Loss of tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels is coupled to persistent neuroinflammation and behavioral deficits in late sepsis

Nwafor, Divine C.; Chakraborty, Sreeparna; Brichacek, Allison L.; Jun, Sujung; Gambill, Catheryne A.; Wang, Wei; Engler-Chiurazzi, Elizabeth B.; Dakhlallah, Duaa; Pinkerton, Anthony B.; Millán, José Luis; Benkovic, Stanley A.; Brown, Candice M.

doi:10.1016/j.bbi.2019.11.016

Cited by 15 publications

(27 citation statements)

References 79 publications

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“…Mice were deeply anesthetized with isoflurane and transcardially perfused (Masterflex 7524-10, Cole-Parmer, Vernon Hills, IL) as described previously [19]. Briefly, blood was removed with 0.9% saline followed by perfusion and fixation with 4% chilled paraformaldehyde (PFA, Fisher Scientific, Pittsburgh, PA).…”

Section: Tissue Collecting and Processingmentioning

confidence: 99%

Brain endothelial cell tissue-nonspecific alkaline phosphatase (TNAP) activity promotes maintenance of barrier integrityviathe ROCK pathway

Nwafor

Brichacek

Wang

et al. 2021

Preprint

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Blood-brain barrier (BBB) dysfunction is a key feature in many neuroinflammatory diseases. Yet, no therapies exist to effectively mitigate BBB dysfunction. A strategy to bridge this knowledge gap requires an examination of proteins localized to brain microvascular endothelial cells (BMECs) and evaluating their role in preserving barrier integrity. Tissue-nonspecific alkaline phosphatase (TNAP) is highly abundant in brain microvascular endothelial cells (BMECs); however, its function in BMECs remains unclear. We hypothesized that a loss or inhibition of TNAP activity on BMECs would impair barrier integrity through increased cytoskeletal remodeling driven by the Rho-associated protein kinase (ROCK) pathway. First, we examined barrier integrity in hCMEC/D3 cells treated with a TNAP inhibitor (TNAPi) and in primary BMECs (pBMECs) via the conditional deletion of TNAP in endothelial cells. Our results showed that both pharmacological inhibition and genetic conditional loss of TNAP significantly worsened endothelial barrier integrity compared to controls. Next, we examined the mechanisms through which TNAP activity exerts a protective phenotype on BMECs. Our results showed that hCMEC/D3 cells treated with TNAPi displayed remarkable phalloidin and vimentin cytoskeletal remodeling compared to control. We then examined the role of ROCK, a key player in cytoskeletal remodeling. Our results showed that TNAPi increased the expression of ROCK 1/2. Furthermore, inhibition of ROCK 1/2 with fasudil mitigated TNAPi-induced and VE-cKO barrier dysfunction. Collectively, our results support a novel mechanism through which loss of TNAP activity results in cerebrovascular dysfunction, and selective modulation of TNAP activity in BMECs may be a therapeutic strategy to improve BBB function.

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Section: Tissue Collecting and Processingmentioning

confidence: 99%

Brain endothelial cell tissue-nonspecific alkaline phosphatase (TNAP) activity promotes maintenance of barrier integrityviathe ROCK pathway

Nwafor

Brichacek

Wang

et al. 2021

Preprint

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“…Mouse brains were analyzed as free-floating sections to localize immunoreactivity for GFAP (Z0334, Dako/Agilent; 1:10,000 primary, 1:10,000 secondary), Iba-1 (019-19741, Wako; 1:2,000 primary, 1:1,000 secondary), ALPL (702454, Invitrogen, 1:100 primary, 1:1,000 secondary), and CD11b (ab 133357 Abcam, 1:500 primary, 1:1,000 secondary) using a modified ABC procedure (Vector Laboratories, Burlingame, CA) as previously described [28,29]. Briefly, sections were treated to remove endogenous peroxidases followed by permeabilization.…”

Section: Immunohistochemistrymentioning

confidence: 99%

Experimental Stroke Induces Chronic Gut Dysbiosis and Neuroinflammation in Male Mice

Brichacek¹,

Nwafor²,

Benkovic³

et al. 2020

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Recent literature implicates gut epithelia mucosa and intestinal microbiota as important players in post-stroke morbidity and mortality. As most studies have focused on the acute effects of stroke on gut dysbiosis, our study objective was to measure chronic, longitudinal changes in the gut microbiota and intestinal pathology following ischemic stroke. We hypothesized that mice with experimental ischemic stroke would exhibit chronic gut dysbiosis and intestinal pathology up to 36 days post-stroke compared to sham controls. Male C57BL/6J mice were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) or sham surgery. To determine the long-term effects of tMCAO on gut dysbiosis, fecal boli were collected pre- and post-tMCAO on days 0, 3, 14, and 28. Bioinformatics analysis demonstrate significant differences in abundance among Firmicutes and Bacteroidetes taxa at the phylum, family, and species levels in tMCAO compared to sham mice that persisted up to one month post-stroke. The most persistent changes in post-stroke microbial abundance were a decrease in bacteria family S24-7 and significant increases in Ruminococcaceae. Overall, these changes resulted in a persistently increased Firmicutes:Bacteroidetes ratio in stroke animals. Intestinal histopathology showed evidence of chronic intestinal inflammation that included marked increases in immune cell infiltration with mild-moderate epithelial hyperplasia and villous blunting. Increased astrocyte and microglial activity were also detected one-month post-stroke. These results demonstrate that acute, post-stroke disruption of the gut-brain-microbiota axis progresses to chronic gut dysbiosis, intestinal inflammation, and chronic neuroinflammation.Clinical PerspectivesThe microbiota-gut-brain axis, recently implicated in several neurological disorders, remains largely unexplored at chronic time points post-tMCAO.Our results demonstrate chronic gut dysbiosis, prolonged behavioral deficits, and persistent cerebral and intestinal inflammation post-tMCAO in male C57BL/6J mice.These results suggest that manipulation of microbiota may help reduce poor outcomes after stroke and lead to improved post-stroke functional recovery.

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“…In an elegant in vitro experiment, it was demonstrated that an experimental cytokine mixture, which mimicked plasma obtained from patients with severe sepsis, led to enhanced human leukocytes adhesion to human cerebrovascular endothelial cells (hCMEC/D3) [ 42 ]. Similarly, increased leukocyte and platelet adhesion to cerebral vessels is a common finding in animal models of SAE [ 43 – 47 ]. Corroborating those results, we observed sepsis increased both leukocyte and platelet adhesion to pial vessels of mice.…”

Section: Discussionmentioning

confidence: 99%

Gold nanoparticles reduce inflammation in cerebral microvessels of mice with sepsis

et al. 2021

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Background Sepsis is an emergency medical condition that can lead to death and it is defined as a life-threatening organ dysfunction caused by immune dysregulation in response to an infection. It is considered the main killer in intensive care units. Sepsis associated-encephalopathy (SAE) is mostly caused by a sepsis-induced systemic inflammatory response. Studies report SAE in 14–63% of septic patients. Main SAE symptoms are not specific and usually include acute impairment of consciousness, delirium and/or coma, along with electroencephalogram (EEG) changes. For those who recover from sepsis and SAE, impaired cognitive function, mobility and quality of life are often observed months to years after hospital discharge, and there is no treatment available today to prevent that. Inflammation and oxidative stress are key players for the SAE pathophysiology. Gold nanoparticles have been demonstrated to own important anti-inflammatory properties. It was also reported 20 nm citrate-covered gold nanoparticles (cit-AuNP) reduce oxidative stress. In this context, we tested whether 20 nm cit-AuNP could alleviate the acute changes caused by sepsis in brain of mice, with focus on inflammation. Sepsis was induced in female C57BL/6 mice by cecal ligation and puncture (CLP), 20 nm cit-AuNP or saline were intravenously (IV) injected 2 h after induction of sepsis and experiments performed 6 h after induction. Intravital microscopy was used for leukocyte and platelet adhesion study in brain, blood brain barrier (BBB) permeability carried out by Evans blue assay, cytokines measured by ELISA and real time PCR, cell adhesion molecules (CAMs) by flow cytometry and immunohistochemistry, and transcription factors, by western blotting. Results 20 nm cit-AuNP treatment reduced leukocyte and platelet adhesion to cerebral blood vessels, prevented BBB failure, reduced TNF- concentration in brain, and ICAM-1 expression both in circulating polymorphonuclear (PMN) leukocytes and cerebral blood vessels of mice with sepsis. Furthermore, 20 nm cit-AuNP did not interfere with the antibiotic effect on the survival rate of mice with sepsis. Conclusions Cit-AuNP showed important anti-inflammatory properties in the brain of mice with sepsis, being a potential candidate to be used as adjuvant drug along with antibiotics in the treatment of sepsis to avoid SAE

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Loss of tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels is coupled to persistent neuroinflammation and behavioral deficits in late sepsis

Cited by 15 publications

References 79 publications

Brain endothelial cell tissue-nonspecific alkaline phosphatase (TNAP) activity promotes maintenance of barrier integrityviathe ROCK pathway

Brain endothelial cell tissue-nonspecific alkaline phosphatase (TNAP) activity promotes maintenance of barrier integrityviathe ROCK pathway

Experimental Stroke Induces Chronic Gut Dysbiosis and Neuroinflammation in Male Mice

Gold nanoparticles reduce inflammation in cerebral microvessels of mice with sepsis

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