let-7 microRNAs: Their Role in Cerebral and Cardiovascular Diseases, Inflammation, Cancer, and Their Regulation

The let-7 family is among the first microRNAs found. Recent investigations have indicated that it is highly expressed in many systems, including cerebral and cardiovascular systems. Numerous studies have implicated the aberrant expression of let-7 members in cardiovascular diseases, such as stroke, myocardial infarction (MI), cardiac fibrosis, and atherosclerosis as well as in the inflammation related to these diseases. Furthermore, the let-7 microRNAs are involved in development and differentiation of embryonic stem cells in the cardiovascular system. Numerous genes have been identified as target genes of let-7, as well as a number of the let-7’ regulators. Further studies are necessary to identify the gene targets and signaling pathways of let-7 in cardiovascular diseases and inflammatory processes. The bulk of the let-7’ regulatory proteins are well studied in development, proliferation, differentiation, and cancer, but their roles in inflammation, cardiovascular diseases, and/or stroke are not well understood. Further knowledge on the regulation of let-7 is crucial for therapeutic advances. This review focuses on research progress regarding the roles of let-7 and their regulation in cerebral and cardiovascular diseases and associated inflammation

Blocking of P2X7r Reduces Mitochondrial Stress Induced by Alcohol and Electronic Cigarette Exposure in Brain Microvascular Endothelial Cells

Studies in both humans and animal models demonstrated that chronic alcohol/e-cigarette (e-Cig) exposure affects mitochondrial function and impairs barrier function in brain microvascular endothelial cells (BMVECs). Identification of the signaling pathways by which chronic alcohol/e-Cig exposure induces mitochondrial damage in BMVEC is vital for protection of the blood–brain barrier (BBB). To address the issue, we treated human BMVEC [hBMVECs (D3 cell-line)] with ethanol (ETH) [100 mM], acetaldehyde (ALD) [100 μM], or e-cigarette (e-Cig) [35 ng/mL of 1.8% or 0% nicotine] conditioned medium and showed reduced mitochondrial oxidative phosphorylation (OXPHOS) measured by a Seahorse analyzer. Seahorse data were further complemented with the expression of mitochondrial OXPHOS proteins detected by Western blots. We also observed cytosolic escape of ATP and its extracellular release due to the disruption of mitochondrial membrane potential caused by ETH, ALD, or 1.8% e-Cig exposure. Moreover ETH, ALD, or 1.8% e-Cig treatment resulted in elevated purinergic P2X7r and TRPV1 channel gene expression, measured using qPCR. We also demonstrated the protective role of P2X7r antagonist A804598 (10 μM) in restoring mitochondrial oxidative phosphorylation levels and preventing extracellular ATP release. In a BBB functional assay using trans-endothelial electrical resistance, we showed that blocking the P2X7r channel enhanced barrier function. In summary, we identified the potential common pathways of mitochondrial injury caused by ETH, ALD, and 1.8% e-Cig which allow new protective interventions. We are further investigating the potential link between P2X7 regulatory pathways and mitochondrial health

Curcumin and Carnosic Acid Cooperate to Inhibit Proliferation and Alter Mitochondrial Function of Metastatic Prostate Cancer Cells

Anticancer activities of plant polyphenols have been demonstrated in various models of neoplasia. However, evidence obtained in numerous in vitro studies indicates that proliferation arrest and/or killing of cancer cells require quite high micromolar concentrations of polyphenols that are difficult to reach in vivo and can also be (geno)toxic to at least some types of normal cells. The ability of certain polyphenols to synergize with one another at low concentrations can be used as a promising strategy to effectively treat human malignancies. We have recently reported that curcumin and carnosic acid applied at non-cytotoxic concentrations synergistically cooperate to induce massive apoptosis in acute myeloid leukemia cells, but not in normal hematopoietic and non-hematopoietic cells, via sustained cytosolic calcium overload. Here, we show that the two polyphenols can also synergistically suppress the growth of DU145 and PC-3 metastatic prostate cancer cell cultures. However, instead of cell killing, the combined treatment induced a marked inhibition of cell proliferation associated with G0/G1 cell cycle arrest. This was preceded by transient elevation of cytosolic calcium levels and prolonged dissipation of the mitochondrial membrane potential, without generating oxidative stress, and was associated with defective oxidative phosphorylation encompassing mitochondrial dysfunction. The above effects were concomitant with a significant downregulation of mRNA and protein expression of the oncogenic kinase SGK1, the mitochondria-hosted mTOR component. In addition, a moderate decrease in SGK1 phosphorylation at Ser422 was observed in polyphenol-treated cells. The mTOR inhibitor rapamycin produced a similar reduction in SGK1 mRNA and protein levels as well as phosphorylation. Collectively, our findings suggest that the combination of curcumin and carnosic acid at potentially bioavailable concentrations may effectively target different types of cancer cells by distinct modes of action. This and similar combinations merit further exploration as an anticancer modality

Inhibition of glycogen synthase kinase 3β promotes tight junction stability in brain endothelial cells by half-life extension of occludin and claudin-5.

Neuroinflammatory conditions often involve dysfunction of the Blood-Brain Barrier (BBB). Therefore, identifying molecular targets that can maintain barrier fidelity is of clinical importance. We have previously reported on the anti-inflammatory effects that glycogen synthase kinase 3β (GSK3β) inhibition has on primary human brain endothelial cells. Here we show that GSK3β inhibitors also promote barrier tightness by affecting tight junction (TJ) protein stability. Transendothelial electrical resistance (TEER) was used to evaluate barrier integrity with both pharmacological inhibitors and mutants of GSK3β. Inhibition of GSK3β produced a gradual and sustained increase in TEER (as much as 22% over baseline). Analysis of subcellular membrane fractions revealed an increase in the amount of essential tight junction proteins, occludin and claudin-5, but not claudin-3. This phenomenon was attributed to a decrease in TJ protein turnover and not transcriptional regulation. Using a novel cell-based assay, inactivation of GSK3β significantly increased the half-life of occludin and claudin-5 by 32% and 43%, respectively. A correlation was also established between the enhanced association of β-catenin with ZO-1 as a function of GSK3β inhibition. Collectively, our findings suggest the possibility of using GSK3β inhibitors as a means to extend the half-life of key tight junction proteins to promote re-sealing of the BBB during neuroinflammation

CCL8/MCP-2 is a target for mir-146a in HIV-1-infected human microglial cells

MicroRNA-mediated regulation of gene expression appears to be involved in a variety of cellular processes, including development, differentiation, proliferation, and apoptosis. Mir-146a is thought to be involved in the regulation of the innate immune response, and its expression is increased in tissues associated with chronic inflammation. Among the predicted gene targets for mir-146a, the chemokine CCL8/MCP-2 is a ligand for the CCR5 chemokine receptor and a potent inhibitor of CD4/CCR5-mediated HIV-1 entry and replication. In the present study, we have analyzed changes in the expression of mir-146a in primary human fetal microglial cells upon infection with HIV-1 and found increased expression of mir-146a. We further show that CCL8/MCP-2 is a target for mir-146a in HIV-1 infected microglia, as overexpression of mir-146a prevented HIV-induced secretion of MCP-2 chemokine. The clinical relevance of our findings was evaluated in HIV-encephalitis (HIVE) brain samples in which decreased levels of MCP-2 and increased levels of mir-146a were observed, suggesting a role for mir-146a in the maintenance of HIV-mediated chronic inflammation of the brain.—Rom, S., Rom, I., Passiatore, G., Pacifici, M., Radhakrishnan, S., Del Valle, L., Piña-Oviedo, S., Khalili, K., Eletto, D., Peruzzi, F. CCL8/MCP-2 is a target for mir-146a in HIV-1 infected human microglial cells

Dysfunction of brain pericytes in chronic neuroinflammation.

Brain pericytes are uniquely positioned within the neurovascular unit to provide support to blood brain barrier (BBB) maintenance. Neurologic conditions, such as HIV-1-associated neurocognitive disorder, are associated with BBB compromise due to chronic inflammation. Little is known about pericyte dysfunction during HIV-1 infection. We found decreased expression of pericyte markers in human brains from HIV-1-infected patients (even those on antiretroviral therapy). Using primary human brain pericytes, we assessed expression of pericyte markers (α1-integrin, α-smooth muscle actin, platelet-derived growth factor-B receptor β, CX-43) and found their downregulation after treatment with tumor necrosis factor-α (TNFα) or interleukin-1 β (IL-1β). Pericyte exposure to virus or cytokines resulted in decreased secretion of factors promoting BBB formation (angiopoietin-1, transforming growth factor-β1) and mRNA for basement membrane components. TNFα and IL-1β enhanced expression of adhesion molecules in pericytes paralleling increased monocyte adhesion to pericytes. Monocyte migration across BBB models composed of human brain endothelial cells and pericytes demonstrated a diminished rate in baseline migration compared to constructs composed only of brain endothelial cells. However, exposure to the relevant chemokine, CCL2, enhanced the magnitude of monocyte migration when compared to BBB models composed of brain endothelial cells only. These data suggest an important role of pericytes in BBB regulation in neuroinflammation

Secoisolariciresinol diglucoside is a blood-brain barrier protective and anti-inflammatory agent: implications for neuroinflammation

Abstract Background Secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, is known for its beneficial effects in inflammation, oxidative stress, heart disease, tumor progression, atherosclerosis, and diabetes. SDG might be an attractive natural compound that protects against neuroinflammation. Yet, there are no comprehensive studies to date investigating the effects of SDG on brain endothelium using relevant in vivo and in vitro models. Methods We evaluated the effects of orally administered SDG on neuroinflammatory responses using in vivo imaging of the brain microvasculature during systemic inflammation and aseptic encephalitis. In parallel, the anti-inflammatory actions of SDG on brain endothelium and monocytes were evaluated in vitro blood-brain barrier (BBB) model. Multiple group comparisons were performed by one-way analysis of variance with Dunnet’s post hoc tests. Results We found that SDG diminished leukocyte adhesion to and migration across the BBB in vivo in the setting of aseptic encephalitis (intracerebral TNFα injection) and prevented enhanced BBB permeability during systemic inflammatory response (LPS injection). In vitro SDG pretreatment of primary human brain microvascular endothelial cells (BMVEC) or human monocytes diminished adhesion and migration of monocytes across brain endothelial monolayers in conditions mimicking CNS inflammatory responses. Consistent with our in vivo observations, SDG decreased expression of the adhesion molecule, VCAM1, induced by TNFα, or IL-1β in BMVEC. SDG diminished expression of the active form of VLA-4 integrin (promoting leukocyte adhesion and migration) and prevented the cytoskeleton changes in primary human monocytes activated by relevant inflammatory stimuli. Conclusion This study indicates that SDG directly inhibits BBB interactions with inflammatory cells and reduces the inflammatory state of leukocytes. Though more work is needed to determine the mechanism by which SDG mediates these effects, the ability of SDG to exert a multi-functional response reducing oxidative stress, inflammation, and BBB permeability makes it an exciting potential therapeutic for neuroinflammatory diseases. SDG can serve as an anti-inflammatory and barrier-protective agent in neuroinflammation

Additional file 1: Figure S1. of PARP inhibition in leukocytes diminishes inflammation via effects on integrins/cytoskeleton and protects the blood-brain barrier

PARPi decrease PARP activity in a dose-dependent manner. PARP activity of primary monocytes treated with different concentrations of PARPi AIQ (A) and olaparib (B). Results are presented as the mean ± SEM (*P < 0.05, **P < 0.01 vs. untreated control) from two independent experiments for at least three replicates. Figure S2. PARP activity significantly diminished in ex vivo treated mouse leukocytes. (A) PARP activity was measured in freshly isolated and ex vivo PARPi-treated mouse leukocytes. Data are presented as mean ± SEM for at least three replicates from three/four donor mice. ***P < 0.005 indicate significance vs. non-treated. (B) Flow cytometry data presenting leukocyte profile of isolated leukocytes with or without PARPi treatment