Matrix-specific protein kinase A signaling regulates p21-activated kinase activation by flow in endothelial cells.

RATIONALE: Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and nuclear factor (NF)-kappaB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-kappaB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-kappaB. OBJECTIVE: To elucidate the mechanisms regulating matrix-specific PAK activation. METHODS AND RESULTS: We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead, basement membrane proteins enhance flow-induced protein kinase (PK)A activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-kappaB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the prostacyclin analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKA inhibitor (PKI) injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI. CONCLUSIONS: Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK.

Considerations for Conducting Bring Your Own "Device" (BYOD) Clinical Studies.

Background: Digital health technologies are attracting attention as novel tools for data collection in clinical research. They present alternative methods compared to in-clinic data collection, which often yields snapshots of the participants' physiology, behavior, and function that may be prone to biases and artifacts, e.g., white coat hypertension, and not representative of the data in free-living conditions. Modern digital health technologies equipped with multi-modal sensors combine different data streams to derive comprehensive endpoints that are important to study participants and are clinically meaningful. Used for data collection in clinical trials, they can be deployed as provisioned products where technology is given at study start or in a bring your own "device" (BYOD) manner where participants use their technologies to generate study data. Summary: The BYOD option has the potential to be more user-friendly, allowing participants to use technologies that they are familiar with, ensuring better participant compliance, and potentially reducing the bias that comes with introducing new technologies. However, this approach presents different technical, operational, regulatory, and ethical challenges to study teams. For example, BYOD data can be more heterogeneous, and recruiting historically underrepresented populations with limited access to technology and the internet can be challenging. Despite the rapid increase in digital health technologies for clinical and healthcare research, BYOD use in clinical trials is limited, and regulatory guidance is still evolving. Key Messages: We offer considerations for academic researchers, drug developers, and patient advocacy organizations on the design and deployment of BYOD models in clinical research. These considerations address: (1) early identification and engagement with internal and external stakeholders; (2) study design including informed consent and recruitment strategies; (3) outcome, endpoint, and technology selection; (4) data management including compliance and data monitoring; (5) statistical considerations to meet regulatory requirements. We believe that this article acts as a primer, providing insights into study design and operational requirements to ensure the successful implementation of BYOD clinical studies.

The subendothelial extracellular matrix modulates JNK activation by flow.

Atherosclerosis begins as local inflammation of artery walls at sites of disturbed flow. JNK (c-Jun NH(2)-terminal kinase) is thought to be among the major regulators of flow-dependent inflammatory gene expression in endothelial cells in atherosclerosis. We now show that JNK activation by both onset of laminar flow and long-term oscillatory flow is matrix-specific, with enhanced activation on fibronectin compared to basement membrane protein or collagen. Flow-induced JNK activation on fibronectin requires new integrin ligation and requires both the mitogen-activated protein kinase kinase MKK4 and p21-activated kinase. In vivo, JNK activation at sites of early atherogenesis correlates with the deposition of fibronectin. Inhibiting p21-activated kinase reduces JNK activation in atheroprone regions of the vasculature in vivo. These results identify JNK as a matrix-specific, flow-activated inflammatory event. Together with other studies, these data elucidate a network of matrix-specific pathways that determine inflammatory events in response to fluid shear stress.

Focal adhesion kinase modulates activation of NF-kappaB by flow in endothelial cells.

Atherogenesis involves activation of NF-kappaB in endothelial cells by fluid shear stress. Because this pathway involves integrins, we investigated the involvement of focal adhesion kinase (FAK). We found that FAK was not required for flow-stimulated translocation of the p65 NF-kappaB subunit to the nucleus but was essential for phosphorylation of p65 on serine 536 and induction of ICAM-1, an NF-kappaB-dependent gene. NF-kappaB activation by TNF-alpha or hydrogen peroxide was FAK independent. Events upstream of NF-kappaB, including integrin activation, Rac activation, reactive oxygen production, and degradation of IkappaB, were FAK independent. FAK therefore regulates NF-kappaB phosphorylation and transcriptional activity in response to flow by a novel mechanism.

Expression of striatal adenosine and dopamine receptors in mice deficient in the p50 subunit of NF-kappaB.

The striatal dopamine D2 receptor (D2R) and adenosine A2A receptor (A2AAR) exhibit mutually antagonistic effects through physical interactions and by differential modulation of post-receptor signaling pathways. The expression of the A2AAR and the D2R is differentially regulated by nuclear factor-kappaB (NF-kappaB). In this report, we determined the role of NF-kappaB in regulation of these receptors by comparing mice deficient in the NF-kappaB p50 subunit (p50 KO) with genetically intact B6129PF2/J (F2) mice. Quantification of adenosine receptor (AR) subtypes in mouse striatum by real time PCR, immunocytochemistry and radioligand binding assays showed more A2AAR but less A1AR in p50 KO mice as compared with F2 mice. Striata from p50 KO mice also had less D2R mRNA and [(3)H]-methylspiperone binding than did striata from F2 mice. G(alphaolf) and G(alphas) proteins, which are transducers of A2AAR signals, were also present at a higher level in striata from the p50 KO versus F2 mice. In contrast, the G(alphai1) protein, which transduces signals from the A1AR and D2R, was significantly reduced in striata from p50 KO mice. Behaviorally, p50 KO mice exhibited increased locomotor activity relative to that of F2 mice after caffeine ingestion. These data are consistent with a role for the NF-kappaB in the regulation of A1AR, A2AAR, D2R and possibly their coupling G proteins in the striatum. Dysregulation of these receptors in the striata of p50 KO mice might sensitize these animals to locomotor stimulatory action of caffeine.

Sleep mechanisms in health and disease.

Excess sleepiness, abnormal sleep patterns, non-restorative sleep, and fatigue are becoming increasingly pervasive in modern society. Identifying substances and mechanisms that modulate sleep and vigilance during health and disease is a critical prelude to eventual development of interventions to prevent or alleviate these disabling problems. A unified interdisciplinary approach that includes neurophysiology, neuroanatomy, neurochemistry, and molecular biology will promote elucidation of the complex biology of sleep. Integration of basic sleep physiology with modern genetic techniques will eventually lead to identification of specific genes and substances involved in regulation of various facets of sleep. The review presented here highlights recent progress in defining the anatomy and physiology of sleep-wake regulatory systems, delineating the role of homeostatic and circadian process in regulating sleep and wakefulness, and establishing the relationship of sleep and sleep disorders to other medical conditions. Particular emphasis is placed on reviewing the interactions between sleep, infectious challenge, and host defense response, and on identifying mechanisms that contribute to variation in sleep patterns among various strains of inbred mice.

Nuclear Factor κB and Adenosine Receptors: Biochemical and Behavioral Profiling.

Adenosine is produced primarily by the metabolism of ATP and mediates its physiological actions by interacting primarily with adenosine receptors (ARs) on the plasma membranes of different cell types in the body. Activation of these G protein-coupled receptors promotes activation of diverse cellular signaling pathways that define their tissue-specific functions. One of the major actions of adenosine is cytoprotection, mediated primarily via two ARs - A(1) (A(1)AR) and A(3) (A(3)AR). These ARs protect cells exposed to oxidative stress and are also regulated by oxidative stress. Stress-mediated regulation of ARs involves two prominent transcription factors - activator protein-1 (AP-1) and nuclear factor (NF)-κB - that mediate the induction of genes important in cell survival. Mice that are genetically deficient in the p50 subunit of NF-κB (i.e., p50 knock-out mice) exhibit altered expression of A(1)AR and A(2A)AR and demonstrate distinct behavioral phenotypes under normal conditions or after drug challenges. These effects suggest an important role for NF-κB in dictating the level of expression of ARs in vivo, in regulating the cellular responses to stress, and in modifying behavior.

Activation of the adenosine A1 receptor inhibits HIV-1 tat-induced apoptosis by reducing nuclear factor-kappaB activation and inducible nitric-oxide synthase.

Human immunodeficiency virus dementia (HIV-D) is a nonfocal central nervous system manifestation characterized by cognitive, behavioral, and motor abnormalities. The pathophysiology of neuronal damage in HIV-D includes a direct toxic effect of viral proteins on neuronal cells and an indirect effect caused by the release of inflammatory mediators and neurotoxins by activated macrophages/microglia and astrocytes, culminating into neuronal apoptosis. Previous studies have documented that the nucleoside adenosine mediates neuroprotection by activating adenosine A(1) receptor subtype (A(1)AR) linked to suppression of neuronal excitability. In this study, we show that A(1)AR activation protects against HIV-1 Tat-induced toxicity in primary cultures of rat cerebellar granule neurons and in rat pheochromocytoma (PC12) cell. In PC12 cells, HIV-1 Tat increased [Ca(2+)](i) levels, release of nitric oxide (NO), and expression of inducible nitric-oxide synthase (iNOS) and A(1)AR. Activation of A(1)AR suppressed Tat-mediated increases in [Ca(2+)](i) and NO. Furthermore, A(1)AR agonists inhibited iNOS expression in a nuclear factor-kappaB (NF-kappaB)-dependent manner. It is noteworthy that activation of the A(1)AR or inhibition of NOS protected against Tat-induced apoptosis in PC12 cells and cerebellar granule cells. Moreover, activation of the A(1)AR-inhibited Tat-induced increases in the levels of proapoptotic proteins Bax and caspase-3. Taken together, our results demonstrate that the A(1)AR protects against HIV-1 toxicity by inhibiting NF-kappaB, thereby reducing the expression of iNOS and NO radicals and neuronal apoptosis.

Nitric oxide serves as an endogenous regulator of neuronal adenosine A1 receptor expression.

Nitric oxide (NO) radicals are produced during normal cellular function, after tissue injury, and in response to immune system activation during infection. The transformation of NO to peroxynitrite is essential for mediating some of its physiological and/or cytotoxic actions. As the expression of the adenosine A1 receptor (A1AR) is regulated by oxidative stress, we evaluated the role of NO in the regulation of A1AR expression, a G protein-coupled receptor involved in cytoprotection in the central nervous system. Administration of the NO donor, S-nitrosylpenicillamine (SNAP), to pheochromocytoma 12 (PC12) cells increased A1AR protein in a time- and dose-dependent manner, with maximal induction observed with 20 microm SNAP at 24 h. The response to SNAP was attenuated by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (C-PTIO), and by the inhibition of nuclear factor-kappaB (NF-kappaB), implicating this transcription factor in the regulatory process. In addition SNAP also increased the degradation of Inhibitory kappaB-alpha (IkappaB-alpha), a marker of NF-kappaB activation. Furthermore, the induction of inducible nitric oxide synthase (iNOS) by lipopolysaccharide increased A1AR in PC12 cells and in mice, whereas the inhibition of NOS activity suppressed this response. We conclude that NO, via the activation of NF-kappaB, serves as an endogenous regulator of A1AR, and speculate that the induction of the A1AR could counteract the cytotoxicity of NO.