Reduced brain UCP2 expression mediated by microRNA-503 contributes to increased stroke susceptibility in the high-salt fed stroke-prone spontaneously hypertensive rat.
ABSTRACT: UCP2 maps nearby the lod score peak of STR1-stroke QTL in the SHRSP rat strain. We explored the potential contribution of UCP2 to the high-salt diet (JD)-dependent increased stroke susceptibility of SHRSP. Male SHRSP, SHRSR, two reciprocal SHRSR/SHRSP-STR1/QTL stroke congenic lines received JD for 4 weeks to detect brain UCP2 gene/protein modulation as compared with regular diet (RD). Brains were also analyzed for NF-κB protein expression, oxidative stress level and UCP2-targeted microRNAs expression level. Next, based on knowledge that fenofibrate and Brassica Oleracea (BO) stimulate UCP2 expression through PPARα activation, we monitored stroke occurrence in SHRSP receiving JD plus fenofibrate versus vehicle, JD plus BO juice versus BO juice plus PPARα inhibitor. Brain UCP2 expression was markedly reduced by JD in SHRSP and in the (SHRsr.SHRsp-(D1Rat134-Mt1pa)) congenic line, whereas NF-κB expression and oxidative stress level increased. The opposite phenomenon was observed in the SHRSR and in the (SHRsp.SHRsr-(D1Rat134-Mt1pa)) reciprocal congenic line. Interestingly, the UCP2-targeted rno-microRNA-503 was significantly upregulated in SHRSP and decreased in SHRSR upon JD, with consistent changes in the two reciprocal congenic lines. Both fenofibrate and BO significantly decreased brain microRNA-503 level, upregulated UCP2 expression and protected SHRSP from stroke occurrence. In vitro overexpression of microRNA-503 in endothelial cells suppressed UCP2 expression and led to a significant increase of cell mortality with decreased cell viability. Brain UCP2 downregulation is a determinant of increased stroke predisposition in high-salt-fed SHRSP. In this context, UCP2 can be modulated by both pharmacological and nutraceutical agents. The microRNA-503 significantly contributes to mediate brain UCP2 downregulation in JD-fed SHRSP.
Project description:BACKGROUND:The genetic basis of stroke susceptibility remains to be elucidated. STR1 quantitative trait locus (STR1/QTL) was identified on rat chromosome 1 of stroke-prone spontaneously hypertensive rat (SHRSP) upon Japanese-style stroke-permissive diet (JD), and it contributes to 20% of the stroke phenotype variance. METHODS AND RESULTS:Nine hundred eighty-six probe sets mapping on STR1 were selected from the Rat RAE230A array and screened through a microarray differential expression analysis in brains of SHRSP and stroke-resistant SHR (SHRSR) fed with either regular diet or JD. The gene encoding Ndufc2 (NADH dehydrogenase [ubiquinone] 1 subunit), mapping 8 Mb apart from STR1/QTL Lod score peak, was found significantly down-regulated under JD in SHRSP compared to SHRSR. Ndufc2 disruption altered complex I assembly and activity, reduced mitochondrial membrane potential and ATP levels, and increased reactive oxygen species production and inflammation both in vitro and in vivo. SHRSR carrying heterozygous Ndufc2 deletion showed renal abnormalities and stroke occurrence under JD, similarly to SHRSP. In humans, T allele variant at NDUFC2/rs11237379 was associated with significant reduction in gene expression and with increased occurrence of early-onset ischemic stroke by recessive mode of transmission (odds ratio [OR], 1.39; CI, 1.07-1.80; P=0.012). Subjects carrying TT/rs11237379 and A allele variant at NDUFC2/rs641836 had further increased risk of stroke (OR=1.56; CI, 1.14-2.13; P=0.006). CONCLUSIONS:A significant reduction of Ndufc2 expression causes complex I dysfunction and contributes to stroke susceptibility in SHRSP. Moreover, our current evidence may suggest that Ndufc2 can contribute to an increased occurrence of early-onset ischemic stroke in humans.
Project description:The stroke-prone spontaneously hypertensive rat (SHRSP) is known to have exaggerated sympathetic nerve activity to various types of stress, which might contribute to the pathogenesis of severe hypertension and stroke observed in this strain. Previously, by using a congenic strain (called SPwch1.72) constructed between SHRSP and the normotensive Wistar-Kyoto rat (WKY), we showed that a 1.8-Mbp fragment on chromosome 1 (Chr1) of SHRSP harbored the responsible gene(s) for the exaggerated sympathetic response to stress. To further narrow down the candidate region, in this study, another congenic strain (SPwch1.71) harboring a smaller fragment on Chr1 including two functional candidate genes, Phox2a and Ship2, was generated. Sympathetic response to cold and restraint stress was compared among SHRSP, SPwch1.71, SPwch1.72 and WKY by three different methods (urinary norepinephrine excretion, blood pressure measurement by the telemetry system and the power spectral analysis on heart rate variability). The results indicated that the response in SPwch1.71 did not significantly differ from that in SHRSP, excluding Phox2a and Ship2 from the candidate genes. As the stress response in SPwch1.72 was significantly less than that in SHRSP, it was concluded that the 1.2-Mbp congenic region covered by SPwch1.72 (and not by SPwch1.71) was responsible for the sympathetic stress response. The sequence analysis of 12 potential candidate genes in this region in WKY/Izm and SHRSP/Izm identified a nonsense mutation in the stromal interaction molecule 1 (Stim1) gene of SHRSP/Izm which was shared among 4 substrains of SHRSP. A western blot analysis confirmed a truncated form of STIM1 in SHRSP/Izm. In addition, the analysis revealed that the protein level of STIM1 in the brainstem of SHRSP/Izm was significantly lower when compared with WKY/Izm. Our results suggested that Stim1 is a strong candidate gene responsible for the exaggerated sympathetic response to stress in SHRSP.
Project description:By means of introgressing a loss-of-function mutation in the p22phox gene from the Matsumoto Eosinophilia Shinshu (MES) rat to stroke-prone spontaneously hypertensive rats (SHRSP), we constructed the SHRSP-based congenic strain lacking the P22PHOX expression (i.e., lacking NADPH oxidases [NOX] activities) (SHRSP.MES-Cyba(mes)/Izm; hereafter referred to as SP.MES). To examine the effects of Nox activities on the focal ischemic injury or stroke, we performed middle cerebral artery (MCA) occlusion in this new congenic strain; the distal MCA was occluded by 561-nm laser-driven photothrombosis. Resting mean arterial blood pressure was significantly lower in SP.MES when compared with the control PM0/SHRSP (150±11 mmHg vs. 166±11 mmHg). Cerebral blood flow decreased to 37±13% in SP.MES and 35±17% in PM0/SHRSP at 10 min after MCA occlusion (not significant). Infarct volume determined at 24 h after MCA occlusion in SP.MES was 89±39 mm3, which was not significantly different from 83±35 mm3 in PM0/SHRSP. The distal MCA pattern was more complex in SP.MES (median 3, IQR 3-5) than PM0/SHRSP (median 2, IQR 1-3) (p = 0.001). Because more complex distal MCA is known to produce larger infarction after distal MCA occlusion in SHR, we adjusted for the branching pattern in an ANCOVA. The adjusted mean of infarct volume was significantly smaller in SP.MES compared with that in PM0/SHRSP (67 [95% CI 46 to 87] mm3 vs. 100 [95% CI 82 to 118] mm3, p = 0.032). Elimination of the P22PHOX expression induced complex distal MCA, which would suggest the presence of 'loss of complexity' induced by enhanced oxidative stress in SHRSP; infarct size in SP.MES--when adjusted for distal MCA complexity--was significantly attenuated compared with that in PM0/SHRSP. Therefore, the present results suggest that Nox is harmful for ischemic brain tissue.
Project description:The identification of the mechanisms predisposing to stroke may improve its preventive and therapeutic strategies in patients with essential hypertension. The role of macroautophagy/autophagy in the development of hypertension-related stroke needs to be clarified. We hypothesized that a defective autophagy may favor hypertension-related spontaneous stroke by promoting mitochondrial dysfunction. We studied autophagy in the stroke-prone spontaneously hypertensive (SHRSP) rat, which represents a clinically relevant model of stroke associated with high blood pressure. We assessed autophagy, mitophagy and NAD<sup>+</sup>:NADH levels in brains of SHRSP and stroke-resistant SHR fed with high salt diet. Vascular smooth muscle cells silenced for the mitochondrial complex I subunit <i>Ndufc2</i> gene (NADH:ubiquinone oxidoreductase subunit C2) and cerebral endothelial cells isolated from SHRSP were also used to assess autophagy/mitophagy and mitochondrial function in response to high salt levels. We found a reduction of autophagy in brains of high salt-fed SHRSP. Autophagy impairment was associated with NDUFC2 downregulation, mitochondrial dysfunction and NAD<sup>+</sup> depletion. Restoration of NAD<sup>+</sup> levels by nicotinamide administration reactivated autophagy and reduced stroke development in SHRSP. A selective reactivation of autophagy/mitophagy by Tat-Beclin 1 also reduced stroke occurrence, restored autophagy/mitophagy and improved mitochondrial function. Endothelial progenitor cells (EPCs) from subjects homozygous for the thymine allele variant at <i>NDUFC2</i>/rs11237379, which is associated with <i>NDUFC2</i> deficiency and increased stroke risk, displayed an impairment of autophagy and increased senescence in response to high salt levels. EPC senescence was rescued by Tat-Beclin 1. Pharmacological activation of autophagy may represent a novel therapeutic strategy to reduce stroke occurrence in hypertension.<h4>Abbreviations</h4>10 VSMCs: aortic vascular smooth muscle cells; COX4I1/COX IV: cytochrome c oxidase subunit 4I1; ECs: endothelial cells; EPCs: endothelial progenitor cells; JD: Japanese-style diet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NAD: nicotinamide adenine dinucleotide; NDUFC2: NADH:ubiquinone oxidoreductase subunit C2; NMN: nicotinamide mononucleotide; RD: regular diet; SHRSP: stroke-prone spontaneously hypertensive rat; SHRSR: stroke-resistant spontaneously hypertensive rat.
Project description:Oxidative stress in the rostral ventrolateral medulla (RVLM), a sympathetic center in the brainstem, was implicated in the regulation of sympathetic activity in various hypertensive models including stroke-prone spontaneously hypertensive rats (SHRSP). In this study, we evaluated the role of the NADPH oxidases (NOX) in the blood pressure (BP) regulation in RVLM in SHRSP. The P22PHOX-depleted congenic SHRSP (called SP.MES) was constructed by introducing the mutated p22phox gene of Matsumoto Eosinophilic Shinshu rat. BP response to glutamate (Glu) microinjection into RVLM was compared among SHRSP, SP.MES, SHR and Wistar Kyoto (WKY); the response to Glu microinjection was significantly greater in SHRSP than in SP.MES, SHR and WKY. In addition, tempol, losartan and apocynin microinjection reduced the response to Glu significantly only in SHRSP. The level of oxidative stress, measured in the brainstem using lucigenin and dihydroethidium, was reduced in SP.MES than in SHRSP. BP response to cold stress measured by telemetry system was also blunted in SP.MES when compared with SHRSP. The results suggested that oxidative stress due to the NOX activation in RVLM potentiated BP response to Glu in SHRSP, which might contribute to the exaggerated response to stress in this strain.
Project description:We previously revealed that two major quantitative trait loci (QTLs) for stroke latency of the stroke-prone spontaneously hypertensive rat (SHRSP) under salt-loading were located on chromosome (Chr) 1 and 18. Here, we attempted further dissection of the stroke-QTLs using multiple congenic strains between SHRSP and a stroke-resistant hypertensive rat (SHR). Cox hazard model among subcongenic strains harboring a chromosomal fragment of Chr-1 QTL region showed that the most promising region was a 2.1 Mbp fragment between D1Rat177 and D1Rat97. The QTL region on Chr 18 could not be narrowed down by the analysis, which may be due to multiple QTLs in this region. Nonsynonymous sequence variations were found in four genes (Cblc, Cxcl17, Cic, and Ceacam 19) on the 2.1 Mbp fragment of Chr-1 QTL by whole-genome sequence analysis of SHRSP/Izm and SHR/Izm. Significant changes in protein structure were predicted in CBL-C and CXCL17 using I-TASSER. Comprehensive gene expression analysis in the kidney with a cDNA microarray identified three candidate genes (LOC102548695 (Zinc finger protein 45-like, Zfp45L), Ethe1, and Cxcl17). In conclusion, we successfully narrowed down the QTL region on Chr 1, and identified six candidate genes in this region.
Project description:We have previously confirmed the importance of rat chromosome 3 (RNO3) genetic loci on blood pressure elevation, pulse pressure (PP) variability and renal pathology during salt challenge in the stroke-prone spontaneously hypertensive (SHRSP) rat. The aims of this study were to generate a panel of RNO3 congenic sub-strains to genetically dissect the implicated loci and identify positional candidate genes by microarray expression profiling and analysis of next-generation sequencing data.A panel of congenic sub-strains were generated containing Wistar-Kyoto (WKY)-introgressed segments of varying size on the SHRSP genetic background, focused within the first 50?Mbp of RNO3. Haemodynamic profiling during salt challenge demonstrated significantly reduced systolic blood pressure, diastolic blood pressure and PP variability in SP.WKYGla3a, SP.WKYGla3c, SP.WKYGla3d and SP.WKYGla3e sub-strains. Only SBP and DBP were significantly reduced during salt challenge in SP.WKYGla3b and SP.WKYGla3f sub-strains, whereas SP.WKYGla3g rats did not differ in haemodynamic response to SHRSP. Those sub-strains demonstrating significantly reduced PP variability during salt challenge also demonstrated significantly reduced renal pathology and proteinuria. Microarray expression profiling prioritized two candidate genes for blood pressure regulation (Dnm1, Tor1b), localized within the common congenic interval shared by SP.WKYGla3d and SP.WKYGla3f strains, and one candidate gene for salt-induced PP variability and renal pathology (Rabgap1), located within the region unique to the SP.WKYGla3d strain. Comparison of next-generation sequencing data identified variants within additional positional genes that are likely to affect protein function.This study has identified distinct intervals on RNO3-containing genes that may be important for blood pressure regulation and renal pathology during salt challenge.
Project description:Similar to humans, the risk of cerebrovascular disease in stroke-prone spontaneously hypertensive rats (SHR-A3/SHRSP) arises from naturally occurring genetic variation. In the present study, we show the involvement of genetic variation affecting the store-operated calcium signaling gene, Stim1, in the pathogenesis of stroke in SHR. Stim1 is a key lymphocyte activation signaling molecule and contains functional variation in SHR-A3 that diverges from stroke-resistant SHR-B2. We created a SHR-A3 congenic line in which Stim1 was substituted with the corresponding genomic segment from SHR-B2. Compared with SHR-A3 rats, Stim1 congenic SHR-A3 (SHR-A3(Stim1-B2)) have reduced cerebrovascular disease in response to salt loading including lower neurological deficit scores and cerebral edema. Microbleeds and major hemorrhages occurred in over half of SHR-A3 rats. These lesions were absent in SHR-A3(Stim1-B2) rats. Loss of Stim1 function in mice and humans is associated with antibody-mediated autoimmunity due to defects in T lymphocyte helper function to B cells. We investigated autoantibody formation using a high-density protein array to detect the presence of IgG and IgM autoantibodies in SHR-A3. Autoantibodies to key cerebrovascular stress proteins were detected that were reduced in the congenic line.
Project description:The present study aimed to integrate the mRNA and microRNA (miRNA) expression profiles of spontaneously hypertensive rats (SHR rats) and stroke?prone spontaneously hypertensive rats (SHRSP rats) to screen for potential therapeutic targets for hypertension and stroke. The datasets GSE41452, GSE31457, GSE41453 and GSE53363 were collected from the Gene Expression Omnibus (GEO) database to screen differentially expressed genes (DEGs). The GSE53361 dataset was obtained to analyze differentially expressed miRNAs (DEMs). The DEGs and DEMs were identified between SHR (or SHRSP) rats and normotensive Wistar?Kyoto (WKY) rats using the Linear Models for Microarray (limma) data method. Venn diagrams were used to show the SHR?specific, SHRSP?specific and SHR?SHRSP shared DEGs and DEMs, and these were utilized to construct the protein?protein interaction (PPI) and miRNA?mRNA regulatory networks. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to explore the function of the genes. Subsequently, the connectivity Map (CMAP) database was searched to identify small?molecule drugs. Comparisons between the GSE41452?GSE31457?GSE41453 merged and GSE53363 datasets identified 2 SHR?specific, 8 SHRSP?specific and 15 SHR?SHRSP shared DEGs. Function enrichment analysis showed that SHRSP?specific D?box binding PAR bZIP transcription factor (Dbp) was associated with circadian rhythm, and SHR?SHRSP shared carnitine O?octanoyltransferase (Crot) was involved in fatty acid metabolic processes or the inflammatory response via interacting with epoxide hydrolase 2 (EPHX2). SHR?SHRSP shared mitochondrial ribosomal protein L4 (Mrpl4) may exert roles by interacting with the threonine?tRNA ligase, TARS2. The miRNA regulatory network predicted that upregulated Dbp could be regulated by rno?miR?126a?5p, whereas downregulated Crot and Mrpl4 could be modulated by rno?miR?31a. The CMAP database predicted that small?molecule drugs, including botulin, Gly?His?Lys, and podophyllotoxin, may possess therapeutic potential. In conclusion, the present study has identified Dbp, Crot and Mrpl4 as potential targets for the treatment of hypertension and stroke. Furthermore, the expression of these genes may be reversed by the above miRNAs or drugs.
Project description:Uncoupling protein (UCP) 2 is a widely expressed mitochondrial protein whose precise function is still unclear but has been linked to mitochondria-derived reactive oxygen species production. Thus, the chronic absence of UCP2 has the potential to promote persistent reactive oxygen species accumulation and an oxidative stress response. Here, we show that Ucp2-/- mice on three highly congenic (N >10) strain backgrounds (C57BL/6J, A/J, 129/SvImJ), including two independently generated sources of Ucp2-null animals, all exhibit increased oxidative stress. Ucp2-null animals exhibit a decreased ratio of reduced glutathione to its oxidized form in blood and tissues that normally express UCP2, including pancreatic islets. Islets from Ucp2-/- mice exhibit elevated levels of numerous antioxidant enzymes, increased nitrotyrosine and F4/80 staining, but no change in insulin content. Contrary to results in Ucp2-/- mice of mixed 129/B6 strain background, glucose-stimulated insulin secretion in Ucp2-/- islets of each congenic strain was significantly decreased. These data show that the chronic absence of UCP2 causes oxidative stress, including in islets, and is accompanied by impaired glucose-stimulated insulin secretion.