Poly(ADP-ribose) Polymerase-1 Inhibition in Brain Endothelium Protects the Blood—Brain Barrier under Physiologic and Neuroinflammatory Conditions

Abstract

Blood-brain barrier (BBB) dysfunction seen in neuroinflammation contributes to mortality and morbidity in multiple sclerosis, encephalitis, traumatic brain injury, and stroke. Identification of molecular targets maintaining barrier function is of clinical relevance. We used a novel in vivo model of localized aseptic meningitis where tumor necrosis factor alpha (TNFα) was introduced intracerebrally and surveyed cerebral vascular changes and leukocyte-endothelium interactions by intravital videomicroscopy. Poly (ADP-ribose) polymerase-1 (PARP) inhibition significantly reduced leukocyte adhesion to and migration across brain endothelium in cortical microvessels. PARP inactivation diminished BBB permeability in an in vivo model of systemic inflammation. PARP suppression in primary human brain microvascular endothelial cells (BMVEC), an in vitro model of BBB, enhanced barrier integrity and augmented expression of tight junction proteins. PARP inhibition in BMVEC diminished human monocyte adhesion to TNFα-activated BMVEC (up to 65%) and migration (80-100%) across BBB models. PARP suppression decreased expression of adhesion molecules and decreased activity of GTPases (controlling BBB integrity and monocyte migration across the BBB). PARP inhibitors down-regulated expression of inflammatory genes and dampened secretion of pro-inflammatory factors increased by TNFα in BMVEC. These results point to PARP suppression as a novel approach to BBB protection in the setting of endothelial dysfunction caused by inflammation. PARP-1 is a member of a family of NAD-dependent enzymes that is responsible for~80% of cellular poly(ADP-ribose) formation. 1 Poly(ADP-ribosyl)ation is a post-translational modification of proteins with widespread effects on diverse cellular functions including gene expression, differentiation and cell death. 2 As a scaffold protein and as an enzyme, PARP-1 (further referred to in the text as PARP) is a key component of the transcriptional machinery that controls chromatin architecture, histone shuttling, and spatial organization of transcription-regulating supramolecular complexes, 2 thereby regulating inflammationassociated genes.In this article, for the first time, we show mechanisms of the modulatory effects of PARP inhibition in brain endothelium. Previous studies have demonstrated anti-inflammatory effects of PARP suppression in animal models of traumatic brain injury, multiple sclerosis (MS), meningitis, and stroke, [3][4][5] where PARP inhibitors reduced edema, leukocyte infiltration and neuroinflammation, and decreased intercellular adhesion molecule 1 (ICAM-1) expression. 4,6 PARP inhibitors have now reached the stage of

miR-98 and let-7g* Protect the Blood-Brain Barrier Under Neuroinflammatory Conditions

“…Intravital videomicroscopy for in vivo leukocyte adhesion was performed in mice that underwent craniotomy, cranial window, and cannula implantation. 15,16 Six-day recovery period was allowed between implantation of the cannula and IC injections. Intravital videomicroscopy for in vivo leukocyte adhesion was performed on animals with cranial windows.…”

“…Intravital videomicroscopy for in vivo leukocyte adhesion was performed on animals with cranial windows. 15,16 Before IVM, animals were IC injected with TNFα (0.5 μg/mouse). The total maximum volume injected into the mouse brain was 5 μL, injecting 1 μL at a time with a 5-minute waiting period between injections as described.…”

“…The total maximum volume injected into the mouse brain was 5 μL, injecting 1 μL at a time with a 5-minute waiting period between injections as described. 16 The dose (0.5 μg/mouse) was established in dose-response experiments (data not shown), where consistent leukocyte infiltration was produced accompanied by mild brain edema (known markers of meningitis 18 ). No adverse effects (high fever, seizures, locomotor impairment, and/or death) were noted.…”

“…Leukocyte adhesion was detected in cerebral vessels through the cranial window using a Stereo Discovery V20 epifluorescence microscope (Carl Zeiss Microimaging Inc., Thornwood, NY, USA) equipped with a AxioCam MR digital camera as previously described. 15,16 Transmigrated leukocytes were enumerated in an area covering a distance of 10 μm from the pial and parenchymal vessel wall by IVM and by Leica SP5 II 2-photon microscopy (Leica Microsystems Gmbh, Mannheim, Germany), respectively. The number of extravasated leukocytes was counted and normalized with respect to the immediate perivascular area surrounding the microvessel (MV) (10 μm from the endothelium × 100 μm long along the vessel).…”

“…The number of extravasated leukocytes was counted and normalized with respect to the immediate perivascular area surrounding the microvessel (MV) (10 μm from the endothelium × 100 μm long along the vessel). 16 In Vitro and In Vivo miRNA Transfection Brain microvascular endothelial cells were transfected at 2 × 10 6 cells/mL with miRNA oligos at 50 nmol/L with the Neon Transfection System (Life Technologies) according to the manufacturer's instructions. Cells were collected at 24, 48, 72, and 96 hours after transfection and levels of transfected miRNAs were estimated by qPCR as described below.…”