Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Inositol 1,4,5‐Trisphosphate Receptors in Endothelial Cells Play an Essential Role in Vasodilation and Blood Pressure Regulation

doi: 10.1161/JAHA.118.011704

Figure Lengend Snippet: Endothelial cell–specific deletion of inositol 1,4,5‐trisphosphate receptor 1 ( IP 3 R1) in adult mice had no major effects on blood pressure and vasodilation. A , mRNA levels of IP 3 Rs in isolated endothelial cells from control and endothelial cell–specific IP 3 R1 knockout ( ECR 1 KO ) mice. n=3 to 5 (with endothelial cells from 3 mice pooled as 1 sample) per group. B , Confocal Ca 2+ imaging of isolated endothelial cells using Fluo‐4‐AM. Intracellular Ca 2+ mobilization was elicited by 10 μmol/L acetylcholine. Top , Sequential confocal images of endothelial cells at the time points of time 0, time 1, and time 2, as indicated by the arrows at the bottom . Bar=20 μm. Bottom , Representative traces of Ca 2+ signals in control (black) and ECR 1 KO (gray) endothelial cells. F0, the Fluo‐4 fluorescence at rest. F/F0, normalized fluorescence. C , The amplitude of Ca 2+ signals induced by 10 μmol/L acetylcholine. n=20 to 30 cells from 3 independent experiments per group. D , Systolic blood pressure ( SBP ) measured in control and ECR 1 KO mice at 2, 3, and 4 months after tamoxifen injection using the tail‐cuff system. n=11 to 12 mice per group. E , Vascular reactivity in response to endothelium‐dependent agonist acetylcholine in ECR 1 aortas and mesenteric arteries showed a slight trend toward reduced vasodilation that was not statistically significant. F , Vascular reactivity in response to endothelium‐independent agonist sodium nitroprusside (SNP) in aortas and mesenteric arteries. The vessels were preconstricted by 10 μmol/L phenylephrine, and the vasorelaxing effects of acetylcholine and sodium nitroprusside ( SNP ) were presented as a percentage of phenylephrine‐induced contraction. n=6 mice per group. G , Quantitative real‐time polymerase chain reaction analysis of the expression of NOS3 and major acetylcholine receptors, including CHRM 1 , CHRM 2 , CHRM 3 , CHRM 4 , CHRM 5 , and CHRNA 7 , in control and ECR 1 KO endothelial cells. n=3 (with endothelial cells from 3 mice pooled as 1 sample) per group. Significance was determined using a 2‐tailed, unpaired, Student t test or 2‐way ANOVA analysis with Bonferroni post hoc test. Error bars represent mean±SEM. * P <0.05, *** P <0.001 vs control.

Article Snippet: To generate endothelial cell–specific single IP 3 R1 knockout and triple IP 3 R knockout mice, the 7‐ to 8‐week‐old iCre + IP 3 R1 floxed (IP 3 R1 f/f ) and iCre + IP 3 R1 f/f IP 3 R2 f/f IP 3 R3 f/f mice were intraperitoneally injected with tamoxifen (50 mg/kg per day; Sigma‐Aldrich) for 5 consecutive days, and they were considered as endothelial cell–specific IP 3 R1 knockout (ECR1KO) and endothelial cell–specific IP 3 R triple knockout (ECTKO) mice, respectively.

Techniques: Isolation, Knock-Out, Imaging, Fluorescence, Injection, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Inositol 1,4,5‐Trisphosphate Receptors in Endothelial Cells Play an Essential Role in Vasodilation and Blood Pressure Regulation

doi: 10.1161/JAHA.118.011704

Figure Lengend Snippet: Endothelial cell–specific deletion of all 3 subtypes of inositol 1,4,5‐trisphosphate receptors ( IP 3 Rs) in adult mice increased systolic blood pressure (SBP). A , Quantitative real‐time polymerase chain reaction analysis of the 3 IP 3 R subtypes in isolated endothelial cells from control and endothelial cell–specific IP 3 R triple knockout ( ECTKO) mice. n=3 (with endothelial cells from 3 mice pooled as 1 sample) per group. B , Confocal Ca 2+ imaging of endothelial cells isolated from control and ECTKO mice. Top , Sequential confocal images of endothelial cells at the time point of time 0, time 1, and time 2, as indicated by the arrows at the bottom . Bar=20 μm. Bottom , Representative traces of Ca 2+ signals in control (black) and ECTKO (gray) endothelial cells. F0, the Fluo‐4 fluorescence at rest. F/F0, normalized fluorescence. C , The amplitude of Ca 2+ signals induced by 10 μmol/L acetylcholine in control and ECTKO endothelial cells. n=20 to 30 cells from 3 independent experiments per group. D , SBP measured in control and ECTKO mice at 2, 3, and 4 months after tamoxifen administration using the tail‐cuff system. n=13 to 17 mice per group. E , Representative hematoxylin and eosin–stained sections of the second‐order mesenteric arteries isolated from control and ECTKO mice 4 months after tamoxifen administration. Bar=40 μm. F , The wall thickness and the ratio of medial/luminal area were calculated in control and ECTKO mice. n=5 to 6 mice per group. G , The concentration of nitrite and nitrate ( NO x) in the serum measured in control and ECTKO mice 4 months after tamoxifen administration. n=6 mice per group. Significance was determined using a 2‐tailed, unpaired, Student t test. Error bars represent mean± SEM . * P <0.05, ** P <0.01, *** P <0.001 vs control.

Article Snippet: To generate endothelial cell–specific single IP 3 R1 knockout and triple IP 3 R knockout mice, the 7‐ to 8‐week‐old iCre + IP 3 R1 floxed (IP 3 R1 f/f ) and iCre + IP 3 R1 f/f IP 3 R2 f/f IP 3 R3 f/f mice were intraperitoneally injected with tamoxifen (50 mg/kg per day; Sigma‐Aldrich) for 5 consecutive days, and they were considered as endothelial cell–specific IP 3 R1 knockout (ECR1KO) and endothelial cell–specific IP 3 R triple knockout (ECTKO) mice, respectively.

Techniques: Real-time Polymerase Chain Reaction, Isolation, Triple Knockout, Imaging, Fluorescence, Staining, Concentration Assay

Journal: Human Molecular Genetics

Article Title: Genetic ablation of IP 3 receptor 2 increases cytokines and decreases survival of SOD1 G93A mice

doi: 10.1093/hmg/ddw190

Figure Lengend Snippet: Relative IP 3 R2 gene expression is increased in models of neurodegeneration and inflammation. ( A ) Relative gene expression of IP 3 R2 assessed by qPCR on ventral spinal cords of non-transgenic (ntg; n = 6), SOD1 WT (wt; n = 6), pre-symptomatic SOD1 G93A (pre-s; n = 6), symptomatic SOD1 G93A (s; n = 5) and end stage SOD1 G93A (es; n = 5) mice (ANOVA, Bonferroni post-hoc). ( B ) Relative IP 3 R2 gene expression analysed in the lumbar spinal cord of severely affected EAE-mice ( n = 3) and control mice ( n = 6) by qPCR (unpaired t- test). ( C ) Relative IP 3 R2 gene expression analysed in the penumbra zone of stroke in mice ( n = 4) and a similar region in the contralateral side of the brain by qPCR (paired t -test). ( D ) Relative IP 3 R2 gene expression in ventral spinal cord astrocytes in vitro by 24 h LPS application ( n = 8) or vehicle ( n = 8; unpaired t -test). ( E ) Relative IP 3 R2 gene expression in murine macrophages ( n = 4; Wilcoxon signed rank test compared to 1.0, two-tailed). The dotted line reflects the normalising vehicle condition set at 1. Mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: IP 3 R2 knockout mice, described previously , were intercrossed with high-copy number SOD1 G93A (The Jackson Laboratory, Bar Harbor, USA).

Techniques: Gene Expression, Transgenic Assay, Control, In Vitro, Two Tailed Test, Standard Deviation

Journal: Human Molecular Genetics

Article Title: Genetic ablation of IP 3 receptor 2 increases cytokines and decreases survival of SOD1 G93A mice

doi: 10.1093/hmg/ddw190

Figure Lengend Snippet: IP 3 R2 knockout exacerbates disease in SOD1 G93A mice. Relative gene expression of IP 3 R1 ( A ), IP 3 R2 ( B ) and IP 3 R3 ( C ) assessed by qPCR in ventral spinal cords of IP 3 R2 +/+ ( n = 6), IP 3 R2 +/- ( n = 6) and IP 3 R2 -/- ( n = 6) mice. ( D ) Early symptom onset as determined by the hanging wire test between IP 3 R2 +/+ SOD1 G93A ( n = 6; 129.7 ± 8.6 days), IP 3 R2 +/- SOD1 G93A ( n = 7; 126.8 ± 9.1 days) and IP 3 R2 -/- SOD1 G93A mice ( n = 7; 126.4 ± 8.1 days, Log-rank, P = 0.88). ( E ) Late symptom onset as determined by the rotarod test between IP 3 R2 +/+ SOD1 G93A ( n = 6; 141.2 ± 5.1 days), IP 3 R2 +/- SOD1 G93A ( n = 7; 142.6 ± 5.4 days) and IP 3 R2 -/- SOD1 G93A mice ( n = 7; 138.7 ± 6.0 days, Log-rank, P = 0.55). ( F ) Survival analysis by determining the age of end stage of IP 3 R2 +/+ SOD1 G93A ( n = 19; 170.7 ± 9.6 days), IP 3 R2 +/- SOD1 G93A (n= 17; 162.8 ± 9.6 days) and IP 3 R2 -/- SOD1 G93A ( n = 18; 153.2 ± 12.5 days; Log-rank, P < 0.0001). ( G-H ) Disease progression as measured by grip strength of IP 3 R2 +/+ SOD1 G93A mice ( n = 5), IP 3 R2 +/- SOD1 G93A mice ( n = 7), IP 3 R2 -/- SOD1 G93A mice ( n = 6) and IP 3 R2 -/- mice ( n = 4) for the fore limbs (G) and all limbs (H). ( I ) Quantification of neurons from lumbar spinal cord in adult IP 3 R2 +/+ ( n = 3) , IP 3 R2 -/- ( n = 3) and 145 day old IP 3 R2 +/+ SOD1 G93A ( n = 3) and IP 3 R2 -/- SOD1 G93A mice ( n = 2; 2-way ANOVA disease stage P = 0.0061). ( J ) The viability of murine motor neurons isolated from IP 3 R2 -/- ( n = 4) and non-transgenic ( n = 4) plated on non-transgenic rat astrocytic feeder layers in serum-enriched media. Mean ± standard deviation.

Article Snippet: IP 3 R2 knockout mice, described previously , were intercrossed with high-copy number SOD1 G93A (The Jackson Laboratory, Bar Harbor, USA).

Techniques: Knock-Out, Gene Expression, Biomarker Discovery, Isolation, Transgenic Assay, Standard Deviation