Transcriptional activation of endothelial cells by TGFβ coincides with acute microvascular plasticity following focal spinal cord ischaemia/reperfusion injury

Abstract

Microvascular dysfunction, loss of vascular support, ischaemia and sub-acute vascular instability in surviving blood vessels contribute to secondary injury following SCI (spinal cord injury). Neither the precise temporal profile of the cellular dynamics of spinal microvasculature nor the potential molecular effectors regulating this plasticity are well understood. TGFβ (transforming growth factor β) isoforms have been shown to be rapidly increased in response to SCI and CNS (central nervous system) ischaemia, but no data exist regarding their contribution to microvascular dysfunction following SCI. To examine these issues, in the present study we used a model of focal spinal cord ischaemia/reperfusion SCI to examine the cellular response(s) of affected microvessels from 30 min to 14 days post-ischaemia. Spinal endothelial cells were isolated from affected tissue and subjected to focused microarray analysis of TGFβ-responsive/related mRNAs 6 and 24 h post-SCI. Immunohistochemical analyses of histopathology show neuronal disruption/loss and astroglial regression from spinal microvessels by 3 h post-ischaemia, with complete dissolution of functional endfeet (loss of aquaporin-4) by 12 h post-ischaemia. Coincident with this microvascular plasticity, results from microarray analyses show 9 out of 22 TGFβ-responsive mRNAs significantly up-regulated by 6 h post-ischaemia. Of these, serpine 1/PAI-1 (plasminogen-activator inhibitor 1) demonstrated the greatest increase (>40-fold). Furthermore, uPA (urokinase-type plasminogen activator), another member of the PAS (plasminogen activator system), was also significantly increased (>7.5-fold). These results, along with other select up-regulated mRNAs, were confirmed biochemically or immunohistochemically. Taken together, these results implicate TGFβ as a potential molecular effector of the anatomical and functional plasticity of microvessels following SCI.

Figure 1. Temporal course of neuronal loss following focal ischaemic SCI

Intact spinal grey matter staining in the rostral lumbar spinal cord of a sham-control rat (A). As early as 3 h following ET-1 microinjection, significant loss of Map2-immunoreactive tissue in focal grey matter supplied by targeted supply microvessels is observed (B). At higher magnification, a dense network of neuronal processes in ventral grey matter can be seen (C), a pattern that is diminished by 3 h post-ischaemia (E). Furthermore, NeuN-immunoreactive motor neurons are apparent in control tissue (D; arrowheads), but are lost by 3 h following ET-1 microinjection (F). Nissl staining of adjacent spinal cord sections (G and H) corroborates this neuronal loss. Many Nissl-positive neurons are apparent in control tissue (I) with obvious loss of these profiles in ventral grey matter 3 h following ET-1-induced focal ischaemia (J). Scale bar = 50 μm.

Figure 2. Acute astroglial loss from ischaemic microvessels

In intact spinal grey matter, Aqp-4- and GFAP-immunoreactive astrocytic endfeet are associated with perfused microvessels (A–D; arrowheads). By 3 h post-ischaemia, significant loss of GFAP-immunoreactivity is observed in affected tissue (E), with no detectable Aqp-4 staining associated (F) with microvascular elements (G). By 6 h post-ischaemia, all GFAP- (I) and Aqp-4- (J) immunoreactivity is lost in affected grey matter, despite a preservation of intact microvessels (K and L). Scale bar = 50 μm (C–F).

Figure 3. Tissue levels of uPA enzymatic activity are significantly increased in response to spinal cord ischaemia

At 3 and 6 h post-ET-1 microinjection, 5 mm of experimental spinal cords (centered on the microinjection sites) were rapidly isolated, ventral spinal tissue was dissected, and 10 μg of total protein was analysed by uPA zymography (A). Densitometric analysis of zymographic results revealed that by 3 h, levels of uPA enzymatic activity were significantly increased compared with sham-injected tissue (B). Levels of uPA activity are further increased by 6 h post-ischaemia, and were significantly higher than both sham and 3 h levels (B). Quantitative data are the mean uPA activity±S.D. (*P≤0.05, **P≤0.01 and ***P≤0.001; F = 48.79, df = 9).

Figure 4. Microvascular MMP-9 expression is induced by ischaemic SCI

In control ventral grey matter, no detectable MMP-9 immunoreactivity is present in smvECs (A and B). By 12 h post-ischaemia, significant MMP-9-immunoreactvity is observed in affected spinal microvessels (C and D), although a subset of microvessels does not express MMP-9 (D; arrowheads). This increase in MMP-9 immunoreactivity is maintained 24 h post-ischaemia (E and F), with all microvessels in affected tissue expressing detectable levels of MMP-9. Confocal analysis confirms co-localization of RECA-1 and MMP-9 in the xz and yz planes (D and F; arrows). Scale bar = 50 μm (A–F).

Figure 5. smvEC expression of IL-6 and IGFBP-3 after SCI

Basal levels of expression for both IL-6 (A and B; arrowheads) and IGFBP-3 (C and D; arrows) were detected in the vasculature in control spinal grey matter. Increased IL-6 immunoreactivity was observed in smvECs at 6 h post-ischaemia (E and F; arrowheads). Interestingly, this expression appeared to exhibit a biphasic pattern, with qualitative levels diminished at 1 day (I and J), but reappearing in activated microvascular profiles by 3 days post-ischaemia (M and N; arrowheads). Similar to IL-6 staining, levels of IGFBP-3 immunoreactivity were intensified by 6 h post-ET-1 microinjection (G and H; arrows). However, levels were then found to diminish progressively beginning at 2 days post-ischaemia (K and L; arrows), with little IGFBP-3 immunoreactivity associated with perfused microvessels 5 days post-ischaemia (O and P; arrow). Scale bar = 50 μm (A–P).

Figure 6. smvEC expression of PAI-1 is rapidly induced by ischaemic SCI

No detectable PAI-1 immunoreactivity was observed in control spinal grey matter (B). As early as 3 h post-ischaemia, robust PAI-1 immunoreactivity is observed in affected smvECs (C and D; arrowheads). This expression level is maintained in smvECs for up to 3 days post-ischaemia (E–H; arrowheads). By 5 days post-ischaemia, PAI-1 immunoreactivity returned to basal levels in perfused spinal microvessels (I and J). Confocal imaging definitively co-localizes PAI-1 to FITC–LEA activated/perfused spinal microvessels in the xz and yz planes (F and H; arrows). Scale bar = 50 μm (A–J).