Effects of repeated sleep deprivation on brain pericytes in mice.

The damaging effects of sleep deprivation (SD) on brain parenchyma have been extensively studied. However, the specific influence of SD on brain pericytes, a primary component of the blood-brain barrier (BBB) and the neurovascular unit (NVU), is still unclear. The present study examined how acute or repeated SD impairs brain pericytes by measuring the cerebrospinal fluid (CSF) levels of soluble platelet-derived growth factor receptor beta (sPDGFRß) and quantifying pericyte density in the cortex, hippocampus, and subcortical area of the PDGFRß-P2A-CreERT2/tdTomato mice, which predominantly express the reporter tdTomato in vascular pericytes. Our results showed that a one-time 4 h SD did not significantly change the CSF sPDGFRß level. In contrast, repeated SD (4 h/day for 10 consecutive days) significantly elevated the CSF sPDGFRß level, implying explicit pericyte damages due to repeated SD. Furthermore, repeated SD significantly decreased the pericyte densities in the cortex and hippocampus, though the pericyte apoptosis status remained unchanged as measured with Annexin V-affinity assay and active Caspase-3 staining. These results suggest that repeated SD causes brain pericyte damage and loss via non-apoptosis pathways. These changes to pericytes may contribute to SD-induced BBB and NVU dysfunctions. The reversibility of this process implies that sleep improvement may have a protective effect on brain pericytes.

cAMP-induced decrease in cell-surface laminin receptor and cellular prion protein attenuates amyloid-ß uptake and amyloid-ß-induced neuronal cell death.

Previous studies have shown that amyloid-ß oligomers (AßO) bind with high affinity to cellular prion protein (PrPC ). The AßO-PrPC complex binds to cell-surface co-receptors, including the laminin receptor (67LR). Our current studies revealed that in Neuroscreen-1 cells, 67LR is the major co-receptor involved in the cellular uptake of AßO and AßΟ-induced cell death. Both pharmacological (dibutyryl-cAMP, forskolin and rolipram) and physiological (pituitary adenylate cyclase-activating polypeptide) cAMP-elevating agents decreased cell-surface PrPC and 67LR, thereby attenuating the uptake of AßO and the resultant neuronal cell death. These cAMP protective effects are dependent on protein kinase A, but not dependent on the exchange protein directly activated by cAMP. Conceivably, cAMP protects neuronal cells from AßO-induced cytotoxicity by decreasing cell-surface-associated PrPC and 67LR.

Mild pericyte deficiency is associated with aberrant brain microvascular flow in aged PDGFRß+/- mice.

The receptor tyrosine kinase PDGFRß is essential for pericyte migration to the endothelium. In mice lacking one allele of PDGFRß (PDGFRß+/-), previous reports have described an age-dependent loss of pericytes in the brain, leading to cerebrovascular dysfunction and subsequent neurodegeneration reminiscent of that seen in Alzheimer's disease and vascular dementia. We examined 12-20-month-old PDGFRß+/- mice to better understand how pericyte loss affects brain microvascular structure and perfusion in vivo. We observed a mild reduction of cortical pericyte number in PDGFRß+/- mice (27% fewer cell bodies) compared to controls, but no decrease in pericyte coverage of the endothelium. This mild degree of pericyte loss caused no discernable change in cortical microvascular density, length, basal diameter or reactivity to hypercapnia. Yet, it was associated with an increase in basal blood cell velocity, primarily in pre-capillary arterioles. Taken together, our results suggest that mild pericyte loss can lead to aberrant cerebral blood flow despite a lack of apparent effect on microvascular structure and reactivity.

Organizational hierarchy and structural diversity of microvascular pericytes in adult mouse cortex.

Smooth muscle cells and pericytes, together called mural cells, coordinate many distinct vascular functions. Canonically, smooth muscle cells are ring-shaped and cover arterioles with circumferential processes, whereas pericytes extend thin processes that run longitudinally along capillaries. In between these canonical mural cell types are cells with features of both smooth muscle cells and pericytes. Recent studies suggest that these transitional cells are critical for controlling blood flow to the capillary bed during health and disease, but there remains confusion on how to identify them and where they are located in the brain microvasculature. To address this issue, we measured the morphology, vascular territory, and α-smooth muscle actin content of structurally diverse mural cells in adult mouse cortex. We first imaged intact 3D vascular networks to establish the locations of major gradations in mural cell appearance as arterioles branched into capillaries. We then imaged individual mural cells occupying the regions within these gradations. This revealed two transitional cells that were often similar in appearance, but with sharply contrasting levels of α-smooth muscle actin. Our findings highlight the diversity of mural cell morphologies in brain microvasculature, and provide guidance for identification and categorization of mural cell types.

Pericyte Structural Remodeling in Cerebrovascular Health and Homeostasis.

The biology of brain microvascular pericytes is an active area of research and discovery, as their interaction with the endothelium is critical for multiple aspects of cerebrovascular function. There is growing evidence that pericyte loss or dysfunction is involved in the pathogenesis of Alzheimer's disease, vascular dementia, ischemic stroke and brain injury. However, strategies to mitigate or compensate for this loss remain limited. In this review, we highlight a novel finding that pericytes in the adult brain are structurally dynamic in vivo, and actively compensate for loss of endothelial coverage by extending their far-reaching processes to maintain contact with regions of exposed endothelium. Structural remodeling of pericytes may present an opportunity to foster pericyte-endothelial communication in the adult brain and should be explored as a potential means to counteract pericyte loss in dementia and cerebrovascular disease. We discuss the pathophysiological consequences of pericyte loss on capillary function, and the biochemical pathways that may control pericyte remodeling. We also offer guidance for observing pericytes in vivo, such that pericyte structural remodeling can be more broadly studied in mouse models of cerebrovascular disease.

Dynamic Remodeling of Pericytes In Vivo Maintains Capillary Coverage in the Adult Mouse Brain.

Direct contact and communication between pericytes and endothelial cells is critical for maintenance of cerebrovascular stability and blood-brain barrier function. Capillary pericytes have thin processes that reach hundreds of micrometers along the capillary bed. The processes of adjacent pericytes come in close proximity but do not overlap, yielding a cellular chain with discrete territories occupied by individual pericytes. Little is known about whether this pericyte chain is structurally dynamic in the adult brain. Using in vivo two-photon imaging in adult mouse cortex, we show that while pericyte somata were immobile, the tips of their processes underwent extensions and/or retractions over days. The selective ablation of single pericytes provoked exuberant extension of processes from neighboring pericytes to contact uncovered regions of the endothelium. Uncovered capillary regions had normal barrier function but were dilated until pericyte contact was regained. Pericyte structural plasticity may be critical for cerebrovascular health and warrants detailed investigation.

Basal hyperinsulinemia beyond a threshold predicts major adverse cardiac events at 1 year after coronary angiogram in type 2 diabetes mellitus: a retrospective cohort study.

BACKGROUND: There is a substantial reduction in cardiovascular related morbidity and mortality in the general population attributed to improved treatment of cardiac risk factors and disease, the same magnitude of benefit has not been observed in those with diabetes mellitus. The aim of the present study was to evaluate factors associated with the cardiac outcome at 1 year after coronary angiogram in patients with type 2 diabetes mellitus and to compare the outcomes with nondiabetics. METHODS: A retrospective cohort study was carried out in subjects who underwent coronary angiogram for an evaluation of CAD, with follow-up data available for period of 12 months. The data consisted of 208 type 2 diabetic and 75 non-diabetic patients. Clinical, anthropometric and other biochemical risk factors of the study participants were recorded. Univariate and multivariate cox proportional hazard regression analyses were performed to evaluate the relation between the cardiovascular risk factors and major adverse cardiac events (MACE). RESULTS: At 1 year, MACE was observed in 50 (24.04%) type 2 diabetic subjects, which included non-fatal myocardial infarction 24 (11.54%), target vessel revascularization 15 (7.21%) and death 11 (5.29%). The area under the curve for insulin in predicting MACE was found to be 0.81 (95% CI 0.73-0.88) with sensitivity and specificity of 88% (95% CI 0.71-0.96) and 74% (95% CI 0.65-0.81) respectively. After adjustment for potential confounders hyperinsulinemia (>20 µIU/ml) was significantly associated with MACE [adjusted hazard ratio (HR): 3.03, 95% CI 1.41-6.54, p = 0.005]. Interestingly, the MACE rate in type 2 diabetics with insulin levels <20 µIU/ml (10.2%) and non-diabetics (12%) (p = 0.676) appears to be same. CONCLUSIONS: In addition to severity of the CAD at the baseline, basal hyperinsulinemia beyond a threshold strongly predicts adverse cardiac events at 1 year in type 2 diabetes mellitus. Those below the threshold, appears to be having a risk equivalent to non-diabetics.

Manipal diabetes coronary artery severity score.

AIMS: To develop a risk score, for identifying severe and complex CAD in patients with type 2 diabetes mellitus. METHODS: In this cross sectional study, 179 patients with type 2 diabetes mellitus undergoing coronary angiogram for the evaluation of suspected coronary artery disease (CAD) were recruited at a tertiary-care hospital. Patients were divided into developmental (n=124) and validation (n=55) cohorts. Biochemical and anthropometric parameters were analysed. Predictors of severe and complex CAD (SYNTAX Score>22) were identified by multiple logistic regression analysis. RESULTS: Insulin resistance>3.4 (OR: 21.26, 95% CI: 5.71-79.09), duration of diabetes>5years (OR: 13.50, 95% CI: 3.13-58.25), total cholesterol/HDL-C ratio>5 (OR: 2.75, 95% CI: 0.66-11.55) and waist circumference>96cm (OR: 5.08, 95% CI: 1.27-20.42) were independent predictors of severe and complex CAD, and Manipal Diabetes Coronary Artery Severity Score was developed. CONCLUSIONS: The prediction of severe and complex CAD was achieved with this simple score, and thus enabling effective identification of patients beforehand, who are not likely to be suitable for angioplasty.

Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendroglioma cell line.

Th1 pro-inflammatory cytokines, i.e., TNF-α and IFN-γ, in combination are known to induce cell death in several cell types, including oligodendrocytes, but the mechanism of their synergistic cytotoxicity is unclear. Although ceramide (Cer) has been implicated in cytokine- and stress-induced cell death, its intracellular levels alone cannot explain cytokine synergy. We considered the possibility that Cer released as part of extracellular vesicles may contribute to cytokine-induced synergistic cell death. Using a human oligodendroglioma (HOG) cell line as a model, here we show that exosomes derived from TNF-α-treated "donor" cells, while being mildly toxic to fresh cultures (similar to individual cytokines), induce enhanced cell death when added to IFN-γ-primed target cultures in a fashion resembling the effect of cytokine combination. Further, the sphingolipid profiles of secreted exosomes, as determined by HPLC-MS/MS, revealed that the treatment with the cytokines time-dependently induced the formation and exosomal release, in particular of C16-, C24-, and C24:1-Cer species; C16-, C24-, and C24:1-dihydroCer species; and C16-, C24-, and C24:1-SM species. Finally, exogenous C6-Cer or C16-Cer mimicked and enhanced the cytotoxic effects of the cytokines upon HOG cells, thereby supporting the cell death-signaling role of extracellular Cer.

Severity of coronary artery disease in type 2 diabetes mellitus: Does the timing matter?

AIM: The aim of our study was to compare the angiographic changes in 53 nondiabetic patients, 54 type 2 diabetic patients of less than 5 years of duration, 41 patients with 5-10 years of diabetes, and 27 with more than 10 years of diabetic duration. METHODS: In this cross-sectional study, 175 patients, who underwent coronary angiogram for the evaluation of the coronary artery disease (CAD), were recruited. Based on the angiographic findings, syntax score, vessel score, and coronary collaterals grading were analyzed. The biochemical analysis was done by using the auto analyzer. RESULTS: A significant increase in the mean syntax score (p=0.019), vessel score (p=0.007), and coronary collateral grade (p=0.008) was observed in the patients with 5-10 years of diabetes when compared to those with less than 5 years of diabetic duration. There was no significant difference in the mean syntax score (p=0.979), vessel score (p=0.299), and collateral grade (p=0.842) between the patients with 5-10 years and more than 10 years of diabetes. The difference in the mean syntax score (p=0.791), vessel score (p=0.098), and collateral grade (p=0.661) between the nondiabetic and the patients with less than 5 years of diabetes was not significant. CONCLUSION: A significant structural change in the coronary arteries was found among the patients with 5-10 years of diabetes.

Optogenetic stimulation of astrocytes in the posterior hypothalamus increases sleep at night in C57BL/6J mice.

A distributed network of neurons regulates wake, non-rapid eye movement (NREM) sleep, and REM sleep. However, there are also glia in the brain, and there is growing evidence that neurons and astroglia communicate intimately to regulate behaviour. To identify the effect of optogenetic stimulation of astrocytes on sleep, the promoter for the astrocyte-specific cytoskeletal protein, glial fibrillary acidic protein (GFAP) was used to direct the expression of channelrhodopsin-2 (ChR2) and the linked reporter gene, enhanced yellow fluorescent protein (EYFP), in astrocytes. rAAV-GFAP-ChR2 (H134R)-EYFP or rAAV-GFAP-EYFP was microinjected (750 nL) into the posterior hypothalamus (bilateral) of mice. Three weeks later baseline sleep was recorded (0 Hz) and 24 h later optogenetic stimulation applied during the first 6 h of the lights-off period. Mice with ChR2 were given 5, 10 or 30 Hz stimulation for 6 h (10-ms pulses; 1 mW; 1 min on 4 min off). At least 36 h elapsed between the stimulation periods (5, 10, 30 Hz) and although 0 Hz was always first, the order of the other three stimulation rates was randomised. In mice with ChR2 (n = 7), 10 Hz, but not 5 or 30 Hz stimulation increased both NREM and REM sleep during the 6-h period of stimulation. Delta power did not increase. In control mice (no ChR2; n = 5), 10 Hz stimulation had no effect. This study demonstrates that direct stimulation of astrocytes powerfully induces sleep during the active phase of the sleep-wake cycle and underlines the inclusion of astrocytes in network models of sleep-wake regulation.

Factors associated with no apparent coronary artery disease in patients with type 2 diabetes mellitus for more than 10 years of duration: a case control study.

BACKGROUND: Type 2 diabetes mellitus is an important risk factor in the development of coronary artery disease (CAD) and is often associated with severe disease. However, this risk is not uniform, some patients remain free of CAD even after many years of treatment for diabetes. The present study was aimed to identify the factors that are associated with a favorable CAD profile. METHODS: A case-control study of 76 patients with type 2 diabetes mellitus who were on treatment for more than 10 years duration and undergoing a coronary angiogram for the evaluation of clinically suspected CAD at a tertiary care hospital were recruited for the study. The presence and absence of significant CAD was determined after a coronary angiogram. Clinical history, and anthropometric and biochemical parameters were analyzed. Insulin resistance was determined by the Homeostasis Model Assessment. Multiple logistic regressions were done to find out the factors associated for a favorable CAD profile. RESULTS: The difference in HOMA-IR (2.37 ± 0.69 VS 3.77 ± 1.64, p < 0.001) and urine microalbumin (24.15 ± 32.16 VS 82.72 ± 117.70, p = 0.004) were found to be statistically significant among those who did not have CAD when compared to those who had CAD. The difference in lipid profile, HbA1C, fasting blood sugar, BMI, waist hip ratio, waist and hip circumference was not significant. The adjusted odds ratio for insulin resistance less than 2.5 (OR 9.09, 95 % CI 1.91-41.83, p = 0.005), females (OR 7.91, 95% CI 1.55-40.38, p = 0.013) and microalbumin <20 mg/l (OR 4.57, 95% CI 1.17-17.85, p = 0.029) were independently associated with normal coronaries. The adjusted odds ratio for lipid profile, BMI, blood pressure and HbA1C were not significant. CONCLUSIONS: HOMA-IR less than 2.5, microalbuminuria less than 20 mg/l and females are the factors appear to be associated with no apparent CAD.

Vasculoprotection as a Convergent, Multi-Targeted Mechanism of Anti-AD Therapeutics and Interventions.

Using a variety of animal models of Alzheimer's disease (AD), there have been a number of recent studies reporting varying degrees of success with anti-AD therapeutics. The efficacies are often discussed in terms of the modulatory effects of the compounds tested on identified or assumed targets among the known (or proposed) pathogenic and neuroprotective mechanisms, largely within the context of the dominant amyloid cascade hypothesis. However, it is clear that several of the relatively more efficacious treatments tend to be multifunctional and target multiple pathological processes associated with AD including most commonly, oxidative and metabolic stress and neuroinflammation. Increasing evidence suggests that vascular and neurodegenerative pathologies often co-exist and that neurovascular dysfunction plays a critical role in the development or progression of AD. In this review, we will discuss the significance of vasculoprotection or neurovascular unit integrity as a common, multi-targeted mechanism underlying the reported efficacy of a majority of anti-AD therapeutics--amyloid-targeted or otherwise--while providing a strong support for future neurovascular-based treatment strategies and interventions.

The role of tumor progression locus 2 protein kinase in glial inflammatory response.

Tumor progression locus 2 (Tpl2)/cancer Osaka thyroid kinase is a newer member of MAP3K family that is now known for its essential role in tumor necrosis factor-aplha (TNFα) expression in macrophages, but its pro-inflammatory signaling, if any, in glia is unknown. When cultures of murine microglia and astrocytes were exposed to lipopolysaccharide, there was a rapid activation (i.e., phosphorylation) of Tpl2 in parallel to the activation of down-stream effector MAPKs, that is, extracellular signal regulated kinase (ERK), p38 MAPK and C-Jun N-terminal kinase (JNK). Pre-incubation of the cultures with a Tpl2 inhibitor selectively suppressed the activation of the primary down-stream target, that is, ERK relative to p38 MAPK and JNK. That Tpl2 activation was functionally involved in glial inflammatory response was indicated by a reduced release of the cytokines, i.e. TNFα and the expression of inducible nitric oxide synthase in the presence of the kinase inhibitor. Furthermore, over-expression of a wild-type Tpl2 construct in C-6 glia resulted in an enhanced transcriptional activation of inducible nitric oxide synthase, while transfection with a dominant negative form of Tpl-2 had the opposite effect. The findings assign an important pro-inflammatory signaling function for Tpl2 pathway in glial cells.

Increased tau phosphorylation and impaired brain insulin/IGF signaling in mice fed a high fat/high cholesterol diet.

Previous studies demonstrated that a high fat/high cholesterol (HFC) diet results in a loss of working memory in mice correlated with neuroinflammatory changes and increased AßPP processing (Thirumangalakudi et al. (2008) J Neurochem 106, 475-485). To further explore the nature of the molecular correlates of cognitive impairment, in this study, we examined changes in tau phosphorylation, insulin/IGF-1 signaling (IIS) including GSK3, and levels of specific synaptic proteins. Immunoblot analysis of hippocampal tissue from C57BL/6 mice fed HFC for 2 months with anti-phospho-tau (i.e., PHF1 and phospho-Thr-231 tau) antibodies demonstrated the presence of hyperphosphorylated tau. The tau phosphorylation correlated with activated GSK3, a prominent tau kinase normally kept inactive under the control of IIS. That IIS itself was impaired due to the hyperlipidemic diet was confirmed by a down-regulation of insulin receptor substrate-1 and phospho-Akt levels. Although no significant changes in the levels of the pre-synaptic protein (i.e., synaptophysin) in response to HFC were apparent in immunoblot analysis, there was a clear down-regulation of the post-synaptic protein, PSD95, and drebrin, a dendritic spine-specific protein, indicative of altered synaptic plasticity. The results, in concert with previous findings with the same model, suggest that high dietary fat/cholesterol elicits brain insulin resistance and altered IIS leading to Alzheimer's disease-like cognitive impairment in 'normal' mice.

Metabolic stress modulates Alzheimer's ß-secretase gene transcription via SIRT1-PPARγ-PGC-1 in neurons.

Classic cardio-metabolic risk factors such as hypertension, stroke, diabetes, and hypercholesterolemia all increase the risk of Alzheimer's disease. We found increased transcription of ß-secretase/BACE1, the rate-limiting enzyme for Aß generation, in eNOS-deficient mouse brains and after feeding mice a high-fat, high-cholesterol diet. Up- or downregulation of PGC-1α reciprocally regulated BACE1 in vitro and in vivo. Modest fasting in mice reduced BACE1 transcription in the brains, which was accompanied by elevated PGC-1 expression and activity. Moreover, the suppressive effect of PGC-1 was dependent on activated PPARγ, likely via SIRT1-mediated deacetylation in a ligand-independent manner. The BACE1 promoter contains multiple PPAR-RXR sites, and direct interactions among SIRT1-PPARγ-PGC-1 at these sites were enhanced with fasting. The interference on the BACE1 gene identified here represents a unique noncanonical mechanism of PPARγ-PGC-1 in transcriptional repression in neurons in response to metabolic signals that may involve recruitment of corepressor NCoR.

Engineering an in situ crosslinkable hydrogel for enhanced remyelination.

Remyelination has to occur to fully regenerate injured spinal cords or brain tissues. A growing body of evidence has suggested that exogenous cell transplantation is one promising strategy to promote remyelination. However, direct injection of neural stem cells or oligodendrocyte progenitor cells (OPCs) to the lesion site may not be an optimal therapeutic strategy due to poor viability and functionality of transplanted cells resulted from the local hostile tissue environment. The overall objective of this study was to engineer an injectable biocompatible hydrogel system as a supportive niche to provide a regeneration permissive microenvironment for transplanted OPCs to survive, functionally differentiate, and remyelinate central nervous system (CNS) lesions. A highly biocompatible hydrogel, based on thiol-functionalized hyaluronic acid and thiol-functionalized gelatin, which can be crosslinked by poly-(ethylene glycol) diacrylate (PEGDA), was used. These hydrogels were optimized first regarding cell adhesive properties and mechanical properties to best support the growth properties of OPCs in culture. Transplanted OPCs with the hydrogels optimized in vitro exhibited enhanced survival and oligodendrogenic differentiation and were able to remyelinate demyelinated axons inside ethidium bromide (EB) demyelination lesion in adult spinal cord. This study provides a new possible therapeutic approach to treat CNS injuries in which cell therapies may be essential.

Vitamin D3-enriched diet correlates with a decrease of amyloid plaques in the brain of AßPP transgenic mice.

In addition to its function in calcium and bone metabolism, vitamin D is neuroprotective and important for mitigating inflammation. Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the central nervous system, characterized by neuronal loss in many areas of the brain, and the formation of senile (neuritic) plaques, which increase in number and size over time. The goal of this project was to investigate whether vitamin D3 supplementation would affect amyloid plaque formation in amyloid-ß protein precursor (AßPP) transgenic mice that spontaneously develop amyloid plaques within 3-4 months of birth. AßPP mice were fed control, vitamin D3-deficient or vitamin D3-enriched diets for five months, starting immediately after weaning. At the end of the study, the animals were subjected to behavioral studies, sacrificed, and examined for bone changes and brain amyloid load, amyloid-ß (Aß) peptide levels, inflammatory changes, and nerve growth factor (NGF) content. The results obtained indicate that a vitamin D3-enriched diet correlates with a decrease in the number of amyloid plaques, a decrease in Aß peptides, a decrease in inflammation, and an increase in NGF in the brains of AßPP mice. These observations suggest that a vitamin D3-enriched diet may benefit AD patients.