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.

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.

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.

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.

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.

Purification and cDNA cloning of inducible antibacterial peptides from Protaetia brevitarsis (Coleoptera).

Three antibacterial peptides, named protaetins 1, 2, and 3, were purified and characterized from immunized larval hemolymph of Protaetia brevitarsis, a fruit tree pest in Korea. Also, protaetin 1 was cloned. Acid extraction, gel filtration, preparative acid-urea PAGE, and reversed-phase FPLC were used for purification of peptides. Protaetins 1 and 3 had molecular masses of 7.5 and 12 kDa on Tricine SDS-PAGE, respectively, and the molecular mass of protaetin 2 was 9,283.95 Da as determined by MALDI-TOF mass spectrometry. In an antibacterial assay, protaetins showed antibacterial activities against a panel of Gram-positive and -negative bacteria. For the RT-PCR (reverse transcription polymerase chain reaction) to obtain the complete primary sequence, the primer was designed according to the N-terminal amino acid sequence of protaetin 1. Amino acid sequence homology of protaetin 1 with holotricin 2, an antibacterial peptide from Holotrichia diomphalia, showed 99% identity. Northern blot analysis showed that the protaetin 1 gene was strongly expressed in the fat body after Escherichia coli injection, but not in normal fat body. Also, it was expressed in the gut, but was much weaker after immunization.