Role of endothelial nitric oxide synthetase in arteriogenesis after stroke in mice

Abstract

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 a nitric oxide (NO) donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] 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 h) right middle cerebral artery occlusion (MCAo) and were treated with or without DETA-NONOate (0.4 mg/kg) 24 h 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.

Fig.1. Schematic image and functional tests

A: Schematic representation of a coronal brain section shows eight fields selected along the ischemic boundary zone (IBZ) for quantitative measurements of αSMA-positive artery density and Ki67-positive VSMC proliferation. B: modification of neurological severity score (mNSS) test. C: Foot-fault test.

Fig.2. eNOS^-/- decreases arterial density and diameter and VSMC proliferation in the ischemic brain after MCAo in mice, and treatment of eNOS^-/- with DETA-NONOate increases density and diameter and VSMC proliferation

A-D: αSMA-immunoreactive positive arteries present in the leptomeninge of ischemic brain at 14 days after MCAo (A: WT; B: eNOS^-/-; C: eNOS^-/- + DETA-NONOate; D: Quantitative diameter data). E-H: αSMA-immunoreactive positive arteries present in the IBZ at 14 days after MCAo (E: WT; F: eNOS^-/-; G: eNOS^-/- + DETA-NONOate; H: Quantitative density data). I-M: αSMA and Ki67 double-immunoflureoscent staining in the arteries in the IBZ in mice after MCAo (I: αSMA immunostaining shows VSMCs; J: Ki67 immunostaining shows the proliferated VSMC; K: DAPI staining shows all cell neuclei; L: merged image). M: quantitative data of VSMC proliferation in the IBZ in WT and eNOS^-/- and DETA-NONO-treatment eNOS^-/- mice after stroke. Scale bar = 100 μm. n = 12 / group.

Fig.3. eNOS^-/- reduced artery cell and VSMC migration after stroke, DETA-NONOate treatment enhanced artery cell and VSMC migration

A-D: Common carotid artery cell migration derived from WT (A), eNOS^-/- (B), eNOS^-/- + DETA-NONOate-treatment (C), mice and quantitative data (D) at 3 days after MCAo. E-I: VSMC migration in control (E), DETA-NONOate treatment (F), L-NAME treatment (G) and L-NAME+DETA-NONOate treatment (H) groups, and the quantitative data (I) after 24 hours culture. n = 6 / group.