The primary findings in the present study are: 1) Treatment of simvastatin promotes angiogenesis and evokes the functional recovery after TBI; 2) Simvastatin stimulates in vitro angiogenesis after OGD, which could be suppressed by inhibition of Akt or blockage of VEGFR-2; and 3) Simvastatin-induced pro-angiogenetic effects may be related to the activation of VEGFR-2/PI3K/Akt/eNOS pathway in endothelial cells.
One of the most prominent pathophysiological changes after TBI is the ischemia and hypoxia in the lesion boundary area.22
Early after the injury, the direct trauma causes necrotic neuronal cell death, followed by increased neuronal apoptosis hours and days later as a result of secondary injury.23
The post-traumatic ischemia plays an important role in secondary injury after TBI. Regional ischemia early after TBI has been shown to correlate with neurological outcome,1
which suggests that TBI and ischemic stroke share fundamental mechanisms of cell damage.22
To date, drugs that solely target neuroprotection have not been proven efficacious for TBI amelioration.3
The failure of therapies directed only to neuronal protection is, in part, attributable to the lack of concomitant effects on cerebral blood vessels after injury. The ‘neurovascular unit’ has emerged as a new paradigm for understanding the pathology of CNS disease24
To improve neurological function after TBI, one may need not only to promote neuroprotection but also to enhance angiogenesis.
Recent studies show significant benefit of statins in models of TBI26
and related disease processes, including cerebral ischemia,7
and subarachnoid hemorrhage.28
We reported earlier that simvastatin increases neurogenesis and reduces neuronal apoptosis in TBI rats. Simvastatin also increases angiogenesis in limb ischemic rabbits13
and stroke rats.7
Therefore, simvastatin possesses the potential to promote angiogenesis after TBI. In the current study, fluorescent double staining with BrdU and vWF was performed to identify the newly generated endothelial cells. Our data show that TBI-alone induces angiogenesis in the lesion boundary zone and ipsilateral hippocampus, which is consistent with the previous study.2
However, simvastatin enhanced the TBI-induced angiogenesis identified by increasing endothelial cell proliferation and vascular length (). These data are consistent with the previous findings using atorvastatin.8
In this study, simvastatin induces angiogenesis and promotes functional recovery (), although recovery of neurological function after TBI is mediated by many coupled events, including vascular remodeling, neurogenesis, and synaptogenesis. In the treatment of simvastatin, it is possible that other restorative events, in addition to angiogenesis, contribute to recovery of function.
To investigate the mechanism of the pro-angiogenic effects of simvastatin on TBI, endothelial cell tube formation was used as an in vitro
Since ischemia is a fundamental pathophysiological process after TBI,22
OGD was utilized to induce the injury. Simvastatin significantly increases the RBMVECs capillary-like tube formation compared with OGD. Simvastatin-induced tube formation is inhibited by LY29402, a PI3K/Akt inhibitor; enhanced with LiCl, a GSK-3 inhibitor; and blocked by SU1498, a VEGFR-2 antagonist (). These data suggest that simvastatin-induced angiogenesis is related to the activation of the VEGFR-2/Akt/GSK-3 signaling pathway. Endothelial cell capillary tube formation is enhanced by activating VEGFR-2, and Akt is involved in these activities.13, 30
Akt plays an important role in ischemic and VEGF-mediated angiogenesis.31
It regulates many aspects of endothelial cellular functions including apoptosis, cell motility, and tube formation.32
Our previous studies also demonstrate that simvastatin activates Akt in the rat brain after TBI.10
VEGFR-2 mediates the majority of the downstream angiogenic effects of VEGF, including microvascular permeability, endothelial cell proliferation, migration and survival.33
VEGFR-2 transduces VEGF signals through several intracellular signaling pathways including the Raf-Mek-Erk and the PI3K/Akt pathway.34
In light of the critical role of VEGFR-2 on angiogenesis, VEGFR-2 expression was examined both in the brain tissues post TBI and in the cultured RBMVECs subjected to OGD. Our results show that there is an upregulation of VEGFR-2 following TBI, which is consistent with the previous report about VEGFR-2 expression in brain injury,35
and simvastatin further enhances the level of VEGFR-2 in the injured cortex. A similar pattern is seen in the cultured RBMVECs. Hypoxia induces expression of VEGFR-2 in RBMVECs, and simvastatin promotes this induction both in the cytoplasm and in the nucleus of RBMVECs (). Recent reports show that VEGFR-2 can internalize and extend its proliferative signaling activity for longer periods within intracellular compartments.36
Simvastatin-induced intracellular distribution of VEGFR-2 may also contribute to the enhanced angiogenesis, as the intracellular receptor has longer signal transduction capacity. Simvastatin-induced upregulation of VEGFR-2 expression may be partially related to Akt activity, as LY29402 attenuates this effect (). These data indicate that simvastatin modulates VEGFR-2 expression in RBMECs both in vitro
and in vivo
, which results in an increased endothelial cell proliferation and OGD-resistance that may enhance angiogenesis in the injured brain.
eNOS is a downstream mediator of VEGFR-2 and is critical for angiogenesis.21
Enhanced p-eNOS induces a broad range of effects, including the promotion of angiogenesis and mural cell recruitment to immature angiogenic sprouts.37
Our data show that treatment of simvastatin activates Akt/GSK-3 and promotes phosphorylation of eNOS in the TBI brain. Simvastatin treatment of RBMVECs significantly increases p-eNOS activity after OGD compared with controls, which could be suppressed by inhibition of PI3K/Akt and blockade of VEGFR-2 (). These results suggest that simvastatin evokes phosphorylation of eNOS in a VEGFR-2/ PI3K/Akt dependent manner and that eNOS plays an important role in simvastatin-induced angiogenesis after TBI.
Studies reported here show an association between increased overall expressions of VEGFR-2, elevated Akt/eNOS phosphorylation levels, and higher angiogenesis rates in simvastatin treatment compared with control.