Sodium-dependent vitamin C transporter 2 (SVCT2) expression and activity in brain capillary endothelial cells after transient ischemia in mice

Abstract

Expression and transport activity of Sodium-dependent Vitamin C Transporter 2 (SVCT2) was shown in various tissues and organs. Vitamin C was shown to be cerebroprotective in several animal models of stroke. Data on expression, localization and transport activity of SVCT2 after cerebral ischemia, however, has been scarce so far. Thus, we studied the expression of SVCT2 after middle cerebral artery occlusion (MCAO) in mice by immunohistochemistry. We found an upregulation of SVCT2 after stroke. Co-stainings with Occludin, Von-Willebrand Factor and CD34 demonstrated localization of SVCT2 in brain capillary endothelial cells in the ischemic area after stroke. Time-course analyses of SVCT2 expression by immunohistochemistry and western blots showed upregulation in the subacute phase of 2-5 days. Radioactive uptake assays using (14)C-labelled ascorbic acid showed a significant increase of ascorbic acid uptake into the brain after stroke. Taken together, these results provide evidence for the expression and transport activity of SVCT2 in brain capillary endothelial cells after transient ischemia in mice. These results may lead to the development of novel neuroprotective strategies in stroke therapy.

Figure 1. SVCT2 is upregulated after stroke and mainly localized in endothelial structures.

Sections of brain tissue from mice with MCAO were stained with SVCT2 (red, left column) and co-stained with occludin (B, B'), NeuN (E, E'), GFAP (H, H'). Intense SVCT2- immunoreactivity was found in the infarct area (ipsilateral), compared to only weak expression on the contralateral side (compare A to A', D to D', G to G'). Co-immunohistochemistry with occludin showed colocalisation in linear, occasionally branched structures reminiscent of capillaries (A–C, D–F, G–I, arrows). Co-stainings with NeuN showed weak SVCT2-immunoreactivity in NeuN positive cells in both hemispheres (D–F, D'–F', arrowheads). Co-stainings with GFAP showed no SVCT2-immunoreactivity in GFAP-positive cells (G–I, G'–I'). Size bars: 10 µm.

Figure 2. SVCT2 is localized in brain capillary endothelial cells after murine stroke.

Brain sections of mice with focal cerebral ischemia were stained with SVCT2 and Von-Willebrand factor (vWF) antibodies (A–C) or SVCT2 and CD34 antibodies (D–F). SVCT2 colocalised with both vWF and CD34, indicating a localization in brain capillary endothelial cells. Size bars: 10 µm.

Figure 3. Time-course of SVCT2 immunohistochemistry after murine stroke.

Brains of mice with a focal cerebral ischemia were dissected at day 0, 1, 2, 4, 5 and 7 after stroke and stained for SVCT2 (red) and nuclei (DAPI, blue). Ipsilateral (A–B) and contralateral (A'–B') hemispheres of day 1 and 5 are shown. An increase of SVCT2 immunoreactivity was found in the ipsilateral hemisphere at day 5 (B). Contralateral hemispheres (A'–B') showed only weak background staining. Quantification of immunohistochemistry showed a significant increase at day 4, a peak at day 5 and a decline at day 7 (C). Size bar: 10 µm. (** p<0.01, n = 4).

Figure 4. Time-course of SVCT2 protein levels after stroke.

To assess the levels of SVCT2 on a whole-protein level, brains of stroke mice were dissected and lysed at day 0, day 2 and day 5 after stroke and analysed by western blot. Western blots showed only very weak SVCT2 bands in animals without stroke and on day 0 after stroke. An increase in SVCT2 bands can be seen in ipsilateral hemispheres 2 days after stroke and a further increase 5 days after stroke (A). Actin was used as a marker for protein loading (A). SVCT2 and actin bands were measured using image analysis software ImageJ (NIH). Intensities of SVCT2 bands were normalized by the corresponding actin band. Semi-quantitative analysis of western blot signals showed a significant increase of SVCT2 at days 2 and 5 in ipsilateral hemispheres (B). (** p<0.01, *** p<0.001, n = 3).

Figure 5. Uptake of 14C-labelled ascorbic acid into the brain is increased 5 days after stroke.

Animals with focal cerebral ischemia and control animals without stroke were injected with 14C-labelled ascorbic acid or dehydroascorbate and 3H-labelled inulin. Radioactive uptake assays showed a significantly increased ascorbic acid uptake 5 days after stroke compared to animals without stroke or at day 0 after stroke. Dehydroascorbate was transported into the brain at similar rates before, at day 0 and day 5 after stroke. At day 5 ascorbic acid transport was not significantly lower than dehydroascorbate. Inulin uptake, as a marker for blood-brain-barrier integrity showed an insignificant trend towards an increase after stroke. (*** p<0.001, ns  =  non-significant, n = 4).