cAMP: A master regulator of cadherin‐mediated binding in endothelium, epithelium and myocardium

Regulation of cadherin-mediated cell adhesion is crucial not only for maintaining tissue integrity and barrier function in the endothelium and epithelium but also for electromechanical coupling within the myocardium. Therefore, loss of cadherin-mediated adhesion causes various disorders, including vascular inflammation and desmosome-related diseases such as the autoimmune blistering skin dermatosis pemphigus and arrhythmogenic cardiomyopathy. Mechanisms regulating cadherin-mediated binding contribute to the pathogenesis of diseases and may also be used as therapeutic targets. Over the last 30 years, cyclic adenosine 3′,5′-monophosphate (cAMP) has emerged as one of the master regulators of cell adhesion in endothelium and, more recently, also in epithelial cells as well as in cardiomyocytes. A broad spectrum of experimental models from vascular physiology and cell biology applied by different generations of researchers provided evidence that not only cadherins of endothelial adherens junctions (AJ) but also desmosomal contacts in keratinocytes and the cardiomyocyte intercalated discs are central targets in this scenario. The molecular mechanisms involve protein kinase A- and exchange protein directly activated by cAMP-mediated regulation of Rho family GTPases and S665 phosphorylation of the AJ and desmosome adaptor protein plakoglobin. In line with this, phosphodiesterase 4 inhibitors such as apremilast have been proposed as a therapeutic strategy to stabilize cadherin-mediated adhesion in pemphigus and may also be effective to treat other disorders where cadherin-mediated binding is compromised

Role of Dsg1-and Dsg3-Mediated Signaling in Pemphigus Autoantibody-Induced Loss of Keratinocyte Cohesion

Pemphigus is an autoimmune dermatosis in which mucocutaneous blisters are induced primarily by autoantibodies against Desmoglein (Dsg) 1 and 3. Pemphigus vulgaris (PV) usually is associated with autoantibodies against Dsg3 whereas pemphigus foliaceus (PF) patients present autoantibodies against Dsg1. Several signaling pathways were proposed to cause loss of keratinocyte adhesion. However, relevance of different signaling pathways and role of Dsg1 and 3 to trigger signaling are not fully understood. Here, we show that Ca2+ chelation reduced PV-IgG-and PF-IgG-mediated loss of HaCaT keratinocyte cohesion whereas EGFR inhibition did not inhibit effects of PF-IgG. PV-IgG activated EGFR in a Src-dependent manner whereas both PV-IgG and PF-IgG caused Ca2+ influx independent of EGFR. ERK activation was Src-dependent in response to PV-IgG but not PF-IgG. To delineate the roles of Dsg isoforms to trigger signaling pathways, Dsg3-and Dsg2-deficient HaCaT keratinocyte cell lines were generated using CRISPR/Cas9. Dsg3-but not Dsg2-deficient cells were protected against PV-IgG-induced loss of cell adhesion. Ca2+ influx and ERK activation in response to PF-IgG were preserved in both cell lines

Dsg1 and Dsg3 Composition of Desmosomes Across Human Epidermis and Alterations in Pemphigus Vulgaris Patient Skin

Desmosomes are important epidermal adhesion units and signalling hubs, which play an important role in pemphigus pathogenesis. Different expression patterns of the pemphigus autoantigens desmoglein (Dsg)1 and Dsg3 across different epidermal layers have been demonstrated. However, little is known about changes in desmosome composition in different epidermal layers or in patient skin. The aim of this study was thus to characterize desmosome composition in healthy and pemphigus skin using super-resolution microscopy. An increasing Dsg1/Dsg3 ratio from lower basal (BL) to uppermost granular layer (GL) was observed. Within BL desmosomes, Dsg1 and Dsg3 were more homogeneously distributed whereas superficial desmosomes mostly comprised one of the two molecules or domains containing either one but not both. Extradesmosomal, desmoplakin (Dp)-independent, co-localization of Dsg3 with plakoglobin (Pg) was found mostly in BL and extradesmosomal Dsg1 co-localization with Pg in all layers. In contrast, in the spinous layer (SL) most Dsg1 and Dsg3 staining was confined to desmosomes, as revealed by the co-localization with Dp. In pemphigus patient skin, Dsg1 and Dsg3 immunostaining was altered especially along blister edges. The number of desmosomes in patient skin was reduced significantly in basal and spinous layer keratinocytes with only few split desmosomes found. In addition, Dsg1-Pg co-localization at the apical BL and Dsg3-Pg co-localization in SL were significantly reduced in patients, suggesting that that extradesmosomal Dsg molecules were affected. These results support the hypothesis that pemphigus is a desmosome assembly disease and may help to explain histopathologic differences between pemphigus phenotypes

Atomic Force Microscopy Provides New Mechanistic Insights into the Pathogenesis of Pemphigus

Autoantibodies binding to the extracellular domains of desmoglein (Dsg) 3 and 1 are critical in the pathogenesis of pemphigus by mechanisms leading to impaired function of desmosomes and blister formation in the epidermis and mucous membranes. Desmosomes are highly organized protein complexes which provide strong intercellular adhesion. Desmosomal cadherins such as Dsgs, proteins of the cadherin superfamily which interact via their extracellular domains in Ca2+ -dependent manner, are the transmembrane adhesion molecules clustered within desmosomes. Investigations on pemphigus cover a wide range of experimental approaches including biophysical methods. Especially atomic force microscopy (AFM) has recently been applied increasingly because it allows the analysis of native materials such as cultured cells and tissues under near-physiological conditions. AFM provides information about the mechanical properties of the sample together with detailed interaction analyses of adhesion molecules. With AFM, it was recently demonstrated that autoantibodies directly inhibit Dsg interactions on the surface of living keratinocytes, a phenomenon which has long been considered the main mechanism causing loss of cell cohesion in pemphigus. In addition, AFM allows to study how signaling pathways altered in pemphigus control binding properties of Dsgs. More general, AFM and other biophysical studies recently revealed the importance of keratin filaments for regulation of Dsg binding and keratinocyte mechanical properties. In this mini-review, we reevaluate AFM studies in pemphigus and keratinocyte research, recapitulate what is known about the interaction mechanisms of desmosomal cadherins and discuss the advantages and limitations of AFM in these regards

Dsg2 Upregulation as a Rescue Mechanism in Pemphigus

In pemphigus vulgaris (PV), autoantibodies directed against the desmosomal cadherin desmoglein (Dsg) 3 cause loss of intercellular adhesion. It is known that Dsg3 interactions are directly inhibited by autoantibody binding and that Dsg2 is upregulated in epidermis of PV patients. Here, we investigated whether heterophilic Dsg2-Dsg3 interactions occur and would modulate PV pathogenesis. Dsg2 was upregulated in PV patients’ biopsies and in a human ex vivo pemphigus skin model. Immunoprecipitation and cell-free atomic force microscopy (AFM) experiments demonstrated heterophilic Dsg2-Dsg3 interactions. Similarly, in Dsg3-deficient keratinocytes with severely disturbed intercellular adhesion Dsg2 was upregulated in the desmosome containing fraction. AFM revealed that Dsg2-Dsg3 heterophilic interactions showed binding frequency, strength, Ca2+-dependency and catch-bond behavior comparable to homophilic Dsg3-Dsg3 or homophilic Dsg2-Dsg2 interactions. However, heterophilic Dsg2-Dsg3 interactions had a longer lifetime compared to homophilic Dsg2-Dsg2 interactions and PV autoantibody-induced direct inhibition was significantly less pronounced for heterophilic Dsg2-Dsg3 interactions compared to homophilic Dsg3 interactions. In contrast, a monoclonal anti-Dsg2 inhibitory antibody reduced heterophilic Dsg2-Dsg3 and homophilic Dsg2-Dsg2 binding to the same degree and further impaired intercellular adhesion in Dsg3-deficient keratinocytes. Taken together, the data demonstrate that Dsg2 undergoes heterophilic interactions with Dsg3, which may attenuate autoantibody-induced loss of keratinocyte adhesion in pemphigus

Water-content related alterations in macro and micro scale tendon biomechanics

Though it is known that the water content of biological soft tissues alters mechanical properties, little attempt has been made to adjust the tissue water content prior to biomechanical testing as part of standardization procedures. The objective of this study was to examine the effects of altered water content on the macro and micro scale mechanical tissues properties. Human iliotibial band samples were obtained during autopsies to osmotically adapt their water content. Macro mechanical tensile testing of the samples was conducted with digital image correlation, and micro mechanical tests using atomic force microscopy. Analyses were conducted for elastic moduli, tensile strength, and strain at maximum force, and correlations for water content, anthropometric data, and post-mortem interval. Different mechanical properties exist at different water concentrations. Correlations to anthropometric data are more likely to be found at water concentrations close to the native state. These data underline the need for adapting the water content of soft tissues for macro and micro biomechanical experiments to optimize their validity. The osmotic stress protocol provides a feasible and reliable standardization approach to adjust for water content-related differences induced by age at death, post-mortem interval and tissue processing time with known impact on the stress-strain properties

Biomechanical Properties of the Internal Limiting Membrane after Intravitreal Ocriplasmin Treatment

Purpose: To assess the stiffness of the human internal limiting membrane (ILM) and evaluate potential changes of mechanical properties following intravitreal ocriplasmin injection for vitreomacular traction. Methods: This is an interventional comparative case series of 12 surgically excised ILM specimens consecutively obtained from 9 eyes of 9 patients after unsuccessful pharmacologic vitreolysis with ocriplasmin. During the same time period, 16 specimens from 13 other eyes without ocriplasmin treatment were harvested during vitrectomy and served as controls. All patients presented with macular holes or vitreomacular traction and underwent vitrectomy with ILM peeling either with or without brilliant blue (BB) staining. All specimens were analyzed using atomic force microscopy with scan regions of 25 x 25 mu m. In all specimens, both the retinal side and vitreal side of the ILM were analyzed. Results: Atomic force microscopy revealed no significant differences in elasticity of ILM specimens removed from eyes with or without ocriplasmin treatment. Undulated areas of the retinal side presented stiffer than the vitreal side of the ILM. Topographical mapping of both the vitreal and retinal side of the ILM showed no apparent alteration of the morphology in ocriplasmin-treated eyes compared to untreated eyes. Staining with BB resulted in an increase of tissue stiffness. Conclusions: Intravitreal injection of ocriplasmin does not change biomechanical properties of the human ILM. There is no evidence of a potential enzymatic effect of ocriplasmin interfering with the stiffness of this basement membrane. (C) 2016 S. Karger AG, Base

Desmosomal Hyper-adhesion Affects Direct Inhibition of Desmoglein Interactions in Pemphigus

During differentiation, keratinocytes acquire a strong, hyper-adhesive state, where desmosomal cadherins interact calcium ion independently. Previous data indicate that hyper-adhesion protects keratinocytes from pemphigus vulgaris autoantibody–induced loss of intercellular adhesion, although the underlying mechanism remains to be elucidated. Thus, in this study, we investigated the effect of hyper-adhesion on pemphigus vulgaris autoantibody–induced direct inhibition of desmoglein (DSG) 3 interactions by atomic force microscopy. Hyper-adhesion abolished loss of intercellular adhesion and corresponding morphological changes of all pathogenic antibodies used. Pemphigus autoantibodies putatively targeting several parts of the DSG3 extracellular domain and 2G4, targeting a membrane-proximal domain of DSG3, induced direct inhibition of DSG3 interactions only in non-hyper-adhesive keratinocytes. In contrast, AK23, targeting the N-terminal extracellular domain 1 of DSG3, caused direct inhibition under both adhesive states. However, antibody binding to desmosomal cadherins was not different between the distinct pathogenic antibodies used and was not changed during acquisition of hyper-adhesion. In addition, heterophilic DSC3–DSG3 and DSG2–DSG3 interactions did not cause reduced susceptibility to direct inhibition under hyper-adhesive condition in wild-type keratinocytes. Taken together, the data suggest that hyper-adhesion reduces susceptibility to autoantibody-induced direct inhibition in dependency on autoantibody-targeted extracellular domain but also demonstrate that further mechanisms are required for the protective effect of desmosomal hyper-adhesion in pemphigus vulgaris