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 IC50 value of 7.4 μM, a level three times greater than the positive control, L-NG-monomethyl arginine citrate, followed by 6 (sigmosceptrellin A, IC50 = 9.9 μM), whereas other compounds exhibited only modest activity (Table 1). These compounds did not show appreciable cytotoxicity at their IC50 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-3β 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

Mechanisms of DLL4/Notch and BMP9 Mediated Endothelial Cell Quiescence

Endothelial cell quiescence and homeostasis is required for maintaining vascular function, but various pathological states and therapeutic interventions disrupt this homeostasis and exacerbate vascular dysfunction and disease. Notch and BMP signaling are critical regulators of endothelial cells, and the ligands DLL4 and BMP9 are key ligands that cooperate in activating these pathways and inducing endothelial quiescence. The mechanisms of DLL4 and BMP9 cooperation are yet to be fully elucidated. The goal of this project was to uncover some of the mechanisms by which DLL4 and BMP9 signaling overlap for cooperativity, and the downstream effectors that mediate their homeostatic phenotype. Biochemical and molecular mechanisms were utilized for in vitro characterization of the effects of DLL4 and BMP9 stimulation of human endothelial cells, including changes in proliferation, cell cycle regulators, morphology, migration, feedback mechanisms, and cross-regulatory controls of the two pathways. P27KIP1 was identified as a major mediator of endothelial cell quiescence, and a suppression of the expression of multiple growth factor receptors was observed. A proteomic approach was taken to identify systematic changes in endothelial cells following DLL4 and BMP9 stimulation. We found changes in extracellular matrix proteins, among which thrombospondin-1 was identified as another major contributor to quiescence. To study these pathways in vivo, we characterized the blood vessels of a Dll4 heterozygous knockout mouse model (Dll4+/-). In the viable Dll4+/- mice we found splenomegaly, irregular patterning of the lung vasculature, and an upregulation of phospho-SMAD1/5/8 in endothelial cells, which was postulated to compensate for the decreased signaling from Dll4. Dll4+/- mouse aortae had increased endothelial cell density, enlarged lumens and thickened vessel walls. Together, our in vivo and in vitro results indicate that DLL4 and BMP9 mediate cooperative and interdependent signaling mechanisms which regulate multiple downstream effectors of quiescence. Future studies or therapies targeting DLL4 or BMP9 in endothelial cells or in the vasculature should take both pathways into consideration for increased efficacy and to prevent systemic loss of vascular homeostasis

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

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 protein–protein 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

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

Characterizing living ocular bacterial communities and the effects of antibiotic perturbation in house finches

Abstract DNA‐based methods to measure the abundance and relative abundance of bacterial taxa can be skewed by the presence of dead or transient bacteria. Consequently, the active, functional members of the community may be a small subset of the detected bacterial community. This mismatch can make inferences about the roles of communities in host health difficult and can be particularly problematic for low‐abundance microbiomes, such as those on conjunctival surfaces. In this study, we manipulated bacterial communities on bird conjunctiva with a bacteriostatic antibiotic, reducing bacterial activity while preserving viability, to identify the living and active conjunctival communities using comparisons of 16S ribosomal DNA and RNA in paired samples. DNA amplicons included many more sequence variants than RNA amplicons from the same communities, with consequent differences in diversity. While we found that changes in communities in DNA samples broadly represent shifts in the living (RNA‐amplicon) communities, assessments of community function may be better described by RNA samples, reducing background noise from dead cells. We further used these data to test RNA:DNA ratios, used in other microbiological contexts, to detect shifts in bacterial activity after antibiotic disruption but were unable to detect changes in bacterial activity with this method

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 β-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 β-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

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\u27s 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

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\u27s 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