomal system in subarachnoid haemorrhage

The autophagy–lysosomal pathway is a self‐catabolic process by which dysfunctional or unnecessary intracellular components are degraded by lysosomal enzymes. Proper function of this pathway is critical for maintaining cell homeostasis and survival. Subarachnoid haemorrhage (SAH) is one of the most devastating forms of stroke. Multiple pathogenic mechanisms, such as inflammation, apoptosis, and oxidative stress, are all responsible for brain injury and poor outcome after SAH. Most recently, accumulating evidence has demonstrated that the autophagy–lysosomal pathway plays a crucial role in the pathophysiological process after SAH. Appropriate activity of autophagy–lysosomal pathway acts as a pro‐survival mechanism in SAH, while excessive self‐digestion results in cell death after SAH. Consequently, in this review article, we will give an overview of the pathophysiological roles of autophagy–lysosomal pathway in the pathogenesis of SAH. And approaching the molecular mechanisms underlying this pathway in SAH pathology is anticipated, which may ultimately allow development of effective therapeutic strategies for SAH patients through regulating the autophagy–lysosomal machinery.     

Apolipoprotein E modulates the CNS response to injury

Apolipoprotein E (apoE) plays an important role in recovery from acute brain injury. One potential mechanism for this is that apoE down‐regulates glial activation and subsequent secretion of inflammatory mediators. Following a pneumatic impact to the closed skull of anesthetized apoE deficient and wildtype mice, MRI was performed to quantify the effacement of the lateral ventricles as a radiographic surrogate for cerebral edema. At 24 hours following injury, apoE deficient animals had a greater degree of cerebral edema as compared to matched controls. This increase in edema was associated with enhanced up‐regulation of TNFα, a pro‐inflammatory cytokine believed to play an important role in mediating blood–brain barrier breakdown and the development of cerebral edema. We have previously demonstrated that a peptide derived from the receptor binding region of apoE retained the anti‐inflammatory activities of the holoprotein in vitro, and we next investigated whether a single intravenous administration of this apoE‐mimetic peptide could exert neuroprotective effects. Thirty minutes after closed head injury, mice were randomized to receive low dose peptide (203 mg/kg); high dose peptide (406 mg/kg), or vehicle. At 24 h post injury, the saline injected animals had a profound deficit in rotorod testing which was associated with weight loss. A single intravenous injection of apoE‐mimetic peptide 30 min following closed head injury protected against motor deficit, weight loss, and neurocognitive deficit in a dose dependent fashion. This suggests that apoE may modify prognosis in brain injury by down‐regulating the CNS inflammatory response, and that mimicking the biological activity of apoE may lead to novel therapeutic strategies in the setting.     

Renalase contributes to the renal protection of delayed ischaemic preconditioning via the regulation of hypoxia‐inducible factor‐1α

Ischaemic preconditioning (IPC) attenuates acute kidney injury (AKI) from renal ischaemia reperfusion. Renalase, an amine oxidase secreted by the proximal tubule, not only degrades circulating catecholamines but also protects against renal ischaemia reperfusion injury. Here, it has been suggested that the renoprotective effect of renal IPC is partly mediated by renalase. In a model of brief intermittent renal IPC, the increased cortex renalase expression was found to last for 48 hrs. IPC significantly reduced renal tubular inflammation, necrosis and oxidative stress following renal ischaemia reperfusion injury. Such effects were attenuated by blocking renalase with an anti‐renalase monoclonal antibody. We further demonstrated that renalase expression was up‐regulated by hypoxia in vitro via an hypoxia‐inducible factor (HIF)‐1α mechanism. The IPC‐induced up‐regulation of renalase in vivo was also reduced by pre‐treatment with an HIF‐1α inhibitor, 3‐(5′‐Hydroxymethyl‐2′‐furyl)‐1‐benzyl indazole. In summary, the renoprotective effect of IPC is partly dependent on the renalase expression, which may be triggered by hypoxia via an HIF‐1α mechanism. Endogenous renalase shows potential as a therapeutic agent for the prevention and treatment of AKI.     

CRISPR/Cas9‐mediated whole genomic wide knockout screening identifies mitochondrial ribosomal proteins involving in oxygen‐glucose deprivation/reperfusion resistance

Recanalization therapy by intravenous thrombolysis or endovascular therapy is critical for the treatment of cerebral infarction. However, the recanalization treatment will also exacerbate acute brain injury and even severely threatens human life due to the reperfusion injury. So far, the underlying mechanisms for cerebral ischaemia‐reperfusion injury are poorly understood and effective therapeutic interventions are yet to be discovered. Therefore, in the research, we subjected SK‐N‐BE(2) cells to oxygen‐glucose deprivation/reperfusion (OGDR) insult and performed a pooled genome‐wide CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated protein 9) knockout screen to discover new potential therapeutic targets for cerebral ischaemia‐reperfusion injury. We used Metascape to identify candidate genes which might involve in OGDR resistance. We found that the genes contributed to OGDR resistance were primarily involved in neutrophil degranulation, mitochondrial translation, and regulation of cysteine‐type endopeptidase activity involved in apoptotic process and response to oxidative stress. We then knocked down some of the identified candidate genes individually. We demonstrated that MRPL19, MRPL32, MRPL52 and MRPL51 inhibition increased cell viability and attenuated OGDR‐induced apoptosis. We also demonstrated that OGDR down‐regulated the expression of MRPL19 and MRPL51 protein. Taken together, our data suggest that genome‐scale screening with Cas9 is a reliable tool to analyse the cellular systems that respond to OGDR injury. MRPL19 and MRPL51 contribute to OGDR resistance and are supposed to be promising targets for the treatment of cerebral ischaemia‐reperfusion damage.     

Osteopontin stimulates autophagy via integrin/CD44 and p38 MAPK signaling pathways in vascular smooth muscle cells

Osteopontin (OPN) exerts pro‐inflammatory effect and is associated with the development of abdominal aortic aneurysm (AAA). However, the molecular mechanism underlying this association remains obscure. In the present study, we compared gene expression profiles of AAA tissues using microarray assay, and found that OPN was the highest expressed gene (>125‐fold). Furthermore, the expression of LC3 protein and autophagy‐related genes including Atg4b, Beclin1/Atg6, Bnip3, and Vps34 was markedly upregulated in AAA tissues. To investigate the ability of OPN to stimulate autophagy as a potential mechanism involved in the pathogenesis of this disease, we treated vascular smooth muscle cells (SMCs) with OPN, and found that OPN significantly increased the formation of autophagosomes, expression of autophagy‐related genes and cell death, whereas blocking the signal by anti‐OPN antibody markedly inhibited OPN‐induced autophagy and SMC death. Furthermore, inhibition of integrin/CD44 and p38 MAPK signaling pathways markedly abrogated the biological effects of OPN on SMCs. These data for the first time demonstrate that OPN sitmulates autophagy directly through integrin/CD44 and p38 MAPK‐mediated pathways in SMCs. Thus, inhibition of OPN‐induced autophagy might be a potential therapeutic target in the treatment of AAA disease. J. Cell. Physiol. 227: 127–135, 2012. © 2011 Wiley Periodicals, Inc.