p120-Catenin suppresses NLRP3 inflammasome activation in macrophages.

Inflammasome activation is of central importance for the process of generation of overwhelming inflammatory response and the pathogenesis of sepsis. The intrinsic molecular mechanism for controlling inflammasome activation is still poorly understood. Here we investigated the role of p120-catenin expression in macrophages in regulating nucleotide-binding oligomerization domain (NOD) and leucine-rich repeat (LRR)- and pyrin domain-containing proteins 3 (NLRP3) inflammasome activation. Depletion of p120-catenin in murine bone marrow-derived macrophages enhanced caspase-1 activation and secretion of active interleukin (IL)-1ß in response to ATP stimulation following LPS priming. Coimmunoprecipitation analysis showed that p120-catenin deletion promoted NLRP3 inflammasome activation by accelerating the assembly of the inflammasome complex comprised of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1. Depletion of p120-catenin also increased the production of mitochondrial reactive oxygen species. Pharmacological inhibition of mitochondrial reactive oxygen species nearly completely abolished NLRP3 inflammasome activation, caspase-1 activation, and the production of IL-1ß in p120-catenin-depleted macrophages. Furthermore, p120-catenin ablation significantly disrupted mitochondrial function, evidenced by decreased mitochondrial membrane potential and lower production of intracellular ATP. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of p120-catenin-deficient macrophages dramatically enhanced the accumulation of IL-1ß and IL-18 in bronchoalveolar lavage fluid. These results demonstrate that p120-catenin prevents NLRP3 inflammasome activation in macrophages by maintaining mitochondrial homeostasis and reducing the production of mitochondrial reactive oxygen species in response to endotoxin insult. Thus, inhibition of NLRP3 inflammasome activation by stabilization of p120-catenin expression in macrophages may be a novel strategy to prevent an uncontrolled inflammatory response in sepsis.

Neutralization of excessive CCL28 improves wound healing in diabetic mice.

Introduction: Chronic, non-healing skin wounds such as diabetic foot ulcers (DFUs) are common in patients with type 2 diabetes mellitus (T2DM) and often result in limb amputation and even death. However, mechanisms by which T2DM and inflammation negatively impact skin wound healing remains poorly understood. Here we investigate a mechanism by which an excessive level of chemokine CCL28, through its receptor CCR10, impairs wound healing in patients and mice with T2DM. Methods & Results: Firstly, a higher level of CCL28 was observed in skin and plasma in both patients with T2DM, and in obesity-induced type 2 diabetic db/db mice. Compared with WT mice, adipose tissue from db/db mice released 50% more CCL28, as well as 2- to 3-fold more IL-1ß, IL-6, and TNF-α, and less VEGF, as determined by ELISA measurements. Secondly, overexpression of CCL28 with adenovirus (Adv-CCL28) caused elevation of proinflammatory cytokines as well as CCR10 expression and also reduced eNOS expression in the dorsal skin of WT mice as compared with control Adv. Thirdly, topical application of neutralizing anti-CCL28 Ab dose-dependently accelerated wound closure and eNOS expression, and decreased IL-6 level, with an optimal dose of 1 µg/wound. In addition, mRNA levels of eNOS and anti-inflammatory cytokine IL-4 were increased as shown by real-time RT-PCR. The interaction between eNOS and CCR10 was significantly reduced in diabetic mouse wounds following application of the optimal dose of anti-CCL28 Ab, and eNOS expression increased. Finally, enhanced VEGF production and increased subdermal vessel density as indicated by CD31 immunostaining were also observed with anti-CCL28 Ab. Discussion: Taken together, topical application of neutralizing anti-CCL28 Ab improved dorsal skin wound healing by reducing CCR10 activation and inflammation in part by preventing eNOS downregulation, increasing VEGF production, and restoring angiogenesis. These results indicate anti-CCL28 Ab has significant potential as a therapeutic strategy for treatment of chronic non-healing diabetic skin wounds such as DFUs.

Hypoxia-induced pulmonary hypertension upregulates eNOS and TGF-ß contributing to sex-linked differences in BMPR2 +/R899X mutant mice.

Dysfunctional bone morphogenetic protein receptor 2 (BMPR2) and endothelial nitric oxide synthase (eNOS) have been largely implicated in the pathogenesis of pulmonary arterial hypertension (PAH); a life-threatening cardiopulmonary disease. Although the incident of PAH is about three times higher in females, males with PAH usually have a worse prognosis, which seems to be dependent on estrogen-associated cardiac and vascular protection. Here, we evaluated whether hypoxia-induced pulmonary hypertension (PH) in humanized BMPR2+/R899X loss-of-function mutant mice contributes to sex-associated differences observed in PAH by altering eNOS expression and inducing expansion of hyperactivated TGF-ß-producing pulmonary myofibroblasts. To test this hypothesis, male and female wild-type (WT) and BMPR2+/R899X mutant mice were kept under hypoxic or normoxic conditions for 4 weeks, and then right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) were measured. Chronic hypoxia exposure elevated RVSP, inducing RVH in both groups, with a greater effect in BMPR2+/R899X female mice. Lung histology revealed no differences in vessel thickness/area between sexes, suggesting RVSP differences in this model are unlikely to be in response to sex-dependent vascular narrowing. On the other hand, hypoxia exposure increased vascular collagen deposition, the number of TGF-ß-associated α-SMA-positive microvessels, and eNOS expression, whereas it also reduced caveolin-1 expression in the lungs of BMPR2+/R899X females compared to males. Taken together, this brief report reveals elevated myofibroblast-derived TGF-ß and eNOS-derived oxidants contribute to pulmonary microvascular muscularization and sex-linked differences in incidence, severity, and outcome of PAH.

A requirement for Krüppel Like Factor-4 in the maintenance of endothelial cell quiescence.

Rationale and Goal: Endothelial cells (ECs) are quiescent and critical for maintaining homeostatic functions of the mature vascular system, while disruption of quiescence is at the heart of endothelial to mesenchymal transition (EndMT) and tumor angiogenesis. Here, we addressed the hypothesis that KLF4 maintains the EC quiescence. Methods and Results: In ECs, KLF4 bound to KLF2, and the KLF4-transctivation domain (TAD) interacted directly with KLF2. KLF4-depletion increased KLF2 expression, accompanied by phosphorylation of SMAD3, increased expression of alpha-smooth muscle actin (αSMA), VCAM-1, TGF-ß1, and ACE2, but decreased VE-cadherin expression. In the absence of Klf4, Klf2 bound to the Klf2-promoter/enhancer region and autoregulated its own expression. Loss of EC-Klf4 in Rosa mT/mG ::Klf4 fl/fl ::Cdh5 CreERT2 engineered mice, increased Klf2 levels and these cells underwent EndMT. Importantly, these mice harboring EndMT was also accompanied by lung inflammation, disruption of lung alveolar architecture, and pulmonary fibrosis. Conclusion: In quiescent ECs, KLF2 and KLF4 partnered to regulate a combinatorial mechanism. The loss of KLF4 disrupted this combinatorial mechanism, thereby upregulating KLF2 as an adaptive response. However, increased KLF2 expression overdrives for the loss of KLF4, giving rise to an EndMT phenotype.

Pharmacological Activation of Potassium Channel Kv11.1 with NS1643 Attenuates Triple Negative Breast Cancer Cell Migration by Promoting the Dephosphorylation of Caveolin-1.

The prevention of metastasis is a central goal of cancer therapy. Caveolin-1 (Cav-1) is a structural membrane and scaffolding protein shown to be a key regulator of late-stage breast cancer metastasis. However, therapeutic strategies targeting Cav-1 are still lacking. Here, we demonstrate that the pharmacological activation of potassium channel Kv11.1, which is uniquely expressed in MDA-MB-231 triple negative breast cancer cells (TNBCs) but not in normal MCF-10A cells, induces the dephosphorylation of Cav-1 Tyr-14 by promoting the Ca2+-dependent stimulation of protein tyrosine phosphatase 1B (PTP1B). Consequently, the dephosphorylation of Cav-1 resulted in its disassociation from ß-catenin, which enabled the accumulation of ß-catenin at cell borders, where it facilitated the formation of cell-cell adhesion complexes via interactions with R-cadherin and desmosomal proteins. Kv11.1 activation-dependent Cav-1 dephosphorylation induced with NS1643 also reduced cell migration and invasion, consistent with its ability to regulate focal adhesion dynamics. Thus, this study sheds light on a novel pharmacological mechanism of promoting Cav-1 dephosphorylation, which may prove to be effective at reducing metastasis and promoting contact inhibition.

Inhibition of CCL28/CCR10-Mediated eNOS Downregulation Improves Skin Wound Healing in the Obesity-Induced Mouse Model of Type 2 Diabetes.

Chronic, nonhealing skin wounds, such as diabetic foot ulcers (DFUs), are common in patients with type 2 diabetes. Here, we investigated the role of chemokine (C-C motif) ligand 28 (CCL28) and its receptor C-C chemokine receptor type 10 (CCR10) in downregulation of endothelial nitric (NO) oxide synthase (eNOS) in association with delayed skin wound healing in the db/db mouse model of type 2 diabetes. We observed reduced eNOS expression and elevated CCL28/CCR10 levels in dorsal skin of db/db mice and subdermal leg biopsy specimens from human subjects with type 2 diabetes. Further interrogation revealed that overexpression of CCR10 reduced eNOS expression, NO bioavailability, and tube formation of human dermal microvascular endothelial cells (HDMVECs) in vitro, which was recapitulated in mouse dorsal skin. In addition, incubation of HDMVECs with CCL28 led to internalization of the CCR10/eNOS complex and colocalization with lysosome-associated membrane protein 1. Finally, topical application of myristoylated CCR10 binding domain 7 amino acid (Myr-CBD7) peptide prevented CCR10-eNOS interaction and subsequent eNOS downregulation, enhanced eNOS/NO levels, eNOS/VEGF-R2+ microvessel density, and blood perfusion, reduced inflammatory cytokine levels, and importantly, decreased wound healing time in db/db mice. Thus, endothelial cell CCR10 activation in genetically obese mice with type 2 diabetes promotes eNOS depletion and endothelial dysfunction, and targeted disruption of CCR10/eNOS interaction improves wound healing.

Caveolin-1 controls mitochondrial damage and ROS production by regulating fission - fusion dynamics and mitophagy.

As essential regulators of mitochondrial quality control, mitochondrial dynamics and mitophagy play key roles in maintenance of metabolic health and cellular homeostasis. Here we show that knockdown of the membrane-inserted scaffolding and structural protein caveolin-1 (Cav-1) and expression of tyrosine 14 phospho-defective Cav-1 mutant (Y14F), as opposed to phospho-mimicking Y14D, altered mitochondrial morphology, and increased mitochondrial matrix mixing, mitochondrial fusion and fission dynamics as well as mitophagy in MDA-MB-231 triple negative breast cancer cells. Further, we found that interaction of Cav-1 with mitochondrial fusion/fission machinery Mitofusin 2 (Mfn2) and Dynamin related protein 1 (Drp1) was enhanced by Y14D mutant indicating Cav-1 Y14 phosphorylation prevented Mfn2 and Drp1 translocation to mitochondria. Moreover, limiting mitochondrial recruitment of Mfn2 diminished formation of the PINK1/Mfn2/Parkin complex required for initiation of mitophagy resulting in accumulation of damaged mitochondria and ROS (mtROS). Thus, these studies indicate that phospho-Cav-1 may be an important switch mechanism in cancer cell survival which could lead to novel strategies for complementing cancer therapies.

Tyrosine phosphorylation of S1PR1 leads to chaperone BiP-mediated import to the endoplasmic reticulum.

Cell surface G protein-coupled receptors (GPCRs), upon agonist binding, undergo serine-threonine phosphorylation, leading to either receptor recycling or degradation. Here, we show a new fate of GPCRs, exemplified by ER retention of sphingosine-1-phosphate receptor 1 (S1PR1). We show that S1P phosphorylates S1PR1 on tyrosine residue Y143, which is associated with recruitment of activated BiP from the ER into the cytosol. BiP then interacts with endocytosed Y143-S1PR1 and delivers it into the ER. In contrast to WT-S1PR1, which is recycled and stabilizes the endothelial barrier, phosphomimicking S1PR1 (Y143D-S1PR1) is retained by BiP in the ER and increases cytosolic Ca2+ and disrupts barrier function. Intriguingly, a proinflammatory, but non-GPCR agonist, TNF-α, also triggered barrier-disruptive signaling by promoting S1PR1 phosphorylation on Y143 and its import into ER via BiP. BiP depletion restored Y143D-S1PR1 expression on the endothelial cell surface and rescued canonical receptor functions. Findings identify Y143-phosphorylated S1PR1 as a potential target for prevention of endothelial barrier breakdown under inflammatory conditions.

TLR4 is required for macrophage efferocytosis during resolution of ventilator-induced lung injury.

Mechanical ventilation is a life-sustaining therapy for patients with respiratory failure but can cause further lung damage known as ventilator-induced lung injury (VILI). However, the intrinsic molecular mechanisms underlying recovery of VILI remain unknown. Phagocytosis of apoptotic cells (also known as efferocytosis) is a key mechanism orchestrating successful resolution of inflammation. Here we show the positive regulation of macrophage Toll-like receptor (TLR) 4 in efferocytosis and resolution of VILI. Mice were depleted of alveolar macrophages and then subjected to injurious ventilation (tidal volume, 20 mL/kg) for 4 h. On day 1 after mechanical ventilation, Tlr4+/+ or Tlr4-/- bone marrow-derived macrophages (BMDMs) were intratracheally administered to alveolar macrophage-depleted mice. We observed that mice depleted of alveolar macrophages exhibited defective resolution of neutrophilic inflammation, exuded protein, lung edema, and lung tissue injury after ventilation, whereas these delayed responses were reversed by administration of Tlr4+/+ BMDMs. Importantly, these proresolving effects by Tlr4+/+ BMDMs were abolished in mice receiving Tlr4-/- BMDMs. The number of macrophages containing apoptotic cells or bodies in bronchoalveolar lavage fluid was much less in mice receiving Tlr4-/- BMDMs than that in those receiving Tlr4+/+ BMDMs. Macrophage TLR4 deletion facilitated a disintegrin and metalloprotease 17 maturation and enhanced Mer cleavage in response to mechanical ventilation. Heat shock protein 70 dramatically increased Mer tyrosine kinase surface expression, phagocytosis of apoptotic neutrophils, and rescued the inflammatory phenotype in alveolar macrophage-depleted mice receiving Tlr4+/+ BMDMs, but not Tlr4-/- BMDMs. Our results suggest that macrophage TLR4 promotes resolution of VILI via modulation of Mer-mediated efferocytosis.

Compensatory expression of NRF2-dependent antioxidant genes is required to overcome the lethal effects of Kv11.1 activation in breast cancer cells and PDOs.

Potassium channels are important regulators of cellular homeostasis and targeting these proteins pharmacologically is unveiling important mechanisms in cancer cell biology. Here we demonstrate that pharmacological stimulation of the Kv11.1 potassium channel activity results in mitochondrial reactive oxygen species (ROS) production and fragmentation in breast cancer cell lines and patient-derived organoids independent of breast cancer subtype. mRNA expression profiling revealed that Kv11.1 activity significantly altered expression of genes controlling the production of ROS and endoplasmic-reticulum (ER) stress. Characterization of the transcriptional signature of breast cancer cells treated with Kv11.1 potassium channel activators strikingly revealed an adaptive response to the potentially lethal augmentation of ROS by increasing Nrf2-dependent transcription of antioxidant genes. Nrf2 in this context was shown to promote survival in breast cancer, whereas knockdown of Nrf2 lead to Kv11.1-induced cell death. In conclusion, we found that the Kv11.1 channel activity promotes oxidative stress in breast cancer cells and that suppression of the Nrf2-mediated anti-oxidant survival mechanism strongly sensitized breast cancer cells to a lethal effect of pharmacological activation of Kv11.1.

Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function.

Caveolin-1 (Cav-1) is a scaffolding protein and a major component of caveolae/lipid rafts. Previous reports have shown that endothelial dysfunction in Cav-1-deficient (Cav-1-/-) mice is mediated by elevated oxidative stress through endothelial nitric oxide synthase (eNOS) uncoupling and increased NADPH oxidase. Oxidant stress is the net balance of oxidant generation and scavenging, and the role of Cav-1 as a regulator of antioxidant enzymes in vascular tissue is poorly understood. Extracellular SOD (SOD3) is a copper (Cu)-containing enzyme that is secreted from vascular smooth muscle cells/fibroblasts and subsequently binds to the endothelial cells surface, where it scavenges extracellular [Formula: see text] and preserves endothelial function. SOD3 activity is dependent on Cu, supplied by the Cu transporter ATP7A, but whether Cav-1 regulates the ATP7A-SOD3 axis and its role in oxidative stress-mediated vascular dysfunction has not been studied. Here we show that the activity of SOD3, but not SOD1, was significantly decreased in Cav-1-/- vessels, which was rescued by re-expression of Cav-1 or Cu supplementation. Loss of Cav-1 reduced ATP7A protein, but not mRNA, and this was mediated by ubiquitination of ATP7A and proteasomal degradation. ATP7A bound to Cav-1 and was colocalized with SOD3 in caveolae/lipid rafts or perinucleus in vascular tissues or cells. Impaired endothelium-dependent vasorelaxation in Cav-1-/- mice was rescued by gene transfer of SOD3 or by ATP7A-overexpressing transgenic mice. These data reveal an unexpected role of Cav-1 in stabilizing ATP7A protein expression by preventing its ubiquitination and proteasomal degradation, thereby increasing SOD3 activity, which in turn protects against vascular oxidative stress-mediated endothelial dysfunction.

PV1 in Caveolae Controls Lung Endothelial Permeability.

Caveolae are prominent plasmalemmal invaginations in endothelial cells, especially in the lung vasculature, which comprises a vast surface area. PV1 (plasmalemmal vesicle-associated protein-1), a 60-kD glycoprotein expressed in endothelial cells, is essential for generating spoke-like diaphragmatic structures that span the neck region of endothelial caveolae. However, their role in caveolae-mediated uptake and endothelial-barrier function is unknown. Here, we generated mice with endothelial cell-specific deletion of PV1 through tamoxifen-induced Cdh5.Cre.ERT2 (endothelial-specific vascular cadherin.Cre.estrogen receptor 2)-mediated excision of the floxed PV1 allele. We observed that loss of PV1 specifically in endothelial cells increased lung vascular permeability of fluid and protein, indicating that PV1 is required for maintenance of lung vascular-barrier integrity. Endothelial-specific PV1 deletion also increased caveolae-mediated uptake of tracer albumin compared with controls, promoted Au-albumin accumulation in the bulb of caveolae, and induced caveolar swelling. In addition, we observed the progressive loss of plasma proteins from the circulation and reduced arterial pressure resulting from transudation of water and protein as well as edema formation in multiple tissues, including lungs. These changes seen after endothelial-specific PV1 deletion occurred in the absence of disruption of endothelial junctions. We demonstrated that exposure of wild-type mice to endotoxin, which is known to cause acute lung injury and increase protein permeability, also significantly reduced PV1 protein expression. We conclude that the key function of PV1 is to regulate lung endothelial permeability through its ability to restrict the entry of plasma proteins such as albumin into caveolae and their transport through the endothelial barrier.

P2X7 receptor activation increases expression of caveolin-1 and formation of macrophage lipid rafts, thereby boosting CD39 activity.

Macrophages are tissue-resident immune cells that are crucial for the initiation and maintenance of immune responses. Purinergic signaling modulates macrophage activity and impacts cellular plasticity. The ATP-activated purinergic receptor P2X7 (also known as P2RX7) has pro-inflammatory properties, which contribute to macrophage activation. P2X7 receptor signaling is, in turn, modulated by ectonucleotidases, such as CD39 (also known as ENTPD1), expressed in caveolae and lipid rafts. Here, we examined P2X7 receptor activity and determined impacts on ectonucleotidase localization and function in macrophages primed with lipopolysaccharide (LPS). First, we verified that ATP boosts CD39 activity and caveolin-1 protein expression in LPS-primed macrophages. Drugs that disrupt cholesterol-enriched domains - such as nystatin and methyl-ß-cyclodextrin - decreased CD39 enzymatic activity in all circumstances. We noted that CD39 colocalized with lipid raft markers (flotillin-2 and caveolin-1) in macrophages that had been primed with LPS followed by treatment with ATP. P2X7 receptor inhibition blocked these ATP-mediated increases in caveolin-1 expression and inhibited the colocalization with CD39. Further, we found that STAT3 activation is significantly attenuated caveolin-1-deficient macrophages treated with LPS or LPS+BzATP. Taken together, our data suggest that P2X7 receptor triggers the initiation of lipid raft-dependent mechanisms that upregulates CD39 activity and could contribute to limit macrophage responses restoring homeostasis.

Sevoflurane Promotes Bactericidal Properties of Macrophages through Enhanced Inducible Nitric Oxide Synthase Expression in Male Mice.

BACKGROUND: Sevoflurane with its antiinflammatory properties has shown to decrease mortality in animal models of sepsis. However, the underlying mechanism of its beneficial effect in this inflammatory scenario remains poorly understood. Macrophages play an important role in the early stage of sepsis as they are tasked with eliminating invading microbes and also attracting other immune cells by the release of proinflammatory cytokines such as interleukin-1ß, interleukin-6, and tumor necrosis factor-α. Thus, the authors hypothesized that sevoflurane mitigates the proinflammatory response of macrophages, while maintaining their bactericidal properties. METHODS: Murine bone marrow-derived macrophages were stimulated in vitro with lipopolysaccharide in the presence and absence of 2% sevoflurane. Expression of cytokines and inducible NO synthase as well as uptake of fluorescently labeled Escherichia coli (E. coli) were measured. The in vivo endotoxemia model consisted of an intraperitoneal lipopolysaccharide injection after anesthesia with either ketamine and xylazine or 4% sevoflurane. Male mice (n = 6 per group) were observed for a total of 20 h. During the last 30 min fluorescently labeled E. coli were intraperitoneally injected. Peritoneal cells were extracted by peritoneal lavage and inducible NO synthase expression as well as E. coli uptake by peritoneal macrophages was determined using flow cytometry. RESULTS: In vitro, sevoflurane enhanced lipopolysaccharide-induced inducible NO synthase expression after 8 h by 466% and increased macrophage uptake of fluorescently labeled E. coli by 70% compared with vehicle-treated controls. Inhibiting inducible NO synthase expression pharmacologically abolished this increase in bacteria uptake. In vivo, inducible NO synthase expression was increased by 669% and phagocytosis of E. coli by 49% compared with the control group. CONCLUSIONS: Sevoflurane enhances phagocytosis of bacteria by lipopolysaccharide-challenged macrophages in vitro and in vivo via an inducible NO synthase-dependent mechanism. Thus, sevoflurane potentiates bactericidal and antiinflammatory host-defense mechanisms in endotoxemia.

Depletion of Caveolin-1 in Type 2 Diabetes Model Induces Alzheimer's Disease Pathology Precursors.

Type 2 diabetes mellitus (T2DM) is a risk factor for the development of late-onset Alzheimer's disease (AD). However, the mechanism underlying the development of late-onset AD is largely unknown. Here we show that levels of the endothelial-enriched protein caveolin-1 (Cav-1) are reduced in the brains of T2DM patients compared with healthy aging, and inversely correlated with levels of ß-amyloid (Aß). Depletion of Cav-1 is recapitulated in the brains of db/db (Leprdb ) diabetic mice and corresponds with recognition memory deficits as well as the upregulation of amyloid precursor protein (APP), BACE-1, a trending increase in ß-amyloid Aß42/40 ratio and hyperphosphorylated tau (p-tau) species. Importantly, we show that restoration of Cav-1 levels in the brains of male db/db mice using adenovirus overexpressing Cav-1 (AAV-Cav-1) rescues learning and memory deficits and reduces pathology (i.e., APP, BACE-1 and p-tau levels). Knocking down Cav-1 using shRNA in HEK cells expressing the familial AD-linked APPswe mutant variant upregulates APP, APP carboxyl terminal fragments, and Aß levels. In turn, rescue of Cav-1 levels restores APP metabolism. Together, these results suggest that Cav-1 regulates APP metabolism, and that depletion of Cav-1 in T2DM promotes the amyloidogenic processing of APP and hyperphosphorylation of tau. This may suggest that depletion of Cav-1 in T2DM underlies, at least in part, the development of AD and imply that restoration of Cav-1 may be a therapeutic target for diabetic-associated sporadic AD.SIGNIFICANCE STATEMENT More than 95% of the Alzheimer's patients have the sporadic late-onset form (LOAD). The cause for late-onset Alzheimer's disease is unknown. Patients with Type 2 diabetes mellitus have considerably higher incidence of cognitive decline and AD compared with the general population, suggesting a common mechanism. Here we show that the expression of caveolin-1 (Cav-1) is reduced in the brain in Type 2 diabetes mellitus. In turn, reduced Cav-1 levels induce AD-associated neuropathology and learning and memory deficits. Restoration of Cav-1 levels rescues these deficits. This study unravels signals underlying LOAD and suggests that restoration of Cav-1 may be an effective therapeutic target.

The GTPase Rab1 Is Required for NLRP3 Inflammasome Activation and Inflammatory Lung Injury.

Uncontrolled inflammatory response during sepsis predominantly contributes to the development of multiorgan failure and lethality. However, the cellular and molecular mechanisms for excessive production and release of proinflammatory cytokines are not clearly defined. In this study, we show the crucial role of the GTPase Ras-related protein in brain (Rab)1a in regulating the nucleotide binding domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation and lung inflammatory injury. Expression of dominant negative Rab1 N124I plasmid in bone marrow-derived macrophages prevented the release of IL-1ß and IL-18, NLRP3 inflammasome activation, production of pro-IL-1ß and pro-IL-18, and attenuated TLR4 surface expression and NF-кB activation induced by bacterial LPS and ATP compared with control cells. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of Rab1a-inactivated macrophages by expression of Rab1 N124I plasmid dramatically reduced the release of IL-1ß and IL-18, neutrophil count in bronchoalveolar lavage fluid, and inflammatory lung injury. Rab1a activity was elevated in alveolar macrophages from septic patients and positively associated with severity of sepsis and respiratory dysfunction. Thus, inhibition of Rab1a activity in macrophages resulting in the suppression of NLRP3 inflammasome activation may be a promising target for the treatment of patients with sepsis.

Caveolin and Endothelial NO Signaling.

Pulmonary vascular diseases are associated with several factors including infection, cigarette smoking, abuse of dietary suppressants and drugs, prolonged exposure to high altitude, and other causes which in part induce significant oxidative stress resulting in endothelial cell injury, apoptosis, hyperproliferation, and vaso-occlusive disease. Maintenance of normal endothelial cell function is a critical role of endothelial nitric oxide synthase (eNOS) activity and physiologic nitric oxide (NO) signaling in the vascular wall. eNOS expression and activity is regulated by the membrane-associated scaffolding protein caveolin-1 (Cav-1), the main protein constituent of caveolae. This chapter summarizes the literature and highlights unanswered questions related to how inflammation-associated oxidative stress affects Cav-1 expression and regulatory functions, and how dysregulated eNOS enzymatic activity promotes endothelial dysfunction. Focus is given to how the conversion of eNOS from a NO-producing enzyme to a transient oxidant-generating system is associated twith Cav-1 depletion, endothelial cell injury, and pulmonary vascular diseases. Importantly, the vascular defects observed in absence of Cav-1 that give rise to injured or hyperproliferative endothelial cells and promote remodeled vasculature can be rescued by "re-coupling," inhibiting, or genetically deleting eNOS, supporting the notion that strict control of Cav-1 expression and eNOS activity and signaling is critical for maintaining pulmonary vascular homeostasis.

Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

Reciprocal regulation of eNOS and caveolin-1 functions in endothelial cells.

We hypothesized that the maintenance of vascular homeostasis is critically dependent on the expression and reciprocal regulation of caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs). Skeletal muscle biopsies from subjects with type 2 diabetes showed 50% less Cav-1 and eNOS than those from lean healthy controls. The Cav-1:eNOS expression ratio was 200:1 in primary culture human ECs. Cav-1 small interfering RNA (siRNA) reduced eNOS protein and gene expression in association with a twofold increase in eNOS phosphorylation and nitrate production per molecule of eNOS, which was reversed in cells overexpressing Adv-Cav-1-GFP. Upon addition of the Ca2+ ionophore A23187 to activate eNOS, we observed eNOS Ser1177 phosphorylation, its translocation to ß-catenin-positive cell-cell junctions, and increased colocalization of eNOS and Cav-1 within 5 min. We also observed Cav-1 S-nitrosylation and destabilization of Cav-1 oligomers in cells treated with A23187 as well as insulin or albumin, and this could be blocked by L-NAME, PP2, or eNOS siRNA. Finally, caveola-mediated endocytosis of albumin or insulin was reduced by Cav-1 or eNOS siRNA, and the effect of Cav-1 siRNA was rescued by Adv-Cav-1-GFP. Thus, Cav-1 stabilizes eNOS expression and regulates its activity, whereas eNOS-derived NO promotes caveola-mediated endocytosis.

CD39 limits P2X7 receptor inflammatory signaling and attenuates sepsis-induced liver injury.

BACKGROUND & AIMS: The severity of sepsis can be linked to excessive inflammatory responses resulting in hepatic injury. P2X7 receptor activation by extracellular ATP (eATP) exacerbates inflammation by augmenting cytokine production; while CD39 (ENTPD1) scavenges eATP to generate adenosine, thereby limiting P2X7 activation and resulting in A2A receptor stimulation. We aim to determine how the functional interaction of P2X7 receptor and CD39 control the macrophage response, and consequently impact on sepsis and liver injury. METHODS: Sepsis was induced by cecal ligation and puncture in C57BL/6 wild-type (WT) and CD39-/- mice. Several in vitro assays were performed using peritoneal or bone marrow derived macrophages to determine CD39 ectonucleotidase activity and its role in sepsis-induced liver injury. RESULTS: CD39 expression in macrophages limits ATP-P2X7 receptor pro-inflammatory signaling. P2X7 receptor paradoxically boosts CD39 activity. Inhibition and/or deletion of P2X7 receptor in LPS-primed macrophages attenuates cytokine production and inflammatory signaling as well as preventing ATP-induced increases in CD39 activity. Septic CD39-/- mice exhibit higher levels of inflammatory cytokines and show more pronounced liver injury than WT mice. Pharmacological P2X7 blockade largely prevents tissue damage, cell apoptosis, cytokine production, and the activation of inflammatory signaling pathways in the liver from septic WT, while only attenuating these outcomes in CD39-/- mice. Furthermore, the combination of P2X7 blockade with adenosine A2A receptor stimulation completely inhibits cytokine production, the activation of inflammatory signaling pathways, and protects septic CD39-/- mice against liver injury. CONCLUSIONS: CD39 attenuates sepsis-associated liver injury by scavenging eATP and ultimately generating adenosine. We propose boosting of CD39 would suppress P2X7 responses and trigger adenosinergic signaling to limit systemic inflammation and restore liver homeostasis during the acute phase of sepsis. Lay summary: CD39 expression in macrophages limits P2X7-mediated pro-inflammatory responses, scavenging extracellular ATP and ultimately generating adenosine. CD39 genetic deletion exacerbates sepsis-induced experimental liver injury. Combinations of a P2X7 antagonist and adenosine A2A receptor agonist are hepatoprotective during the acute phase of abdominal sepsis.