Growth differentiation factor 7 pretreatment enhances the therapeutic capacity of bone marrow-derived mesenchymal stromal cells against cerebral ischemia-reperfusion injury.

Bone marrow-derived mesenchymal stem cells (BMSCs) transplantation is a promising therapeutic strategy for cerebral ischemia/reperfusion (I/R) injury; however, the clinical outcome is barely satisfactory and demands further improvement. The present study aimed to investigate whether preconditioning of BMSCs by recombinant human growth differentiation factor 7 (rhGDF7) could enhance its therapeutic capacity against cerebral I/R injury. Mouse BMSCs and primary neurons were co-cultured and exposed to oxygen glucose deprivation/reperfusion (OGD/R) stimulation. To investigate the role of exosomal microRNA-369-3p (miR-369-3p), inhibitors, RNAi and the miR-369-3p antagomir were used. Meanwhile, mice were intravenously injected with rhGDF7-preconditioned BMSCs and then received cerebral I/R surgery. Markers of inflammation, oxidative stress and neural damage were evaluated. To inhibit AMP-activated protein kinase (AMPK), compound C was used in vivo and in vitro. Compared with cell-free transwell or vehicle-preconditioned BMSCs, rhGDF7-preconditioned BMSCs significantly prevented OGD/R-induced inflammation, oxidative stress and neural damage in vitro. Meanwhile, rhGDF7-preconditioned BMSCs could prevent I/R-induced cerebral inflammation and oxidative stress in vivo. Mechanistically, rhGDF7 preconditioning significantly increased exosomal miR-369-3p expression in BMSCs and then transferred exosomal miR-369-3p to primary neurons, where it bound to phosphodiesterase 4 D (Pde4d) 3'-UTR and downregulated PDE4D expression, thereby preventing I/R-induced inflammation, oxidative stress and neural damage through activating AMPK pathway. Our study identify GDF7 pretreatment as a promising adjuvant reagent to improve the therapeutic potency of BMSCs for cerebral I/R injury and ischemic stroke.

MicroRNA-32-3p facilitates cerebral ischemia/reperfusion injury through inhibiting Cab39/AMPK.

Oxidative stress is a key pathogenic factor of cerebral ischemia/reperfusion (I/R) injury. MicroRNA-32-3p (miR-32-3p) plays critical roles in regulating ischemic diseases; however, its role in oxidative stress and cerebral I/R injury remains elusive. Primary cortical neurons and rats were treated with the agomir, antagomir and matched controls of miR-32-3p, and then received oxygen glucose deprivation/reperfusion (OGD/R) or I/R stimulation. To investigate the involvement of AMP-activated protein kinase (AMPK) and calcium-binding protein 39 (Cab39), a pharmacological inhibitor and small interfering RNA were used in vivo and in vitro. Herein, we found that miR-32-3p was upregulated in OGD/R-treated neurons and I/R-injured brains, and that inhibiting miR-32-3p by the miR-32-3p antagomir dramatically alleviated oxidative stress and neural death in OGD/R-stimulated primary cortical neurons. Conversely, overexpressing miR-32-3p by the miR-32-3p agomir further aggravated OGD/R-induced neural death and oxidative damage in primary cortical neurons. Meanwhile, we observed that the miR-32-3p antagomir prevented, while the miR-32-3p agomir facilitated neural death, oxidative damage and cerebral I/R injury in vivo. Mechanistically, miR-32-3p bound to the 3'-untranslated regions of Cab39, inhibited its protein level and subsequently inactivated AMPK. Conversely, treatment with the miR-32-3p antagomir upregulated Cab39 and activated AMPK, thereby attenuating oxidative damage and cerebral I/R injury. Moreover, inhibiting AMPK or Cab39 dramatically blocked the miR-32-3p antagomir-mediated beneficial effects against cerebral I/R injury in vivo and in vitro. miR-32-3p plays critical roles in neural death and oxidative damage upon I/R stimulation, and it is a novel target to treat cerebral I/R injury.

circHIPK3 regulates apoptosis and mitochondrial dysfunction induced by ischemic stroke in mice by sponging miR-148b-3p via CDK5R1/SIRT1.

Circular RNAs (circRNAs) are aberrantly expressed in the brain and play a role in a variety of central nervous system diseases. However, the essential role and therapeutic potential of circRNAs in ischemic stroke (IS) are poorly understood. Here, using circRNA sequencing, we showed that circRNA homeodomain-interacting protein kinase 3 (circHIPK3) was abundantly expressed in ischemic brain tissues in transient middle cerebral artery occlusion (tMCAO)-evoked stroke model mice. Knockdown of circHIPK3 markedly reduced the infarct volume, brain water content, neurological deficit scores, and blood-brain permeability and ameliorated brain microvascular endothelial cell (BMEC) apoptosis and mitochondrial dysfunction in tMCAO mice. Gain- and loss-of-function experiments were performed to verify the effects of miR-148b-3p on oxygen-glucose deprivation (OGD)-induced BMEC apoptosis and mitochondrial dysfunction. Mechanistically, circHIPK3 functions as an endogenous sponge of miR-148b-3p to decrease its activity, resulting in upregulation of CDK5R1 and CDK5 expression, downregulation of SIRT1 expression and subsequent BMEC apoptosis and mitochondrial dysfunction. Collectively, our findings suggest that circHIPK3 and its coupling mechanism are implicated in IS, providing translational evidence that circHIPK3 could be a key therapeutic target for IS.

N6-methyladenosine modulation classes and immune microenvironment regulation in ischemic stroke.

N6-methyladenosine (m6A) modifications play an important role in the differentiation and regulation of immune cells. However, research on m6A in ischemic stroke (IS) is still in its infancy, and their role of the immune microenvironment remains unknown. In this study, we systematically assessed the modification classes of m6A regulators in IS based on the GEO database (GSE16561 and GSE22255). We found that in IS patients, IGF2BP2, IGF2BP1, and YTHDF2 expression was significantly upregulated, and ELAVL1, LRPPRC, METTL3, ALKBH5, CBLL1, and METTL14 expression was significantly downregulated. Seven IS-related genes (ELAVL1, IGF2BP2, LRPPRC, YTHDF2, ALKBH5, METTL14, and YTHDC1) were finally screened by logistic and least absolute shrinkage and selection operator (LASSO) regressions, and the AUC of the riskScore was 0.942, which was a good classification. For immune infiltration, there were highly significant differences in memory B cells, CD8 T cells, monocytes, activated dendritic cells, and mast cells between IS and normal samples. The IS samples were grouped into three classes by consistent clustering, and 15 m6A genes were differentially expressed in the different classes. Multiple infiltrating immune cells, immune-associated genes, and HLA-associated genes differed significantly across m6A modification classes, indicating the diversity and complexity of m6A modifications in the immune microenvironment of IS. Finally, 487 genes associated with the m6A modification class were identified, and 227 potential drugs were found. Our findings demonstrated that m6A modification plays a crucial role in the immune regulation of IS.

Bioinformatics Identification of Ferroptosis-Related Biomarkers and Therapeutic Compounds in Ischemic Stroke.

Background: Stroke is one of the most common deadly diseases with an estimated 780,000 new cases globally, of which ischemic stroke accounts for over 80% of all cases. Ferroptosis is a new form of programmed cell death that plays a vital role in many diseases, including ischemic stroke and heart diseases. The role of the ferroptosis-related gene in the diagnosis, prognosis, or therapy of ischemic stroke was not fully clarified. Methods: Ferroptosis-related differentially expressed genes (DEGs) in ischemic stroke were identified by bioinformatic analysis of the GSE16561 and GSE22255 datasets. Subsequently, receiver operator characteristic (ROC) monofactor analysis was performed to evaluate the diagnostic value of ferroptosis-related biomarkers in ischemic stroke. Results: A total of 10 ferroptosis-related DEGs were identified in ischemic stroke vs. normal control. GO and KEGG analysis revealed that these 10 ferroptosis-related DEGs were mainly enriched in response to oxidative stress, HIF-1 signaling pathway, ferroptosis, lipid, and atherosclerosis. Moreover, the random forest model suggested three ferroptosis-related biomarkers, namely, PTGS2, MAP1LC3B, and TLR4, for ischemic stroke. Interestingly, the expression of PTGS2, MAP1LC3B, and TLR4 was upregulated in ischemic stroke. ROC monofactor analysis demonstrated a good performance of MAP1LC3B, PTGS2, and TLR4 in the diagnosis of ischemic stroke. The expression and diagnostic value of MAP1LC3B, PTGS2, and TLR4 in ischemic stroke were also verified using GSE22255. We also revealed the transcription factor regulation network and co-expressed protein network of ferroptosis-related biomarkers. Several potential therapeutic compounds corresponding to MAP1LC3B, PTGS2, and TLR4 were also identified for ischemic stroke, including Zinc12503187 (Conivaptan), Zinc3932831 (Avodart), Zinc64033452 (Lumacaftor), Zinc11679756 (Eltrombopag), Zinc100378061 (Naldemedine), and Zinc3978005 (Dihydroergotamine). Conclusion: Our results suggested MAP1LC3B, PTGS2, and TLR4 as potential diagnostic biomarkers for ischemic stroke, providing more evidence about the vital role of ferroptosis in ischemic stroke.

Integrative bioinformatics analysis of a prognostic index and immunotherapeutic targets in renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the most common malignancies. The immunogenomic landscape signature significantly correlates with the progression and prognosis of RCC. Novel therapeutic targets and prognostic indices in RCC are highly desirable. The TCGA database enables comprehensive immunogenomic landscape analysis. Differentially expressed immune-related genes (IRGs) were obtained from TCGA and GO analyses, and KEGG pathway analyses were performed to explore their functions and molecular mechanisms. Multivariable Cox analysis was utilized to calculate the risk score of each patient and locate survival-associated IRGs, thereby constructing a novel immune-related gene-based prognostic index (IRGPI). The correlation between IRGPI and immune cell infiltration was also investigated. A total of 41 differentially expressed IRGs were notably related to prognosis in RCC. GO functions and KEGG pathway analyses demonstrated that these genes were primarily associated with the tumour immune response and cytokine-cytokine receptor interaction pathway. An IRGPI based on seventeen survival-associated differentially expressed IRGs was constructed and exhibited a moderate predictive value in the prognosis of RCC patients and a powerful identification ability in refining the risk stratification of RCC patients. A close correlation was found between IRGPI and specific clinicopathological parameters, including age, gender, pathological stage, tumour stage, lymph node metastasis and distant metastasis. A positive correlation was found between IRGPI and the infiltration levels of neutrophils, dendritic cells, CD8+ T cells and B cells. Our results demonstrated the clinical significance and potential function of IRGs, providing additional data for prognostic risk prediction and immunotherapeutic target selection in RCC.

Remote ischemic postconditioning protects the brain from focal ischemia/reperfusion injury by inhibiting autophagy through the mTOR/p70S6K pathway.

OBJECTIVE: Remote ischemic postconditioning (RIPostC) has been recognized as an applicable strategy for protecting against cerebral ischemia/reperfusion (I/R) injury. This study was performed to examine the effect of RIPostC on cerebral I/R and to explore its underlying mechanism. METHODS: Healthy male SD rats (N = 36) were assigned randomly into 3 groups of 12 each: sham group, I/R model group and RIPostC group. Animal models were performed by filament insertion for 2 h with middle cerebral artery occlusion(MCAO) followed by 24 h of reperfusion. RIPostC was induced by 15 min occlusion of femoral arteries followed by 15 min of reperfusion for 3 cycles at the beginning of middle cerebral artery reperfusion. The neurological deficits, infarct size and brain edema were determined. Autophagy was examined by transmission electron microscopy (TEM). The protein levels of microtubule-associated protein light chain 3 (LC3-II), mammalian target of rapamycin (mTOR), serine/threonine kinase p70S6 kinase (p70S6K), and their phosphorylation (p-mTOR and p-p70S6K) in the brain tissue of the rats were determined by western blotting. RESULTS: Our results suggested that RIPostC significantly reduced I/R-induced brain injury, as exhibited by a significantly decreased infarct size, mitigated brain edema and improved neurological deficits. RIPostC also significantly reduced the LC3-II/LC3-I ratio and protein expression of Beclin 1. Much less severe neuronal injury and fewer autophagosomes were observed by TEM in the RIPostC group. CONCLUSIONS: These results suggest that RIPostC attenuates cerebral I/R injury by inhibiting autophagy through the activation of the mTOR/p70S6K signaling pathway.

Effects of Campylobacter jejuni lipopolysaccharide on axonal injury in the spinal cord in rats.

To explore the effects of Campylobacter jejuni lipopolysaccharide (Cj-LPS) on axonal injury in the spinal cord. Wistar rats were divided into the control (NC) group, model group (Cj-LPS), and LPS antibody group (Anti-LPS). Rats in the NC group were injected with a mixture of normal saline and complete Freund's adjuvant (CFA) while those in Cj-LPS group were injected with Cj-LPS, composed of LPS, CFA, and saline. Rats were sacrificed at 4th week and 6th week after injection, and hematoxylin and eosin (HE) staining was performed on the spinal cord sections. Real time-reverse transcription(RT-PCR) was used to detect mRNA expression of the axonal nutrition factor neurotrophin-3 (NT-3) with its receptor tropomyosin receptor kinase C (TrkC) and axon inhibitory factor of NogoA/NgR (Nogo receptor). The results indicated that Cj-LPS induce axonal injury in the rat spinal cord, decreased the mRNA expression of the axonal nutrition factor NT-3/TrkC, and increased the mRNA expression of the inhibitory factor NogoA/NgR. However, anti-LPS ameliorated axonal injury in the rat spinal cord induced by Cj-LPS.

Cyclooxygenase-2 genetic polymorphism and stroke subtypes in Chinese.

BACKGROUND: Cyclooxygenase-2 (COX-2) catalyzes the formation of prostaglandins that contribute to the inflammation in atherosclerosis. The aim of the present study was to investigate the relationship between two polymorphisms (-1195G>A and -765G>C) in the COX-2 gene and subtypes of ischemic stroke in a Chinese population. METHODS: Genomic DNA of 224 patients with large artery atherosclerosis (LAA), 329 patients with small vessel occlusion (SVO), and 450 controls were genotyped for the COX-2 1195G>A (rs689466) and -765G>C (rs20417) polymorphisms using polymerase chain reaction-restriction fragment length polymorphism. Chi-square test and logistic regression analysis were performed for association analysis. RESULTS: The frequencies of variant allele with -1195G>A and -765G>C polymorphisms were 0.46 and 0.22, respectively. The -1195GA genotype and 1195A allele carriers were identified independently to be related with ischemic stroke (adjusted OR = 1.51, 95 % CI: 1.09-2.10, P = 0.02; OR = 1.45, 95 % CI: 1.06-1.97, P = 0.02) and SVO (adjusted OR = 1.57, 95 % CI: 1.07-2.30, P = 0.02; OR = 1.50, 95 % CI: 1.05-2.16, P = 0.03). In contrast, the 1195G>A polymorphism was not associated with LAA. No relationship between the -765G>C polymorphism and risk of either ischemic stroke was observed. The linkage disequilibrium analysis showed that -1195G>A and -765G>C SNPs are moderate linkage disequilibrium in this study population (D' = 0.72, r (2) = 0.16). Compared with G-1195-G-765 haplotype, the haplotype of A-1195-G-765 showed significant increased risk of ischemic stroke (OR = 1.27, 95 % CI: 1.05-1.54, P = 0.02) and SVO (OR = 1.27, 95 % CI: 1.02-1.58, P = 0.03) but not LAA. CONCLUSIONS: In conclusion, we found that -1195G>A polymorphism and A-1195-G-765 haplotype of COX-2 were associated with susceptibility to ischemic stroke in a Chinese population. The effects were confined to SVO among the stroke subtypes rather than to LAA.

Cyclooxygenase 2 genetic polymorphism may increase the risk of developing leukoaraiosis in Chinese.

Cyclooxygenase-2 (COX-2) is a key enzyme involved in the conversion of arachidonic acid into prostaglandins, which are important mediators of inflammation. To clarify the role of inflammation in the pathogenesis of cerebral small vessel disease (SVD), we investigate the possible modulating effect of the functional COX-2 polymorphisms -1195G > A (rs689466) and -765G > C (rs20417) on the risk for development of cerebral SVD in a Chinese population. Genomic DNA of 116 patients with lacunar infarction (LI), 334 patients with leukoaraiosis (LA) and 450 control subjects was genotyped for the COX-2 -1195G > A and -765G > C polymorphisms using polymerase chain reaction-restriction fragment length polymorphism. Distribution of genotypes and haplotypes in patients and controls were compared. The genotype distribution of the -765G > C polymorphism was not different between the patients with LI or LA and the control group. The 1195A allele carriers was identified independently to be related with LA (adjusted OR = 1.41, 95 % confidence interval (CI) = 1.09-2.10, P = 0.03) but not associated with LI. The linkage disequilibrium analysis showed that -1195G > A and -765G > C SNPs are moderate linkage disequilibrium in this study population (D' = 0.70, r(2) = 0.16). Compared with G-1195-G-765 haplotype, the haplotype of A-1195-G-765 showed significantly increased the risk of LA (OR = 1.24, 95 % CI = 1.10-1.55, P = 0.04) but not LI. In conclusion, we found that -1195G > A polymorphism and A-1195-G-765 haplotype of COX-2 were associated with susceptibility to LA in a Chinese population.

The haplotype of the TGFß1 gene associated with cerebral infarction in Chinese.

BACKGROUND: Transforming growth factor beta1 (TGFß1) is a multifunctional cytokine involved in inflammation and pathogenesis of atherosclerosis. The aim of the present study was to investigate the relationship between human TGFß1 gene +869T>C (rs1800470), -509C>T (rs1800469) single nucleotide polymorphisms (SNPs) and haplotypes and cerebral infarction (CI) in a Chinese population. METHODS: The genetic association study was performed in 450 Chinese patients (306 male and 144 female) with CI and 450 control subjects (326 male and 124 female). TGFß1 gene +869T>C and -509C>T polymorphisms were identified with amplification refractory mutation system polymerase chain reaction and DNA sequencing method. RESULTS: The individual SNPs analysis showed the +869T and -509C in an additive model (+869T vs +869C; -509 C vs T), +869TT genotype in a recessive model (TT vs TC+CC) and 509CC genotype in a dominant model (CC+ CT vs TT) were identified to be related to CI (P<0.05). +869T>C and -509C>T SNPs were in strong linkage disequilibrium (d'=0.87, R2=0.75). Haplotype analysis showed that +869C/-509T haplotype was associated with a significant decreased risk of CI (OR= 0.86, 95%CI, 0.70-0.92; P=0.007). Furthermore,+869T/-509C haplotype was associated with a significant increased risk of CI (OR=1.31, 95%CI, 1.10-2.03; P=0.019). CONCLUSIONS: The results of this study indicate that polymorphisms and the haplotypes in the TGFß1 gene might be genetic markers for CI in the Chinese population.

TGF-beta 1 codon 10 polymorphism is associated with cerebral SVD.

BACKGROUND: To clarify the role of inflammation in the pathogenesis of cerebral small vessel disease (SVD), we investigated whether the gene encoding transforming growth factor-beta 1(TGF-beta 1) is a risk factor for cerebral SVD as a whole, and for two different SVD subtypes. METHODS: TGF-beta 1 codon10 (T+29C) genotype was determined in 441 Chinese patients (313 male and 128 female) with cerebral SVD and 450 control subjects (326 male and 124 female). Cerebral SVD patients were retrospectively classified into two groups based on neuroimaging findings: lacunar infarction group with 112 patients and ischaemic leukoaraiosis group with 329 patients. RESULTS: Subjects carrying TT homozygote were susceptible to cerebral SVD [adjusted odds ratio (OR) =1.44, 95% confidence interval (CI), 1.05-1.98; P=0.026]. Further analysis of SVD subtypes revealed a moderate association with the ischaemic leukoaraiosis group [OR= 1.60, 95% CI, 1.14-2.25; P=0.007]. CONCLUSIONS: Codon 10 of TGF-beta 1 might be a risk factor for SVD, specifically in ischaemic leukoaraiosis phenotype.

TGF-ß1 869T/C polymorphism and ischemic stroke: sex difference in Chinese.

BACKGROUND: Inflammation plays a pivotal role in the pathogenesis of atherosclerosis and of cerebrovascular complications. Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine with a central role in inflammation. To investigate whether polymorphisms of the TGF-ß1 gene can modify the risk of ischemic stroke (IS) in Chinese population, we conduct this hospital-based, case-control study. METHODS: Transforming growth factor-ß1 genotype was determined in 450 Chinese patients (306 male and 144 female) with IS and 450 control subjects (326 male and 124 female). RESULTS: Subjects carrying 869TT were susceptible to IS (odds ratio [OR] =1.58; P=0.003). Further analysis of IS data partitioned by gender revealed the female-specific association with 869T/C (OR=2.64; P=0.001). CONCLUSIONS: Findings suggest that the TT genotype of 869T/C might be a risk factor of IS in Chinese, especially in females.

Endothelial NO synthase gene polymorphisms and risk of ischemic stroke: a meta-analysis.

Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene may influence the risk of ischemic stroke, but data from published studies with individually low statistical power are conflicting. To evaluate the role of eNOS gene polymorphisms in ischemic stroke, we considered all available studies in a meta-analysis. Case-control studies evaluating the association between the G894T, 4b/a polymorphisms and ischemic stroke were searched in MEDLINE, EMBASE, HuGEnet, CBMdisc and CNKI up to December 2008. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) from random- and fixed-effect models were calculated. Data were available for 5516 cases and 6150 controls from 18 studies. We found that homozygosity for the 4a allele of the eNOS gene was not associated with increase in the risk of ischemic stroke (OR, 1.67; 95% CI, 0.81-3.45). A marginal association was observed for homozygosity for the 894T allele with ischemic stroke (OR, 1.14; 95% CI, 0.99-1.31). This analysis provides strong evidence that the eNOS 4b/a gene polymorphisms is not associated with ischemic stroke, the G894T polymorphisms might be associated with ischemic stroke. Further research is warranted to clarify the relevance of G894T polymorphisms to ischemic stroke.

Meta-analysis of the ACE gene polymorphism in cerebral infarction.

BACKGROUND: The angiotensin-1 converting enzyme (ACE) gene is known to have two polymorphic alleles insertion/deletion (I/D). People with the DD genotype have been shown to be at greater risk of cerebral infarction, but only in some studies. Identification of cerebral infarction susceptibility genes and quantification of associated risks have been hampered by conflicting results from underpowered case-control studies. This meta-analysis was made to look specifically into the genetics of cerebral infarction among Han Chinese population. METHODS: Genetic associations studies published from January 1, 1990 to December 30, 2007 were collected from databases of MEDLINE, EMBASE, CBM and CNKI. Data were extracted using standardised forms and pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. RESULTS: Twenty-nine original case-control studies of Han Chinese population, comprising 3654 patients with cerebral infarction and 3058 controls were included in the meta-analysis. Using the random effects model, the pooled ORs of ACE DD genotype VS ID+ II was 1.91 (95% CI 1.56 to 2.34, P<0.00001). CONCLUSIONS: These data suggest that the ACE DD genotype may be a risk factor for cerebral infarction in Han Chinese population. A large scale case-control study is needed to clarify the functional effect of the polymorphism of the ACE I/D gene in the pathogenesis of cerebral infarction in Han Chinese population.