Plaque Proteins Research Articles

Identification of Mature Atherosclerotic Plaque Proteome Signatures Using Data-Independent Acquisition Mass Spectrometry.

Atherosclerosis is a chronic inflammatory disease with complex pathobiology and one of the most common causes of cardiovascular events. The process is characterized by complex vascular remodeling processes that require the actions of numerous proteins. The composition of atherosclerotic plaque is increasingly recognized as a major factor governing the occurrence of cardiovascular or neurological symptoms. To gain deeper insights into the composition of atherosclerotic plaques, we created quantitative proteome profiles of advanced plaque tissues of six male patients undergoing carotid endarterectomy for stroke prevention. Using a quantitative, data-independent proteome approach, we identified 4181 proteins with an average protein coverage of 45%. An analysis of the quantitative composition of the tissue revealed key players of vascular remodeling processes. Moreover, compared with proximal arterial tissue, 20 proteins in mature plaques were enriched, whereas 52 proteins were found in lower quantities. Among the proteins with increased abundance were prominent extracellular matrix proteins such as biglycan and lumican, whereas cytoskeletal markers for contractile smooth muscle cells (SMCs) were decreased. Taken together, this study provides the most comprehensive quantitative assessment of mature human plaque tissue to date, which indicates a central role of SMCs in the structure of advanced atherosclerotic plaques.

Alzheimer’s Dementia: An Overview

Alzheimer's disease (AD), the most common cause of dementia, is a chronic illness characterized by cognitive symptoms, behavioural and psychological symptoms and difficulty in performing activities of daily living. Mild cognitive impairment (MCI) is regarded as a transitional state between healthy cognitive ageing and dementia and around 12-15% of individuals with MCI progress to dementia annually. This provides unique opportunity to initiate treatments targeted to delay or prevent the onset of AD. Based on the age at onset, AD is divided into two broad categories: early onset and late onset. Early onset AD follows the classical mendelian pattern of inheritance, whereas the late onset AD has a complex interplay of gene-environment interaction, with several lifestyle-related risk factors strongly implicated in the pathogenesis of this degenerative condition. The onset of AD pathology predates the clinical symptoms by several years. The neurodegenerative processes in AD is related to the accumulation of abnormally folded A beta and tau proteins in amyloid plaques and neuronal tangles. Diagnosis of AD is by presence of cluster of clinical features and aided by biomarkers such as CSF A beta 42 and PET amyloid imaging, CSF tau and tau imaging, 18fluorodeoxyglucose uptake on PET and atrophy on structural magnetic resonance imaging increase the diagnostic certainty. In the absence of curative treatments, the management of AD involves pharmacological treatment to delay the onset or progression of AD and supportive care by family members. Targeting these lifestyle-related factors in young adulthood and middle age may be protective against AD. High educational achievement in early life, involvement in cognitively stimulating activity, physical activity and social engagement including rich social network have been associated with reduced risk of late-life dementia and AD.

Plakophilin3 increases desmosome assembly, size and stability by increasing expression of desmocollin2

Previous reports show that the desmosomal plaque protein plakophilin3 (PKP3) is essential for desmosome formation. Here, we report that PKP3 over-expression decreases calcium dependency for de novo desmosome formation and makes existing cell-cell adhesion junctions more resilient in low calcium medium due to an increase in desmocollin2 expression. PKP3 overexpression increases the stability of other desmosomal proteins independently of the increase in DSC2 levels and regulates desmosome formation and stability by a multimodal mechanism affecting transcription, protein stability and cell border localization of desmosomal proteins.

Genetic, Transcriptome, Proteomic, and Epidemiological Evidence for Blood-Brain Barrier Disruption and Polymicrobial Brain Invasion as Determinant Factors in Alzheimer's Disease.

Diverse pathogens are detected in Alzheimer’s disease (AD) brains. A bioinformatics survey showed that AD genome-wide association study (GWAS) genes (localized in bone marrow, immune locations and microglia) relate to multiple host/pathogen interactomes (Candida albicans, Cryptococcus neoformans, Bornavirus, Borrelia burgdorferri, cytomegalovirus, Ebola virus, HSV-1, HERV-W, HIV-1, Epstein-Barr, hepatitis C, influenza, Chlamydia pneumoniae, Porphyrymonas gingivalis, Helicobacter pylori, Toxoplasma gondii, Trypanosoma cruzi). These interactomes also relate to the AD hippocampal transcriptome and to plaque or tangle proteins. Upregulated AD hippocampal genes match those upregulated by multiple bacteria, viruses, fungi, or protozoa in immunocompetent cells. AD genes are enriched in GWAS datasets reflecting pathogen diversity, suggesting selection for pathogen resistance, as supported by the old age of AD patients, implying resistance to earlier infections. APOE4 is concentrated in regions of high parasitic burden and protects against childhood tropical infections and hepatitis C. Immune/inflammatory gain of function applies to APOE4, CR1, and TREM2 variants. AD genes are also expressed in the blood-brain barrier (BBB), which is disrupted by AD risk factors (age, alcohol, aluminum, concussion, cerebral hypoperfusion, diabetes, homocysteine, hypercholesterolemia, hypertension, obesity, pesticides, pollution, physical inactivity, sleep disruption, smoking) and by pathogens, directly or via olfactory routes to basal-forebrain BBB control centers. The BBB benefits from statins, NSAIDs, estrogen, melatonin, memantine, and the Mediterranean diet. Polymicrobial involvement is supported by upregulation of bacterial, viral, and fungal sensors/defenders in the AD brain, blood, or cerebrospinal fluid. AD serum amyloid-β autoantibodies may attenuate its antimicrobial effects favoring microbial survival and cerebral invasion leading to activation of neurodestructive immune/inflammatory processes, which may also be augmented by age-related immunosenescence. AD may thus respond to antibiotic, antifungal, or antiviral therapy.

Microtubule-organizing centers of Aspergillus nidulans are anchored at septa by a disordered protein.

Microtubule-organizing centers (MTOCs) are large, multi-subunit protein complexes. Schizosaccharomyces pombe harbors MTOCs at spindle pole bodies, transient MTOCs in the division plane (eMTOCs) and nuclear-envelope associated MTOCs in interphase cells (iMTOCs). In the filamentous fungus Aspergillus nidulans SPBs and septum-associated MTOCs were described. Although comparable to S. pombe eMTOCs, A. nidulans sMTOCS are permanent septum-associated structures. The composition of sMTOCs is poorly understood and how they are targeted to septa was unknown. Here, we show that in A. nidulans several SPB outer plaque proteins also locate to sMTOCs while other SPB proteins do not, including SfiA, a protein required for SPB duplication in Saccharomyces cerevisiae and S. pombe and PcpA, the anchor for γ-TuSCs at the SPB inner plaque. The A. nidulans disordered protein Spa18Mto2 and the centrosomin-domain containing protein ApsBMto1 were required for recruiting the γ-TuRC component GcpC to sMTOCs and for seeding MT formation from septa. Testing different septum-associated proteins for a role in sMTOC function, Spa10 was identified. It forms a septal pore disc structure, recruits Spa18 and ApsB to septa and is required for sMTOC activity. This is the first evidence for a septum-specific protein, Spa10, as anchor for a specific class of MTOCs.

Cell-specific diversity in the expression and organization of cytoplasmic plaque proteins of apical junctions.

Tight and adherens junctions play critical roles in the barrier, adhesion, and signaling functions of epithelial and endothelial cells. How the molecular organization of these junctions is tuned to the widely diverse physiological requirements of each tissue type is not well understood. Here, we address this question by examining the expression, localization, and interactions of major cytoplasmic plaque proteins of tight and adherens junctions in different cultured epithelial and endothelial cell lines. Immunoblotting and immunofluorescence analyses show that the expression profiles of cingulin, paracingulin, ZO-1, ZO-2, ZO-3, PLEKHA7, afadin, PDZD11, p120-catenin, and α-catenin, as well as the transmembrane junctional proteins occludin, E-cadherin, and VE-cadherin, are significantly diverse when comparing kidney cells (MDCK, mCCD), keratinocytes (HaCaT), lung carcinoma (A427, A549), and endothelium-derived cells (bEnd.3, meEC, H5V). Proximity ligation and co-immunoprecipitation assays show that PLEKHA7 and PDZD11 are significantly more associated with the tight junction proteins cingulin and ZO-1 in aortic endothelium-derived (meEC) cells but not kidney collecting duct epithelial (mCCD) cells. These results provide evidence that the cytoplasmic plaques of tight and adherens junctions are diverse in their composition and molecular architecture and establish a conceptual framework by which we can rationally address the mechanisms of tissue-dependent junction physiology and signaling by cytoplasmic junctional proteins.

Metformin suppressed human cytomegalovirus (hCMV) replication and its potential molecular mechanisms in human fibroblasts

Abstract Background Chronic hCMV infection may contribute to T-cell immunosenenscence and chronic inflammation, and is shown to be associated with frailty and mortality in old adults. Metformin is a first-line treatment for type 2 diabetes mellitus. Emerging data have shown its additional beneficial effects including cardiovascular protection as well as anti-inflammatory, anti-cancer, and anti-aging activities. The objective of this study was to investigate the effect of metformin on hCMV infection in human fibroblasts and its underlying mechanisms. Methods Metformin was added (at concentration of 0, 1mM, 3mM, or 5mM) 2 h before inoculation of hCMV (Towne strain) at multiplicity of infection (MOI) of 0.01 or 0.1 to 80% confluent MRC-5 culture. Cells were cultured for additional 24h to 96 h. Viral plaques were observed under microscope, replication measured by qPCR (primers targeted to hCMV UL123 gene), cell cycle effect determined using flow cytometer, and hCMV protein expression (IE1/IE2, pp28, ICP36) and signaling pathway molecules analyzed by Western blot. Results and discussion Metformin at 3mM potently suppressed hCMV viral plaque formation, replication, and protein production. While hCMV infection promoted MRC-5 cells from G2 to M and S phase, metformin treatment led to G0/G1 arrest. Metformin suppressed expression of cyclin D, CDK4, and p21. It had no effect on CK2 or CDk6 expression. While AMPK pathway antagonist compound C suppressed AMPKa phosphorylation from both hCMV and metformin, it did not antagonize suppressive effect of metformin on hCMV protein expression. Taken together, metformin may suppress hCMV infection in MRC-5 cells via cell cycle regulation and molecular mechanisms independent of AMPK pathway.

Adrenergic Signaling Strengthens Cardiac Myocyte Cohesion

The sympathetic nervous system is a major mediator of heart function. Intercalated discs composed of desmosomes, adherens junctions, and gap junctions provide the structural backbone for coordinated contraction of cardiac myocytes. Gap junctions dynamically remodel to adapt to sympathetic signaling. However, it is unknown whether such rapid adaption also occurs for the adhesive function provided by desmosomes and adherens junctions. Atomic force microscopy revealed that β-adrenergic signaling enhances both the number of desmoglein 2-specific interactions along cell junctions and the mean desmoglein 2-mediated binding forces, whereas N-cadherin-mediated interactions were not affected. This was accompanied by increased cell cohesion in cardiac myocyte cultures and murine heart slices. Enhanced desmoglein 2-positive contacts and increased junction length as revealed by immunofluorescence and electron microscopy reflected cAMP-induced reorganization of intercellular contacts. The mechanism underlying cAMP-mediated strengthening of desmoglein 2 binding was dependent on expression of the intercalated disc plaque protein plakoglobin (Pg) and direct phosphorylation at S665 by protein kinase A: Pg deficiency as well as overexpression of the phospho-deficient Pg-mutant S665A abrogated both cAMP-mediated junctional remodeling and increase of cohesion. Moreover, Pg knockout hearts failed to functionally adapt to adrenergic stimulation. Taken together, we provide first evidence for positive adhesiotropy as a new cardiac function of sympathetic signaling. Positive adhesiotropy is dependent on Pg phosphorylation at S665 by protein kinase A. This mechanism may be of high medical relevance because loss of junctional Pg is a hallmark of arrhythmogenic cardiomyopathy.

Expression of miRNA-155 in carotid atherosclerotic plaques of apolipoprotein E knockout (ApoE−/−) mice and the interventional effect of rapamycin

Carotid atherosclerosis (AS) is an inflammatory process and is the primary pathogenesis of cerebrovascular disease. Many factors are responsible for development of atherosclerosis such as inflammation and autophagy. It is reported that microRNAs (miRNAs) could regulate the development of atherosclerosis through targeting autophagy-related genes. Many studies have demonstrated that miRNA-155 could regulate autophagy in macrophages or tumor cells. However, the role of miRNA-155 on autophagy in carotid plaques is not yet known. In this study, we explore the expression of miRNA-155 and autophagy-related proteins in carotid plaques of ApoE−/− mice and the interventional effect of rapamycin. We compared the expression of miRNA-155 and autophagy-related proteins between the control, model and rapamycin groups using qRT-PCR and western blot. Compared to the control group, we found the miRNA-155 and LC3-II expression was up-regulated (P<0.05), expression ratio of phosphorylated mammalian target of rapamycin to total mammalian target of rapamycin (p-mTOR/mTOR) was down-regulated in model group (P<0.05), but atherosclerotic lesions were still aggravated. These results following rapamycin group indicated that miRNA-155 and LC3-II expression was significantly up-regulated (P<0.05), the expression ratio of p-mTOR/mTOR was significantly down-regulated (P<0.05), and atherosclerotic lesions were reduced. Our results showed in the early stages of atherosclerotic plaques development, effective autophagy could attenuate atherosclerosis in ApoE−/− mice. Furthermore, our results also demonstrated that rapamycin might promote the activation of the autophagy by enhancing the expression of miRNA-155, which delays the development of atherosclerotic plaques.

March 2017: This Month in JoVE: Human Embryo Vitrification, Paper Microfluidic Devices, Virtual Hand/Face Illusions, and Quantification of Amyloid-Beta for Alzheimer's Diagnosis

Here's a look at what's coming up in the March 2017 issue of JoVE: The Journal of Visualized Experiments. As the start of spring brings new life, we begin the month investigating exciting new developments in embryo storage for in-vitro fertility treatment. In JoVE Developmental Biology, our authors present a modified technique for vitrification and preservation of human embryos. This aseptic technique uses all FDA-compliant materials including ionomeric resin embryo straws and sterile flexipettes, and allows fast, safe, and repeatable embryo storage independent of operator variation. In addition, with a greater than 95% embryo survival-rate the method contributes to excellent pregnancy outcomes. Paper cards...printers, it's all so 1990's, right? Well, our next video highlight might make you reconsider. In JoVE Bioengineering this month, our authors reveal 3-dimensional paper microfluidic devices for immunoassays. By patterning paper with hydrophobic barriers, the transfer of fluids can be controlled and directed. Stacking multiple layers of patterned paper creates sophisticated 3-D microfluidic networks that can be utilized in cheap and simple bioanalytical assays, such as the detection of Human Chorionic Gonadotriphin, or hCG - the pregnancy hormone. So don't throw out those old Valentine's Day cards yet - upcycle! In JoVE Medicine this month, our authors reveal an accurate procedure for quantifying the plaque-forming amyloid-beta protein in cerebral spinal fluid to provide diagnoses in suspected Alzheimer's cases. Current diagnosis is mostly based on mental state and cognitive testing, with some studies beginning to examine amyloid-beta levels, but this is hampered by variability and reproducibility issues. Here, our authors use a combination of solid-phase extraction and liquid chromatography tandem mass spectrometry to quantify amyloid-beta levels to a high degree of accuracy, quantifying the deposition of the protein in plaques in the brain and enhancing the power for accurate diagnoses. Our final video highlight this month comes from JoVE Behavior, and showcases a new technique to create human body illusions. Here, our authors demonstrate a computer-based virtual hand and face that move in synchrony with the operator's action, creating an illusion that the model is an extension of the participant's own body. This is a useful tool in the study of self-representation, and is an advance over traditional studies requiring demonstrators to mimic actions on participants in time with actions performed on a physical model body part. Ah, just one small step until I realize my dream of becoming a super-fast blue hedgehog. You've just had a sneak peek of the March 2017 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

An Improved Drugs Screening System Reveals that Baicalein Ameliorates the Aβ/AMPA/NMDA-Induced Depolarization of Neurons

The presence of amyloid-β (Aβ) plaque and tau protein hyperphosphorylation in brain tissue is the pathological hallmark of Alzheimer's disease (AD). At least some Aβ neurotoxicity is caused by the presence of excess glutamate that has been induced by Aβ accumulation. Memantine is currently the only NMDA receptor inhibitor approved for treating moderate-to-severe AD patients. We utilized primary cortical neurons and DiBAC4(3), aslow-response voltage sensitive fluorescence dye, to create anovel system for screening herbal medicines that allows the identification of pure compounds able to ameliorate Aβ-induced abnormal depolarization. The intensity of DiBAC4(3) fluorescence was increased when primary neurons were stimulated by Aβ; furthermore, pre-treatment with memantine abolished this change. Using this system, we identified six crude extracts made from herbal medicines that effectively alleviated this Aβ-induced abnormal depolarization. Among these herbal medicines, one pure compound, baicalein, which was known to be present in Scutellaria baricalensis and is known to improve memory using an AD mouse model, was identified by our assay. However, the compound's molecular mechanism remained unknown. We found that baicalein, in addition to inhibiting Aβ-induced depolarization, possibly functions as an antagonist of AMPA and NMDA receptors. Taken together, we have established asystem/platform to identify herbal medicines that ameliorate Aβ-induced depolarization of neurons. Equally important, baicalein is acandidate drug with great potential for the treatment of AD patients.

Up-regulation of plakophilin-2 is correlated with the progression of glioma.

Glioma is the most common type of primary brain tumor in the CNS. Due to its poor prognosis and high mortality rates, it is urgent to find out more effective therapies. Plakophilin-2 (PKP2) is a widespread desmosomal plaque protein. Recently, the important roles of PKP2 in the proliferation and migration of cancer cells and tumor progression has been shown. However, the expression and potential function of PKP2 in glioma was still unclear. In this study, we demonstrated that PKP2 protein expression level was increased in glioma tissues compared with normal brain tissues, and its level was significantly associated with the Ki-67 expression and WHO grade by Western blot analysis and immunohistochemistry. Clinically, high PKP2 expression was tightly related to poor prognosis of glioma patients. Interestingly, we found that down-regulated PKP2 expression was shown to inhibit the migration of cells in glioma. Moreover, cell counting kit (CCK)-8 and colony formation analyses proved that reduced expression of PKP2 could weaken glioma cell proliferation. Taken together, these data uncover a potential role for PKP2 in the pathogenic process of glioma, suggesting that PKP2 may be a promising therapeutic target of glioma.

Effects of Folic Acid on Secretases Involved in Aβ Deposition in APP/PS1 Mice.

Alzheimer’s disease (AD) is the most common type of dementia. Amyloid-β protein (Aβ) is identified as the core protein of neuritic plaques. Aβ is generated by the sequential cleavage of the amyloid precursor protein (APP) via the APP cleaving enzyme (α-secretase, or β-secretase) and γ-secretase. Previous studies indicated that folate deficiency elevated Aβ deposition in APP/PS1 mice, and this rise was prevented by folic acid. In the present study, we aimed to investigate whether folic acid could influence the generation of Aβ by regulating α-, β-, and γ-secretase. Herein, we demonstrated that folic acid reduced the deposition of Aβ42 in APP/PS1 mice brain by decreasing the mRNA and protein expressions of β-secretase [beta-site APP-cleaving enzyme 1 (BACE1)] and γ-secretase complex catalytic component—presenilin 1 (PS1)—in APP/PS1 mice brain. Meanwhile, folic acid increased the levels of ADAM9 and ADAM10, which are important α-secretases in ADAM (a disintegrin and metalloprotease) family. However, folic acid has no impact on the protein expression of nicastrin (Nct), another component of γ-secretase complex. Moreover, folic acid regulated the expression of miR-126-3p and miR-339-5p, which target ADAM9 and BACE1, respectively. Taken together, the effect of folic acid on Aβ deposition may relate to making APP metabolism through non-amyloidogenic pathway by decreasing β-secretase and increasing α-secretase. MicroRNA (miRNA) may involve in the regulation mechanism of folic acid on secretase expression.

Low vitamin D-modulated calcium-regulating proteins in psoriasis vulgaris plaques: S100A7 overexpression depends on joint involvement.

Psoriasis is an inflammatory skin disease with or without joint involvement. In this disease, the thickened epidermis and impaired barrier are associated with altered calcium gradients. Calcium and vitamin D are known to play important roles in keratinocyte differentiation and bone metabolism. Intracellular calcium is regulated by calcium-sensing receptor (CASR), calcium release-activated calcium modulator (ORAI) and stromal interaction molecule (STIM). Other proteins modulated by vitamin D play important roles in calcium regulation e.g., calbindin 1 (CALB1) and transient receptor potential cation channel 6 (TRPV6). In this study, we aimed to investigate the expression of calcium-regulating proteins in the plaques of patients with psoriasis vulgaris with or without joint inflammation. We confirmed low calcium levels, keratinocyte hyperproliferation and an altered epidermal barrier. The CASR, ORAI1, ORAI3, STIM1, CALB1 and TRPV6 mRNA, as well as the sterol 27-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1-α-hydroxylase (CYP27B1) and 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) protein levels were low in the plaques of patients with psoriasis. We demonstrated S100 calcium-binding protein A7 (S100A7) overexpression in the plaques of patients with psoriasis vulgaris with joint inflammation, compared with those without joint involvement. We suggest an altered capacity to regulate the intracellular Ca2+ concentration ([Ca2+]i), characterized by a reduced expression of CASR, ORAI1, ORAI3, STIM1, CALB1 and TRPV6 associated with diminished levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which may be associated with an altered balance between keratinocyte proliferation and differentiation in the psoriatic epidermis. Additionally, differences in S100A7 expression depend on the presence of joint involvement.

Mapping Alzheimer's disease

Alzheimer's Disease Alzheimer's disease (AD) is a chronic neurodegenerative disorder that progresses slowly into dementia and short-term memory loss. AD is thought to be caused by aggregation of Ab and tau proteins in plaques and tangles. Vendruscolo et al. investigated why aberrant protein aggregations are found in some tissues but not in others. It turns out that failure of protein homeostasis elevates expression of a specific subset of proteins that can be aggregated in AD. Thus, a tissue vulnerability map based on Ab and tau homeostasis in healthy tissue might be predictive for AD. Sci. Adv. 10.1126.sciadv.1600947 (2016).

Abstract 5052: Pro-oncogenic role of desmosomal plaque-related proteins in non-small cell lung cancer (NSCLC)

Abstract Squamous cell carcinomas and adenocarcinomas are the most frequent forms of lung carcinoma, and have different prognoses and therapeutic approaches. During recent years, accumulating evidence has supported an important role of several junctional proteins in carcinogenesis, tumor invasion and metastasis. Contact between epithelial cells is mediated by several types of cell-cell junction, which are composed by complex arrays of transmembrane and plaque proteins and are connected typically to cytoskeletal components. Desmosomes are cell-cell complexes found primarily in epithelial tissues. In addition to the well-known structural role of desmosome genes, evidences such as their dual subcellular location, the interaction with single-stranded DNA and translation initiation factors, etc. suggest there is a poorly understood role of these proteins in signalling pathways. Desmosomal components have been traditionally considered as tumoral suppressors although current evidences suggest potential pro-oncogenic functions of desmosomal-plaque proteins, probably due to an unknown regulatory role in intracellular signaling -such as in Wnt and Akt pathways. Previously, our group showed differentially expressed gene sequences corresponding to several desmosomal plaque-related proteins as a function of tumor type, stage and differentiation grade in NSCLC. The staining pattern of some junctional proteins also differed between squamous-cell carcinomas and adenocarcinomas. Expression of these proteins marked intercellular junctions that are characteristic of the squamous stratum of stratified flattened epithelium and of neoplasias with this type of differentiation. We observed more intense staining of these proteins in better-differentiated areas. In order to gain greater insight into the specific function of some desmosomal components in NSCLC tumors, we have performed several functional assays, as cell cycle and proliferation among others, in selected SCC lung cancer cell lines. Furthermore, we are developing stable cell lines with over-expression and knockdown of some desmosomal proteins by lentiviral vectors and inducible clonal cell lines; and also carrying out knockout cell lines using CRISPR-Cas9 system. Presumably, they will allow us to perform long-term functional assays and expression arrays in order to identify subcellular location and potential pro-oncogenic roles of desmosomal proteins in NSCLC carcinogenesis which may lead to improvements in the treatment and prevention of this disease. Our preliminary results indicate some desmosomal proteins as potential targets for NSCLC diagnosis and treatment. In a future perspective, xenografts will lead to understand the molecular origins and progression of lung cancer in regard to structural components of the desmosome. Corresponding authors: pedromedina@ugr.es, efarez@ugr.es Citation Format: Pedro P. Medina, Laura Boyero, Joel Martin-Padrón, Esther Farez. Pro-oncogenic role of desmosomal plaque-related proteins in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5052.

Involvement of Tight Junction Plaque Proteins in Cancer

Here we review the molecular organization and protein–protein interactions of the following plaque proteins of tight junction (TJ): MAGI-1, -2 and -3, the polarity complex Par3/Par6/aPKC, afadin, MUPP1, PATJ, Pals1, cingulin, paracingulin, and JEAP. We analyze the status of these proteins in cancer tissue and their association to tumor suppressor proteins, kinases, and viral oncoproteins. Zonula occludens plaque proteins of the TJ are discussed separately in the preceding review within this issue. The expression of the above-mentioned TJ plaque proteins is frequently altered in cancer. However, while the loss of some of these proteins correlates with cancer development and low sensitivity to apoptosis, the overexpression of others is associated with poor patient survival rates. Some of these proteins are associated with tumor suppressor proteins like PTEN or to kinases activated in cancer like Src, while others are the target of oncoviral proteins or interact with signaling pathways involved in cell proliferation and transformation. TJ present at the apical junctional complex of epithelial cells control the passage of ions and molecules through the paracellular route and block the movement of proteins and lipids within the plasma membrane from the apical to the basolateral surfaces. TJ are constituted by integral proteins linked to a vast group of plaque proteins, which form a scaffold associated with the actomyosin cytoskeleton. In cancerous tissues some of these plaque proteins are silenced while others are overexpressed.

Distinctive Effects of Cytochalasin B in Chick Primary Myoblasts and Fibroblasts.

Actin-based structures play fundamental roles in cellular functions. However it remains controversial how cells cope with the absence of F-actin structures. This report focuses on short- and long-term effects of cytochalasin B (CB) on actin-complexes in fibroblasts and myoblasts. Thirty min of CB treatment dispersed subplasma actin cortices, lamellipodia, ruffled membranes, stress fibers and adhesion plaques into actin patches in fibroblasts and muscle cells. In contrast, 72 hrs CB treatment showed distinct morphological effects. Fibroblasts became giant multinucleated-finger shaped with 5 to 10 protrusions, 3–8 μm in width, and >200 μm in length. They lacked cortical actin, stress fibers, adhesion plaques and ruffled membranes but contained immense lamelliopodia with abnormal adhesion plaque protein complexes. Muscle cells transformed into multinucleated globular-shaped but contained normal I-Z-I and A-bands, indicating that CB did not interfere with the assembly of myofibrils. Within 30 min after CB removal, finger-shaped fibroblasts returned to their original shape and actin-containing structures rapidly reappeared, whereas muscle cells respond slowly to form elongated myotubes following CB washout. The capacity to grow, complete several nuclear cycles, assemble intermediate filaments and microtubules without a morphologically recognizable actin cytoskeleton raises interesting issues related to the role of the actin compartments in eukaryotic cells.

Cell interaction with modified nanotubes formed on titanium alloy Ti-6Al-4V

Nanotubes with diameters ranging from 40 to 60nm were prepared by electrochemical oxidation of the Ti-6Al-4V alloy in electrolyte containing ammonium sulphate and ammonium fluoride. The nanotubes were further modified with calcium and phosphate ions or were heat treated. Polished Ti-6Al-4V alloy served as a reference sample. The spreading of human osteoblast-like cells was similar on all nanotube samples but lower than on polished samples. The number of initially adhered cells was higher on non-modified nanotubes, but the final cell number was the highest on Ca-enriched nanotubes and the lowest on heat-treated nanotubes. However, these differences were relatively small and less pronounced than the differences in the concentration of specific molecular markers of cell adhesion and differentiation, estimated by their intensity of immunofluorescence staining. The concentration of vinculin, i.e. a protein of focal adhesion plaques, was the lowest on nanotubes modified with calcium. Collagen I, an early marker of osteogenic cell differentiation, was also the lowest on samples modified with calcium and was highest on polished samples. Alkaline phosphatase, a middle marker of osteogenic differentiation, was observed in lowest concentration on nanotubes modified with phosphorus and the highest on heat-treated samples. Osteocalcin concentrations, a late marker of osteogenic cell differentiation, were similar on all tested samples, although they tended to be the highest on heat-treated samples. Thus, osteogenic differentiation can be modulated by various additional treatments of nanotube coatings on Ti-6Al-4V implants.

Sympathetic Signaling Regulates Cardiomyocyte Cohesion via the Plaque Protein Plakoglobin

The intercalated disc (ICD), composed of desmosomes, adherens junctions and gap junctions, provides the structural backbone for coordinated contraction and structural integrity of the heart. Recently we have reported sympathetic signaling to regulate cell cohesion of cardiomyocytes. Increased cAMP levels strengthened desmoglein 2 (Dsg2)‐mediated adhesion paralleled with a reorganization of the ICD. Here we demonstrate this adrenergic effect to be strictly dependent on the plaque protein plakoglobin (Pg). Similar to Dsg2, Pg staining was enhanced along intercellular junctions following adrenergic stimulation, while distribution of other desmosomal proteins was not altered. Silencing Pg abrogated cAMP‐dependent enhancement of Dsg2‐mediated binding events at cell junctions as revealed by atomic force microscopy (AFM). To further investigate the role of Pg, we established a cardiac specific Pg knock‐out (Pg KO) mouse model. Deleting Pg blocked the positive effect of adrenergic stimulation on cardiomyocyte cohesion and prevented the positive inotropic and chronotropic impact of sympathetic signaling in the ex vivo Langendorff setting. Furthermore, ICDs of Pg KO hearts challenged by adrenergic stimulation were ruptured compared to challenged wild type hearts. Interestingly, in Pg KO hearts all other ICD components were present whereas Dsg2 was selectively reduced before onset of dilatation and fibrosis. These data indicate that strengthening of cell cohesion via Pg and Dsg2 is required for a sufficient response to adrenergic stimulation and maintenance of ICD integrity. Because reduction of Pg at the ICD has been reported to be a feature of arrythmogenic cardiomyopathy, this mechanism may be of high medical relevance.