Topical nicotinic receptor activation improves wound bacterial infection outcomes and TLR2-mediated inflammation in diabetic mouse wounds.

The cholinergic anti-inflammatory pathway can directly affect skin antibacterial responses via nicotinic acetylcholine receptors (nAChRs). In particular, α7 nAChR (CHRNA7) present in the epidermis modulates the host response to wounding and/or wound bacterial infection. While physiologic inflammation is required to initiate normal wound repair and can be triggered by Toll-like receptor (TLR) activation, chronic inflammation is frequently observed in diabetic wounds and can occur, in part, via excessive TLR2 activation or production. Consequently, this can delay physiologic wound healing responses and increase diabetic host susceptibility to bacterial infection. In this study, we demonstrate that topical nAChR activation diminishes bacterial survival and systemic dissemination in a mouse model of diabetic wound infection, while reducing wound TLR2 production, relative to control mice. We further determined that the antimicrobial peptide activity of diabetic mouse wounds is increased compared to control mice, but this effect is lost following topical nAChR activation. Finally, we observed that human diabetic wounds exhibit impaired α7 nAChR (CHRNA7) abundance and localization relative to human control (nondiabetic) skin. These findings suggest that topical administration of nAChR agonists may improve wound healing and infection outcomes in diabetic wounds by dampening TLR2-mediated inflammation and antimicrobial peptide response, and that the diabetic microenvironment may promote aberrant CHRNA7 production/activation that likely contributes to diabetic wound pathogenesis.

The Effect of Subclinical Varicocele on Pregnancy Rates and Semen Parameters: a Systematic Review and Meta-Analysis.

PURPOSE OF REVIEW: Current guidelines recommend against surgical repair of subclinical varicoceles (SCVs) for infertility; several studies demonstrate mixed fertility results after SCV correction. To determine whether surgical correction of SCV improves semen parameters and/or reproductive outcomes, we performed a systematic review and meta-analysis. Seven biomedical literature databases were searched through January 2018 for studies that assessed reproductive outcomes and/or change in semen parameters in men with corrected SCV compared to either (1) uncorrected SCV or (2) corrected clinical varicocele. Estimates were pooled using random-effects meta-analysis. RECENT FINDINGS: Data were extracted from 13 studies involving 1357 men. Overall, the risk of bias for included studies was high and without a consistent SCV definition across studies. Surgical correction of SCV was associated with a minor increase in sperm density and total motile sperm count (TMSC) compared to uncorrected SCV. This increase in semen parameters is not clinically significant, as men prior to varicocelectomy were on average normospermic nor was correction of a SCV associated with an increase in pregnancy rates when compared to men with uncorrected SCV. Comparing corrected SCV to corrected clinical varicocele, SCV correction resulted in a smaller increase in TMSC but no difference in average annual pregnancy rate. The risk of bias within and heterogeneity between studies assessing SCV correction are high, yet overall very little clinical benefit is derived from SCV correction.

Keratinocyte nicotinic acetylcholine receptor activation modulates early TLR2-mediated wound healing responses.

The cholinergic anti-inflammatory pathway spans several macro- and micro-environments to control inflammation via α7 nicotinic acetylcholine receptors (nAChRs). Physiologic inflammation is necessary for normal wound repair and is triggered, in part, via Toll-like receptors (TLRs). Here, we demonstrate that keratinocyte nAChR activation dampens TLR2-mediated migration and pro-inflammatory cytokine and antimicrobial peptide (AMP) production, which is restored by a α7-selective nAChR antagonist. The mechanism of this response occurs by blocking the NF-κB and Erk1/2 pathway during early and late wound healing. In a mouse model of Staphylococcus aureus wound infection, topical nAChR activation reduces wound AMP and TLR2 production to augment bacterial survival in wild-type mice. These findings suggest that aberrant α7 nAChR activation may impair normal wound healing responses, and that pharmacologic administration of topical nAChR antagonists may improve wound healing outcomes in wounds necessitating a more robust inflammatory response.

Nicotinic acetylcholine receptor stimulation impairs epidermal permeability barrier function and recovery and modulates cornified envelope proteins.

AIM: To characterize how nicotinic acetylcholine receptors (nAChRs) influence epidermal barrier function and recovery following prolonged stress or direct nAChR activation or antagonism. MAIN METHODS: Mice were subjected to psychological stress or treated topically with nAChR agonist or antagonist for 3 days. We assessed barrier permeability and recovery by measuring transepidermal water loss (TEWL) before and after barrier disruption. In parallel, we analyzed the production and localization of several epidermal cornified envelope proteins in mouse skin and in human EpiDerm™ organotypic constructs stimulated with a nAChR agonist (nicotine) and/or a nAChR selective antagonist (α-bungarotoxin). KEY FINDINGS: We determined that psychological stress in mice impairs barrier permeability function and recovery, an effect that is reversed by application of the α7 selective nAChR antagonist, α-bungarotoxin (Bung). In the absence of stress, both topical nicotine or Bung treatment alone impaired barrier permeability. We further observed that stress, topical nicotine, or topical Bung treatment in mice influenced the abundance and/or localization of filaggrin, loricrin, and involucrin. Similar alterations in these three major cornified envelope proteins were observed in human EpiDerm™ cultures. SIGNIFICANCE: Perceived psychological stress and nicotine usage can both initiate or exacerbate several dermatoses by altering the cutaneous permeability barrier. Modulation of nAChRs by topical agonists or antagonists may be used to improve epidermal barrier function in skin diseases associated with defects in epidermal barrier permeability.

Adenovirus type 5 rupture of lysosomes leads to cathepsin B-dependent mitochondrial stress and production of reactive oxygen species.

In response to viral infection, reactive oxygen species (ROS) mediate innate immune signaling or generate danger signals to activate immune cells. The mechanisms of virally induced ROS are poorly defined, however. We demonstrate that ROS are produced within minutes of adenovirus type 5 (Ad5) infection of macrophages and that oxidative stress supports Ad5-induced cytokine secretion. We show that short hairpin RNA (shRNA) knockdown of TLR9 has no effect on ROS production despite observed decreases in Ad-induced cytokine secretion. A major source of ROS in macrophages is NADPH oxidase. However, shRNA knockdown of the NADPH oxidase subunit NOX2 does not attenuate Ad-induced ROS. Induction of ROS is not observed in cells infected with a temperature-sensitive mutant of Ad2, ts1, which is defective in endosomal membrane penetration during cell entry. Further, Ad5, but not ts1, induces the release of lysosomal cathepsin B into the cytoplasm of infected cells. In agreement with this finding, we observe a loss of mitochondrial membrane potential upon Ad infection which requires Ad endosomal membrane penetration and cathepsin B activity. Overexpression of Bcl-2 attenuates Ad5-induced ROS, further supporting the role for mitochondrial membrane destabilization as the source of ROS in response to Ad5 infection. Together, these data suggest that ROS produced in response to Ad5 infection depends on the virally induced endosomal membrane rupture to release lysosomal cathepsins. Furthermore, the release of cathepsins leads to mitochondrial membrane disruption and thus the release of ROS from the mitochondria.

Influence of PKC-alpha overexpression on HSP70 and cardioprotection.

Recent research has indicated that the protein kinase C (PKC) isoforms and the heat shock proteins (HSPs) are involved in cardioprotection. We have investigated the possible interaction between these two protein families. We have found that adenoviral-mediated expression of PKC-alpha in neonatal rat ventricular myocytes (NRVM) not only increases the expression of HSP70 but also protects against simulated ischemia-reperfusion. In addition, Western blots of PKC-alpha-infected NRVM indicated that other HSPs are not induced in the same manner as HSP70. In an effort to determine the mechanism of induction of HSP70 by PKC-alpha, we tested a chimeric construct that linked the luciferase reporter gene to the 5'-promoter region of HSP70 in myogenic H9c2 cells. When PKC-alpha was expressed, the 5'-promoter region of the HSP70 responded robustly, indicating that PKC-alpha induction of HSP70 expression is through transcription activation. Electrophoretic mobility shift assay determined that overexpression of PKC-alpha, PKC-delta, or PKC-epsilon did not induce activation of heat shock factor-1 (HSF-1). Therefore, induction of HSP70 by PKC-alpha is independent of heat shock factor-1 activation. We also measured cellular injury by assessing creatine kinase (CK) release from NRVM after simulated ischemia to determine cardioprotection. NRVM infected with the wild-type adenoviral construct AdwtPKC-alpha released 54% less CK than control NRVM. Experiments using small interfering RNA against HSP70 indicate that loss of PKC-alpha-induced HSP70 expression results in increased CK release or a loss of protection. Our results show that there is a close interaction between PKC-alpha and HSP70, independent of heat shock factor-1 activation, and that the protection conferred by PKC-alpha overexpression is mediated by the transcriptionally induced expression of HSP70.

Overexpression of inducible 70-kDa heat shock protein in mouse attenuates skeletal muscle damage induced by cryolesioning.

Heat shock protein expression is elevated upon exposure to a variety of stresses and limits the extent of stress-induced damage. To investigate the putative role of inducible 70-kDa heat shock protein (HSP70) in skeletal muscle damage and regeneration, soleus and tibialis anterior (TA) muscles from HSP70-overexpressing transgenic mice were subjected to cryolesioning and analyzed after 1, 10, and 21 days. Histological analysis showed that the muscles from both HSP70 and wild-type mice treated with radicicol (a HSP inducer) had decreased necrosis after cryolesioning compared with controls. The decrease in muscle fiber cross-sectional area in both soleus and TA muscles in 10 days postlesioning was attenuated in HSP70 mice compared with wild-type mice. Glutathione peroxidase activity was increased 1 day after cryolesioning in both HSP70 and control mice and remained elevated for up to 21 days. Immunodetection of neuronal cell adhesion molecule (a satellite cell marker) and developmental/neonatal MHC were significantly lower in cryolesioned HSP70-overexpressing mice than in cryolesioned controls. These results suggest that HSP70 protects skeletal muscle against injury and radicicol might be useful as a skeletal muscle protective agent.

Radicicol activates heat shock protein expression and cardioprotection in neonatal rat cardiomyocytes.

Heat shock proteins (HSPs) constitute an endogenous cellular defense mechanism against environmental stresses. In the past few years, studies have shown that overexpression of HSPs can protect cardiac myocytes against ischemia-reperfusion injury. In an attempt to increase the HSPs in cardiac tissue, we used the compound radicicol that activates HSP expression by binding to the HSP 90 kDa (HSP90). HSP90 is the main component of the cytosolic molecular chaperone complex, which has been implicated in the regulation of the heat shock factor 1 (HSF1). HSF1 is responsible for the transcriptional activation of the heat shock genes. In the present study, we show that radicicol induces HSP expression in neonatal rat cardiomyocytes, and this increase in HSPs confers cardioprotection to these cardiomyocytes. We also show that radicicol induction of the HSP and cardioprotection is dependent on the inhibition of HSP90 in cardiomyocytes. These results indicate that modulation of the active HSP90 protein level plays an important role in cardioprotection. Therefore, compounds, such as radicicol and its possible derivatives that inhibit the function of HSP90 in the cell may represent potentially useful cardioprotective agents.

Altered PYK2 phosphorylation by ANG II in hypertensive vascular smooth muscle.

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit increased cell growth compared with normotensive Wistar-Kyoto rats (WKY). ANG II stimulates growth via G(q)-protein-coupled signaling that involves changes in cytosolic intracellular Ca(2+) concentration ([Ca(2+)](i)) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. This study examines the role of the proline-rich tyrosine kinase 2 (PYK2) in hypertensive VSMC. Basal PYK2 phosphorylation in SHR VSMC was increased compared with WKY (0.44 +/- 0.02 vs. 0.20 +/- 0.02-fold). ANG II-induced activation of PYK2 in SHR VSMC was of greater magnitude (2.2 +/- 0.2-fold in SHR; 1.4 +/- 0.1-fold in WKY) and occurred more rapidly (peak activation at 2 min in SHR vs. 5 min in WKY). This effect was blocked by pretreatment with the [Ca(2+)](i) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or the PKC inhibitor chelerythrine. Basal and ANG II-stimulated c-Fos expression was increased in SHR versus WKY VSMC. PYK2 downregulation with antisense oligonucleotides blocked ANG II-induced c-Fos expression. Increased PYK2 activation may be altered signaling cascades that regulate cell growth in hypertensive VSMC.