Streamlined synthesis of potential dual-emissive fluorescent silicon quantum dots (SiQDs) for cell imaging.

One of the current challenges of working with nanomaterials in bioapplications is having a tool that is biocompatible (non-toxic) and produces stable, intense fluorescence for bioimaging. To address these challenges, we have developed a streamlined and one-pot synthetic route for silicon-based quantum dots (SiQDs) using a hydrothermal method. Part of our unique approach for designing the SiQDs was to incorporate (3-aminopropyl) triethoxysilane (APTES), which is an amphipathic molecule with hydroxyl and amine functional groups available for modification. In order to reduce the toxicity of APTES, we chose glucose as a reducing agent for the reaction. The resulting SiQDs produced potent, stable, potential dual-emissive fluorescence emission peaks in the visible and near-infrared (NIR) ranges. Both peaks could be used as distinguishing fluorescence signals for bioimaging, separately or in combination. The physical and optical properties of the SiQDs were determined under a range of environmental conditions. The morphology, surface composition, and electronic structure of the SiQDs were characterized using high resolution-transmission electronic microscopy (HR-TEM), energy dispersive X-ray spectroscopy (EDS), Fourier-transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). The stability of the SiQDs was evaluated under a wide range of pHs. The biocompatibility and imaging potential of the SiQDs were tested in microvascular endothelial cells (MVEC), neural stem cells (NSC), and RAW 264.7 macrophage cells. The images obtained revealed different subcellular localizations, particularly during cell division, with distinct fluorescence intensities. The results demonstrated that SiQDs are a promising, non-toxic labeling tool for a variety of cell types, with the added advantage of having dual emission peaks both in visible and NIR ranges for bioimaging.

Postnatal exercise protects offspring from high-fat diet-induced reductions in subcutaneous adipocyte beiging in C57Bl6/J mice.

Maternal low-protein and postnatal high-fat (HF) diets program offspring obesity and type 2 diabetes mellitus (T2DM) risk by epigenetically reducing beige adipocytes (BAs) via increased G9a protein expression (Histone3 Lysine9 dimethyl transferase), an inhibitor of the BA marker fibroblast growth factor 21 (FGF21). Conversely, offspring exercise reduces fat mass and white adipocytes, but the mechanisms are not yet understood. This work investigated whether exercise reduces offspring obesity and T2DM risk caused by a maternal HF diet via regulation of G9a and FGF21 expression that would convert white to BA. Two-month-old female C57Bl/6J mice (F0) were fed a 16% (normal fat; NF) or a 45% HF diet for 3 months prior to breeding, and subsequent gestation and lactation. Male offspring (F1) were fed the same NF and HF diets and further divided into either sedentary (S) or voluntary wheel running (Ex) groups for an additional 3 months yielding eight groups: NF (maternal treatment condition)-NF-S (postweaning treatment conditions), NF-HF-S, NF-NF-Ex, NF-HF-Ex, HF-NF-S, HF-HF-S, HF-NF-Ex, and HF-HF-Ex. Subcutaneous adipose tissue was collected for protein and mRNA analysis of FGF21, peroxisome proliferator-activated receptor-gamma coactivator (PGC-1 alpha, inducer of FGF21), G9a, E4BP4 (G9a coactivator), and protein expression of H3K9 demethylases (KDM4C). Postnatal HF diet decreased FGF21 positive BA numbers regardless of maternal diets and postnatal exercise. Under sedentary conditions, postnatal HF diet increased protein expression of FGF21 transcription inhibitors G9a and E4BP4 compared to NF diet resulting in decreased FGF21 expression. In contrast, postnatal HF diet and exercise decreased G9a and E4BP4 protein expression while decreasing FGF21 expression compared to NF diet. Under exercised condition, postnatal HF diet-induced KDM4C protein expression while no changes in KDM4C protein expression were induced by postnatal HF diet under sedentary conditions. These findings suggest that the postnatal diet exerts a greater impact on offspring adiposity and BA numbers than maternal diets. These data also suggest that offspring exercise induces KDM4C to counter the increase in G9a that was triggered by maternal and postnatal HF diets. Future studies need to determine whether KDM4C induces methylation status of G9a to alter thermogenic function of BA.

Nanozymes-Hitting the Biosensing "Target".

Nanozymes are a class of artificial enzymes that have dimensions in the nanometer range and can be composed of simple metal and metal oxide nanoparticles, metal nanoclusters, dots (both quantum and carbon), nanotubes, nanowires, or multiple metal-organic frameworks (MOFs). They exhibit excellent catalytic activities with low cost, high operational robustness, and a stable shelf-life. More importantly, they are amenable to modifications that can change their surface structures and increase the range of their applications. There are three main classes of nanozymes including the peroxidase-like, the oxidase-like, and the antioxidant nanozymes. Each of these classes catalyzes a specific group of reactions. With the development of nanoscience and nanotechnology, the variety of applications for nanozymes in diverse fields has expanded dramatically, with the most popular applications in biosensing. Nanozyme-based novel biosensors have been designed to detect ions, small molecules, nucleic acids, proteins, and cancer cells. The current review focuses on the catalytic mechanism of nanozymes, their application in biosensing, and the identification of future directions for the field.

Development of alveolar-capillary-exchange (ACE) chip and its application for assessment of PM2.5-induced toxicity.

Although standard two-dimensional (2D) cell culture is an effective tool for cell studies, monolayer cultivation can yield imperfect or misleading information about numerous biological functions. In this study, we developed an alveolar-capillary exchange (ACE) chip aiming to simulate the cellular microenvironment at the alveolar-capillary interface. The ACE chip was designed with two chambers for culturing alveolar epithelial cells and vascular endothelial cells separately, which are separated by a microporous polycarbonate film that allows for the exchange of soluble biomolecules. Using this model, we further tested the toxic effects of fine particulate matter (PM2.5), a form of airborne pollutant known to induce adverse effects on human respiratory system. These effects are largely associated with the ability of PM2.5 to penetrate the alveoli, where it negatively affects the pulmonary function. Our results indicate that alveolar epithelial cells cultured in the ACE chip in solo and coculture with vascular endothelial cells underwent oxidative injury-induced apoptosis mediated via the PEAK-eIF2α signaling pathway of endoplasmic reticulum stress. The use of ACE chip in an alveolar epithelial cell-vascular endothelial cell coculture model revealed cellular vulnerability to PM2.5. Therefore, this chip provides a feasible surrogate approach in vitro for investigating and simulating the cellular microenvironment responses associated with ACE in vivo.

The Hox protein conundrum: The "specifics" of DNA binding for Hox proteins and their partners.

Homeotic genes (Hox genes) are homeodomain-transcription factors involved in conferring segmental identity along the anterior-posterior body axis. Molecular characterization of HOX protein function raises some interesting questions regarding the source of the binding specificity of the HOX proteins. How do HOX proteins regulate common and unique target specificity across space and time? This review attempts to summarize and interpret findings in this area, largely focused on results from in vitro and in vivo studies in Drosophila and mouse systems. Recent studies related to HOX protein binding specificity compel us to reconsider some of our current models for transcription factor-DNA interactions. It is crucial to study transcription factor binding by incorporating components of more complex, multi-protein interactions in concert with small changes in binding motifs that can significantly impact DNA binding specificity and subsequent alterations in gene expression. To incorporate the multiple elements that can determine HOX protein binding specificity, we propose a more integrative Cooperative Binding model.

Loss of Slfn3 induces a sex-dependent repair vulnerability after 50% bowel resection.

Bowel resection accelerates enterocyte proliferation in the remaining gut with suboptimal absorptive and digestive capacity because of a proliferation-associated decrease in functional differentiation markers. We hypothesized that although schlafen 3 (Slfn3) is an important regulator of enterocytic differentiation, Slfn3 would have less impact on bowel resection adaptation, where accelerated proliferation takes priority over differentiation. We assessed proliferation, cell shedding, and enterocyte differentiation markers from resected and postoperative bowel of wild-type (WT) and Slfn3-knockout (Slfn3KO) mice. Villus length and crypt depth were increased in WT mice and were even longer in Slfn3KO mice. Mitotic marker, Phh3+, and the proliferation markers Lgr5, FoxL1, and platelet-derived growth factor-α (PDGFRα) were increased after resection in male WT, but this was blunted in male Slfn3KO mice. Cell-shedding regulators Villin1 and TNFα were downregulated in female mice and male WT mice only, whereas Gelsolin and EGFR increased expression in all mice. Slfn3 expression increased after resection in WT mice, whereas other Slfn family members 1, 2, 5, 8, and 9 had varied expressions that were affected also by sex difference and loss of Slfn3. Differentiation markers sucrase isomaltase, Dpp4, Glut2, and SGLT1 were all decreased, suggesting that enterocytic differentiation effort is incompatible with rapid proliferation shift in intestinal adaptation. Slfn3 absence potentiates villus length and crypt depth, suggesting that the differentiating stimulus of Slfn3 signaling may restrain mucosal mass increase through regulating Villin1, Gelsolin, EGFR, TNFα, and proliferation markers. Therefore, Slfn3 may be an important regulator not only of "normal" enterocytic differentiation but also in response to bowel resection.NEW & NOTEWORTHY The differentiating stimulus of Slfn3 signaling restrains an increase in mucosal mass after bowel resection, and there is a Slfn3-sex interaction regulating differentiation gene expression and intestinal adaptation. This current study highlights the combinatory effects of gender and Slfn3 genotype on the gene expression changes that contribute to the adaptation in intestinal cellular milleu (i.e. villus and crypt structure) which are utilized to compensate for the stress-healing response that the animals display in intestinal adaptation.

Effects of Probiotic Supplementation on Short Chain Fatty Acids in the AppNL-G-F Mouse Model of Alzheimer's Disease.

BACKGROUND: The intestinal microbiota and its metabolites, particularly short-chain fatty acids (SCFAs), have been implicated in immune function, host metabolism, and even behavior. OBJECTIVE: This study was performed to investigate whether probiotic administration influences levels of intestinal microbiota and their metabolites in a fashion that may attenuate brain changes in a mouse model of Alzheimer's disease (AD). METHODS: C57BL/6 wild-type (WT) mice were compared to AppNL-G-Fmice. The animals were treated with either vehicle or probiotic (VSL#3) for 8 weeks. Fecal microbiome analysis along with Aß, GFAP, Iba-1, c-Fos, and Ki-67 immunohistochemistry was done. SCFAs were analyzed in serum and brains using UPLC-MS/MS. RESULTS: Probiotic (VSL#3) supplementation for 2 months resulted in altered microbiota in both WT and AppNL-G-Fmice. An increase in serum SCFAs acetate, butyrate, and lactate were found in both genotypes following VSL#3 treatment. Propionate and isobutyrate were only increased in AppNL-G-Fmice. Surprisingly, VSL#3 only increased lactate and acetate in brains of AppNL-G-Fmice. No significant differences were observed between vehicle and VSL#3 fed AppNL-G-Fhippocampal immunoreactivities of Aß, GFAP, Iba-1, and Ki-67. However, hippocampal c-Fos staining increased in VSL#3 fed AppNL-G-Fmice. CONCLUSION: These data demonstrate intestinal dysbiosis in the AppNL-G-Fmouse model of AD. Probiotic VSL#3 feeding altered both serum and brain levels of lactate and acetate in AppNL-G-Fmice correlating with increased expression of the neuronal activity marker, c-Fos.

The longitudinal effects of early developmental cadmium exposure on conditioned place preference and cardiovascular physiology in zebrafish.

Cadmium (Cd) is a naturally occurring trace metal that is widely considered to be highly toxic to aquatic organisms and a significant health hazard to humans (Amzal et al., 2009; Bernhoft 2013; Burger, 2008; Satarug et al., 2009). The zebrafish (Danio rerio) has been used as a model organism for toxicological studies with Cd (Banni et al., 2011; Blechinger et al., 2007; Chow et al., 2009; Chow et al., 2008; Favorito et al., 2011; Kusch et al., 2007; Matz et al., 2007; Wang and Gallagher, 2013). We asked what the lasting longitudinal effects would be from short early developmental Cd exposure (between 24 and 96h post-fertilization) in a range that larvae might experience living atop typical Cd-containing surface sediments (0, 0.01, 0.1, 1.0 and 10µM CdCl2: 1.124, 11.24, 112.4 and 1124µg Cd/L). The goal of this exposure window was to specifically target secondary neurogenesis, monoaminergic differentiation and cardiovascular development, without affecting earlier patterning processes. Developmental abnormalities in body size and CNS morphology increased with concentration, but were statistically significant only at the highest concentration used (10µM). Heart rate for Cd-treated larvae increased with concentration, and was significant even at the lowest concentration used (0.01µM). Longitudinal survival was significantly lower for fish developmentally exposed to the highest concentration. Except for brain weight, overall morphology was not affected by developmental Cd exposure. However, developmental exposure to lower concentrations of Cd (0.01, 0.1, and 1.0µM) progressively lowered cocaine-induced conditioned place preference (CPP), used to measure function of the reward pathways in the brain. Baseline heart rate was significantly lower in longitudinal fish developmentally exposed to 1.0µM Cd. Cardiovascular response to isoproterenol, a potent ß-adrenergic agonist, in longitudinal adults was also significantly affected by developmental exposure to Cd at low doses (0.01, 0.1 and 1.0µM). Surviving longitudinal adult fish exposed to the highest concentration of Cd showed normal CPP and cardiovascular physiology. The data imply that even lower exposure concentrations can potentially result in fitness-affecting parameters without affecting survival in a laboratory setting.

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease.

UNLABELLED: Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid ß (Aß)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP(-/-)) microglial cultures, oligomeric Aß was unable to stimulate increased secretion from mAPP(-/-) cells. This was consistent with an ability of oligomeric Aß to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aß produced less microgliosis in mAPP(-/-) mice compared with wild-type mice. The mAPP(-/-) mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aß plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT: A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid ß (Aß) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aß stimulation of microglial activation is one source of brain inflammatory changes during disease. Aß is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aß are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aß production to drive the microgliosis associated with AD brains.

Longitudinal Effects of Embryonic Exposure to Cocaine on Morphology, Cardiovascular Physiology, and Behavior in Zebrafish.

A sizeable portion of the societal drain from cocaine abuse results from the complications of in utero drug exposure. Because of challenges in using humans and mammalian model organisms as test subjects, much debate remains about the impact of in utero cocaine exposure. Zebrafish offer a number of advantages as a model in longitudinal toxicology studies and are quite sensitive physiologically and behaviorally to cocaine. In this study, we have used zebrafish to model the effects of embryonic pre-exposure to cocaine on development and on subsequent cardiovascular physiology and cocaine-induced conditioned place preference (CPP) in longitudinal adults. Larval fish showed a progressive decrease in telencephalic size with increased doses of cocaine. These treated larvae also showed a dose dependent response in heart rate that persisted 24 h after drug cessation. Embryonic cocaine exposure had little effect on overall health of longitudinal adults, but subtle changes in cardiovascular physiology were seen including decreased sensitivity to isoproterenol and increased sensitivity to cocaine. These longitudinal adult fish also showed an embryonic dose-dependent change in CPP behavior, suggesting an increased sensitivity. These studies clearly show that pre-exposure during embryonic development affects subsequent cocaine sensitivity in longitudinal adults.

Shifts in the vascular endothelial growth factor isoforms result in transcriptome changes correlated with early neural stem cell proliferation and differentiation in mouse forebrain.

Regulation of neural stem cell (NSC) fate decisions is critical during the transition from a multicellular mammalian forebrain neuroepithelium to the multilayered neocortex. Forebrain development requires coordinated vascular investment alongside NSC differentiation. Vascular endothelial growth factor A (Vegf) has proven to be a pleiotrophic gene whose multiple protein isoforms regulate a broad range of effects in neurovascular systems. To test the hypothesis that the Vegf isoforms (120, 164, and 188) are required for normal forebrain development, we analyzed the forebrain transcriptome of mice expressing specific Vegf isoforms, Vegf120, VegfF188, or a combination of Vegf120/188. Transcriptome analysis identified differentially expressed genes in embryonic day (E) 9.5 forebrain, a time point preceding dramatic neuroepithelial expansion and vascular investment in the telencephalon. Meta-analysis identified gene pathways linked to chromosome-level modifications, cell fate regulation, and neurogenesis that were altered in Vegf isoform mice. Based on these gene network shifts, we predicted that NSC populations would be affected in later stages of forebrain development. In the E11.5 telencephalon, we quantified mitotic cells [Phospho-Histone H3 (pHH3)-positive] and intermediate progenitor cells (Tbr2/Eomes-positive), observing quantitative and qualitative shifts in these populations. We observed qualitative shifts in cortical layering at P0, particularly with Ctip2-positive cells in layer V. The results identify a suite of genes and functional gene networks that can be used to further dissect the role of Vegf in regulating NSC differentiation and downstream consequences for NSC fate decisions.

Sulpiride, but not SCH23390, modifies cocaine-induced conditioned place preference and expression of tyrosine hydroxylase and elongation factor 1α in zebrafish.

Finding genetic polymorphisms and mutations linked to addictive behavior can provide important targets for pharmaceutical and therapeutic interventions. Forward genetic approaches in model organisms such as zebrafish provide a potentially powerful avenue for finding new target genes. In order to validate this use of zebrafish, the molecular nature of its reward system must be characterized. We have previously reported the use of cocaine-induced conditioned place preference (CPP) as a reliable method for screening mutagenized fish for defects in the reward pathway. Here we test if CPP in zebrafish involves the dopaminergic system by co-treating fish with cocaine and dopaminergic antagonists. Sulpiride, a potent D2 receptor (DR2) antagonist, blocked cocaine-induced CPP, while the D1 receptor (DR1) antagonist SCH23390 had no effect. Acute cocaine exposure also induced a rise in the expression of tyrosine hydroxylase (TH), an important enzyme in dopamine synthesis, and a significant decrease in the expression of elongation factor 1α (EF1α), a housekeeping gene that regulates protein synthesis. Cocaine selectively increased the ratio of TH/EF1α in the telencephalon, but not in other brain regions. The cocaine-induced change in TH/EF1α was blocked by co-treatment with sulpiride, but not SCH23390, correlating closely with the action of these drugs on the CPP behavioral response. Immunohistochemical analysis revealed that the drop in EF1α was selective for the dorsal nucleus of the ventral telencephalic area (Vd), a region believed to be the teleost equivalent of the striatum. Examination of TH mRNA and EF1α transcripts suggests that regulation of expression is post-transcriptional, but this requires further examination. These results highlight important similarities and differences between zebrafish and more traditional mammalian model organisms.

Long-term retention of knowledge and critical thinking skills in developmental biology.

The primary goal of this project was to assess long-term retention of concepts and critical thinking skills in individuals who completed a Developmental Biology course. Undergraduates who had completed the course between 2006 and 2009 were recently contacted and asked to complete a professional goals survey and a multiple-choice developmental biology assessment test (DBAT) targeting four levels of learning. The DBAT was designed to assess students' retention of knowledge and skills related to factual recall, concept application, data analysis, and experimental design. Performance of the 2006-2009 cohorts was compared to that of students enrolled in 2010 who completed the DBAT at the beginning and the end of the semester. Participants from the 2010 course showed significant learning gains based on pre- and posttest scores overall and for each of the four levels of learning. No significant difference in overall performance was observed for students grouped by year from 2006-2010. Participants from the 2006-2009 cohorts scored slightly, but significantly, higher on average if they enrolled in graduate or professional training. However, performance on individual question categories revealed no significant differences between those participants with and without postundergraduate training. Scores on exams and a primary literature critique assignment were correlated with DBAT scores and thus represent predictors of long-term retention of developmental biology knowledge and skills.

Vascular endothelial growth factor (VEGF) isoform regulation of early forebrain development.

This work was designed to determine the role of the vascular endothelial growth factor A (VEGF) isoforms during early neuroepithelial development in the mammalian central nervous system (CNS), specifically in the forebrain. An emerging model of interdependence between neural and vascular systems includes VEGF, with its dual roles as a potent angiogenesis factor and neural regulator. Although a number of studies have implicated VEGF in CNS development, little is known about the role that the different VEGF isoforms play in early neurogenesis. We used a mouse model of disrupted VEGF isoform expression that eliminates the predominant brain isoform, VEGF164, and expresses only the diffusible form, VEGF120. We tested the hypothesis that VEGF164 plays a key role in controlling neural precursor populations in developing cortex. We used microarray analysis to compare gene expression differences between wild type and VEGF120 mice at E9.5, the primitive stem cell stage of the neuroepithelium. We quantified changes in PHH3-positive nuclei, neural stem cell markers (Pax6 and nestin) and the Tbr2-positive intermediate progenitors at E11.5 when the neural precursor population is expanding rapidly. Absence of VEGF164 (and VEGF188) leads to reduced proliferation without an apparent effect on the number of Tbr2-positive cells. There is a corresponding reduction in the number of mitotic spindles that are oriented parallel to the ventricular surface relative to those with a vertical or oblique angle. These results support a role for the VEGF isoforms in supporting the neural precursor population of the early neuroepithelium.

Endogenous VEGF is required for visual function: evidence for a survival role on müller cells and photoreceptors.

BACKGROUND: Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical. METHODS AND FINDINGS: Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective. CONCLUSIONS: These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.

Engineering vascularized skeletal muscle tissue.

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.

CADASIL mutations impair Notch3 glycosylation by Fringe.

Mutations in the NOTCH3 gene trigger adult-onset stroke and vascular dementia in patients with CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). All CADASIL mutations described to date affect the epidermal growth factor-like (EGF-like) repeats located in the extracellular domain of the Notch3 receptor. These domains are also the target of sequential complex O-linked glycosylation mediated by protein O-fucosyltransferase 1 and Fringe. We investigated whether O-fucosylation or Fringe-mediated elongation of O-fucose on Notch3 is impaired by CADASIL mutations. Biochemical studies of a Notch3 fragment containing the first five EGF-like repeats of Notch3, including the mutational hot spot, showed that CADASIL mutations do not affect the addition of O-fucose but do impair carbohydrate chain elongation by Fringe. CADASIL changes also induced aberrant homodimerization of mutant Notch3 fragments and heterodimerization of mutant Notch3 with Lunatic Fringe itself. Together, these data suggest that Fringe plays a role in CADASIL pathophysiology.

Functional analysis of a mutant form of the receptor tyrosine kinase Tie2 causing venous malformations.

Tie2 is expressed predominantly in endothelial cells and is required for blood vessel formation and maintenance. A missense mutation resulting in an R to W substitution in the kinase domain of Tie2 co-segregates with an autosomal dominantly inherited form of vascular dysmorphogenesis, venous malformation (VM). The mechanism by which this activating mutation leads to vessel dysmorphogenesis in VM is not known. Here we examined Tie2 activation status in VM and found activated receptor in lesional and non-lesional vessels. To gain insight into functional effects of VM mutant Tie2, wild-type and R849W mutant receptor were expressed in cultured human venous endothelial cells. Mutant Tie2 was constitutively phosphorylated in endothelial cells in vivo and caused a marked suppression of apoptosis. The anti-apoptotic kinase Akt was constitutively activated in cells expressing mutant receptor. Dominant-negative Akt inhibited the pro-survival activity of mutant Tie2. Migration of smooth muscle cells induced by conditioned medium from cells expressing mutant receptor was similar to that from cells expressing wild-type receptor. These data suggest that a primary effect of R849W Tie2 in VM is to allow survival of mural cell poor vessels via ligand-independent Tie2 activation of Akt and endothelial survival, rather than to directly induce formation of dysmorphogenic vessels.

Endothelial-mesenchymal interactions in vitro reveal molecular mechanisms of smooth muscle/pericyte differentiation.

Cell-cell interactions are central to vascular development. We have developed an in vitro system in which endothelial cells (EC) are co-cultured with 10T1/2 cells as smooth muscle cell (SMC)/pericyte precursors. 10T1/2 cells, in contact with EC, differentiate to SMC in a process mediated, at least in part, by a transforming growth factor-beta (TGF-beta)-mediated event. Co-culture with EC or TGF-beta treatment induced expression of SM22alpha, with co-culture inducing a significantly greater response. To dissect the molecular mechanisms of SMC/pericyte differentiation, reporter constructs containing the promoter for SM22alpha, a SMC-specific gene, were stably transfected into 10T1/2 cells and response to EC-co-culture and TGFbeta were compared. Co-culture with EC or TGFbeta treatment stimulated activity of a 441-bp SM22-alpha promoter to about the same extent, whereas co-culture induced the activity of a 3.7-kb promoter to about twice that of TGBbeta. Neutralization of TGFbeta in EC-10T1/2 co-cultures partially reduced the 3.7-kb SM22alpha promoter activity in 10T1/2 cells. Previously unidentified CArG and TCE elements near the 5' end of the promoter are responsible for full promoter activity. EC-mesenchymal contact appears to be required for full promoter activity of the SM22alpha gene in 10T1/2 and requires upstream CArG and TCE elements. The 3.7-kb SM22alpha promoter can direct expression of lacZ in vivo to SMC of the large vessels and the smaller intersomitic vessels. We have identified the expression of SM22alpha in pericytes of the retinal microvasculature in developing and remodeling vessels.

Endothelial cell-astrocyte interactions and TGF beta are required for induction of blood-neural barrier properties.

We sought to establish a blood-neural barrier (BNB) model of astrocyte contact with endothelial cells (EC) to test the hypothesis that transforming growth factor beta (TGF beta) promotes an EC barrier-phenotype. Astrocyte-EC contact induced BNB properties in EC. Transendothelial resistance was augmented by direct contact between astrocytes-EC, but not by astrocyte-conditioned medium or astrocyte-EC coculture conditioned medium. Coculture of EC and astrocytes led to significant increase in endothelial occludin levels and junctional localization. EC gamma-glutamyl-transferase (GGT) activity was increased by direct contact with astrocytes, by conditioned medium from cocultures or by TGF beta1. Coculture inhibited EC proliferation with no effect on astrocyte proliferation. A neutralizing antibody to TGF beta decreased GGT activity in cocultures and increased cell number. Whereas total TGF beta was not significantly altered by coculture, activated TGF beta increased in astrocyte-EC cocultures. In summary, astrocyte-EC contact induces BNB characteristics in EC and locally activated TGF beta is responsible for part of the induction.