Vascular Stress Signaling in Hypertension.

Cells respond to stress by activating a variety of defense signaling pathways, including cell survival and cell death pathways. Although cell survival signaling helps the cell to recover from acute insults, cell death or senescence pathways induced by chronic insults can lead to unresolved pathologies. Arterial hypertension results from chronic physiological maladaptation against various stressors represented by abnormal circulating or local neurohormonal factors, mechanical stress, intracellular accumulation of toxic molecules, and dysfunctional organelles. Hypertension and aging share common mechanisms that mediate or prolong chronic cell stress, such as endoplasmic reticulum stress and accumulation of protein aggregates, oxidative stress, metabolic mitochondrial stress, DNA damage, stress-induced senescence, and proinflammatory processes. This review discusses common adaptive signaling mechanisms against these stresses including unfolded protein responses, antioxidant response element signaling, autophagy, mitophagy, and mitochondrial fission/fusion, STING (signaling effector stimulator of interferon genes)-mediated responses, and activation of pattern recognition receptors. The main molecular mechanisms by which the vasculature copes with hypertensive and aging stressors are presented and recent advancements in stress-adaptive signaling mechanisms as well as potential therapeutic targets are discussed.

Glucose consumption of vascular cell types in culture: toward optimization of experimental conditions.

In this study, we have looked for an optimum media glucose concentration and compared glucose consumption in three vascular cell types, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and adventitial fibroblasts (AFs) with or without angiotensin II (AngII) stimulation. In a subconfluent 6-well experiment in 1 mL DMEM with a standard low (100 mg/dL), a standard high (450 mg/dL), or a mixed middle (275 mg/dL) glucose concentration, steady and significant glucose consumption was observed in all cell types. After 48-h incubation, media that contained low glucose was reduced to almost 0 mg/dL, media that contained high glucose remained significantly higher at ∼275 mg/dL, and media that contained middle glucose remained closer to physiological range. AngII treatment enhanced glucose consumption in AFs and VSMCs but not in ECs. Enhanced extracellular acidification rate by AngII was also observed in AFs. In AFs, AngII induction of target proteins at 48 h varied depending on the glucose concentration used. In low glucose media, induction of glucose regulatory protein 78 or hexokinase II was highest, whereas induction of VCAM-1 was lowest. Utilization of specific inhibitors further suggests essential roles of angiotensin II type-1 receptor and glycolysis in AngII-induced fibroblast activation. Overall, this study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Media glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.

Detection of Protein Aggregation and Proteotoxicity Induced by Angiotensin II in Vascular Smooth Muscle Cells.

ABSTRACT: Disruption of protein quality control occurs with aging and cardiovascular pathologies including arterial stiffness and hypertension. Angiotensin II (Ang II) is believed to induce endoplasmic reticulum stress in vascular smooth muscle cells (VSMCs), thus contributing to vascular remodeling and dysfunction. However, whether Ang II increases formation of protein aggregates and mediates proteotoxicity in VSMCs remain obscure. Accordingly, this study aimed to establish a quantitative method of protein aggregate detection induced by Ang II and to investigate their potential involvement in inflammatory and senescence responses. Proteostat staining showed increased aggregate numbers per cell on Ang II exposure. Immunoblot analysis further showed an increase in preamyloid oligomer presence in a detergent insoluble protein fraction purified from VSMCs stimulated with Ang II. Moreover, these responses were attenuated by treatment with chemical chaperone, 4-phenylbutyrate. 4-phenylbutyrate further blocked Ang II-induced senescence associated ß-galactosidase activity and THP-1 monocyte adhesion in VSMCs. These data suggest that Ang II induces proteotoxicity in VSMCs which likely contributes to aging and inflammation in the vasculature.

Zika virus infection in chemosensory cells.

Zika virus (ZIKV) is an emerging virus belonging to the genus Flavivirus. ZIKV infection is a significant health concern, with increasing numbers of reports of microcephaly cases in fetuses and Guillain-Barré syndrome (GBS) in adults. Interestingly, chemosensory disturbances are also reported as one of the manifestations of GBS. ZIKV infects several human tissues and cell types in vitro and in vivo. However, there is no study demonstrating ZIKV infection and replication in chemosensory cells, including olfactory and taste cells. Taste papilla and olfactory cells are chemosensory receptor cells with unique histological, molecular, and physiological characteristics. Here we examined ZIKV infection (PRVABC59) in cultured human olfactory epithelial cells (hOECs) and fungiform taste papilla (HBO) cells in vitro, as well as in vivo mouse taste and olfactory epithelial and olfactory bulb tissues. Interestingly, while HBO cells showed resistance to ZIKV replication, hOECs were highly susceptible for ZIKV infection and replication. Further, we demonstrated the presence of ZIKV particles and expression of viral proteins in olfactory epithelium, as well as in olfactory bulb, but not in taste papillae, of immunocompromised mice (ifnar/-) infected with the PRVABC59 strain of ZIKV. These observations suggest that chemosensory cells in the olfactory neuroepithelium and olfactory bulb may be important tissues for ZIKV replication and dissemination.

Depletion of CD11c+ cell attenuates progression of abdominal aortic aneurysm.

Chronic inflammation of the arterial wall has been implicated in the development of abdominal aortic aneurysm (AAA). However, the detailed molecular mechanism(s) by which inflammatory cells contributes to AAA pathogenesis remains largely unclear. In their article in Clinical Science, Krishna et al. have reported that depletion of CD11c+ dendritic cells inhibited experimental AAA formation in mice. The authors also demonstrated a decrease in CD4 and CD8 positive T cells in the circulation, lower plasma neutrophil elastase activity, and aortic matrix remodeling. These novel findings will help clarify the underlying mechanisms of AAA progression and may provide a new target for future therapeutic research in AAA formation.

Chemokine CCR5 and cocaine interactions in the brain: Cocaine enhances mesolimbic CCR5 mRNA levels and produces place preference and locomotor activation that are reduced by a CCR5 antagonist.

C-C chemokine receptor type 5, also known as CCR5 or CD195, is best known as a viral co-receptor that facilitates entry of HIV into cells. Evidence that CCR5 knockout mice display fewer dopamine neurons, lower striatal dopamine levels, and reduced locomotor activation compared to wild types also suggest a link between CCR5 receptors and cocaine dependence. Here, we tested the hypothesis using male Sprague-Dawley rats that cocaine-induced locomotor activation and conditioned place preference (CPP) are inhibited by a FDA-approved CCR5 antagonist (maraviroc), and that CCR5 gene expression in mesolimbic substrates is enhanced by repeated cocaine exposure. Pretreatment with maraviroc (1, 2.5, 5 mg/kg, IP) reduced hyperlocomotion induced by acute cocaine (10 mg/kg) without affecting spontaneous locomotor activity. For CPP experiments, rats conditioned with cocaine (10 mg/kg × 4 days, IP) were injected with maraviroc (1, 2.5, 5 mg/kg, IP) before each injection of cocaine. Maraviroc dose-dependently inhibited development of cocaine CPP, with a dose of 5 mg/kg producing a significant reduction. In rats treated repeatedly with cocaine (10 mg/kg × 4 days, IP), CCR5 gene expression was upregulated in the nucleus accumbens and ventral tegmental area but mRNA levels of CCR5 ligands (i.e., CCL3, CCL4 and CCL5) were not affected. Our results suggest that mesolimbic CCR5 receptors are dysregulated by cocaine exposure and, similar to CXCR4 and CCR2 receptors, influence behavioral effects related to the abuse liability of cocaine.

Transduction Efficiency of Adenovirus Vectors in Endothelial Cells and Vascular Smooth Muscle Cells.

Adenoviral vectors are useful tools in manipulating a gene of interest in vitro and in vivo, including in the vascular system. The transduction efficiencies of adenoviral vectors in vascular cells such as endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are known to be lower than those in epithelial cell types. The effective entry for adenoviral vectors is primarily mediated through the coxsackievirus and adenovirus receptor (CAR), which has been shown to be expressed in both cell types. Cationic liposomes have been used to enhance adenovirus transduction efficiency in nonepithelial cells. Accordingly, the aim of this study is to obtain new information regarding differences in transduction efficiencies, cationic liposome sensitivity, and CAR expression between ECs and VSMCs. Using cultured rat aortic ECs and VSMCs, here, we have compared transduction efficiency of adenoviruses with or without inclusion of liposomes and CAR expression. A significant increase in basal transduction efficiency was observed in ECs compared with VSMCs. Cationic liposome polybrene enhanced transduction efficiency in VSMCs, whereas decreased efficiency was observed in ECs. Western blotting demonstrated expression of the CAR in ECs but not in VSMCs. Proteomic analysis and mouse aorta immunostaining further suggests significant expression of the CAR in ECs but not in VSMCs. In conclusion, adenoviruses can effectively transduce the gene of interest in aortic ECs likely because of abundant expression of the CAR, whereas cationic liposomes such as polybrene enhance the transduction efficiency in VSMCs lacking CAR expression.

Suppression of Zika Virus Infection in the Brain by the Antiretroviral Drug Rilpivirine.

Zika virus (ZIKV) infection is associated with microcephaly in neonates and Guillain-Barré syndrome in adults. ZIKV produces a class of nonstructural (NS) regulatory proteins that play a critical role in viral transcription and replication, including NS5, which possesses RNA-dependent RNA polymerase (RdRp) activity. Here we demonstrate that rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of HIV-1 infection, inhibits the enzymatic activity of NS5 and suppresses ZIKV infection and replication in primary human astrocytes. Similarly, other members of the NNRTI family, including etravirine and efavirenz, showed inhibitory effects on viral infection of brain cells. Site-directed mutagenesis identified 14 amino acid residues within the NS5 RdRp domain (AA265-903), which are important for the RPV interaction and the inhibition of NS5 polymerase activity. Administration of RPV to ZIKV-infected interferon-alpha/beta receptor (IFN-A/R) knockout mice improved the clinical outcome and prevented ZIKV-induced mortality. Histopathological examination of the brains from infected animals revealed that RPV reduced ZIKV RNA levels in the hippocampus, frontal cortex, thalamus, and cerebellum. Repurposing of NNRTIs, such as RPV, for the inhibition of ZIKV replication offers a possible therapeutic strategy for the prevention and treatment of ZIKV-associated disease.

Targeting Molecular Mechanism of Vascular Smooth Muscle Senescence Induced by Angiotensin II, A Potential Therapy via Senolytics and Senomorphics.

Cardiovascular disease (CVD) is a prevalent issue in the global aging population. Premature vascular aging such as elevated arterial stiffness appears to be a major risk factor for CVD. Vascular smooth muscle cells (VSMCs) are one of the essential parts of arterial pathology and prone to stress-induced senescence. The pervasiveness of senescent VSMCs in the vasculature increases with age and can be further expedited by various stressing events such as oxidative stress, mitochondria dysfunction, endoplasmic reticulum stress, and chronic inflammation. Angiotensin II (AngII) can induce many of these responses in VSMCs and is thus considered a key regulator of VSMC senescence associated with CVD. Understanding the precise mechanisms and consequences of senescent cell accumulation may uncover a new generation of therapies including senolytic and senomorphic compounds against CVD. Accordingly, in this review article, we discuss potential molecular mechanisms of VSMC senescence such as those induced by AngII and the therapeutic manipulations of senescence to control age-related CVD and associated conditions such as by senolytic.

78 kDa Glucose-Regulated Protein Attenuates Protein Aggregation and Monocyte Adhesion Induced by Angiotensin II in Vascular Cells.

Investigations of vascular smooth muscle cell (VSMC) phenotypic modulation due to angiotensin II (AngII) stimulation are important for understanding molecular mechanisms contributing to hypertension and associated vascular pathology. AngII induces endoplasmic reticulum (ER) stress in VSMCs, which has been implicated in hypertensive vascular remodeling. Under ER stress, 78 kDa glucose-regulated protein (GRP78) acts as an endogenous chaperone, as well as a master controller of unfolded protein response (UPR) to maintain protein quality control. However, the potential downstream consequences of ER stress induced by AngII on protein quality control and pro-inflammatory phenotype in VSMCs remain elusive. This study aims to identify protein aggregation as evidence of the disruption of protein quality control in VSMCs, and to test the hypothesis that preservation of proteostasis by overexpression of GRP78 can attenuate the AngII-induced pro-inflammatory phenotype in VSMCs. Increases in protein aggregation and enhanced UPR were observed in VSMCs exposed to AngII, which were mitigated by overexpression of GRP78. Moreover, GRP78 overexpression attenuated enhanced monocyte adhesion to VSMCs induced by AngII. Our results thus indicate that the prevention of protein aggregation can potentially mitigate an inflammatory phenotype in VSMCs, which may suggest an alternative therapy for the treatment of AngII-associated vascular disorders.

Involvement of Senescence and Mitochondrial Fission in Endothelial Cell Pro-Inflammatory Phenotype Induced by Angiotensin II.

Angiotensin II (AngII) has a crucial role in cardiovascular pathologies, including endothelial inflammation and premature vascular aging. However, the precise molecular mechanism underlying aging-related endothelial inflammation induced by AngII remains elusive. Here, we have tested a hypothesis in cultured rat aortic endothelial cells (ECs) that the removal of AngII-induced senescent cells, preservation of proteostasis, or inhibition of mitochondrial fission attenuates the pro-inflammatory EC phenotype. AngII stimulation in ECs resulted in cellular senescence assessed by senescence-associated ß galactosidase activity. The number of ß galactosidase-positive ECs induced by AngII was attenuated by treatment with a senolytic drug ABT737 or the chemical chaperone 4-phenylbutyrate. Monocyte adhesion assay revealed that the pro-inflammatory phenotype in ECs induced by AngII was alleviated by these treatments. AngII stimulation also increased mitochondrial fission in ECs, which was mitigated by mitochondrial division inhibitor-1. Pretreatment with mitochondrial division inhibitor-1 attenuated AngII-induced senescence and monocyte adhesion in ECs. These findings suggest that mitochondrial fission and endoplasmic reticulum stress have causative roles in endothelial senescence-associated inflammatory phenotype induced by AngII exposure, thus providing potential therapeutic targets in age-related cardiovascular diseases.

Endoplasmic reticulum stress and mitochondrial biogenesis are potential therapeutic targets for abdominal aortic aneurysm.

Endoplasmic reticulum (ER) and mitochondria are crucial organelles for cell homeostasis and alterations of these organelles have been implicated in cardiovascular disease. However, their roles in abdominal aortic aneurysm (AAA) pathogenesis remain largely unknown. In a recent issue of Clinical Science, Navas-Madronal et al. ((2019), 133(13), 1421-1438) reported that enhanced ER stress and dysregulation of mitochondrial biogenesis are associated with AAA pathogenesis in humans. The authors also proposed that disruption in oxysterols network such as an elevated concentration of 7-ketocholestyerol in plasma is a causative factor for AAA progression. Their findings highlight new insights into the underlying mechanism of AAA progression through ER stress and dysregulation of mitochondrial biogenesis. Here, we will discuss the background, significance of the study, and future directions.

Glycated collagen and altered glucose increase endothelial cell adhesion strength.

Cell adhesion strength is important to cell survival, proliferation, migration, and mechanotransduction, yet changes in endothelial cell adhesion strength have not yet been examined in diseases such as diabetes with high rates of cardiovascular complications. We therefore investigated porcine aortic endothelial cell adhesion strength on native and glycated collagen-coated substrates and in low, normal, and high glucose culture using a spinning disc apparatus. Adhesion strength increased by 30 dynes/cm(2) in cells on glycated collagen as compared to native collagen. Attachment studies revealed that cells use higher adhesion strength αv ß3 integrins to bind to glycated collagen instead of the typical α2 ß1 integrins used to bind to native collagen. Similarly, endothelial cells cultured in low and high glucose had 15 dynes/cm(2) higher adhesion strength than cells in normal glucose after 2 days. Increased adhesion strength was due to elevated VEGF release and intracellular PKC in low and high glucose cells, respectively. Thus glucose increased endothelial cell adhesion strength via different underlying mechanisms. These adhesion strength changes could contribute to diabetic vascular disease, including accelerated atherosclerosis and disordered angiogenesis.

Angiotensin II Type 1A Receptor Expressed in Smooth Muscle Cells is Required for Hypertensive Vascular Remodeling in Mice Infused With Angiotensin II.

BACKGROUND: Ang II (angiotensin II) type 1 (AT1) receptors play a critical role in cardiovascular diseases such as hypertension. Rodents have 2 types of AT1 receptor (AT1A and AT1B) of which knock-in Tagln-mediated smooth muscle AT1A silencing attenuated Ang II-induced hypertension. Although vascular remodeling, a significant contributor to organ damage, occurs concurrently with hypertension in Ang II-infused mice, the contribution of smooth muscle AT1A in this process remains unexplored. Accordingly, it is hypothesized that smooth muscle AT1A receptors exclusively contribute to both medial thickening and adventitial fibrosis regardless of the presence of hypertension. METHODS: About 1 µg/kg per minute Ang II was infused for 2 weeks in 2 distinct AT1A receptor silenced mice, knock-in Tagln-mediated constitutive smooth muscle AT1A receptor silenced mice, and Myh11-mediated inducible smooth muscle AT1A together with global AT1B silenced mice for evaluation of hypertensive cardiovascular remodeling. RESULTS: Medial thickness, adventitial collagen deposition, and immune cell infiltration in aorta were increased in control mice but not in both smooth muscle AT1A receptor silenced mice. Coronary arterial perivascular fibrosis in response to Ang II infusion was also attenuated in both AT1A receptor silenced mice. Ang II-induced cardiac hypertrophy was attenuated in constitutive smooth muscle AT1A receptor silenced mice. However, Ang II-induced cardiac hypertrophy and hypertension were not altered in inducible smooth muscle AT1A receptor silenced mice. CONCLUSIONS: Smooth muscle AT1A receptors mediate Ang II-induced vascular remodeling including medial hypertrophy and inflammatory perivascular fibrosis regardless of the presence of hypertension. Our data suggest an independent etiology of blood pressure elevation and hypertensive vascular remodeling in response to Ang II.

Infused juice concentrate of Japanese plum Prunus mume attenuates inflammatory vascular remodeling in a mouse model of hypertension induced by angiotensin II.

Fruit from the Prunus mume tree is a traditional food in Japan. Recently, bainiku-ekisu, an infused juice concentrate of Japanese Prunus mume, is attracting attention as a health promoting supplement. Angiotensin II (Ang II) plays a central role in development of hypertension. It has been reported that bainiku-ekisu treatment attenuates the growth-promoting signaling induced by Ang II in vascular smooth muscle cells. However, whether bainiku-ekisu has any effect on an animal model of hypertension remains unknown. Therefore, this study was designed to explore the potential anti-hypertensive benefit of bainiku-ekisu utilizing a mouse model of hypertension with Ang II infusion. Male C57BL/6 mice were infused with Ang II for 2 weeks and given 0.1% bainiku-ekisu containing water or normal water for 2 weeks with blood pressure evaluation. After 2 weeks, mice were euthanized, and the aortas were collected for evaluation of remodeling. Aortic medial hypertrophy was observed in control mice after Ang II infusion, which was attenuated in bainiku-ekisu group with Ang II infusion. Bainiku-ekisu further attenuated aortic induction of collagen producing cells and immune cell infiltration. Development of hypertension induced by Ang II was also prevented by bainiku-ekisu. Echocardiograph indicated protection of Ang II-induced cardiac hypertrophy by bainiku-ekisu. In vascular fibroblasts, bainiku-ekisu attenuated vascular cell adhesion molecule-1 induction, an endoplasmic reticulum stress marker, inositol requiring enzyme-1α phosphorylation, and enhancement in glucose consumption in response to Ang II. In conclusion, Bainiku-ekisu prevented Ang II-induced hypertension and inflammatory vascular remodeling. Potential cardiovascular health benefit to taking bainiku-ekisu should be further studied.

Endoplasmic Reticulum Chemical Chaperone 3-Hydroxy-2-Naphthoic Acid Reduces Angiotensin II-Induced Vascular Remodeling and Hypertension In Vivo and Protein Synthesis In Vitro.

Background Investigations into alternative treatments for hypertension are necessary because current treatments cannot fully reduce the risk for the development of cardiovascular diseases. Chronic activation of unfolded protein response attributable to the endoplasmic reticulum stress has been proposed as a potential therapeutic target for hypertension and associated vascular remodeling. Triggered by the accumulation of misfolded proteins, chronic unfolded protein response leads to downstream signaling of cellular inflammation and dysfunction. Here, we have tested our hypothesis that a novel chemical chaperone, 3-hydroxy-2-naphthoic acid (3HNA) can attenuate angiotensin II (AngII)-induced vascular remodeling and hypertension. Methods and Results Mice were infused with AngII for 2 weeks to induce vascular remodeling and hypertension with or without 3HNA treatment. We found that injections of 3HNA prevented hypertension and increase in heart weight body weight ratio induced by AngII infusion. Histological assessment revealed that 3HNA treatment prevented vascular medial thickening as well as perivascular fibrosis in response to AngII infusion. In cultured vascular smooth muscle cells, 3HNA attenuated enhancement in protein synthesis induced by AngII. In vascular adventitial fibroblasts, 3HNA prevented induction of unfolded protein response markers. Conclusions We present evidence that a chemical chaperone 3HNA prevents vascular remodeling and hypertension in mice with AngII infusion, and 3HNA further prevents increase in protein synthesis in AngII-stimulated vascular smooth muscle cells. Using 3HNA may represent a novel therapy for hypertension with multiple benefits by preserving protein homeostasis under cardiovascular stress.

Targeting mitochondrial fission as a potential therapeutic for abdominal aortic aneurysm.

AIMS: Angiotensin II (AngII) is a potential contributor to the development of abdominal aortic aneurysm (AAA). In aortic vascular smooth muscle cells (VSMCs), exposure to AngII induces mitochondrial fission via dynamin-related protein 1 (Drp1). However, pathophysiological relevance of mitochondrial morphology in AngII-associated AAA remains unexplored. Here, we tested the hypothesis that mitochondrial fission is involved in the development of AAA. METHODS AND RESULTS: Immunohistochemistry was performed on human AAA samples and revealed enhanced expression of Drp1. In C57BL6 mice treated with AngII plus ß-aminopropionitrile, AAA tissue also showed an increase in Drp1 expression. A mitochondrial fission inhibitor, mdivi1, attenuated AAA size, associated aortic pathology, Drp1 protein induction, and mitochondrial fission but not hypertension in these mice. Moreover, western-blot analysis showed that induction of matrix metalloproteinase-2, which precedes the development of AAA, was blocked by mdivi1. Mdivi1 also reduced the development of AAA in apolipoprotein E-deficient mice infused with AngII. As with mdivi1, Drp1+/- mice treated with AngII plus ß-aminopropionitrile showed a decrease in AAA compared to control Drp1+/+ mice. In abdominal aortic VSMCs, AngII induced phosphorylation of Drp1 and mitochondrial fission, the latter of which was attenuated with Drp1 silencing as well as mdivi1. AngII also induced vascular cell adhesion molecule-1 expression and enhanced leucocyte adhesion and mitochondrial oxygen consumption in smooth muscle cells, which were attenuated with mdivi1. CONCLUSION: These data indicate that Drp1 and mitochondrial fission play salient roles in AAA development, which likely involves mitochondrial dysfunction and inflammatory activation of VSMCs.

Alcohol exposure alters pre-mRNA splicing of antiapoptotic Mcl-1L isoform and induces apoptosis in neural progenitors and immature neurons.

Alternative splicing and expression of splice variants of genes in the brain may lead to the modulation of protein functions, which may ultimately influence behaviors associated with alcohol dependence and neurotoxicity. We recently showed that ethanol exposure can lead to pre-mRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by downregulating the expression levels of serine/arginine rich splicing factor 1 (SRSF1). Little is known about the physiological expression of these isoforms in neuronal cells and their role in toxicity induced by alcohol exposure during the developmental period. In order to investigate the impact of alcohol exposure on alternative splicing of Mcl-1 pre-mRNA and its role in neurotoxicity, we developed a unique primary human neuronal culture model where neurospheres (hNSPs), neural progenitors (hNPCs), immature neurons, and mature neurons were cultured from the matching donor fetal brain tissues. Our data suggest that neural progenitors and immature neurons are highly sensitive to the toxic effects of ethanol, while mature neuron cultures showed resistance to ethanol exposure. Further analysis of Mcl-1 pre-mRNA alternative splicing by semi-quantitative and quantitative analysis revealed that ethanol exposure causes a significant decrease in Mcl-1L/Mcl-1S ratio in a dose and time dependent manner in neural progenitors. Interestingly, ectopic expression of Mcl-1L isoform in neural progenitors was able to recover the viability loss and apoptosis induced by alcohol exposure. Altogether, these observations suggest that alternative splicing of Mcl-1 may play a crucial role in neurotoxicity associated with alcohol exposure in the developing fetal brain.

Neuroimmune Regulation of JC Virus by Intracellular and Extracellular Agnoprotein.

JC virus (JCV) is a human polyomavirus and the etiologic agent of the demyelinating disease progressive multifocal leukoencephalopathy (PML). PML is observed in patients with underlying immunocompromising conditions, suggesting that neuro-immune interactions between peripheral immune cells and neuro-glia play an important role in controlling viral reactivation in the brain. There is little known about the immunobiology of JCV reactivation in glial cells and the role of immune, glial, and viral players in this regulation. We have previously showed that agnoprotein, a small JCV regulatory protein, is released from infected cells and internalized by neighboring bystander cells. Here we have investigated the possible role of extracellular and intracellular agnoprotein in the neuroimmune response to JC virus. Our findings suggest that glial cells exposed to agnoprotein secrete significantly less GM-CSF, which is mediated by agnoprotein induced suppression of GM-CSF transcription. Likewise, monocytes treated with agnoprotein showed altered differentiation and maturation. In addition, monocytes and microglial cells exposed to agnoprotein showed a significant reduction in their phagocytic activities. Moreover, when an in vitro blood-brain barrier model was used, agnoprotein treatment resulted in decreased monocyte migration through the endothelial cell layer in response to activated astrocytes. All together, these results have revealed a novel immunomodulatory function of agnoprotein during JCV infection within theCNS and open a new avenue of research to better understand the mechanisms associated with JCV reactivation in patients who are at risk of developing PML.