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NADPH oxidase and angiogenesis following endothelin-1 induced stroke in rats: role for nox2 in brain repair.

Taylor CJ, Weston RM, Dusting GJ, Roulston CL - Brain Sci (2013)

Bottom Line: VEGF mRNA expression was increased in the ipsilateral cortex and striatum between 6 h and 28 days post-stroke concurrently with a marked increase in Nox2 mRNA expression up to 7 days, and increased Nox4 mRNA expression detected between 7 and 28 days.Point counting of blood vessels using Metamorph imaging software showed increased vascular sprouting between 3 and 7 days after stroke with new vascular networks detected in the core infarct region by 14 days.Angiogenic blood vessels 3 and 7 days post-stroke were observed to co-localise with both Nox2 antibody and dihydroethidium fluorescence suggesting a role for Nox2 generated superoxide during the phase of vascular remodeling, whilst Nox4 expression was detected once new cerebral vessels had formed.

View Article: PubMed Central - PubMed

Affiliation: Stroke Injury and Repair Team, O'Brien Institute, 42 Fitzroy St, Fitzroy, Melbourne, Victoria 3065, Australia. cj.taylor@unimelb.edu.au.

ABSTRACT
NADPH oxidases contribute to brain injury, yet they may also have a role in brain repair, particularly in vascular signaling and angiogenesis. This study determined the temporal and spatial profile of NADPH oxidase subunit expression/activity concurrently with angiogenesis in the brain following transient ischemic stroke induced by prolonged constriction of the middle cerebral artery by perivascular injection of endothelin-1 in conscious Hooded Wistar rats (n = 47). VEGF mRNA expression was increased in the ipsilateral cortex and striatum between 6 h and 28 days post-stroke concurrently with a marked increase in Nox2 mRNA expression up to 7 days, and increased Nox4 mRNA expression detected between 7 and 28 days. Point counting of blood vessels using Metamorph imaging software showed increased vascular sprouting between 3 and 7 days after stroke with new vascular networks detected in the core infarct region by 14 days. Angiogenic blood vessels 3 and 7 days post-stroke were observed to co-localise with both Nox2 antibody and dihydroethidium fluorescence suggesting a role for Nox2 generated superoxide during the phase of vascular remodeling, whilst Nox4 expression was detected once new cerebral vessels had formed. These results indicate for the first time that ROS signaling through a cerebrovascular Nox2 NADPH oxidase may be important in initiating brain angiogenesis.

No MeSH data available.


Related in: MedlinePlus

Real-time PCR detection of mRNA for NADPH oxidase subunits, angiogenic factor VEGF and its receptor, Flk, in the striatum 6 h to 28 days after ET-1 induced stroke. Nox2 mRNA expression is increased in the ipsilateral striatum as early as 6 h after stroke and remains elevated for up to 28 days in comparison to the contralateral hemisphere (A–D). Nox4 mRNA expression is also increased at 6 h in the ipsilateral striatum and remains elevated for up to 28 days post-stroke, with increases in both the ipsilateral and contralateral hemispheres detected at 7 and 28 days in comparison to the 6 h group (E–H). An increase in the angiogenic factor VEGF is detected in the ipsilateral striatum at 6 hand remains elevated for up to 28 days (I–L). An increase in VEGF receptor Flt mRNA after stroke is detected 6 h, 14 and 28 days after stroke in the ipsilateral hemisphere (M–P). Expression of mRNA was normalized to the respective GAPDH content for each sample (ΔCt), then expressed relative to the contralateral side of the brain at 1.2 mm from Bregma, 6 h post-stroke (ΔΔCt). Data are presented as mean SEM of 8 samples per region (n = 4 per group). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. contralateral; #p < 0.05; ##p < 0.01; ###p < 0.001 vs. corresponding ipsilateral 6 h value; δ p < 0.05 vs. corresponding contralateral 6 h value (ANOVA).
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brainsci-03-00294-f005: Real-time PCR detection of mRNA for NADPH oxidase subunits, angiogenic factor VEGF and its receptor, Flk, in the striatum 6 h to 28 days after ET-1 induced stroke. Nox2 mRNA expression is increased in the ipsilateral striatum as early as 6 h after stroke and remains elevated for up to 28 days in comparison to the contralateral hemisphere (A–D). Nox4 mRNA expression is also increased at 6 h in the ipsilateral striatum and remains elevated for up to 28 days post-stroke, with increases in both the ipsilateral and contralateral hemispheres detected at 7 and 28 days in comparison to the 6 h group (E–H). An increase in the angiogenic factor VEGF is detected in the ipsilateral striatum at 6 hand remains elevated for up to 28 days (I–L). An increase in VEGF receptor Flt mRNA after stroke is detected 6 h, 14 and 28 days after stroke in the ipsilateral hemisphere (M–P). Expression of mRNA was normalized to the respective GAPDH content for each sample (ΔCt), then expressed relative to the contralateral side of the brain at 1.2 mm from Bregma, 6 h post-stroke (ΔΔCt). Data are presented as mean SEM of 8 samples per region (n = 4 per group). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. contralateral; #p < 0.05; ##p < 0.01; ###p < 0.001 vs. corresponding ipsilateral 6 h value; δ p < 0.05 vs. corresponding contralateral 6 h value (ANOVA).

Mentions: Increased Nox2 mRNA expression was also detected in the ipsilateral striatum in the same brain region (bregma level 1.2) at all times investigated after stroke (Figure 5A–D; p < 0.05, ANOVA). This increase was significantly greatest in the 14 day recovery group when compared to the contralateral control region in the 6 h recovery group (~25 fold increase; p < 0.05, two-way ANOVA). Nox4 mRNA expression was also significantly increased in the ipsilateral striatum at all times investigated after stroke (Figure 5E–H; ~30 fold increase; p < 0.05, ANOVA) with changes in the contralateral hemisphere also detected at both 7 and 28 days in comparison to the contralateral control region in the 6 h recovery group (p < 0.05, two-way ANOVA). Increased levels of VEGF mRNA were detected in the ipsilateral striatum in the same brain region (bregma level 1.2) at 6 h, 14 and 28 day recovery times (Figure 5I–L; ~2 fold increase; p < 0.05, ANOVA). mRNA levels for VEGF receptors Flk and Flt were also investigated with no change in Flk detected at any time after stroke in the striatum. However increased levels of Flt mRNA were detected in the ipsilateral striatum 6 h, 14 and 28 days after stroke when compared to the contralateral control region in the 6 h recovery group (Figure 5 M–P; ~2 fold increase; p < 0.05, ANOVA).


NADPH oxidase and angiogenesis following endothelin-1 induced stroke in rats: role for nox2 in brain repair.

Taylor CJ, Weston RM, Dusting GJ, Roulston CL - Brain Sci (2013)

Real-time PCR detection of mRNA for NADPH oxidase subunits, angiogenic factor VEGF and its receptor, Flk, in the striatum 6 h to 28 days after ET-1 induced stroke. Nox2 mRNA expression is increased in the ipsilateral striatum as early as 6 h after stroke and remains elevated for up to 28 days in comparison to the contralateral hemisphere (A–D). Nox4 mRNA expression is also increased at 6 h in the ipsilateral striatum and remains elevated for up to 28 days post-stroke, with increases in both the ipsilateral and contralateral hemispheres detected at 7 and 28 days in comparison to the 6 h group (E–H). An increase in the angiogenic factor VEGF is detected in the ipsilateral striatum at 6 hand remains elevated for up to 28 days (I–L). An increase in VEGF receptor Flt mRNA after stroke is detected 6 h, 14 and 28 days after stroke in the ipsilateral hemisphere (M–P). Expression of mRNA was normalized to the respective GAPDH content for each sample (ΔCt), then expressed relative to the contralateral side of the brain at 1.2 mm from Bregma, 6 h post-stroke (ΔΔCt). Data are presented as mean SEM of 8 samples per region (n = 4 per group). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. contralateral; #p < 0.05; ##p < 0.01; ###p < 0.001 vs. corresponding ipsilateral 6 h value; δ p < 0.05 vs. corresponding contralateral 6 h value (ANOVA).
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Related In: Results  -  Collection

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Show All Figures
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brainsci-03-00294-f005: Real-time PCR detection of mRNA for NADPH oxidase subunits, angiogenic factor VEGF and its receptor, Flk, in the striatum 6 h to 28 days after ET-1 induced stroke. Nox2 mRNA expression is increased in the ipsilateral striatum as early as 6 h after stroke and remains elevated for up to 28 days in comparison to the contralateral hemisphere (A–D). Nox4 mRNA expression is also increased at 6 h in the ipsilateral striatum and remains elevated for up to 28 days post-stroke, with increases in both the ipsilateral and contralateral hemispheres detected at 7 and 28 days in comparison to the 6 h group (E–H). An increase in the angiogenic factor VEGF is detected in the ipsilateral striatum at 6 hand remains elevated for up to 28 days (I–L). An increase in VEGF receptor Flt mRNA after stroke is detected 6 h, 14 and 28 days after stroke in the ipsilateral hemisphere (M–P). Expression of mRNA was normalized to the respective GAPDH content for each sample (ΔCt), then expressed relative to the contralateral side of the brain at 1.2 mm from Bregma, 6 h post-stroke (ΔΔCt). Data are presented as mean SEM of 8 samples per region (n = 4 per group). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. contralateral; #p < 0.05; ##p < 0.01; ###p < 0.001 vs. corresponding ipsilateral 6 h value; δ p < 0.05 vs. corresponding contralateral 6 h value (ANOVA).
Mentions: Increased Nox2 mRNA expression was also detected in the ipsilateral striatum in the same brain region (bregma level 1.2) at all times investigated after stroke (Figure 5A–D; p < 0.05, ANOVA). This increase was significantly greatest in the 14 day recovery group when compared to the contralateral control region in the 6 h recovery group (~25 fold increase; p < 0.05, two-way ANOVA). Nox4 mRNA expression was also significantly increased in the ipsilateral striatum at all times investigated after stroke (Figure 5E–H; ~30 fold increase; p < 0.05, ANOVA) with changes in the contralateral hemisphere also detected at both 7 and 28 days in comparison to the contralateral control region in the 6 h recovery group (p < 0.05, two-way ANOVA). Increased levels of VEGF mRNA were detected in the ipsilateral striatum in the same brain region (bregma level 1.2) at 6 h, 14 and 28 day recovery times (Figure 5I–L; ~2 fold increase; p < 0.05, ANOVA). mRNA levels for VEGF receptors Flk and Flt were also investigated with no change in Flk detected at any time after stroke in the striatum. However increased levels of Flt mRNA were detected in the ipsilateral striatum 6 h, 14 and 28 days after stroke when compared to the contralateral control region in the 6 h recovery group (Figure 5 M–P; ~2 fold increase; p < 0.05, ANOVA).

Bottom Line: VEGF mRNA expression was increased in the ipsilateral cortex and striatum between 6 h and 28 days post-stroke concurrently with a marked increase in Nox2 mRNA expression up to 7 days, and increased Nox4 mRNA expression detected between 7 and 28 days.Point counting of blood vessels using Metamorph imaging software showed increased vascular sprouting between 3 and 7 days after stroke with new vascular networks detected in the core infarct region by 14 days.Angiogenic blood vessels 3 and 7 days post-stroke were observed to co-localise with both Nox2 antibody and dihydroethidium fluorescence suggesting a role for Nox2 generated superoxide during the phase of vascular remodeling, whilst Nox4 expression was detected once new cerebral vessels had formed.

View Article: PubMed Central - PubMed

Affiliation: Stroke Injury and Repair Team, O'Brien Institute, 42 Fitzroy St, Fitzroy, Melbourne, Victoria 3065, Australia. cj.taylor@unimelb.edu.au.

ABSTRACT
NADPH oxidases contribute to brain injury, yet they may also have a role in brain repair, particularly in vascular signaling and angiogenesis. This study determined the temporal and spatial profile of NADPH oxidase subunit expression/activity concurrently with angiogenesis in the brain following transient ischemic stroke induced by prolonged constriction of the middle cerebral artery by perivascular injection of endothelin-1 in conscious Hooded Wistar rats (n = 47). VEGF mRNA expression was increased in the ipsilateral cortex and striatum between 6 h and 28 days post-stroke concurrently with a marked increase in Nox2 mRNA expression up to 7 days, and increased Nox4 mRNA expression detected between 7 and 28 days. Point counting of blood vessels using Metamorph imaging software showed increased vascular sprouting between 3 and 7 days after stroke with new vascular networks detected in the core infarct region by 14 days. Angiogenic blood vessels 3 and 7 days post-stroke were observed to co-localise with both Nox2 antibody and dihydroethidium fluorescence suggesting a role for Nox2 generated superoxide during the phase of vascular remodeling, whilst Nox4 expression was detected once new cerebral vessels had formed. These results indicate for the first time that ROS signaling through a cerebrovascular Nox2 NADPH oxidase may be important in initiating brain angiogenesis.

No MeSH data available.


Related in: MedlinePlus