Infection promotes Ser-214 phosphorylation important for generation of cytotoxic tau variants.

Patients who recover from hospital-acquired pneumonia exhibit a high incidence of end-organ dysfunction following hospital discharge, including cognitive deficits. We have previously demonstrated that pneumonia induces the production and release of cytotoxic oligomeric tau from pulmonary endothelial cells, and these tau oligomers can enter the circulation and may be a cause of long-term morbidities. Endothelial-derived oligomeric tau is hyperphosphorylated during infection. The purpose of these studies was to determine whether Ser-214 phosphorylation of tau is a necessary stimulus for generation of cytotoxic tau variants. The results of these studies demonstrate that Ser-214 phosphorylation is critical for the cytotoxic properties of infection-induced oligomeric tau. In the lung, Ser-214 phosphorylated tau contributes to disruption of the alveolar-capillary barrier, resulting in increased permeability. However, in the brain, both the Ser-214 phosphorylated tau and the mutant Ser-214-Ala tau, which cannot be phosphorylated, disrupted hippocampal long-term potentiation suggesting that inhibition of long-term potentiation was relatively insensitive to the phosphorylation status of Ser-214. Nonetheless, phosphorylation of tau is essential to its cytotoxicity since global dephosphorylation of the infection-induced cytotoxic tau variants rescued long-term potentiation. Collectively, these data demonstrate that multiple forms of oligomeric tau are generated during infectious pneumonia, with different forms of oligomeric tau being responsible for dysfunction of distinct end-organs during pneumonia.

Cytotoxic tau released from lung microvascular endothelial cells upon infection with Pseudomonas aeruginosa promotes neuronal tauopathy.

Patients who recover from nosocomial pneumonia oftentimes exhibit long-lasting cognitive impairment comparable with what is observed in Alzheimer's disease patients. We previously hypothesized that the lung endothelium contributes to infection-related neurocognitive dysfunction, because bacteria-exposed endothelial cells release a form(s) of cytotoxic tau that is sufficient to impair long-term potentiation in the hippocampus. However, the full-length lung and endothelial tau isoform(s) have yet to be resolved and it remains unclear whether the infection-induced endothelial cytotoxic tau triggers neuronal tau aggregation. Here, we demonstrate that lung endothelial cells express a big tau isoform and three additional tau isoforms that are similar to neuronal tau, each containing four microtubule-binding repeat domains, and that tau is expressed in lung capillaries in vivo. To test whether infection elicits endothelial tau capable of causing transmissible tau aggregation, the cells were infected with Pseudomonas aeruginosa. The infection-induced tau released from endothelium into the medium-induced neuronal tau aggregation in reporter cells, including reporter cells that express either the four microtubule-binding repeat domains or the full-length tau. Infection-induced release of pathological tau variant(s) from endothelium, and the ability of the endothelial-derived tau to cause neuronal tau aggregation, was abolished in tau knockout cells. After bacterial lung infection, brain homogenates from WT mice, but not from tau knockout mice, initiated tau aggregation. Thus, we conclude that bacterial pneumonia initiates the release of lung endothelial-derived cytotoxic tau, which is capable of propagating a neuronal tauopathy.

FAK Activation Promotes SMC Dedifferentiation via Increased DNA Methylation in Contractile Genes.

RATIONALE: Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. OBJECTIVE: Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. METHODS AND RESULTS: Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA-sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared with vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 (TNF [tumor necrosis factor] receptor-associated factor 6) drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. CONCLUSIONS: This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.

Pneumonia initiates a tauopathy.

Pneumonia causes short- and long-term cognitive dysfunction in a high proportion of patients, although the mechanism(s) responsible for this effect are unknown. Here, we tested the hypothesis that pneumonia-elicited cytotoxic amyloid and tau variants: (1) are present in the circulation during infection; (2) lead to impairment of long-term potentiation; and, (3) inhibit long-term potentiation dependent upon tau. Cytotoxic amyloid and tau species were recovered from the blood and the hippocampus following pneumonia, and they were present in the extracorporeal membrane oxygenation oxygenators of patients with pneumonia, especially in those who died. Introduction of immunopurified blood-borne amyloid and tau into either the airways or the blood of uninfected animals acutely and chronically impaired hippocampal information processing. In contrast, the infection did not impair long-term potentiation in tau knockout mice and the amyloid- and tau-dependent disruption in hippocampal signaling was less severe in tau knockout mice. Moreover, the infection did not elicit cytotoxic amyloid and tau variants in tau knockout mice. Therefore, pneumonia initiates a tauopathy that contributes to cognitive dysfunction.

Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-Mediated Cyclin D1 Transcription.

RATIONALE: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase). OBJECTIVE: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. METHODS AND RESULTS: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (Myh11-Cre-ERT2) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4- (GATA-binding protein 4) cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter, and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitant increase in GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control mice, but not in VS-4718-treated and SMC-specific FAK kinase-dead mice. Finally, lentiviral shGATA4 knockdown in the wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared with control mice. CONCLUSIONS: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.

Ablation of Endothelial TRPV4 Channels Alters the Dynamic Ca2+ Signaling Profile in Mouse Carotid Arteries.

Transient receptor potential vanilloid 4 channels (TRPV4) are pivotal regulators of vascular homeostasis. Altered TRPV4 signaling has recently been implicated in various cardiovascular diseases, including hypertension and atherosclerosis. These versatile nonselective cation channels increase endothelial Ca2+ influx in response to various stimuli including shear stress and G protein-coupled receptor (GPCR) activation. Recent findings suggest TRPV4 channels produce localized Ca2+ transients at the endothelial cell plasma membrane that may allow targeted effector recruitment and promote large-scale Ca2+ events via release from internal stores (endoplasmic reticulum). However, the specific impact of TRPV4 channels on Ca2+ signaling in the intact arterial intima remains unknown. In the current study, we employ an endothelium-specific TRPV4 knockout mouse model (ecTRPV4-/-) to identify and characterize TRPV4-dependent endothelial Ca2+ dynamics. We find that carotid arteries from both ecTRPV4-/- and WT mice exhibit a range of basal and acetylcholine (ACh)-induced Ca2+ dynamics, similar in net frequency. Analysis of discrete Ca2+ event parameters (amplitude, duration, and spread) and event composite values reveals that while ecTRPV4-/- artery endothelium predominantly produces large Ca2+ events comparable to and in excess of those produced by WT endothelium, they are deficient in a particular population of small events, under both basal and ACh-stimulated conditions. These findings support the concept that TRPV4 channels are responsible for generating a distinct population of focal Ca2+ transients in the intact arterial endothelium, likely underlying their essential role in vascular homeostasis.

Extracellular vesicles: another compartment for the second messenger, cyclic adenosine monophosphate.

The second messenger, cAMP, is highly compartmentalized to facilitate signaling specificity. Extracellular vesicles (EVs) are submicron, intact vesicles released from many cell types that can act as biomarkers or be involved in cell-to-cell communication. Although it is well recognized that EVs encapsulate functional proteins and RNAs/miRNAs, currently it is unclear whether cyclic nucleotides are encapsulated within EVs to provide an additional second messenger compartment. Using ultracentrifugation, EVs were isolated from the culture medium of unstimulated systemic and pulmonary endothelial cells. EVs were also isolated from pulmonary microvascular endothelial cells (PMVECs) following stimulation of transmembrane adenylyl cyclase (AC) in the presence or absence of the phosphodiesterase 4 inhibitor rolipram over time. Whereas cAMP was detected in EVs isolated from endothelial cells derived from different vascular beds, it was highest in EVs isolated from PMVECs. Treatment of PMVECs with agents that increase near-membrane cAMP led to an increase in cAMP within corresponding EVs, yet there was no increase in EV number. Elevated cell cAMP, measured by whole cell measurements, peaked 15 min after treatment, yet in EVs the peak increase in cAMP was delayed until 60 min after cell stimulation. Cyclic AMP was also increased in EVs collected from the perfusate of isolated rat lungs stimulated with isoproterenol and rolipram, thus corroborating cell culture findings. When added to unperturbed confluent PMVECs, EVs containing elevated cAMP were not barrier disruptive like cytosolic cAMP but maintained monolayer resistance. In conclusion, PMVECs release EVs containing cAMP, providing an additional compartment to cAMP signaling.

Changes in vascular reactivity and endothelial Ca2+ dynamics with chronic low flow.

Disruption of blood flow promotes endothelial dysfunction and predisposes vessels to remodeling and atherosclerosis. Recent findings suggest that spatial and temporal tuning of local Ca2+ signals along the endothelium is vital to vascular function. In this study, we examined whether chronic flow disruption causes alteration of dynamic endothelial Ca2+ signal patterning associated with changes in vascular structure and function. For these studies, we performed surgical PL of the left carotid arteries of mice to establish chronic low flow for 2 weeks; right carotid arteries remained open and served as controls (C). Histological sections showed substantial remodeling of PL compared to C arteries, including formation of neointima. Isometric force measurements revealed increased PE-induced contractions and decreased KCl-induced contractions in PL vs C arteries. Endothelium-dependent vasorelaxation in response to ACh; 10-8 to 10-5  mol/L) was significantly impaired in PL vs C vessels. Evaluation of endothelial Ca2+ using confocal imaging and custom analysis exposed distinct impairment of Ca2+ dynamics in PL arteries, characterized by reduction in active sites and truncation of events, corresponding to attenuated vasorelaxation. Our findings suggest that endothelial dysfunction in developing vascular disease may be characterized by distinct shifts in the spatial and temporal patterns of localized Ca2+ signals.

Pulmonary endothelial cells from different vascular segments exhibit unique recovery from acidification and Na+/H+ exchanger isoform expression.

Sodium-hydrogen exchangers (NHEs) tightly regulate intracellular pH (pHi), proliferation, migration and cell volume. Heterogeneity exists between pulmonary endothelial cells derived from different vascular segments, yet the activity and isoform expression of NHEs between these vascular segments has not been fully examined. Utilizing the ammonium-prepulse and recovery from acidification technique in a buffer lacking bicarbonate, pulmonary microvascular and pulmonary artery endothelial cells exhibited unique recovery rates from the acid load dependent upon the concentration of the sodium transport inhibitor, amiloride; further, pulmonary artery endothelial cells required a higher dose of amiloride to inhibit sodium-dependent acid recovery compared to pulmonary microvascular endothelial cells, suggesting a unique complement of NHEs between the different endothelial cell types. While NHE1 has been described in pulmonary endothelial cells, all NHE isoforms have not been accounted for. To address NHE expression in endothelial cells, qPCR was performed. Using a two-gene normalization approach, Sdha and Ywhag were identified for qPCR normalization and analysis of NHE isoforms between pulmonary microvascular and pulmonary artery endothelial cells. NHE1 and NHE8 mRNA were equally expressed between the two cell types, but NHE5 expression was significantly higher in pulmonary microvascular versus pulmonary artery endothelial cells, which was confirmed at the protein level. Thus, pulmonary microvascular and pulmonary artery endothelial cells exhibit unique NHE isoform expression and have a unique response to acid load revealed through recovery from cellular acidification.

Restricted Intimal Ca2+ Signaling Associated With Cardiovascular Disease.

Endothelial dysfunction is a key feature of cardiovascular disease (CVD) including atherosclerosis. Impaired endothelial signaling leads to plaque formation, vascular wall remodeling and widespread cardiovascular dysregulation. The specific changes along the vascular intima associated with atherosclerosis, including the vulnerable circulation downstream of the flow obstruction, remain poorly understood. Previous findings from animal models suggest that preservation of a distinct Ca2+ signaling profile along the arterial endothelial network is crucial for maintaining vasculature homeostasis and preventing arterial disease. Ca2+ signaling in the intact human artery intima has not been well characterized. Here, we employed confocal imaging and a custom analysis algorithm to assess the spatially and temporally dynamic Ca2+ signaling profiles of human peripheral arteries isolated from the amputated legs of patients with advanced CVD (peripheral artery disease and/or diabetes) or patients who had lost limbs due to non-cardiovascular trauma. In all tibial artery branches (0.5-5 mm diameter) assessed, the intima consistently elicited a broad range of basal Ca2+ signals ranging from isolated focal transients to broad waves. Arteries from patients with existing CVD displayed a restricted intimal Ca2+ signaling pattern characterized by diminished event amplitude and area. Stimulation of type-4 vanilloid transient receptor potential channels (TRPV4) amplified endothelial Ca2+ signals; however, these signals remained smaller and spatially confined in arteries from patients with CVD verses those without CVD. Our findings reveal a characteristic underlying basal Ca2+ signaling pattern within the intima of human peripheral arteries and suggest a distinct truncation of the inherent Ca2+ profile with CVD.

cGMP-dependent protein kinase and the regulation of vascular smooth muscle cell gene expression: possible involvement of Elk-1 sumoylation.

Although the regulation of smooth muscle cell (SMC) gene expression by cGMP-dependent protein kinase (PKG) is now recognized, the mechanisms underlying these effects are not fully understood. In this study, we report that PKG-I stimulates myocardin/serum response factor (SRF)-dependent gene expression in vascular SMCs. The expression of PKG in PKG-deficient cells enhanced myocardin-induced SM22 promoter activity in a concentration-dependent fashion. However, neither SRF nor myocardin expression was affected. To investigate alternative mechanisms, we examined whether PKG affects the phosphorylation of E26-like protein-1 (Elk-1), a SRF/myocardin transcription antagonist. The activation of PKG caused an increase in a higher molecular mass form of phospho-Elk-1 that was determined to be small ubiquitin-related modifier (sumo)ylated Elk-1. PKG increased Elk-1 sumoylation twofold compared with the PKG-deficient cells, and Elk-1 sumoylation was reduced using dominant-negative sumo-conjugating enzyme, DN-Ubc9, confirming PKG-dependent sumoylation of phospho-Elk-1 in vascular SMCs. In addition, PKG stimulated Elk-1 sumoylation in COS-7 cells overexpressing Elk-1, sumo-1, and PKG-I. The increased expression of PKG in vascular SMCs inhibited Elk-1 binding to SMC-specific promoters, SM22 and smooth muscle myosin heavy chain, as measured by EMSA and chromatin immunoprecipitation assay, and PKG suppressed the Elk-1 inhibition of SM22 reporter gene expression. Taken together, these data suggest that PKG-I decreases Elk-1 activity by sumo modification of Elk-1, thereby increasing myocardin-SRF activity on SMC-specific gene expression.

Regulation of vascular smooth muscle cell phenotype by cyclic GMP and cyclic GMP-dependent protein kinase.

This basic science review examines the role of cGMP and cGMP-dependent protein kinase (PKG) in the regulation of vascular smooth muscle cell (VSMC) phenotype. The first such studies suggested a role for nitric oxide (NO) and atrial natriuretic peptides (ANP), and the downstream second messenger cGMP, in the inhibition of VSMC proliferation. Subsequently, many laboratories confirmed the anti-proliferative effects of the cGMP pathway in cultured cells and the anti-atherosclerotic effects of the pathway in in vivo animal models. Other studies suggested that the cGMP target, PKG, mediated the anti-proliferative effects of cGMP although other laboratories have not consistently observed these effects. On the other hand, PKG mediates cGMP-dependent increases in smooth muscle-specific gene expression, and in vivo studies suggest that PKG expression itself reduces vascular lesions. The mechanisms by which PKG regulates gene expression are addressed, but it still unknown how the cGMP-PKG pathway is involved in smooth muscle-specific gene expression and phenotype.

Smooth muscle specific overexpression of p22phox potentiates carotid artery wall thickening in response to injury.

We hypothesized that transgenic mice overexpressing the p22(phox) subunit of the NADPH oxidase selectively in smooth muscle (Tg(p22smc)) would exhibit an exacerbated response to transluminal carotid injury compared to wild-type mice. To examine the role of reactive oxygen species (ROS) as a mediator of vascular injury, the injury response was quantified by measuring wall thickness (WT) and cross-sectional wall area (CSWA) of the injured and noninjured arteries in both Tg(p22smc) and wild-type animals at days 3, 7, and 14 after injury. Akt, p38 MAPK, and Src activation were evaluated at the same time points using Western blotting. WT and CSWA following injury were significantly greater in Tg(p22smc) mice at both 7 and 14 days after injury while noninjured contralateral carotids were similar between groups. Apocynin treatment attenuated the injury response in both groups and rendered the response similar between Tg(p22smc) mice and wild-type mice. Following injury, carotid arteries from Tg(p22smc) mice demonstrated elevated activation of Akt at day 3, while p38 MAPK and Src activation was elevated at day 7 compared to wild-type mice. Both increased activation and temporal regulation of these signaling pathways may contribute to enhanced vascular growth in response to injury in this transgenic model of elevated vascular ROS.

Transcriptional and post-transcriptional regulation of cGMP-dependent protein kinase (PKG-I): pathophysiological significance.

The ability of the endothelium to produce nitric oxide, which induces generation of cyclic guanosine monophosphate (cGMP) that activates cGMP-dependent protein kinase (PKG-I), in vascular smooth muscle cells (VSMCs), is essential for the maintenance of vascular homeostasis. Yet, disturbance of this nitric oxide/cGMP/PKG-I pathway has been shown to play an important role in many cardiovascular diseases. In the last two decades, in vitro and in vivo models of vascular injury have shown that PKG-I is suppressed following nitric oxide, cGMP, cytokine, and growth factor stimulation. The molecular basis for these changes in PKG-I expression is still poorly understood, and they are likely to be mediated by a number of processes, including changes in gene transcription, mRNA stability, protein synthesis, or protein degradation. Emerging studies have begun to define mechanisms responsible for changes in PKG-I expression and have identified cis- and trans-acting regulatory elements, with a plausible role being attributed to post-translational control of PKG-I protein levels. This review will focus mainly on recent advances in understanding of the regulation of PKG-I expression in VSMCs, with an emphasis on the physiological and pathological significance of PKG-I down-regulation in VSMCs in certain circumstances.

Stabilization of cGMP-dependent protein kinase G (PKG) expression in vascular smooth muscle cells: contribution of 3'UTR of its mRNA.

The type-I cGMP-dependent protein kinase (PKG-I) expression regulation is not yet completely understood. In this study, we examined the role of 3'-untranslated region (3'UTR)-PKG-I messenger RNA (mRNA) in the control of PKG-I expression in vascular smooth muscle cells (VSMCs). Using a 3'-rapid amplification of cDNA ends (RACE) for the amplification of complementary DNA (cDNA) ends, we generated and cloned a 1.2-kb-3'UTR mRNA PKG-I in pGL3 control vector downstream of the luciferase reporter gene. Serial deletions and functional studies revealed that among the deleted constructs, only the 1.2-kb-3'UTR PKG-I mRNA possesses the highest activity in transfected VSMC. Kinetic luciferase assays in the presence of actinomycin D showed that this construct stabilizes luciferase activity compared to the control vector. Sequence analysis of 3'UTR-PKG-I mRNA revealed the existence of four AU-rich regions (AU1 through AU4) in addition to a potential poly(A) site. Different riboprobes were generated either by 5'-end-labelling of designed ribonucleotides, containing individual AU-rich regions or by in vitro transcription assay using cloned 1.2-kb cDNA as a template. RNA-electrophoretic mobility shift assay (EMSA) and ultra-violet cross-linking (UV-CL) assays showed that AU1, AU3, AU4 and 1.2-kb probes were able to retard cytosolic and nuclear proteins. Taken together, these data suggest that PKG-I expression is subjected to post-transcriptional regulation in VSMC through the 3'UTR of its mRNA.

Upstream stimulatory factors (USF-1/USF-2) regulate human cGMP-dependent protein kinase I gene expression in vascular smooth muscle cells.

Cyclic GMP-dependent protein kinase I plays a pivotal role in regulating smooth muscle cell relaxation, growth, and differentiation. Expression of the enzyme varies greatly in smooth muscle and in other tissues and cell types, yet little is known regarding the mechanisms regulating cGMP-dependent protein kinase gene expression. The present work was undertaken to characterize the mechanisms controlling kinase gene expression in vascular smooth muscle cells. A 2-kb human cGMP-dependent protein kinase I 5'-noncoding promoter sequence was characterized by serial deletion, and functional studies demonstrated that a 591-bp 5'-promoter construct possessed the highest activity compared with all other constructs generated from the larger promoter. Analysis of the sequence between -472 and -591 bp from the transcriptional start site revealed the existence of two E-like boxes known to bind upstream stimulatory factors. Electrophoretic mobility shift assays and functional studies using luciferase reporter gene assays identified upstream stimulatory factors as the transcription factors bound to the E-boxes in the 591-bp promoter. Site-directed mutagenesis of the E-boxes abolished the binding of upstream stimulatory factor proteins and decreased the activity of the cGMP-dependent protein kinase I 591-bp promoter, thus confirming the involvement of these transcription factors in mediating gene expression. Cotransfection experiments demonstrated that overexpression of upstream stimulatory factors 1 and 2 increased cGMP-dependent protein kinase I promoter activity. Collectively, these data suggest that the human proximal cGMP-dependent protein kinase I promoter is regulated by tandem E-boxes that bind upstream stimulatory factors.