Understanding Mechanisms Underlying Non-Alcoholic Fatty Liver Disease (NAFLD) in Mental Illness: Risperidone and Olanzapine Alter the Hepatic Proteomic Signature in Mice.

Patients with severe mental illness have increased mortality, often linked to cardio-metabolic disease. Non-alcoholic fatty liver disease (NAFLD) incidence is higher in patients with schizophrenia and is exacerbated with antipsychotic treatment. NAFLD is associated with obesity and insulin resistance, both of which are induced by several antipsychotic medications. NAFLD is considered an independent risk factor for cardiovascular disease, the leading cause of death for patients with severe mental illness. Although the clinical literature clearly defines increased risk of NAFLD with antipsychotic therapy, the underlying mechanisms are not understood. Given the complexity of the disorder as well as the complex pharmacology associated with atypical antipsychotic (AA) medications, we chose to use a proteomic approach in healthy mice treated with a low dose of risperidone (RIS) or olanzapine (OLAN) for 28 days to determine effects on development of NAFLD and to identify pathways impacted by AA medications, while removing confounding intrinsic effects of mental illness. Both AA drugs caused development of steatosis in comparison with vehicle controls (p < 0.01) and affected multiple pathways relating to energy metabolism, NAFLD, and immune function. AA-associated alteration in autonomic function appears to be a unifying theme in the regulation of hepatic pathology.

DLL4/Notch1 and BMP9 Interdependent Signaling Induces Human Endothelial Cell Quiescence via P27KIP1 and Thrombospondin-1.

OBJECTIVE: Bone morphogenetic protein-9 (BMP9)/activin-like kinase-1 and delta-like 4 (DLL4)/Notch promote endothelial quiescence, and we aim to understand mechanistic interactions between the 2 pathways. We identify new targets that contribute to endothelial quiescence and test whether loss of Dll4(+/-) in adult vasculature alters BMP signaling. APPROACH AND RESULTS: Human endothelial cells respond synergistically to BMP9 and DLL4 stimulation, showing complete quiescence and induction of HEY1 and HEY2. Canonical BMP9 signaling via activin-like kinase-1-Smad1/5/9 was disrupted by inhibition of Notch signaling, even in the absence of exogenous DLL4. Similarly, DLL4 activity was suppressed when the basal activin-like kinase-1-Smad1/5/9 pathway was inhibited, showing that these pathways are interdependent. BMP9/DLL4 required induction of P27(KIP1) for quiescence, although multiple factors are involved. To understand these mechanisms, we used proteomics data to identify upregulation of thrombospondin-1, which contributes to the quiescence phenotype. To test whether Dll4 regulates BMP/Smad pathways and endothelial cell phenotype in vivo, we characterized the vasculature of Dll4(+/-) mice, analyzing endothelial cells in the lung, heart, and aorta. Together with changes in endothelial structure and vascular morphogenesis, we found that loss of Dll4 was associated with a significant upregulation of pSmad1/5/9 signaling in lung endothelial cells. Because steady-state endothelial cell proliferation rates were not different in the Dll4(+/-) mice, we propose that the upregulation of pSmad1/5/9 signaling compensates to maintain endothelial cell quiescence in these mice. CONCLUSIONS: DLL4/Notch and BMP9/activin-like kinase-1 signaling rely on each other's pathways for full activity. This represents an important mechanism of cross talk that enhances endothelial quiescence and sensitively coordinates cellular responsiveness to soluble and cell-tethered ligands.

A receptor-specific function for Notch2 in mediating vascular smooth muscle cell growth arrest through cyclin-dependent kinase inhibitor 1B.

RATIONALE: Deregulated vascular smooth muscle cell (VSMC) proliferation contributes to multiple vascular pathologies, and Notch signaling regulates VSMC phenotype. OBJECTIVE: Previous work focused on Notch1 and Notch3 in VSMC during vascular disease; however, the role of Notch2 is unknown. Because injured murine carotid arteries display increased Notch2 in VSMC as compared with uninjured arteries, we sought to understand the impact of Notch2 signaling in VSMCs. METHODS AND RESULTS: In human primary VSMCs, Jagged-1 (Jag-1) significantly reduced proliferation through specific activation of Notch2. Increased levels of p27(kip1) were observed downstream of Jag-1/Notch2 signaling and were required for cell cycle exit. Jag-1 activation of Notch resulted in increased phosphorylation on serine 10, decreased ubiquitination, and prolonged half-life of p27(kip1). Jag-1/Notch2 signaling robustly decreased S-phase kinase-associated protein, an F-box protein that degrades p27(kip1) during G1. Overexpression of S-phase kinase-associated protein before Notch activation by Jag-1 suppressed the induction of p27(kip1). Additionally, increased Notch2 and p27(kip1) expression was colocalized to the nonproliferative zone of injured arteries as indicated by co-staining with proliferating cell nuclear antigen, whereas Notch3 was expressed throughout normal and injured arteries, suggesting Notch2 may negatively regulate lesion formation. CONCLUSIONS: We propose a receptor-specific function for Notch2 in regulating Jag-1-induced p27(kip1) expression and growth arrest in VSMCs. During vascular remodeling, colocalization of Notch2 and p27(kip1) to the nonproliferating region supports a model where Notch2 activation may negatively regulate VSMC proliferation to lessen the severity of the lesion. Thus, Notch2 is a potential target for control of VSMC hyperplasia.

RhoA-mediated signaling in Notch-induced senescence-like growth arrest and endothelial barrier dysfunction.

OBJECTIVE: Notch signaling has a critical role in vascular development and morphogenesis. Activation of Notch in endothelial cells led to a senescence-like phenotype with loss of barrier function. Our objective was to understand the molecular pathways mediating this phenotype. METHODS AND RESULTS: Human primary endothelial cells increase expression of Notch receptors and ligands during propagation in vitro toward natural senescence. This senescence was induced at low passage with Notch activation. We characterized the pathways activated downstream of Notch signaling. Notch was activated by Delta-like 4 ligand or constitutively active Notch receptors and measured for cell proliferation, migration, and sprouting. Notch signaling triggered early senescence in low-passage cells, characterized by increased p53 and p21 expression. The senescence phenotype was associated with hyperpermeability of the monolayer, with disrupted vascular endothelial cadherin and ß-catenin levels and localization. Consistent with changes in cell shape and contact, we demonstrated that Notch activation increases myosin light chain phosphorylation by activating Rho kinase. Inhibition of Rho abrogated Notch-induced myosin light chain phosphorylation and led to enhanced barrier function by reorganizing F-actin to ß-catenin-containing cell-cell adherens junctions. CONCLUSIONS: Our findings show that RhoA/Rho kinase regulation by Notch signaling in endothelial cells triggers a senescence phenotype associated with endothelial barrier dysfunction.

Polystyrene and Polyethylene Microplastics Decrease Cell Viability and Dysregulate Inflammatory and Oxidative Stress Markers of MDCK and L929 Cells In Vitro.

Microplastics are ubiquitous environmental pollutants that are a growing concern to many ecosystems, as well as human health. Many of the effects of microplastics on mammalian cells and tissues remain unknown. To address this, we treated L929 murine fibroblasts and Madin-Darby canine kidney (MDCK) epithelial cell lines with 1 µg/mL, 10 µg/mL, or 20 µg/mL of polyethylene (PE) or polystyrene (PS) microspheres in vitro for 6 and 24 h and measured the resulting changes in cell viability, metabolism, and transcriptional expression of inflammatory cytokines and antioxidant enzymes. We observed dose-dependent decreases in cell viability corresponding to increases in doses of both PE and PS. We conducted cell metabolism assays and observed dose-dependent increases in metabolism per cell with increasing doses of both PE and PS. Similarly, we also observed increased expression of the superoxide dismutase-3 gene (SOD3), indicating oxidative stress caused by the microplastics treatments. We also observed increased expression of TNFα, but decreased expression of IFNß, suggesting different mechanisms by which the microplastics regulate inflammatory responses in mammalian cells. Our results contribute new data to the growing understanding of the effects of microplastics on mammalian cells and indicate complex cellular stress responses to microplastics in the environment.

VDR haploinsufficiency impacts body composition and skeletal acquisition in a gender-specific manner.

The vitamin D receptor (VDR) is crucial for virtually all of vitamin D's actions and is thought to be ubiquitously expressed. We hypothesized that disruption of one allele of the VDR gene would impact bone development and would have metabolic consequences. Body composition and bone mass (BMD) in VDR heterozygous (VDR HET) mice were compared to those obtained in male and female VDR KO and WT mice at 8 weeks of age. Male mice were also evaluated at 16 weeks, and bone marrow mesenchymal stem cell (MSC) differentiation was evaluated in VDR female mice. Additionally, female VDR HET and WT mice received intermittent PTH treatment or vehicle (VH) for 4 weeks. BMD was determined at baseline and after treatment. MRI was done in vivo at the end of treatment; µCT and bone histomorphometry were performed after killing the animals. VDR HET male mice had normal skeletal development until 16 weeks of age but showed significantly less gain in fat mass than WT mice. In contrast, female VDR HET mice showed decreased total-body BMD at age 8 weeks but had a normal skeletal response to PTH. MSC differentiation was also impaired in VDR HET female mice. Thus, female VDR HET mice show early impairment in bone acquisition, while male VDR HET mice exhibit a lean phenotype. Our results indicate that the VDR HET mouse is a useful model for studying the metabolic and skeletal impact of decreased vitamin D sensitivity.

Experimental test of microbiome protection across pathogen doses reveals importance of resident microbiome composition.

The commensal microbes inhabiting a host tissue can interact with invading pathogens and host physiology in ways that alter pathogen growth and disease manifestation. Prior work in house finches (Haemorhous mexicanus) found that resident ocular microbiomes were protective against conjunctival infection and disease caused by a relatively high dose of Mycoplasma gallisepticum. Here, we used wild-caught house finches to experimentally examine whether protective effects of the resident ocular microbiome vary with the dose of invading pathogen. We hypothesized that commensal protection would be strongest at low M. gallisepticum inoculation doses because the resident microbiome would be less disrupted by invading pathogen. Our five M. gallisepticum dose treatments were fully factorial with an antibiotic treatment to perturb resident microbes just prior to M. gallisepticum inoculation. Unexpectedly, we found no indication of protective effects of the resident microbiome at any pathogen inoculation dose, which was inconsistent with the prior work. The ocular bacterial communities at the beginning of our experiment differed significantly from those previously reported in local wild-caught house finches, likely causing this discrepancy. These variable results underscore that microbiome-based protection in natural systems can be context dependent, and natural variation in community composition may alter the function of resident microbiomes in free-living animals.

Effects of Vancomycin on Persistent Pain-Stimulated and Pain-Depressed Behaviors in Female Fischer Rats With or Without Voluntary Access to Running Wheels.

The present experiments determined the effects of the narrow-spectrum antibiotic vancomycin on inflammatory pain-stimulated and pain-depressed behaviors in rats. Persistent inflammatory pain was modeled using dilute formalin (0.5%). Two weeks of oral vancomycin administered in drinking water attenuated Phase II formalin pain-stimulated behavior, and prevented formalin pain-depressed wheel running. Fecal microbiota transplantation produced a non-significant trend toward reversal of the vancomycin effect on pain-stimulated behavior. Vancomycin depleted Firmicutes and Bacteroidetes populations in the gut while having a partial sparing effect on Lactobacillus species and Clostridiales. The vancomycin treatment effect was associated with an altered profile in amino acid concentrations in the gut with increases in arginine, glycine, alanine, proline, valine, leucine, and decreases in tyrosine and methionine. These results indicate that vancomycin may have therapeutic effects against persistent inflammatory pain conditions that are distal to the gut. PERSPECTIVE: The narrow-spectrum antibiotic vancomycin reduces pain-related behaviors in the formalin model of inflammatory pain. These data suggest that manipulation of the gut microbiome may be one method to attenuate inflammatory pain amplitude.

Inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells by the norsesterterpene peroxide, epimuqubilin A.

Seven norsesterterpene peroxides: epimuqubilin A (1), muqubilone B (2), unnamed cyclic peroxide ester (3), epimuqubilin B (4), sigmosceptrellin A methyl ester (5), sigmosceptrellin A (6), and sigmosceptrellin B methyl ester (7), isolated from the marine sponge Latrunculia sp., were examined with regard to their effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells. The results indicated epimuqubilin A (1) possessed potent NO inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide release with an IC(50) value of 7.4 microM, a level three times greater than the positive control, L-N(G)-monomethyl arginine citrate, followed by 6 (sigmosceptrellin A, IC(50) = 9.9 microM), whereas other compounds exhibited only modest activity (Table 1). These compounds did not show appreciable cytotoxicity at their IC(50) values for NO-inhibitory activity. The structure-activity upon NO inhibition could be summarized as follows: (1) a monocyclic carbon skeleton framework was essential for activity, (2) free acids gave higher activity, (3) the orientation of H3-22 with an equatorial position increased activity, and (4) a bicyclic structure reduced activity. This is the first report of a norsesterterpene peroxide with NO-inhibitory activity. In addition, compounds 1-7 were also evaluated for their inhibitory activities in the yeast glycogen synthase kinase-3beta assay. In summary, several norsesterterpene peroxides showed novel biological activities of inhibition in NO production, suggesting that these might provide leads for anti-inflammatory or cancer chemopreventive agents.

Antipsychotic-induced immune dysfunction: A consideration for COVID-19 risk.

Patients with severe mental illness are more susceptible to infections for a variety of reasons, some associated with the underlying disease and some due to environmental factors including housing insecurity, smoking, poor access to healthcare, and medications used to treat these disorders. This increased susceptibility to respiratory infections may contribute to risk of COVID-19 infection in patients with severe mental illness or those in inpatient settings. Atypical antipsychotic (AA) medications are FDA approved to treat symptoms associated with schizophrenia, bipolar disorder, depression and irritability associated with autism. Our team and others have shown that AA may have anti-inflammatory properties that may contribute to their efficacy in the treatment of mental health disorders. Additionally, AA are widely prescribed off-label for diverse indications to non-psychotic patients including older adults, who are also at increased risk for COVID-19 complications and mortality. The aim of this study was to determine if AA medications such as risperidone (RIS) alter the ability to mount an appropriate response to an acute inflammatory or adaptive immune challenge using a preclinical model. Short-term treatment of healthy mice with a dose of RIS that achieves plasma concentrations within the low clinical range resulted in disrupted response to an inflammatory (LPS) challenge compared to vehicle controls. Furthermore, RIS also prevented treated animals from mounting an antibody response following vaccination with Pneumovax23®. These data indicate that short-to intermediate-term exposure to clinically relevant levels of RIS dysregulate innate and adaptive immune responses, which may affect susceptibility to respiratory infections, including COVID-19.

Notch signal integration in the vasculature during remodeling.

Notch signaling plays many important roles in homeostasis and remodeling in the vessel wall, and serves a critical role in the communication between endothelial cells and smooth muscle cells. Within blood vessels, Notch signaling integrates with multiple pathways by mechanisms including direct protein­protein interaction, cooperative or synergistic regulation of signal cascades, and co-regulation of transcriptional targets. After establishment of the mature blood vessel, the spectrum and intensity of Notch signaling change during phases of active remodeling or disease progression. These changes can be mediated by regulation via microRNAs and protein stability or signaling, and corresponding changes in complementary signaling pathways. Notch also affects endothelial cells on a system level by regulating key metabolic components. This review will outline the most recent findings of Notch activity in blood vessels, with a focus on how Notch signals integrate with other molecular signaling pathways controlling vascular phenotype.

Effect of soluble Jagged1-mediated inhibition of Notch signaling on proliferation and differentiation of an adipocyte progenitor cell model.

Adipose tissue development is dependent on multiple signaling mechanisms and cell-cell interactions that regulate adipogenesis, angiogenesis and extracellular remodeling. The Notch signaling pathway is an important cell-fate determinant whose role in adipogenesis is not clearly defined. To address this issue, we examined the effect of inhibition of Notch signaling by soluble-Jagged1 in the 3T3-L1 preadipocyte line. In vitro, soluble-Jagged1 expression in 3T3-L1 cells altered cell morphology, increased the rate of cell proliferation and induced an early transcriptional response to differentiation stimuli. However, these cells did not form mature adipocytes due to their inability to exit the cell-cycle in response to serum-starvation and glucocorticoid-induced cell-cycle arrest. In contrast, subcutaneous allografts of soluble-Jagged1 cells formed larger fat pads containing lipid-filled adipocytes with improved neovascularization compared with controls. Since adipogenesis is tightly associated with angiogenesis, we evaluated the influence of soluble-Jagged1 on endothelial cells by culturing them in cell-free conditioned media from preadipocytes. Soluble Jagged1-mediated inhibition of Notch signaling increased levels of secreted cytokines, potentially contributing to the improved cell growth and proliferation observed in these cultures. Our findings demonstrate an initial requirement of Notch signaling inactivation for preadipocyte cell commitment and support the hypothesis that cell-to-cell crosstalk between the preadipocytes and endothelial cells is required for neovascularization and remodeling of the tissue to promote hyperplasia and hypertrophy of differentiating adipocytes.

Cancer chemopreventive and anticancer evaluation of extracts and fractions from marine macro- and microorganisms collected from Twilight Zone waters around Guam.

The cancer chemopreventive and cytotoxic properties of 50 extracts derived from Twilight Zone (50-150 m) sponges, gorgonians and associated bacteria, together with 15 extracts from shallow water hard corals, as well as 16 fractions derived from the methanol solubles of the Twilight Zone sponge Suberea sp, were assessed in a series of bioassays. These assays included: Induction of quinone reductase (QR), inhibition of TNF-alpha activated nuclear factor kappa B (NFkappaB), inhibition of aromatase, interaction with retinoid X receptor (RXR), inhibition of nitric oxide (NO) synthase, inhibition 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH), and inhibition of HL-60 and MCF-7 cell proliferation. The results of these assays showed that at least 10 extracts and five fractions inhibited NFkappaB by greater than 60%, two extracts and two fractions inhibited DPPH by more than 50%, nine extracts and two fractions affected the survival of HL-60 cells, no extracts or fractions affected RXR, three extracts and six fractions affected quinone reductase (QR), three extracts and 12 fractions significantly inhibited aromatase, four extracts and five fractions inhibited nitric oxide synthase, and one extract and no fractions inhibited the growth of MCF-7 cells by more than 95%. These data revealed the tested samples to have many and varied activities, making them, as shown with the extract of the Suberea species, useful starting points for further fractionation and purification. Moreover, the large number of samples demonstrating activity in only one or sometimes two assays accentuates the potential of the Twilight Zone, as a largely unexplored habitat, for the discovery of selectively bioactive compounds. The overall high hit rate in many of the employed assays is considered to be a significant finding in terms of "normal" hit rates associated with similar samples from shallower depths.