Rac1 is a critical mediator of endothelium-derived neurotrophic activity

Abstract

The therapeutic potential of neurotrophic factors has been hampered by their inability to achieve adequate tissue penetration. Brain blood vessels, however, could be an alternative target for neurosalvage therapies by virtue of their close proximity to neurons. Here we show that hemizygous deletion of Rac1 in mouse endothelial cells (ECs) attenuates brain injury and edema after focal cerebral ischemia. Microarray analysis of Rac1(+/-) ECs revealed enrichment of stress response genes, basement membrane components, and neurotrophic factors that could affect neuronal survival. Consistent with these expression profiles, endothelial Rac1 hemizygosity enhanced antioxidative and endothelial barrier capacities and potentiated paracrine neuroprotective activities through the up-regulation of the neurotrophic factor, artemin. Endothelial Rac1, therefore, could be an important therapeutic target for promoting endothelial barrier integrity and neurotrophic activity.

Fig. 1

Haploinsufficiency of endothelial Rac1 in mice leads to neuroprotection. EC-Rac1^+/− and control mice underwent 2-hour MCAo followed by 22-hour reperfusion. (A) Representative brain sections stained with 2,3,5-triphenyltetrazolium chloride visualizing the infarct area. (B and C) Infarct (B) and edema (C) volumes of the ischemic hemisphere. (D) Neurological deficit score (n = 19 to 23 mice; *P < 0.05, Mann-Whitney test). (E) Change in CBF in the ischemic hemisphere at the end of 2-hour MCAo. (B, C, and E) Values are percent of the nonischemic hemisphere and shown as means ± SEM of 7 to 10 mice (*P < 0.05, ANOVA). NS, not significant.

Fig. 2

Expression profiling of Rac1^+/− ECs. Differential mRNA expression between Rac1^+/− and Rac1^+/+ mouse ECs was explored by microarray analysis. (A) qRT-PCR, immunoblotting, and GST-PAK (glutathione S-transferase–p21-activated protein kinase) pull-down assays showing Rac1 expression and activity in Rac1^+/− and Rac1^+/+ ECs. (B and C) The up-regulated (red) and down-regulated (green) genes in Rac1^+/− ECs. The scatter plot (B) shows 12,089 gene features with the signal intensity >200. (D and E) Functional annotation clustering of the down-regulated (D) and up-regulated (E) genes. (F and G) GSEA showing enrichment of cell cycle–related genes in Rac1^+/+ (F) and TGF-β–inducible genes in Rac1^+/− ECs (G). Each plot displays (top) progression of the running enrichment score; (middle) “hits” in the gene set against the ranked list of all genes in the data; (bottom) histogram for the ranked list. (H and I) qRT-PCR (H) and correlation analysis (I) to validate the microarray data. (J) Crystallin αB immunostaining of Rac1^+/− versus Rac1^+/+ ECs. Scale bar, 25 μm. Values are means ± SEM of three to four replicates (*P < 0.05, **P < 0.01; ANOVA).

Fig. 3

Phenotypic profiling of Rac1^+/− ECs. (A, B, D, E, and F) Rac1^+/+ and Rac1^+/− mouse ECs were sequentially exposed to 4-hour hypoxia and 2-hour (A and D) or 20-hour (B, E, and F) reoxygenation (H/R) or treated with normoxia (Norm). (A) NADPH oxidase activity (left) and ROS production (right) as determined by DPI-inhibitable lucigenin chemiluminescence and DCF fluorescence, respectively. (B) Apoptotic death was assessed by flow cytometry (left) as the Annexin V–EGFP–labeled EC fraction (right). (C)ECs at subconfluence (Sub) or prolonged confluence (>4 weeks) (Sheet) were subjected to standard trypsinization until single-cell suspensions were obtained. (D) The permeability of confluent EC monolayers grown on 0.4-μm Transwell inserts was assessed as HRP leak across the membrane during normoxia and H/R. (E) After exposure to normoxia or H/R, ECs were co-cultured with [³H]thymidine-labeled monocytes (THP-1) for 3 hours. After washing, monocyte-EC binding was assessed as the cell lysate radioactivity. (F) VCAM-1 mRNA abundance was determined by qRT-PCR. (A, C to F) Values are means ± SEM of triplicates (*P < 0.05, **P < 0.01; ANOVA).

Fig. 4

Decreased actin polymerization and circumferential redistribution of F-actin to the cortical areas in Rac1^+/− ECs. Mouse ECs cultured in the presence of 20% serum were fixed and stained with (A) phalloidin to visualize F-actin and (B) antibody against β-tubulin for microtubules (MT). The F-actin and MT-labeled areas were quantified and represented as percent of total cell areas. Scale bar, 25 μm. Values are means ± SEM of five to seven replicates (*P < 0.05; ANOVA).

Fig. 5

Rac1^+/− ECs show enhanced neurotrophic activity through paracrine mechanisms. (A) mRNA expression of the potential neurotrophic factors was assessed in the EC-Rac1^+/− and control mouse whole brains by qRT-PCR (n = 7 mouse brains). (B) Schematic of EC-neuron co-culture. (C) Co-culture with Rac1^+/− versus Rac1^+/+ ECs mitigates hypoxia-induced apoptotic death of SH-SY5Y cells, as assessed by Annexin V-EGFP labeling and flow cytometry. (D) Cortical neurons isolated from fetal mouse brains (∼E16) were co-cultured with EC-Rac1^+/− or control MBECs in the presence of artemin-neutralizing antibody (Ab), control IgG, or vehicle in the lower chamber. Control neurons were treated with blank inserts and recombinant artemin proteins. Upper panel, after OGD and reoxygenation, the neurons were double-labeled with DAPI (blue) and cleaved caspase 3 (red). Scale bar, 1 mm. Lower panel, quantification of the cleaved caspase 3–positive fractions. (E) A proposed model of neuroprotection through targeting endothelial Rac1. Values are means ± SEM (*P < 0.05, **P < 0.01; ANOVA).