Disruption of the blood-brain barrier (BBB) is a defining and early feature of multiple sclerosis (MS) that directly damages the central nervous system (CNS), promotes immune cell infiltration, and influences clinical outcomes. There is an urgent need for new therapies to protect and restore BBB function, either by strengthening endothelial tight junctions or suppressing endothelial vesicular transcytosis. Although wingless integrated MMTV (Wnt)/β-catenin signaling plays an essential role in BBB formation and maintenance in healthy CNS, its role in BBB repair in neurologic diseases such as MS remains unclear. Using a Wnt/β-catenin reporter mouse and several downstream targets, we demonstrate that the Wnt/ β-catenin pathway is up-regulated in CNS endothelial cells in both human MS and the mouse model experimental autoimmune encephalomyelitis (EAE). Increased Wnt/β-catenin activity in CNS blood vessels during EAE progression correlates with up-regulation of neuronal Wnt3 expression, as well as breakdown of endothelial cell junctions. Genetic inhibition of the Wnt/β-catenin pathway in CNS endothelium before disease onset exacerbates the clinical presentation of EAE, CD4 + T-cell infiltration into the CNS, and demyelination by increasing expression of vascular cell adhesion molecule-1 and the transcytosis protein Caveolin-1 and promoting endothelial transcytosis. However, Wnt signaling attenuation does not affect the progressive degradation of tight junction proteins or paracellular BBB leakage. These results suggest that reactivation of Wnt/β-catenin signaling in CNS vessels during EAE/MS partially restores functional BBB integrity and limits immune cell infiltration into the CNS.blood-brain barrier | endothelial cell | Wnt/β-catenin signaling | MS | EAE I n both multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), leukocytes infiltrate the central nervous system (CNS) across a damaged blood-brain barrier (BBB) to mediate myelin destruction and neuronal damage (1). BBB breakdown is a contributing factor to the pathogenesis of both MS and EAE (2-4). Structural and functional BBB degradation precedes lesion development in both MS and EAE (5-9), and focal BBB abnormalities correlate with clinical exacerbations in the relapsing-remitting form of MS (10). Moreover, BBB leakage precedes the entry of T cells and monocytes into the brain parenchyma (7, 11) and coincides with early infiltration of neutrophils before the onset of EAE (12). Although the severity of barrier leakage decreases over time for most relapsing-remitting MS lesions, as assessed by gadoliniumenhancing magnetic resonance imaging (7, 13-15), whether BBB recovery is an active process and, if so, which pathways mediate its repair, remain unclear.The BBB achieves its highly selective permeability through the presence of (i) tight junctions (TJs) that prevent paracellular diffusion of small molecules and immune cells between endothelial cells (ECs), (ii) very few endocytotic vesicles that restrict movement of large mo...

Highlights d Macrophages oxidize acetoacetate (AcAc), but not b-hydroxybutyrate d Metabolism of AcAc in macrophages extends into pathways beyond the TCA cycle d Effective AcAc competition with glucose requires its mitochondrial metabolism d Mitochondrial AcAc metabolism in macrophages protects against liver fibrosis

Objective-The presence of apoptotic markers is a prominent histological feature of abdominal aortic aneurysm. To understand the role of apoptosis in the pathogenesis of this common vascular disease, we tested the effect of the pan-caspase inhibitor quinoline-Val-Asp-difluorophenoxymethylketone (Q-VD-OPh) on aneurysm formation using a mouse angiotensin II (Ang II) model. Methods and Results-Ang II in apolipoprotein E-deficient mice significantly induced medial cell apoptosis 3 days after infusion at the aortic region, eventually becoming aneurismal. A daily administration of 20 mg/kg per day Q-VD-OPh starting 6 hours before Ang II infusion reduced aneurysm incidence from 83.3% to 16.7% and maximal aortic diameter from 2.43Ϯ0.29 mm to 1.58Ϯ0.18 mm. The caspase inhibitor treated mice showed profoundly diminished levels of medial apoptosis and inflammation. In contrast, administration of Q-VD-OPh starting 7 days after Ang II infusion had no significant impact on aneurysm development. In vitro, media conditioned by Ang II-treated smooth muscle cells (SMCs) stimulated macrophage chemotaxis in a caspase-dependent manner. Inhibition of monocyte chemoattractant protein-1 (MCP-1) in the conditioned media via a neutralizing antibody completely blocked the ability of conditioned media to attract macrophages. Conclusion-These

Objective The Calcium Chloride (CaCl2) model is a widely accepted rodent model for abdominal aortic aneurysm (AAA). Calcium deposition, mainly consisting of calcium phosphate (CaPO4) crystals, has been reported to exist in both human and experimental aneurysms. CaPO4 crystal has been utilized for in vitro DNA transfection by mixing CaCl2 and Phosphate Buffered Saline (PBS). Here, we describe accelerated aneurysm formation resulting from a modification of the CaCl2 model. Methods The modified CaCl2, the CaPO4 model, was created by applying PBS onto the mouse infrarenal aorta after CaCl2 treatment. Morphological, histological and immunohistochemical analyses were performed on arteries treated with both the CaPO4 model and the conventional CaCl2 model as control. In vitro methods were carried out using a mixture of CaCl2 and PBS to create CaPO4 crystals. CaPO4 induced apoptosis of primary cultured mouse vascular smooth muscle cells (VSMCs) was measured by DNA fragmentation ELISA. Results First, we showed that the CaPO4 model produces AAA, defined as an increase of 50% or greater in the diameter of the aorta; faster than in the CaCl2 model. CaPO4 model showed significantly larger aneurysmal dilation at 7, 28, and 42 days as reflected by a maximum diameter fold change (measured in mm) of 1.69 ± 0.07, 1.99 ± 0.14 and 2.13 ± 0.09 as opposed to 1.22 ± 0.04, 1.48 ± 0.07 and 1.68±0.06 as seen in CaCl2 model, respectively (n=6; P<0.05). A semi-quantitative grading analysis of elastin fiber integrity at 7 days revealed a significant increase in elastin degradation in the CaPO4 model as compared to CaCl2 model (2.7±0.2 vs 1.5±0.2, p<0.05, n=6). Significantly higher level of apoptosis occurred in the CaPO4 model (apoptosis index at 1, 2, and 3 days post-surgery: 0.26 ± 0.14, 0.37± 0.14, and 0.33 ± 0.08 for CaPO4 model and 0.012 ± 0.10, 0.15± 0.02, and 0.12 ± 0.05 for conventional CaCl2 model) (n=3; p<0.05). An enhancement of macrophage infiltration and calcification was also observed at 3 and 7 days in CaPO4. CaPO4 induced approximately 3.7 times more apoptosis in VSMCs when compared to a mixture of CaCl2 (n=4; p<0.0001) in vitro. Conclusion Our data shows that the CaPO4 model accelerates aneurysm formation with the enhancement of apoptosis, macrophage infiltration and calcium deposition. This modified model, with its rapid and robust dilation, can be utilized as a new model for AAA.

Objective Apoptosis of smooth muscle cells (SMCs) is a prominent pathological characteristic of Abdominal Aortic Aneurysm (AAA). We have previously shown that SMC apoptosis stimulates proinflammatory signaling in a mouse model of AAA. Here, we test whether Protein Kinase C-delta (PKCδ), an apoptotic mediator, participates in the pathogenesis of AAA by regulating apoptosis and proinflammatory signals. Methods and Results Mouse experimental AAA is induced by perivascular administration of CaCl2. Mice deficient in PKCδ exhibit a profound reduction in aneurysmal expansion, SMC apoptosis, and transmural inflammation as compared to wildtype littermates. Delivery of PKCδ to the aortic wall of PKCδ−/− mice restores aneurysm, while overexpression of a dominant negative PKCδ mutant in the aorta of wildtype mice attenuates aneurysm. In vitro, PKCδ−/− aortic SMCs exhibit significantly impaired monocyte chemoattractant protein-1 (MCP-1) production. Ectopic administration of recombinant MCP-1 to the arterial wall of PKCδ−/− mice restores inflammatory response and aneurysm development. Conclusions PKCδ is an important signaling mediator for SMC apoptosis and inflammation in a mouse model of AAA. By stimulating MCP-1 expression in aortic SMCs, upregulated PKCδ exacerbates the inflammatory process, in turn perpetuating elastin degradation and aneurysmal dilatation. Inhibition of PKCδ may serve as a potential therapeutic strategy for AAA.

Background Advanced glycation end products (AGEs), formed from proteins and peptides by nonenzymatic glycoxidation after contact with aldose sugars, have been implicated in the pathogenesis of age-related cardiac and vascular dysfunction. Our previous study demonstrated significantly elevated levels of AGE and the receptor for AGE (RAGE) in human abdominal aortic aneurysm (AAA) tissues. Inhibition of AGE signaling by targeted gene deletion of RAGE markedly reduced the development of aneurysm in a mouse model of AAA. We also showed that AGE may stimulate aneurysm formation by promoting metalloproteinase (MMP)-9 expression. In this study, we investigated the molecular mechanism underlying this novel function of AGE. Methods The murine macrophage cell line RAW 264.7 was pretreated with AGE, TGF-β, and MAPK inhibitors. The protein was collected for Western blot analysis. Culture supernatants were collected to determine MMP-9 activity by gelatin zymography. Results We found that AGE induced the production of MMP-9 in macrophages in a dose-dependent manner. This induction of MMP-9 was markedly diminished by pretreatment with TGF-β. To delineate the underlying molecular mechanism, we showed that AGE increased phosphorylation of p44/42 ERK, p38, JNK, and PI3K in macrophages. Moreover, AGE induced active p65 subunit of NF-κB. Inhibition of ERK (UO126) or p38 (SB203580), but not PI3K (LY294002 or wortmannin), blocked AGE-induced MMP-9 expression. In contrast, inhibition of JNK (SP-600125) significantly enhanced the stimulatory effect of AGE on MMP-9. Furthermore, TGF-β suppressed AGE-induced expression of the active p65 subunit of NF-κB. Conclusions Our data indicate that AGE induces MMP-9 through activation of ERK, p38 mitogen-activated protein and NF-κB, a pathway that is antagonized by TGF-β. This finding in conjunction with previously reported AGE functions in inflammation suggests that anti-AGE therapies could be effective in the prevention of human AAA development and progression.

Recent data have shown that preservation of the neuromuscular junction (NMJ) after traumatic nerve injury helps to improve functional recovery with surgical repair via matrix metalloproteinase-3 (MMP3) blockade. As such, we sought to explore additional pathways that may augment this response. Wnt3a has been shown to inhibit acetylcholine receptor (AChR) clustering via β-catenin-dependent signaling in the development of the NMJ. Therefore, we hypothesized that Wnt3a and β-catenin are associated with NMJ destabilization following traumatic denervation. A critical size nerve defect was created by excising a 10-mm segment of the sciatic nerve in mice. Denervated muscles were then harvested at multiple time points for immunofluorescence staining, quantitative real-time PCR, and western blot analysis for Wnt3a and β-catenin levels. Moreover, a novel Wnt/β-catenin transgenic reporter mouse line was utilized to support our hypothesis of Wnt activation after traumatic nerve injury. The expression of Wnt3a mRNA was significantly increased by 2 weeks post-injury and remained upregulated for 2 months. Additionally, β-catenin was activated at 2 months post-injury relative to controls. Correspondingly, immunohistochemical analysis of denervated transgenic mouse line TCF/Lef:H2B-GFP muscles demonstrated that the number of GFP-positive cells was increased at the motor endplate band. These collective data support that post-synaptic AChRs destabilize after denervation by a process that involves the Wnt/β-catenin pathway. As such, this pathway serves as a potential therapeutic target to prevent the motor endplate degeneration that occurs following traumatic nerve injury.

Angiogenesis, or the growth of new blood vessels from existing vasculature, is critical for the proper development of many organs. This process is inhibited and tightly regulated in adults, once endothelial cells have acquired organ-specific properties. Within the central nervous system (CNS), angiogenesis and acquisition of blood–brain barrier (BBB) properties by endothelial cells is essential for CNS function. However, the role of angiogenesis in CNS pathologies associated with impaired barrier function remains unclear. Although vessel abnormalities characterized by abnormal barrier function are well documented in multiple sclerosis (MS), a demyelinating disease of the CNS resulting from an immune cell attack on oligodendrocytes, histological analysis of human MS samples has shown that angiogenesis is prevalent in and around the demyelinating plaques. Experiments using an animal model that mimics several features of human MS, Experimental Autoimmune Encephalomyelitis (EAE), have confirmed these human pathological findings and shed new light on the contribution of pre-symptomatic angiogenesis to disease progression. The CNS-infiltrating inflammatory cells that are a hallmark of both MS and EAE secrete several factors that not only contribute to exacerbating the inflammatory process but also promote and stimulate angiogenesis. Moreover, chemical or biological inhibitors that directly or indirectly block angiogenesis provide clinical benefits for disease progression. While the precise mechanism of action for these inhibitors is unknown, preventing pathological angiogenesis during EAE progression holds great promise for developing effective treatment strategies for human MS.