Current Molecular Medicine (v.14, #9)

Reactive oxygen species (ROS) are now appreciated to play several important roles in a number ofbiological processes and regulate cell physiology and function. ROS are a heterogeneous chemical class thatincludes radicals, such as superoxide ion (O2•-), hydroxyl radical (OH) and nitric oxide (NO), and non-radicals,such as hydrogen peroxide (H2O2), singlet oxygen (1O2), hypochlorous acid (HOCl), and peroxynitrite (NO3-). Inthe cardiovascular system, besides playing a critical role in the development and progression of vasculopathiesand other important pathologies such as congestive heart failure, atherosclerosis and thrombosis, ROS alsoregulate physiological processes. Evidence from a wealth of cardiovascular research studies suggests thatROS act as second messengers and play an essential role in vascular homeostasis by influencing discretesignal transduction pathways in various systems and cell types. They are produced throughout the vascularsystem, regulate differentiation and contractility of vascular smooth muscle cells, control vascular endothelialcell proliferation and migration, mediate platelet activation and haemostasis, and significantly contribute to theimmune response. Our understanding of ROS chemistry and cell biology has evolved to the point of realizingthat different ROS have distinct and important roles in cardiovascular physiology. This review will outlinesources, functions and molecular mechanisms of action of different ROS in the cardiovascular system and willdescribe their emerging role in healthy cardiovascular physiology and homeostasis.

Cholinergic Receptors as Target for Cancer Therapy in a Systems Medicine Perspective by P. Russo, A. Del Bufalo, M. Milic, G. Salinaro, M. Fini, A. Cesario (1126-1138).
Epithelial cells not innervated by cholinergic neurons express nicotinic and muscarinic acetylcholine(ACh) receptors (nAChR, mAChR). nAChR and mAChR are components of the auto-/paracrine-regulatory loopof non-neuronal ACh release. The cholinergic control of non-neuronal cells may be mediated by differenteffects (synergistic, additive, or reciprocal) triggered by these receptors. The ionic events (Ca+2 influx) aregenerated by the ACh-opening of nAChR channels, while the metabolic events by ACh-binding to G-proteincoupledmAChR. Effective inter- and intracellular signaling is crucial for valuable cancer cells proliferation andsurvival. Depending on cancer cell type, different AChR have been identified. The proliferation of airwaysepithelial cancer cells and pancreatic cancer cells may be under the control of α7-nAChR and M3-mAChR,while breast cancer cells and colon cancer cells are regulated by α9-nAChR, and M3-mAChR, respectively. Inturn, these receptors may activate different pathways (Ras-Raf-1-Erk-AKT) as well as other receptors (β-adrenergicR). nAChR or mAChR antagonists may inhibit cancer growth. Inhibition of M3 by antisense orantagonists (Darifenacin, Tiotropium) reduces lung or colon cancer proliferation, as well as inhibition of α9-nAChR [polyphenol (-)-epigallocatechin-3-gallate] diminishes breast cancer cells growth. α7-nAChR silencinginhibits lung cancer proliferation. Moreover, inhibition of the nAChR-β-adrenergicR pathway (β-blockers) couldbe also useful. This review will describe the future translational perspectives of cholinergic receptors druginhibitionin a complex disease such as cancer that poses compelling treatment challenges. Cancer happensas consequence of disease-perturbed molecular networks in relevant organ cells that change duringprogression. The framework for approaching these challenges is a systems approach.

Lactoferricin and lactoferrampin, peptides derived from the whey protein lactoferrin, are antimicrobialagents with a promising prospect and are currently one of the research focuses. In this review, a basicintroduction including location and solution structures of these two peptides is given. Their biological activitiesencompassing antiviral, antibacterial, antifungal and anti-inflammatory activities with possible mechanisms arementioned. In terms of modification studies, research about identification of their active derivatives and crucialamino acid residues is also discussed. Various attempts at modification of lactoferricin and lactoferrampin suchas introducing big hydrophobic side-chains; employing special amino acids for synthesis; N-acetylization,amidation, cyclization and peptide chimera are summarized. The studies on lactoferricin-lactoferrampinchimera are discussed in detail. Future prospects of lactoferricin and lactoferrampin are covered.

Human Cognitive and Neuro-Psychiatric Bio-Markers in the Cardiac Peri-Operative Patient by H.J. Reis, A.C.P. de Oliveira, M.A. Mukhamedyarov, A.L. Zefirov, A.A. Rizvanov, M.E. Yalvac, A.L. Teixeira, Z. Janka, A. Hussain, L.B. Vieira, A. Palotas (1155-1163).
Some of the complexities of surgical interventions include neurological and psychiatric disturbances.Prompt identification and early treatment of these complications are pivotal in achieving excellent clinicalresults. Recognizing major adverse events such as stroke, seizure or delirium is usually straight-forward,however the discovery of less frequent or more subtle post-operative changes such as cognitive dysfunctionmight be delayed due to lack of appropriate diagnostic tools. This review summarizes biological markers thatcan be utilized as surrogates in evaluating surgery-related neuro-psychiatric disorders.

Discovered over a century ago, histones constitute one of the oldest families of proteins and havebeen remarkably conserved throughout eukaryotic evolution. However, only for the past 30 years havehistones demonstrated that their influence extends far beyond packaging DNA. To create the variouschromatin structures that are necessary for DNA function in higher eukaryotes, histones undergo posttranslationalmodifications. While many such modifications are well documented, others, such as histone tailcleavage are less understood. Recent studies have discovered several proteases that cleave histones andhave suggested roles for clipped histones in stem cell differentiation and aging in addition to infection andinflammation; the underlying mechanisms, however, are uncertain. One histone class in particular, histone H3,has received outstanding interest due to its numerous N-terminal modification sites and prevalence inregulating homeostatic processes. Here, with special consideration of H3, we will discuss the novel findingsregarding histone proteolytic cleavage as well as their significance in the studies of immunology andepigenetics.

Prolylcarboxypeptidase Independently Activates Plasma Prekallikrein (Fletcher Factor) by J. Wang, A. Matafonov, H. Madkhali, F. Mahdi, D. Watson, A.H. Schmaier, D. Gailani, Z. Shariat-Madar (1173-1185).
Prolylcarboxypeptidase isoform 1 (PRCP1) is capable of regulating numerous autocrines andhormones, such as angiotensin II, angiotensin III, αMSH1-13, and DesArg9 bradykinin. It does so by cleaving aC-terminal PRO-X bond. Recent work also indicates that the human PRCP1 activates plasma prekallikrein(PK) to kallikrein on endothelial cells through an uncharacterized mechanism. This study aims to identifyPRCP1 binding interaction and cleavage site on PK. Recently, a cDNA encoding a novel splice variant of thehuman PRCP1 was identified. This isoform differed only in the N-terminal region of the deduced amino acidsequence. Using structural and functional studies, a combination of peptide mapping and site-directedmutagenesis approaches were employed to investigate the interaction of PRCP1 with PK. Three PRCPpeptides, in decreasing order of potency, from 1) the N-terminus of the secreted protein, 2) spanning theopening of the active site pocket, and 3) in the dimerization region inhibit PRCP activation of PK on endothelialcells. Investigations also tested the hypothesis that PRCP cleavage site on PK is between its C-terminal Pro637 (P637) and Ala 638 (A638). Recombinant forms of PK with C-terminal alanine mutagenesis or a stop codonis activated equally as wild type PK by PRCP. In conclusion, PRCP1 interacts with PK at multiple sites for PKactivation. PRCP1 also enhances FXIIa activation of PK, suggesting that its activation site on PK is notidentical to that of FXIIa.

Ipsilateral Hippocampal Proteomics Reveals Mitochondrial Antioxidative Stress Impairment in Cortical-Lesioned Chronic Mild Stressed Rats by J. Pan, H. Liu, J. Zhou, Z. Liu, Y. Yang, Y. Peng, H. You, D. Yang, P. Xie (1186-1196).
In this study, a two-dimensional gel-based proteomic approach was applied to profile the proteinalterations underlying the significant adverse effects from post-stroke depression (PSD). In view of the closeassociation between left prefrontal cortical dysfunction and PSD, a PSD rat model was constructed through aleft anterior cortical lesion followed by chronic mild stress (CMS) for three weeks. Through sucrose preferencetesting, PSD rats displayed depression-like behavior during the entire CMS period. In contrast, stroke ratsdisplayed depression-like behavior in the first week post-stroke and recovered in the second week post-stroke.To investigate the PSD-induced protein expression changes, ipsilateral hippocampal protein expression instroke, PSD, and control rats were comparatively analyzed. 46 differential proteins were identified, 22 of whichwere regulated in opposing directions by stroke and post-stroke stress. The majority of these 22 proteins wereinvolved in neurogenesis, cytoskeletal remodeling, and energy metabolism. Additional proteins werefunctionally related to mitochondrial antioxidative stress systems. The differential proteins expressed inopposing directions by stroke and post-stroke stress may play a role in self-repair after adult brain lesions,suggesting that stroke induces self-repair mechanisms, while post-stoke stress mitigates them, in the rathippocampus. Among these differential proteins dysregulated in opposing directions, three mitochondrialproteins involved in mitochondrial antioxidative stress - heat shock 70 kDa protein 9, peroxiredoxin-6, andprohibitin - were validated and may play an important role in stroke-injury self-repair and PSD-induced injury ofhippocampal neurons. These findings offer new insight into deciphering the molecular mechanismsunderpinning PSD's adverse effects on stroke recovery.

The Tumor Suppressor, p53 Regulates the γA-Crystallin Gene During Mouse Lens Development by X.-H. Hu, Q. Nie, M. Yi, T.-T. Li, Z.-F. Wang, Z.-X. Huang, X.-D. Gong, L. Zhou, W.-K. Ji, W.-F. Hu, J.-F. Liu, L. Wang, Z. Woodward, J. Zhu, W.-B. Liu, Q.D. Nguyen, D.W.-C. Li (1197-1204).
The tumor suppressor, p53 regulates a large number of target genes to control cell proliferation andapoptosis. In addition, it is also implicated in the regulation of cell differentiation in muscle, the circulatorysystem and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation.Regarding the mechanism, we reveal that p53 directly regulates several genes including c-Maf and Prox1, twoimportant transcription factors for lens differentiation, and αA and βA3/A1, the lens differentiation markers. Inthe present study, we present evidence to show that the γA-crystallin gene distal promoter and the first intronalso contain p53 binding sites and are capable of mediating p53 control during mouse lens development. First,gel mobility shifting assays revealed that the p53 protein in nuclear extracts from human lens epithelial cells(HLE) directly binds to the p53 binding sites present in the γA-crystallin gene. Second, the exogenous wild typep53 induces the dose-dependent expression of the luciferase reporter gene driven by the basic promotercontaining the γA-crystallin gene p53 binding site. In contrast, the exogenous dominant negative mutant p53causes a dose-dependent inhibition of the same promoter. Third, ChIP assays revealed that p53 binds to theγA-crystallin gene promoter in vivo. Finally, in the p53 knockout mouse lenses, the expression level of the γAcrystallingene was found attenuated in comparison with that in the wild type mouse lenses. Together, ourresults reveal that p53 regulates γA-crystallin gene expression during mouse lens development. Thus, p53directly regulates all 3 types of crystallin genes to control lens differentiation.

Purinergic receptors are implicated in nociceptive signaling in small primary afferents via activationof adenosine triphosphate (ATP). ATP appears to mediate HCl-induced transient receptor potential vanilloidreceptor 1 (TRPV1) activation in esophageal mucosa. Up-regulation of TRPV1 expression in gastroesophagealreflux disease (GERD) is associated with increased nerve growth factor (NGF) and glial derivedneurotrophic factor (GDNF). This study aims to genetically determine the expression of purinergic receptors insevere inflamed human esophagus. Distal esophageal biopsies from the subjects with erosive GERD,asymptomatic patients (AP) and healthy ones were examined. Using real-time qPCR for detecting purinergicreceptors (P2X2, P2X3, P2X7, P2Y1, P2Y2, P2Y4, P2Y6 and P2Y12), TRPV1, TRPV4, NGF, and GDNF wasdone in this study. Both P2X3 and P2X7 mRNA expressions in GERD patients significantly increased thanthose in healthy controls (P < 0.001) and AP (P < 0.001), but P2X2, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12 or P2Y12had no difference within the control, AP or GERD subjects. The well correlated expression in P2X3 gene withTRPV1 (r = 0.46, P = 0.002), NGF (r = 0.54, P = 0.0002), and GDNF (r = 0.64, P = 0.0001) was found. TheP2X7 gene expressions also well correlated with TRPV1 (r = 0.47, P = 0.002), NGF (r = 0.32, P = 0.037), andGDNF (r = 0.42, P = 0.005). These results suggest that chronic esophagitis increases mRNA expressions ofP2X3 and P2X7 receptors accompanied by up-regulation of TRPV1 and neurotrophic factors (NGF and GDNF).These genetical alterations in esophageal mucosa might mediate sensitization of inflamed human esophagus.

Small interfering RNA (siRNA) is a potential agent for the treatment of ocular surface diseases.Previous studies delivered siRNA by directly injecting siRNA into cornea or conjunctiva. In the present studywe sought to explore an alternative approach to deliver siRNA into mouse cornea via eye drops that containscy3-labeled siRNA (cy3-siRNA) and different cationic complexing agents and to evaluate the effects of siRNAtargeting HSV-1 ICP4 gene (ICP4-siRNA) on mouse herpes simplex keratitis (HSK). Cy3-siRNA was mixedwith Lipofectamine 2000, EntransterTM-in vivo, polyethyleneimine (PEI) or PEO-PPO-PEO polymers at differentratios. The efficacy of delivery was analyzed after topical application of the complexes to normal, EDTAtreated, and epithelial scraped cornea of BALB/c mouse eyes. Compared to the other delivery agents andschedules, PEI at 0.75 mg/ml with 20 ?M cy3-siRNA complex delivered eight times daily for two days was themost efficient as revealed by its production of the greatest fluorescence in cornea epithelial cells. In mouseHSK, the application of ICP4-siRNA+PEI eye drops reduced the damage to the corneal epithelia anddecreased viral VP16 expression in the corneal tissue. These results proved the idea that siRNA can beformulated into eye drops with carriers for effective delivery into the cornea and that the formulated eye dropscontaining ICP4-siRNA can inhibit HSV-1 replication in the mouse corneas.

The Effect of Claudin-5 Overexpression on the Interactions of Claudin-1 and -2 and Barrier Function in Retinal Cells by R. Tian, Y. Luo, Q. Liu, M. Cai, J. Li, W. Sun, J. Wang, C. He, Y. Liu, X. Liu (1226-1237).
Claudin-5, one of the dominant tight junctions (TJs) proteins, plays an important role in maintainingthe barrier function in the blood brain and retinal barrier. This study aimed to investigate the effect of claudin-5overexpression on the interactions of claudin-1 and -2 and barrier functions in primary cultured human retinalpigment epithelium cells (HRPECs) and human retina endothelial cells (HRECs). Lentivirus was used tomediate the overexpression of claudin-5 in retinal cells. Significantly increased mRNA and protein levels ofclaudin-5 were detected in the transfection group. After the transfected cells grew on the transwell membranefor three weeks, a stable monolayer cell barrier model was established in vitro. The claudins expressionsanalysis showed that overexpressed claudin-5 significantly increased the expression of claudin-1, while itdecreased the expression of claudin-2 in both mRNA and protein level. Co-IP experiments and barrier functionassay revealed that claudin-5 overexpression promoted the interactions of claudin-1 and claudin-2 andenhanced the barrier function of retinal cells. Intriguingly, the exogenous expression of claudin-5 induced newinteraction pattern between claudin-5 and claudin-1 or -2 in HRPECs, which do not have endogenous claudin-5expression. In addition, claudin-5 overexpression decreased cell mobility and the sprouting capability of vesseltube formation in vitro. This study demonstrated that claudin-5 has a positive regulation in the formation ofretinal barrier. Claudin elements and their interactions can be modulated and that such dynamic properties areimportant for the functions of TJs, ranging from the regulation of retinal barrier integrity to junction-associatedsignaling mechanisms.

The Yin and Yang of Inflammation by M.A. Blackman, J.L. Yates, C.M. Spencer, E.E. Vomhof-DeKrey, A.M. Cooper, E.A. Leadbetter (1238-1243).
Inflammation is an essential protective part of the body's response to infection, yet many diseasesare the product of inflammation. For example, inflammation can lead to autoimmune disease and tissuedamage, and is a key element in chronic health conditions such as heart disease, diabetes, rheumatoidarthritis, and also drives changes associated with aging. Animal models of infectious and chronic disease areimportant tools with which to dissect the pathways whereby inflammatory responses are initiated andcontrolled. Animal models therefore provide a prism through which the role of inflammation in health anddisease can be viewed, and are important means by which to dissect mechanisms and identify potentialtherapies to be tested in the clinic. A meeting, “The Yin and Yang of Inflammation” was organized by TrudeauInstitute and was held between April 4-6, 2014. The main goal was to bring together experts frombiotechnology and academic organizations to examine and describe critical pathways in inflammation andplace these pathways within the context of human disease. A group of ~80 scientists met for three days ofintense formal and informal exchanges. A key focus was to stimulate interactions between basic research andindustry.