Nogo-A targeted therapy promotes vascular repair and functional recovery following stroke

Abstract

Stroke is a major cause of serious disability due to the brain's limited capacity to regenerate damaged tissue and neuronal circuits. After ischemic injury, a multiphasic degenerative and inflammatory response is coupled with severely restricted vascular and neuronal repair, resulting in permanent functional deficits. Although clinical evidence indicates that revascularization of the ischemic brain regions is crucial for functional recovery, no therapeutics that promote angiogenesis after cerebral stroke are currently available. Besides vascular growth factors, guidance molecules have been identified to regulate aspects of angiogenesis in the central nervous system (CNS) and may provide targets for therapeutic angiogenesis. In this study, we demonstrate that genetic deletion of the neurite outgrowth inhibitor Nogo-A or one of its corresponding receptors, S1PR2, improves vascular sprouting and repair and reduces neurological deficits after cerebral ischemia in mice. These findings were reproduced in a therapeutic approach using intrathecal anti-Nogo-A antibodies; such a therapy is currently in clinical testing for spinal cord injury. These results provide a basis for a therapeutic blockage of inhibitory guidance molecules to improve vascular and neural repair after ischemic CNS injuries.

Keywords: CNS; guidance factor; ischemia; revascularization; therapeutic angiogenesis.

Fig. 1.

Angiogenic response after photothrombotic stroke. (A) Location of stroke, time points, and histological stroke size. (Scale bar, 1 mm.) (B) Expression analysis of genes associated with sprouting angiogenesis, vascular maturation, tissue hypoxia, guidance, and Nogo-A pathway at 2 (n = 3), 7 (n = 3), 16 (n = 3), and 28 (n = 4) d postinjury. Data are presented as log expression ratio (-DDCT); purple indicates up-regulation and green indicates down-regulation. (C) Representative immunofluorescence images of Nogo-A (Upper) and S1PR2 (Lower) costained with the vascular endothelium marker Ib4 4 wk after stroke (Left) (Scale bars, 20 µm.) Time series of Rtn4a/Nogo-A and S1pr2 expression after stroke in three mice per time point (Right). (D) Visual representation of the analyzed angiogenesis associated genes according to ontology and function. (E) Representative fluorescence image of hypovascularized 300-µm ischemic border zone stained for the vascular marker CD31 4 wk after stroke with corresponding heat maps of local vascular area fraction. Ibz, ischemic border zone. (Scale bar, 100 µm.) Data are mean ± SEM (one-way ANOVA with Dunnett’s post hoc test). *P < 0.05.

Fig. 2.

Vascular repair in the ischemic border zone. (A) Schematic representation of the experimental timeline in the five experimental conditions: WT (n = 13), S1PR2^−/− (n = 5), Nogo-A^−/− (n = 9), Ctrl Ab (n = 13), and anti–Nogo-A Ab (n = 12). (B) Assessment of the vascular area fraction in the ipsilesional and contralesional sensory-motor cortex. (C) Blood vessels were visualized by CD31 immunostaining. Heat maps show the local area fraction of blood vessels in an area 0 to 300 µm from the stroke core. (Scale bars, 100 µm.) (D) Quantitative evaluation of the vascular area fraction, blood vessel length, number of branches, nearest-neighbor distance (NND), and variability of NND (SD NND) in the ischemic border zone of the different treatment groups. (E) Quantification of newly generated EdU⁺/CD31⁺ endothelial cells. [Scale bars, 20 µm and 5 µm (zoom in).] (F) Ratio of perfused (red) to nonperfused (green) blood vessels in the five experimental groups. (Scale bars, 50 µm.) (G) Relative fluorescence intensity of GFAP and the microglia/macrophage marker Iba1. (H) Pericyte coverage assessed by the ratio of CD13⁺/CD31⁺ cells. Data are mean ± SEM (one-way ANOVA with Dunnett’s post hoc test). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Functional assessment of blood perfusion in the ischemic border zone by laser Doppler flowmetry. (A) Schematic representation of cerebral blood flow measurements in the same animal at sequential time points for WT (n = 4) and Nogo-A^−/− (N = 4) animals after stroke. (B) Heat maps indicate local cerebral blood flow in nine subfields covering the stroke area. (C) Blood perfusion changes in forelimb- and hindlimb-related areas after stroke in Nogo-A^−/− and WT animals. Data are mean ± SEM (Student’s t test). *P < 0.05.

Fig. 4.

Motor performance after photothrombotic stroke. (A) Timeline of behavioral testing after stroke. Motor performance of all animals was tested 3, 7, 14, and 21 d after stroke (arrows) in the horizontal ladder and cylinder test and compared with baseline performances. The groups consist of WT (n = 13), S1PR2^−/− (n = 5), Nogo-A^−/− (n = 9), Ctrl Ab (n = 13), and anti–Nogo-A Ab (n = 12). (B) Ratios of paw preference, dragging, and symmetry in the cylinder test. (C) Sequence of a horizontal ladder run with errors indicated by black bars from representative animals from each group. (D) Ratio of forelimb and hindlimb errors per total number of steps in the horizontal ladder test. (E and F) Correlation between motor function improvement and vascular parameters in single animals of WT, S1PR2^−/−, Nogo-A^−/−, Ctrl Ab, anti–Nogo-A Ab group 3 wk after stroke. Data are mean ± SEM (one-way ANOVA with Dunnett’s post hoc test). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Nonvascular effects of Nogo-A neutralization in the periinfarct zone 3 wk after stroke. The groups consist of animals receiving Ctrl Ab (n = 13) and anti–Nogo-A Ab (n = 12). (A) Number of NeuN⁺ cells per mm². (Scale bar, 100 µm.) (B) Number of apoptotic cells positive for cleaved caspase 3 per mm² (Scale bar, 50 µm). (C) Numbers of PV⁺, SST⁺, and VIP⁺ GABAergic interneurons per mm². (Scale bars, 100 µm.) (D) Relative fluorescence intensity of the synaptic markers synaptophysin, vGat and vGlut2. (Scale bars, 20 µm.) (E) Relative fluorescence intensity for TH/DA and 5-HT. (Scale bars, 100 µm.) (F) Relative fluorescence intensity of Nf160. (Scale bar, 20 µm.) Data are mean ± SEM (Student’s t test). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

Reduction of angiogenesis in the periinfarct region by anti-VEGF reduces the functional recovery effects of Nogo-A deletion. (A) Experimental timeline after stroke. WT or Nogo-A KO animals received either anti-VEGF (Avastin, Av; arrows) or a control Ab (Ctrl) i.v. every other day for 10 d after stroke. (B) Assessment of the vascular area fraction, vascular length and number of branches in the periinfarct region. (Scale bar, 20 µm.) (C) Quantification of EdU⁺/CD31⁺ cells. (Scale bars, 20 µm.) (D and E) Behavioral performance assessed at baseline and 3, 7, 14, and 21 d postinjury in the horizontal ladder (D) and cylinder test (E). Data are mean ± SEM (one-way ANOVA with Dunnett’s post hoc test). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.

Vessel and capillary network formation by HUVECs in vitro is limited by CNS myelin and Nogo-A in a 3D GAG-PEG hydrogel model. (A) Schematic overview of the in situ assembly of GAG-PEG gel-based 3D cultures from reactive polymer conjugates, HUVECs, and heparin-binding growth factors (VEGF, FGF2, and SDF1). (B) Bright-field and fluorescent images of tubular network formation by HUVECs (CD31⁺) in GAG-PEG hydrogels supplemented with the neurite growth inhibitory Nogo-A domain Nogo-A Δ20, the neutralizing Nogo-A Ab 11C7, scrambled Nogo-A Δ20, or CNS myelin extract. (Scale bar, 100 µm.) (C) Quantification of area fraction, diameter, branch number, and length of HUVEC-derived vessels (n = 5 to 7 cultures). Data are mean ± SEM (one-way ANOVA with Dunnett’s post hoc test). *P < 0.05, **P < 0.01, ***P < 0.001.