Search

Endothelial monolayer integrity is required to maintain endothelial barrier functions and has found to be impaired in several disorders like inflammatory edema, allergic shock, or artherosclerosis. Under physiologic conditions in vivo, endothelial cells are exposed to mechanical forces such as hydrostatic pressure, shear stress, and cyclic stretch. However, insight into the effects of hydrostatic pressure on endothelial cell biology is very limited at present. Therefore, in this study, we tested the hypothesis that physiological hydrostatic pressure protects endothelial monolayer integrity in vitro. We investigated the protective efficacy of hydrostatic pressure in microvascular myocardial endothelial (MyEnd) cells and macrovascular pulmonary artery endothelial cells (PAECs) by the application of selected pharmacological agents known to alter monolayer integrity in the absence or presence of hydrostatic pressure. In both endothelial cell lines, extracellular [Ca.sup.2+] depletion by EGTA was followed by a loss of vascular-endothelial cadherin (VE-caherin) immunostaining at cell junctions. However, hydrostatic pressure (15 cm[H.sub.2]O) blocked this effect of EGTA. Similarly, cytochalasin D-induced actin depolymerization and intercellular gap formation and cell detachment in response to the [Ca.sup.2+]/calmodulin antagonist trifluperazine (TFP) as well as thrombin-induced cell dissociation were also reduced by hydrostatic pressure. Moreover, hydrostatic pressure significantly reduced the loss of VE-cadherin-mediated adhesion in response to EGTA, cytochalasin D, and TFP in MyEnd cells as determined by laser tweezer trapping using VE-cadherin-coated microbeads. In caveolin-1-deficient MyEnd cells, which lack caveolae, hydrostatic pressure did not protect mono-layer integrity compromised by EGTA, indicating that caveolae-dependent mechanisms are involved in hydrostatic pressure sensing and signaling. vascular-endothelial cadherin; actin

Regulation of actin dynamics is critical for endothelial barrier functions. We provide evidence that the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) is required for endothelial barrier maintenance. Baseline permeability was significantly increased in VASP-deficient ([VASP.sup.-/-]) microvascular myocardial endothelial cells (MyEnd) in the absence of discernible alterations of immunostaining for adherens and tight junctions. We tested whether VASP is involved in the endothelium-stabilizing effects of cAMP or Rac 1. Forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 (CNF-1) to activate Rac 1 were equally efficient to stabilize barrier functions in [VASP.sup.-/-] and wild-type (wt) cells. In wt cells, VASP was phosphorylated in response to F/R but did not localize to intercellular junctions. In contrast, CNF-1 and expression of constitutively active Rac 1 induced translocation of VASP to cell borders in wt cells, where it colocalized with active Rac 1. In [VASP.sup.-/-] cells, Rac 1 activity was reduced to 0.4 of wt levels in controls and increased ~20-fold in response to CNF-1 compared with 7-fold activation in wt cells. Moreover, inactivation of Rac 1 by lethal toxin led to a greater increase of permeability compared with wt cells. All these data suggest that VASP is involved in the regulation of Rac 1 activity. Taking these findings together, our study indicates that VASP at least in part stabilizes endothelial barrier functions by control of Rho-family GTPases. endothelial barrier functions; vasodilator-stimulated phosphoprotein; Rho guanosine 5'-triphosphatase; adenosine 3',5'-cyclic monophosphate

cAMP enhances endothelial barrier properties and is protective against various inflammatory mediators both in vivo and in vitro. However, the mechanisms whereby cAMP stabilizes the endothelial barrier are largely unknown. Recently we demonstrated that the Rho family GTPase Rac-1 is required for maintenance of endothelial barrier functions in vivo and in vitro. Therefore, in the present study we investigated the effect of forskolin (5 [micro]M)- and rolipram (10 [micro]M)-induced cAMP increase on reduction of barrier functions in response to Rac-1 inhibition by Clostridium sordellii lethal toxin (LT). Forskolin and rolipram treatment blocked LT (200 ng/ml)induced hydraulic conductivity ([L.sub.p]) increase in mesenteric microvessels in vivo. Likewise, LT-induced intercellular gap formation in monolayers of cultured microvascular myocardial endothelial (My-End) cells and LT-induced loss of adhesion of vascular endothelial cadherin-coated microbeads were abolished. Inhibition of PKA by myristoylated inhibitor peptide (14-22) of PKA (100 [micro]M) reduced the protective effect of cAMP on LT-induced [L.sub.p] increase in vivo and gap formation in vitro, indicating that the effect of cAMP on Rac-1 inhibition was PKA dependent. Glucosylation assays demonstrated that cAMP prevents inhibitory Rac-1 glucosylation by LT, indicating that one way that cAMP enhances endothelial barrier functions may be by regulating Rac-1 signaling. Our study suggests that cAMP may provide its well-established protective effects at least in part by regulation of Rho proteins. permeability; Rho proteins; cAMP-dependent protein kinase; vascular endothelial cadherin

We demonstrated previously that inhibition of the small GTPase Rac-1 by Clostridium sordellii lethal toxin (LT) increased the hydraulic conductivity ([L.sub.p]) of rat venular microvessels and induced gap formation in cultured myocardial endothelial cells (MyEnd). In MyEnd cells, we also demonstrated that both LT and cytochalasin D reduced cellular adhesion of vascular endothelial (VE)-cadherin-coated beads. Here we further evaluate the contribution of actin depolymerization, myosin-based contraction, and VE-cadherin linkage to the actin cytoskeleton to LT-induced permeability. The actin-depolymerizing agent cytochalasin D increased [L.sub.p] in single rat mesenteric microvessels to the same extent as LT over 80 min. However, whereas the actin-stabilizing agent jasplakinolide blunted the [L.sub.p] increase due to cytochalasin D by 78%, it had no effect on the LT response. This conforms to the hypothesis that the predominant mechanism whereby Rac-l stabilizes the endothelial barrier in intact microvessels is separate from actin polymerization and likely at the level of the VE-cadherin linkage to the actin cytoskeleton. In intact vessels, neither inhibition of contraction (butanedione monoxime, an inhibitor of myosin ATPase) nor inhibition of Rho kinase (Y-27632) modified the response to LT, even though both inhibitors lowered resting [L.sub.p]. In contrast butanedione monoxime and inhibition of myosin light chain kinase completely inhibited LT-induced intercellular gap formation and largely reduced the LT-induced permeability increase in MyEnd monolayers. These results support the hypothesis that the contractile mechanisms that contribute to the formation of large gaps between cultured endothelial cells exposed to inflammatory conditions do not significantly contribute to increased permeability in intact microvessels. permeability; Rho proteins; actin; stress fibers; vascular endothelial cadherin

Our previous experiments indicated that GTPases, other than RhoA, are important for the maintenance of endothelial barrier integrity in both intact microvessels of rats and mice and cultured mouse myocardial endothelial (MyEnd) cell monolayers (J Physiol 539: 295-308, 2002). In the present study, we inhibited the endothelial GTPase Rac by Clostridium sordellii lethal toxin (LT) and investigated the relation between the degree of inhibition of Rac by glucosylation and increased endothelial barrier permeability. In rat venular microvessels, LT (200 ng/ml) increased hydraulic conductivity from a control value of 2.5 [+ or -] 0.6 to 100.8 [+ or -] 18.7 x [10.sup.-7] cm * [s.sup.-1] * cm[H.sub.2][O.sup.-1] after 80 min. In cultured MyEnd cells exposed to LT (200 ng/ml), up to 60% of cellular Rac was glucosylated after 90 min, resulting in depolymerization of F-actin and interruptions of junctional distribution of vascular endothelial cadherin (VE-cadherin) and [beta]-catenin as well as the formation of intercellular gaps. To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion. LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not. Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion. We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton. vascular endothelial cadherin; adherens junction; permeability; Rho proteins; actin

Summary: Background: Pemphigus is a severe bullous autoimmune skin disease. Pemphigus foliaceus (PF) is characterized by antidesmoglein (Dsg) 1 IgG causing epidermal blistering; mucosal pemphigus vulgaris (mPV) by anti‐Dsg3 IgG inducing erosions in the mucosa; and mucocutaneous pemphigus vulgaris (PV) by affecting both, with autoantibodies targeting Dsg1 and Dsg3. Objectives: To characterize the Ca2+ flux pathway and delineate its importance in pemphigus pathogenesis and clinical phenotypes caused by different antibody profiles. Methods: Immunoprecipitation, Ca2+ flux analysis, Western blotting, immunofluorescence staining, dissociation assays and a human skin ex vivo model were used. Results: PV IgG and PF IgG, but neither Dsg3‐specific monoclonal antibody (AK23) nor mPV IgG, caused Ca2+ influx in primary human keratinocytes. Phosphatidylinositol 4‐kinase α interacts with Dsg1 but not with Dsg3. Its downstream target – phospholipase‐C‐γ1 (PLC) – was activated by PV IgG and PF IgG but not AK23 or mPV IgG. PLC releases inositol 1,4,5‐trisphosphate (IP3) causing IP3 receptor (IP3R) activation and Ca2+ flux from the endoplasmic reticulum into the cytosol, which stimulates Ca2+ release‐activated channels (CRAC)‐mediated Ca2+ influx. Inhibitors against PLC, IP3R and CRAC effectively blocked PV IgG and PF IgG‐induced Ca2+ influx; ameliorated alterations of Dsg1 and Dsg3 localization, and reorganization of keratin and actin filaments; and inhibited loss of cell adhesion in vitro. Finally, inhibiting PLC or IP3R was protective against PV IgG‐induced blister formation and redistribution of Dsg1 and Dsg3 in human skin ex vivo. Conclusions: Ca2+‐mediated signalling is important for epidermal blistering and dependent on the autoantibody profile, which indicates different roles for signalling complexes organized by Dsg1 and Dsg3. Interfering with PLC and Ca2+ signalling may be a promising approach to treat epidermal manifestations of pemphigus. What is already known about this topic?Autoantibody‐induced Ca2+ signalling and activation of phospholipase C in keratinocytes has been reported as the first signalling pathway in pemphigus.Ca2+ influx correlates with the presence of autoantibodies targeting desmoglein (Dsg) 1.The role and exact mechanisms of the Ca2+ signalling pathway and its role in pemphigus pathology are unclear. What does this study add?There is an important role of the phospholipase C/Ca2+ signalling pathway in the pathogenesis of pemphigus.Relevant signal components of the Ca2+ pathway are characterized in detail.Dsg1 and Dsg3 are found to interact with signalling molecules, organizing signalling complexes that differ with respect to the presence of phosphatidylinositol 4‐kinase α, the most upstream activator of the Ca2+‐influx pathway, assigning different roles to Dsg1‐ and Dsg3‐mediated signalling. What is the translational message?Current therapies for pemphigus have a delayed onset of action. Rapidly effective therapeutic approaches that directly stabilize desmosomal adhesion are desirable.While targeting the Ca2+ flux is problematic, the role of phospholipase C in the influx identifies this molecule as a potential target for specific molecular therapy approaches.Elucidating the mechanism of Dsg1‐dependent regulation of Ca2+ influx in keratinocytes helps us to understand the different blistering patterns and the phenotypic variability seen in pemphigus. Plain language summary available online [ABSTRACT FROM AUTHOR]

Summary: Background: Pemphigus vulgaris (PV) is an autoimmune disease characterized by blister formation in the epidermis and oral mucosa due to loss of keratinocyte cohesion. Autoantibodies present in patients with PV (PV‐IgG) are known to primarily target desmoglein (Dsg)1 and Dsg3 in desmosomes. The mucosal‐dominant subtype of PV (mdPV) is caused by PV‐IgG autoantibodies against the cadherin‐type adhesion molecule Dsg3. p38 mitogen‐activated protein kinase (p38MAPK) signalling has been characterized as an important pathway downstream of PV‐IgG binding and its inhibition is protective in ex vivo human skin. However, the role of p38MAPK signalling in mdPV is unknown as no experimental model has been available. Objectives: To establish a human ex vivo oral mucosa culture, and evaluate the p38MAPK dependency of blister formation and of ultrastructural alterations of desmosomes induced by mdPV‐IgG. Methods: Human labial mucosa was injected with mdPV‐IgG as well as AK23, a pathogenic mouse monoclonal Dsg3 antibody, in the presence or absence of p38MAPK inhibitors. Viability was evaluated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and apoptosis by terminal deoxynucleotidyl transferase dUTP nick‐end labelling assay. Blister score was determined following haematoxylin and eosin staining and Dsg3 distribution by immunostaining. Samples were processed for transmission electron microscopy to analyse desmosome ultrastructure. Results: Both AK23 and mdPV‐IgG induced blisters and caused reduction in desmosome size and number in labial mucosa. Inhibition of p38MAPK was not effective in preventing these alterations. Conclusions: In contrast with human epidermis, PV‐IgG and AK23 induce blisters and desmosome ultrastructural changes in labial mucosa via a mechanism not dependent on p38MAPK. What's already known about this topic? Pemphigus vulgaris IgG (PV‐IgG) induces blistering as well as a reduction in desmosome number and size mediated by p38 mitogen‐activated protein kinase (p38MAPK) signalling in ex vivo human skin. What does this study add? This study establishes a new human ex vivo mucosa model to test pathomechanisms mediated by PV‐IgG.The study demonstrates that both AK23 and mucosal‐dominant PV induce blisters and associated ultrastructural changes in labial mucosa via a mechanism not dependent on p38MAPK signalling. What is the translational message? This study highlights the respective tissue‐specific responses of oral mucosa and skin related to PV pathogenesis, similar to the patient situation. Respond to this article Linked Comment:Ludwig. Br J Dermatol 2020; 182:833–834. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pemphigus is a severe blistering disease caused by autoantibodies primarily against the desmosomal cadherins desmoglein (DSG)1 and DSG3, which impair desmosome integrity. Especially for the acute phase, additional treatment options allowing to reduce corticosteroids would fulfill an unmet medical need. In this study, we provide evidence that EGFR inhibition by erlotinib ameliorates pemphigus vulgaris IgG-induced acantholysis in intact human epidermis. Pemphigus vulgaris IgG caused phosphorylation of EGFR (Y845) and Rous sarcoma-related kinase in human epidermis. In line with this, a phosphotyrosine kinome analysis revealed a robust response associated with EGFR and Rous sarcoma-related kinase family kinase signaling in response to pemphigus vulgaris IgG but not to pemphigus foliaceus autoantibodies. Erlotinib inhibited pemphigus vulgaris IgG-induced epidermal blistering and EGFR phosphorylation, loss of desmosomes, as well as ultrastructural alterations of desmosome size, plaque symmetry, and keratin filament insertion and restored the desmosome midline considered as hallmark of mature desmosomes. Erlotinib enhanced both single-molecule DSG3-binding frequency and strength and delayed DSG3 fluorescence recovery, supporting that EGFR inhibition increases DSG3 availability and cytoskeletal anchorage. Our data indicate that EGFR is a promising target for pemphigus therapy owing to its link to several signaling pathways known to be involved in pemphigus pathogenesis., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Patients with eosinophilic esophagitis (EoE) require long-lasting resolution of inflammation to prevent fibrostenosis and dysphagia. However, the dissociation between symptoms and histologic improvement suggests persistent molecular drivers despite histologic remission., Objective: We characterized persisting molecular alterations in pediatric patients with EoE using tissue transcriptomics and proteomics., Methods: Esophageal biopsy samples (n = 247) collected prospectively during 189 endoscopies from pediatric patients with EoE (n = 36, up to 11 follow-up endoscopies) and pediatric controls (n = 44, single endoscopies) were subjected to bulk transcriptomics (n = 96) and proteomics (n = 151). Intercellular junctions (desmoglein-1/3, desmoplakin, E-cadherin) and epithelial-to-mesenchymal transition (vimentin:E-cadherin ratio) were assessed by immunofluorescence staining., Results: Active EoE (≥15 eosinophils per high-power field [eos/hpf]), inactive EoE (<15 eos/hpf), and deep-remission EoE (0 eos/hpf) were diagnosed in 107 of 185, 78 of 185, and 41 of 185 biopsy samples, respectively. Among the dysregulated genes (up-/downregulated 310/112) and proteins (up-/downregulated 68/16) between active EoE and controls, 17 genes, and 6 proteins remained dysregulated in inactive EoE. Using persistently upregulated genes (n = 9) and proteins (n = 3) only, such as ALOX15, CXCL1, CXCL6, CTSG, CDH26, PRRX1, CLC, EPX, and periostin (POSTN), was sufficient to separate inactive EoE and deep-remission biopsy samples from control tissue. While 32 differentially expressed genes persisted in deep-remission EoE compared to controls, the proteome normalized except for persistently upregulated POSTN. Epithelial-to-mesenchymal transition normalized in inactive EoE, whereas desmosome recovery remained impaired as a result of desmoglein-1 downregulation., Conclusion: The analysis of molecular changes shows persistent EoE-associated esophageal dysregulation despite histologic remission. These data expand our understanding of inflammatory processes and possible mechanisms that underlie tissue remodeling in EoE., Competing Interests: Disclosure statement Supported by “Förderprogramm für Forschung und Lehre (FöFoLe)” of the medical faculty, LMU Munich; and the German Research Foundation (DFG; SFB 1371-395357507). The funding source was not involved in the study design, collection, analysis, or interpretation of data or the preparation of the report and its publication. All data supporting our findings are available here and in the Online Repository. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with dataset identifier PXD052250.48 (www.ebi.ac.uk/pride/archive/projects/PXD052250). Raw RNA sequencing data are available on reasonable request from the corresponding author. The data are protected and not publicly available because the research participants are children. Disclosure of potential conflict of interest: T. Schwerd reports receipt of received lecture honoraria from Nutricia and MSD, personal honorarium as member of an advisory board from AstraZeneca, and travel support from AbbVie and Ferring outside the submitted work. H. Hoelz reports receipt of lecture honoraria from Nutricia outside the submitted work. S. Koletzko reports receipt of fees as a member of advisory boards or as speaker from AbbVie, AstraZeneca, Danone, Janssen, Mead Johnson, Nestlé Nutrition, Pfizer, Sanofi, and Takeda outside the submitted work. M. List reports consulting for mbiomics GmbH. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The contribution of Erk1/2 to endothelial barrier regulation is convoluted and differs depending on the vascular bed. We explored the effects of Erk1/2 inhibition on endothelial barrier maintenance and its relationship with cAMP-dependent barrier strengthening. Thus, myocardial endothelial cells (MyEnd) were isolated and protein expression, localization and activity of structural and signaling molecules involved in maintenance of endothelial function were investigated by Western blot, immunostainings and G-LISA, respectively. The transendothelial electrical resistance (TEER) from confluent MyEnd monolayers was measured and used as a direct indicator of barrier integrity in vitro. Miles assay was performed to evaluate vascular permeability in vivo. Erk1/2 inhibition with U0126 affected neither the structural organization of adherens or tight junctions nor the protein level of their components, However, TEER drop significantly upon U0126 application, but the effect was transitory as the barrier function recovered 30 min after treatment. Erk1/2 inhibition delayed cAMP-mediated barrier strengthening but did not prevent barrier fortification despite diminishing Rac1 activation. Moreover, Erk1/2 inhibition, induced vascular leakage that could be prevented by local cAMP elevation in vivo. Our data demonstrate that Erk1/2 is required to prevent vascular permeability but is not critical for cAMP-mediated barrier enhancement.

Endodontic access preparation is one of the initial steps in root canal treatments and can be hindered by the obliteration of pulp canals and formation of tertiary dentin. Until now, methods for direct intraoperative visualization of the 3-dimensional anatomy of teeth have been missing. Here, we evaluate the use of shortwave infrared radiation (SWIR) for navigation during stepwise access preparation. Nine teeth (3 anteriors, 3 premolars, and 3 molars) were explanted en bloc with intact periodontium including alveolar bone and mucosa from the upper or lower jaw of human body donors. Analysis was performed at baseline as well as at preparation depths of 5 mm, 7 mm, and 9 mm, respectively. For reflection, SWIR was used at a wavelength of 1,550 nm from the occlusal direction, whereas for transillumination, SWIR was passed through each sample at the marginal gingiva from the buccal as well as oral side at a wavelength of 1,300 nm. Pulpal structures could be identified as darker areas approximately 2 mm before reaching the pulp chamber using SWIR transillumination, although they were indistinguishable under normal circumstances. Furcation areas in molars appeared with higher intensity than areas with canals. The location of pulpal structures was confirmed by superimposition of segmented micro-computed tomography (µCT) images. By radiomic analysis, significant differences between pulpal and parapulpal areas could be detected in image features. With hierarchical cluster analysis, both segments could be confirmed and associated with specific clusters. The local thickness of µCTs was calculated and correlated with SWIR transillumination images, by which a linear dependency of thickness and intensity could be demonstrated. Lastly, by in silico simulations of light propagation, dentin tubules were shown to be a crucial factor for understanding the visibility of the pulp. In conclusion, SWIR transillumination may allow direct clinical live navigation during endodontic access preparation., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: This article summarizes a global study of the effect of the COVID-19 pandemic on junior health professions students' outlook on medicine. The pandemic has significantly affected health professions education. There is limited understanding of how students' pandemic experiences will affect them, and what impact these events may have on their career paths or the future of the professions. This information is important as it impacts the future of medicine., Methods: In the Fall 2020 semester, 219 health professions students at 14 medical universities worldwide responded to the question: 'Has this experience (with COVID-19) changed your outlook on medicine as a profession?'. Short essay responses were semantically coded and organized into themes and subthemes using an inductive approach to thematic analysis., Results: 145 responses were submitted. Themes were identified: (1) students reflected on the interaction between politics and healthcare; (2) reported becoming more aware of the societal expectations placed on healthcare professionals, including undertaking high risks and the sacrifices that healthcare professionals must make; (3) found reassurance from the recognized importance of healthcare professionals and expressed pride to be entering the profession; and (4) reflected on the current state of healthcare, including its limitations and future., Conclusion: Most students, independent of the extent of the pandemic in their respective countries, noted a change in their outlook regarding medicine. An overall positive outlook was noted in most junior students. Educators need to work on nurturing these sentiments and attitudes to help young students maintain a healthy relationship towards their chosen profession.

Vascular permeability is mediated by Cortactin (Cttn) and regulated by several molecules including cyclic-adenosine-monophosphate, small Rho family GTPases and the actin cytoskeleton. However, it is unclear whether Cttn directly interacts with any of the junctional components or if Cttn intervenes with signaling pathways affecting the intercellular contacts and the cytoskeleton. To address these questions, we employed immortalized microvascular myocardial endothelial cells derived from wild-type and Cttn-knock-out mice. We found that lack of Cttn compromised barrier integrity due to fragmented membrane distribution of different junctional proteins. Moreover, immunoprecipitations revealed that Cttn is within the VE-cadherin-based adherens junction complex. In addition, lack of Cttn slowed-down barrier recovery after Ca 2+ repletion. The role of Cttn for cAMP-mediated endothelial barrier regulation was analyzed using Forskolin/Rolipram. In contrast to Cttn-KO, WT cells reacted with increased transendothelial electrical resistance. Absence of Cttn disturbed Rap1 and Rac1 activation in Cttn-depleted cells. Surprisingly, despite the absence of Cttn, direct activation of Rac1/Cdc42/RhoA by CN04 increased barrier resistance and induced well-defined cortical actin and intracellular actin bundles. In summary, our data show that Cttn is required for basal barrier integrity by allowing proper membrane distribution of junctional proteins and for cAMP-mediated activation of the Rap1/Rac1 signaling pathway., (© 2024. The Author(s).)