Angiogenesis induced by cerebral ischemia contributes to the recovery from ischemic brain injury and is correlated with longer survival in humans (Krupinski et al., 1994
). However, vascular permeability complicated by angiogenesis is inevitable and can exacerbate the subsequent injury. Therefore, identifying the potential mechanisms that regulate angiogenesis and vascular permeability following stroke may provide new insights into the pathogenesis and future therapy.
Based on previous reports that an Src inhibitor efficiently reduced VEGF-induced vascular permeability and infarct volume (Paul et al., 2001
) and protected animals from brain edema and injury (Akiyama et al., 2004
; Lennmyr et al., 2004
), we suggest that Src might modulate angiogenic factors after focal cerebral ischemia. Here we found that Src immunoreactivity was preferentially increased in the astrocyte-like cells surrounding the capillaries, suggesting that Src has a close association with capillaries in cerebral ischemia. Astrocytes, which produce Src, perform an indispensable function in the process of ischemic injury. Src increased during the formation of edema and decreased as the edema receded. Src protein increased significantly in comparison with its mRNA. The results indicate that the increased Src kinase activity was not due solely to the increased Src mRNA expression levels. These findings support the importance of Src kinase activity in ischemic stroke injury.
SSeCKS, an Src-suppressed C kinase substrate, was recently identified and reported to regulate the expression of both VEGF and Ang-1 in astrocytes (Lee et al., 2003
). One study has indicated that activated Src and SSeCKS control similar sets of genes in both human and mouse fibroblasts and epithelial cells (Liu et al., 2006
). In this study we observed that SSeCKS was widespread in normal rat brain and dominantly expressed in astrocyte-like, neuron-like, and endothelial-like cells. We further showed that the temporospatial expression of SSeCKS is reverse that of Src. Indeed, SSeCKS decreased after ischemia and then increased gradually, returning to control level at 1 day, the time at which neovessels were detected. The results indicate that the expression of SSeCKS was markedly decreased by ischemia. It has been reported that SSeCKS can repress angiogenesis and induce maturation and stabilization of permeable vessels by enhancing the expression of tight junction protein in vitro
(Lee et al., 2003
Angiogenesis is a step-wise process. Necessary steps include destabilization of vessels, sprouting and branching of destabilized vessels, proliferation and migration of endothelial cells, and stabilization of neomicrovessels (Conway et al., 2001
; Folkman and D’Amore, 1996
; Risau, 1997
). Many angiogenic factors are involved in this progression, and VEGF, Ang-2, and Ang-1 may play essential roles (Jain, 2003
). Our results show that all these angiogenic factors were highly expressed in neuron-like and glial-like cells in the ischemic penumbra. VEGF and Ang-2 increased significantly in the early period after cerebral ischemia during the formation of brain edema. Ang-2 may stimulate microvessel sprouting and angiogenesis in the presence of VEGF (Zhu et al., 2005
; Lobov et al., 2002
). These results indicate that Ang-2 acts synergistically with VEGF to promote brain angiogenesis and vascular permeability after cerebral ischemia. Ang-1 is a strong anti-permeability factor that can reduce vascular leakage. In this study, we observed that Ang-1 immunoreactivity decreased in the ischemia boundary zone, supporting the leakage-resistant role attributed to Ang-1 (Thurston et al., 1999
). The Ang-1 protein was significantly decreased at 1 day, when cerebral edema was at its greatest. This dynamic expression may explain the progression of cerebral edema after MCAO and reflect the fact that Ang-1 acts at a later stage of vascular stabilization and maturation (Wakui et al., 2006
Angiogenesis has been suggested to contribute to brain repair in stroke (Manoonkitiwongsa et al., 2001
; Wei et al., 2001
; Valable et al., 2005
). Our study described the temporal evolution of vascular remodeling in relation to the expression of Src, SSeCKS, and primary angiogenic factors. Neovessels became clearly apparent in the ischemic boundary area at 1 day after MCAO. However, some of them were dilated and tortuous. With the maturation of neovessels during the later period of ischemia, the cerebral edema decreased significantly. These results suggest that the newly formed microvessels are functional and promote the recovery of ischemic injury.
Although the increase in vascular permeability that accompanies angiogenesis is inevitable after cerebral ischemia (Lee et al., 2004
; Harrigan et al., 2002
), a minor increase in vascular permeability may be necessary for angiogenesis. The increase in edema is likely due, at least in part, to a decrease in the tight junction protein ZO-1 (Dvorak et al., 1999
; Wang et al., 2001
; Fischer et al., 2002
). It has been shown that absence of ZO-1 results in vascular leakage. In this study we found that ZO-1 was expressed around the vessels, especially at sites of endothelial cell–cell contact, suggesting that ZO-1 preserves the integrity of the BBB. We further observed that the expression of ZO-1 was in accordance with that of Ang-1. The decrease in ZO-1 coincided with increases in the extent of cerebral edema.
Our results also showed that the expression of Src and SSeCKS correlates with that of angiogenic factors and the tight junction protein ZO-1 after ischemia. Src correlated positively with VEGF and Ang-2 but negatively with SSeCKS, Ang-1, and ZO-1. The opposite was true of SSeCKS. Src, VEGF, and Ang-2 were increased, whereas SSeCKS, Ang-1, and ZO-1 were decreased during the formation of cerebral edema. These data demonstrate that Src and SSeCKS have opposite effects on angiogenesis and brain edema formation. We further infer that Src inhibits SSeCKS and regulates angiogenesis and vascular permeability by modulating angiogenic factors and tight junction proteins after cerebral ischemia. Src–SSeCKS may be a new signal pathway involved in ischemic injury.
The present study indicates that Src and SSeCKS may play opposite roles in angiogenesis and vascular permeability after focal cerebral ischemia. Angiogenic factors that serve as downstream mediators participate in the process. A detailed understanding of the modulation of angiogenesis and vascular leakage by the Src–SSeCKS pathway will be helpful in the development of therapeutics for focal cerebral ischemia.