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Arteriogenesis supports restored perfusion in the ischemic brain and improves long-term functional outcome after stroke. We investigate the role of endothelial nitric oxide synthetase (eNOS) and an NO donor, [(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) aminio] diazen-1-ium-1, 2-diolate (DETA-NONOate), in promoting arteriogenesis after stroke. Adult wild-type (WT, n=18) and eNOS-knockout (eNOS-/-, n=36) mice were subjected to transient (2.5 hours) right middle cerebral artery occlusion (MCAo) and were treated with or without DETA-NONOate (0.4 mg/kg) 24 hours after MCAo. Functional evaluation was performed. Animals were sacrificed 3 days after MCAo for arterial cell culture studies, or 14 days for immunohistochemical analysis. Consistent with previous studies, eNOS-/- mice exhibited a higher mortality rate (p<0.05, n=18/group) and more severe neurological functional deficit after MCAo than WT mice (p<0.05, n=12/group). Decreased arteriogenesis, was evident in eNOS-/- mice compared with WT mice, as demonstrated by reduced vascular smooth muscle cell (VSMC) proliferation, arterial density and diameter in the ischemic brain. eNOS-/- mice treated with DETA-NONOate had a significantly decreased mortality rate and improved functional recovery, and exhibited enhanced arteriogenesis identified by increased VSMC proliferation, and upregulated arterial density and diameter compared to eNOS-/- mice after stroke (p<0.05, n=12/group). To elucidate the mechanisms underlying eNOS/NO mediated arteriogenesis, VSMC migration was measured in vitro. Arterial cell migration significantly decreased in the cultured common carotid artery (CCA) derived from eNOS-/- mice 3 days after MCAo compared to WT arterial cells. DETA-NONOate-treatment significantly attenuated eNOS-/--induced decrease of arterial cell migration compared to eNOS-/- control artery (p<0.05. n=6/group). Using VSMC culture, DETA-NONOate significantly increased VSMC migration, while inhibition of NOS significantly decreased VSMC migration (p<0.05. n=6/group). Our data indicated that eNOS not only promotes vascular dilation but also increases VSMC proliferation and migration, and thereby enhances arteriogenesis after stroke. Therefore, increase eNOS may play an important role in regulating of arteriogenesis after stroke.

Neurogenesis may contribute to functional recovery after neural injury. Nitric oxide donors such as DETA-NONOate promote functional recovery after stroke. However, the mechanisms underlying functional improvement have not been ascertained. We therefore investigated the effects of DETANONOate on neural progenitor/stem cell neurospheres derived from the subventricular zone from young and retired breeder rat brain. Subventricular zone cells were dissociated from normal young adult male Wistar rats (2-3 months old) and retired breeder rats (14 months old), treated with or without DETA-NONOate. Subventricular zone neurosphere formation, proliferation, telomerase activity, and Neurogenin 1 mRNA expression were significantly decreased and glial fibrillary acidic protein expression was significantly increased in subventricular zone neurospheres from retired breeder rats compared with young rats. Treatment of neurospheres with DETA-NONOate significantly decreased neurosphere formation and telomerase activity, and promoted neuronal differentiation and neurite outgrowth concomitantly with increased N-cadherin and β-catenin mRNA expression in both young and old neurospheres. DETA-NONOate selectively increased Neurogenin 1 and decreased glial fibrillary acidic protein mRNA expression in retired breeder neurospheres. Ncadherin significantly increased Neurogenin 1 mRNA expression in young and old neurospheres. Anti-N-cadherin reversed DETA-NONOate-induced neurosphere adhesion, neuronal differentiation, neurite outgrowth, and β-catenin mRNA expression. Our data indicate that age has a potent effect on the characteristics of subventricular zone neurospheres; neurospheres from young rats show significantly higher formation, proliferation and telomerase activity than older neurospheres. In contrast, older neurospheres exhibit significantly increased glial differentiation than young neurospheres. DETA-NONOate promotes neuronal differentiation and neurite outgrowth in both young and older neurospheres. The molecular mechanisms associated with the DETANONOate modulation of neurospheres from young and older animals as well age dependent effects of neurospheres appear to be controlled by N-cadherin and β-catenin gene expression, which subsequently regulates the neuronal differentiating factor Neurogenin expression in both young and old neural progenitor cells. Neural precursor cells contribute to adult neurogenesis and to the limited attempt of brain repair after injury. Induction of self-renewal or differentiation of neural progenitor cells depends on the interaction of intrinsic and environmental cues. Age is an intrinsic factor, which may influence neurogenesis. However, neurogenesis, which contributes to the ability of the adult brain to function normally and adapt to disease, declines with advancing age (Cameron and McKay, 1999;Kuhn et al., 1996). In addition, accelerated glial reactivity in aged rats also correlates with reduced functional recovery after stroke (Badan et al., 2003a). Glial reactivity increases with age, w...

Neurogenesis declines with advancing age. The mammalian achaete-scute homologue-1 encodes a basic helix-loop-helix transcription factor, which controls neuronal differentiation. In this study, we first tested whether atorvastatin treatment enhances neurological functional outcome and neuronal differentiation after stroke in retired breeder 12 month rats. Rats were subjected to middle cerebral artery occlusion and treated with or without atorvastatin (3 mg/kg) for 7 days. Atorvastatin significantly increased expression of mammalian achaete-scute homologue-1, beta-tubulin III, and vascular endothelial growth factor in the ischemic brain, and concomitantly improved functional outcome compared with middle cerebral artery occlusion control rats. Increased neurogenesis significantly correlated with functional recovery after stroke. To further investigate the mechanisms of atorvastatin-induced neuronal differentiation, experiments were performed on neurospheres derived from retired breeder rat subventricular zone cells. Atorvastatin increased neuronal differentiation and upregulated vascular endothelial growth factor and mammalian achaete-scute homologue-1 gene expression in cultured neurospheres. Vascular endothelial growth factor-treated neurospheres significantly increased mammalian achaete-scute homologue-1 and beta-tubulin III expression. Inhibition of vascular endothelial growth factor decreased atorvastatin-induced mammalian achaete-scute homologue-1 and beta-tubulin III expression. These data indicate that atorvastatin increases neuronal differentiation in retired breeder rats. In addition, atorvastatin upregulation of vascular endothelial growth factor expression, influences mammalian achaete-scute homologue-1 transcription factor, which in turn, facilitates an increase in subventricular zone neuronal differentiation. These atorvastatin-mediated molecular events may contribute to the improved functional outcome in retired breeder rats subjected to stroke.

Human umbilical cord blood cells (HUCBCs) have been employed as a restorative treatment for experimental stroke. In this study, we investigated whether transplantation of sub-therapeutic doses of HUCBCs and Simvastatin enhances cerebral vascular remodeling after stroke. Adult male Wistar rats (n=34) were subjected to transient middle cerebral artery occlusion (MCAo) and treated with: phosphate buffered solution (PBS, gavaged daily for 7 days); Simvastatin (0.5mg/kg, gavaged daily for 7 days); HUCBCs (1x106, injected once via tail vein); and combination Simvasatin with HUCBCs, starting at 24 h after MCAo. There was no significant difference between Simvastatin- or HUCBC-monotherapy and MCAo-alone group. Combination treatment 24 h post-stroke significantly increased the perimeter of von Willebrand factor (vWF)-positive vessels, the diameter and density of alpha smooth muscle actin ( SMA)-positive arteries, and the percentage of BrdU-positive endothelial cells (ECs) in the ischemic boundary zone (IBZ) compared with MCAo-alone or HUCBC-monotherapy 14 days after MCAo (p<0.05, n=8/group); Combination treatment significantly increased the densities of vWF-vessels and SMA-arteries as well as the densities of BrdU-ECs and BrdU-positive smooth muscle cells (SMCs) in vascular walls in the IBZ compared with Simvastatin-monotherapy. Moreover, the increased BrdU-ECs and BrdU-SMCs were significantly correlated with neurological functional outcome 14 days after MCAo. Combination treatment also significantly increased the expression of Angiopoietin-1 (Ang1), Tie2 and Occludin in the IBZ (p<0.05, n=8/group). The in vitro experiments showed that combination treatment and Ang1 significantly increased capillary-like tube formation and arterial cell migration; anti-Ang1 significantly reduced combination treatment induced tube-formation and artery cell migration (p<0.05, n=6/group). These findings indicated that combination of sub-therapeutic doses of Simvastatin and HUCBCs treatment of stroke increases Ang1/Tie2 and Occludin expression in the ischemic brain, amplifies endogenous angiogenesis and arteriogenesis, and enhances vascular remodeling which in concert may contribute to functional outcome after stroke.