G-quadruplexes Stabilization Upregulates CCN1 and Accelerates Aging in Cultured Cerebral Endothelial Cells

Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. However, whether CCN1 contributes to senescence in CEC and how this is regulated requires further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. Stabilization of the G4 structures regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and these mice exhibited enhanced cognitive deficits. A sequence in the 3′-UTR of the human CCN1 mRNA has the ability to fold into G4s in vitro. We hypothesize that G4 stabilization regulates CCN1 in cultured primary CEC and induces endothelial senescence. We used cerebral microvessel fractions and cultured primary CEC from young (4-months old, m/o) and aged (18-m/o) mice to determine CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA-G4s for fluorescence spectra. We found that cultured CEC from aged mice exhibited enhanced levels of SA phenotypes, and higher levels of CCN1 and G4 stabilization. In cultured CEC, CCN1 induced SA phenotypes, such as SA β-galactosidase activity, and double-strand DNA damage. Furthermore, CCN1 levels were upregulated by a G4 ligand, and a G-rich motif in the 3′-UTR of the Ccn1 mRNA was folded into a G4. In conclusion, we demonstrate that CCN1 can induce senescence in cultured primary CEC, and we provide evidence that G4 stabilization is a novel mechanism regulating the SASP component CCN1.

Abstract P725: Cerebral Endothelial Cell Derived Exosomes Promote Neurogenesis and Improve Cognitive Function in Aged Diabetic Rats

Introduction: Middle age and elderly patients with type II diabetes mellitus (DM) are at high risk to develop cognitive decline and dementia. Reduction of hippocampal neurogenesis is highly associated with impairment of cognitive function. Exosomes are small extracellular vesicles that play an important role in intercelluar communication by transferring proteins, lipids, and genomic materials including microRNAs between cells. In the present study, we tested the hypothesis that exosomes derived from healthy cerebral endothelial cells promote hippocampal neurogenesis and ameliorate cognitive impairment in aged DM rats. Methods: DM was induced in middle aged rats (13 month) by co-administration of nicotinamide and streptozotocin (NTM-STZ). Two months (2M) after NTM-STZ injection, rats with confirmed hyperglycemia were treated with exosomes derived from cerebral endothelial cells of healthy young-adult rats (CEC-Exo, 1x10 11 particles, IV, n=10) or saline (n=10), twice a week for 8 consecutive weeks. Bromodeoxyuridine (BrdU, IP) was administered daily for 7 days starting at 2M after NTM-STZ injection. Results: Compared with DM rats treated with saline, treatment of DM rats with CEC-Exo significantly (p<0.05) improved cognitive functions measured by Morris water maze (47±4% vs 40±5% of time spent in the correct quadrant), odor recognition test (56±6% vs 48±7% time spent on new odor), and the social interaction test (67±9% vs 54±14% interaction time with new rat). In addition, CEC-Exo robustly increased number of BrdU + cells by 60% and DCX + cells by 45% in the subgranular zone of the dentate gyrus compared with saline. Moreover, DCX + cells significantly increased their branch numbers by 54% and lengths by 51% in DM rats treated with CEC-Exo, suggesting that CEC-Exo promote DCX + neuroblasts maturation. Concurrently, CEC-Exo treatment significantly reduced numbers of vessels with fibrin deposition (8±3/mm 2 vs 18±7/mm 2 in saline) and extravascular leakage (3±2/mm 2 vs 7±3/mm 2 in saline) in the hippocampus. Conclusion: Treatment with CEC-Exo improves cognitive function in aged DM rats and augments neurogenesis and reduction of vascular thrombosis in the hippocampus, which in concert likely contribute the therapeutic effect of CEC-Exo.

Hyperglycemic Condition Causes Pro-Inflammatory and Permeability Alterations Associated with Monocyte Recruitment and Deregulated NFκB/PPARγ Pathways on Cerebral Endothelial Cells: Evidence for Polyphenols Uptake and Protective Effect

Hyperglycemia alters the function of cerebral endothelial cells from the blood-brain barrier, increasing the risk of cerebrovascular complications during diabetes. This study evaluated the protective effect of polyphenols on inflammatory and permeability markers on bEnd3 cerebral endothelial cells exposed to high glucose concentration. Results show that hyperglycemic condition increased nuclear factor kappa B (NFκB) activity, deregulated the expression of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10) and endothelial-leukocyte adhesion molecule (E-selectin) genes, raised MCP-1 secretion and elevated monocyte adhesion and transendothelial migration. High glucose decreased occludin, claudin-5, zona occludens-1 (ZO-1) and zona occludens-2 (ZO-2) tight junctions production and altered the endothelial permeability. Characterized polyphenolic extracts from the French medicinal plants Antirhea borbonica, Ayapana triplinervis, Dodonaea viscosa and Terminalia bentzoe, and their major polyphenols quercetin, caffeic, chlorogenic and gallic acids limited the pro-inflammatory and permeability alterations caused by high glucose. Peroxisome proliferator-activated receptor gamma (PPARγ) agonist also attenuated these damages while PPARγ antagonist aggravated them, suggesting PPARγ protective action. Interestingly, polyphenols improved PPARγ gene expression lowered by high glucose. Moreover, polyphenols were detected at the intracellular level or membrane-bound to cells, with evidence for breast cancer resistance protein (BCRP) efflux transporter role. Altogether, these findings emphasize the ability of polyphenols to protect cerebral endothelial cells in hyperglycemic condition and their relevance for pharmacological strategies aiming to limit cerebrovascular disorders in diabetes.

AKAP12 Supports Blood-Brain Barrier Integrity against Ischemic Stroke

A-kinase anchor protein 12 (AKAP12) is a scaffolding protein that associates with intracellular molecules to regulate multiple signal transductions. Although the roles of AKAP12 in the central nervous system are still relatively understudied, it was previously shown that AKAP12 regulates blood-retinal barrier formation. In this study, we asked whether AKAP12 also supports the function and integrity of the blood-brain barrier (BBB). In a mouse model of focal ischemia, the expression level of AKAP12 in cerebral endothelial cells was upregulated during the acute phase of stroke. Also, in cultured cerebral endothelial cells, oxygen-glucose deprivation induced the upregulation of AKAP12. When AKAP12 expression was suppressed by an siRNA approach in cultured endothelial cells, endothelial permeability was increased along with the dysregulation of ZO-1/Claudin 5 expression. In addition, the loss of AKAP12 expression caused an upregulation/activation of the Rho kinase pathway, and treatment of Rho kinase inhibitor Y-27632 mitigated the increase of endothelial permeability in AKAP12-deficient endothelial cell cultures. These in vitro findings were confirmed by our in vivo experiments using Akap12 knockout mice. Compared to wild-type mice, Akap12 knockout mice showed a larger extent of BBB damage after stroke. However, the inhibition of rho kinase by Y-27632 tightened the BBB in Akap12 knockout mice. These data may suggest that endogenous AKAP12 works to alleviate the damage and dysfunction of the BBB caused by ischemic stress. Therefore, the AKAP12-rho-kinase signaling pathway represents a novel therapeutic target for stroke.

Medicinal Plant Polyphenols Attenuate Oxidative Stress and Improve Inflammatory and Vasoactive Markers in Cerebral Endothelial Cells during Hyperglycemic Condition

Blood-brain barrier endothelial cells are the main targets of diabetes-related hyperglycemia that alters endothelial functions and brain homeostasis. Hyperglycemia-mediated oxidative stress may play a causal role. This study evaluated the protective effects of characterized polyphenol-rich medicinal plant extracts on redox, inflammatory and vasoactive markers on murine bEnd3 cerebral endothelial cells exposed to high glucose concentration. The results show that hyperglycemic condition promoted oxidative stress through increased reactive oxygen species (ROS) levels, deregulated antioxidant superoxide dismutase (SOD) activity, and altered expression of genes encoding Cu/ZnSOD, MnSOD, catalase, glutathione peroxidase (GPx), heme oxygenase-1 (HO-1), NADPH oxidase 4 (Nox4), and nuclear factor erythroid 2-related factor 2 (Nrf2) redox factors. Cell preconditioning with inhibitors of signaling pathways highlights a causal role of nuclear factor kappa B (NFκB), while a protective action of AMP-activated protein kinase (AMPK) on redox changes. The hyperglycemic condition induced a pro-inflammatory response by elevating NFκB gene expression and interleukin-6 (IL-6) secretion, and deregulated the production of endothelin-1 (ET-1), endothelial nitric oxide synthase (eNOS), and nitric oxide (NO) vasoactive markers. Importantly, polyphenolic extracts from Antirhea borbonica, Ayapana triplinervis, Dodonaea viscosa, and Terminalia bentzoe French medicinal plants, counteracted high glucose deleterious effects by exhibiting antioxidant and anti-inflammatory properties. In an innovative way, quercetin, caffeic, chlorogenic and gallic acids identified as predominant plant polyphenols, and six related circulating metabolites were found to exert similar benefits. Collectively, these findings demonstrate polyphenol protective action on cerebral endothelial cells during hyperglycemic condition.