Hans Schnittler

Actin filament dynamics and endothelial cell junctions: the Ying and Yang between stabilization and motion

The vascular endothelium is a cellular interface between the blood and the interstitial space of tissue, which controls the exchange of fluid, solutes and cells by both transcellular and paracellular means. To accomplish the demands on barrier function, the regulation of the endothelium requires quick and adaptive mechanisms. This is, among others, accomplished by actin dynamics that interdependently interact with both the VE-cadherin/catenin complex, the main components of the adherens type junctions in endothelium and the membrane cytoskeleton. Actin filaments in endothelium are components of super-structured protein assemblies that control a variety of dynamic processes such as endo- and exocytosis, shape change, cell–substrate along with cell–cell adhesion and cell motion. In endothelium, actin filaments are components of: (1) contractile actin bundles appearing as stress fibers and junction-associated circumferential actin filaments, (2) actin networks accompanied by endocytotic ruffles, lamellipodia at leading edges of migrating cells and junction-associated intermittent lamellipodia (JAIL) that dynamically maintain junction integrity, (3) cortical actin and (4) the membrane cytoskeleton. All these structures, most probably interact with cell junctions and cell–substrate adhesion sites. Due to the rapid growth in information, we aim to provide a bird’s eye view focusing on actin filaments in endothelium and its functional relevance for entire cell and junction integrity, rather than discussing the detailed molecular mechanism for control of actin dynamics.

Interplay between neural-cadherin and vascular endothelial-cadherin in breast cancer progression

IntroductionDeregulation of cadherin expression, in particular the loss of epithelial (E)-cadherin and gain of neural (N)-cadherin, has been implicated in carcinoma progression. We previously showed that endothelial cell-specific vascular endothelial (VE)-cadherin can be expressed aberrantly on tumor cells both in human breast cancer and in experimental mouse mammary carcinoma. Functional analyses revealed that VE-cadherin promotes tumor cell proliferation and invasion by stimulating transforming growth factor (TGF)-β signaling. Here, we investigate the functional interplay between N-cadherin and VE-cadherin in breast cancer.MethodsThe expression of N-cadherin and VE-cadherin was evaluated by immunohistochemistry in a tissue microarray with 84 invasive human breast carcinomas. VE-cadherin and N-cadherin expression in mouse mammary carcinoma cells was manipulated by RNA interference or overexpression, and cells were then analyzed by immunofluorescence, reverse transcriptase-polymerase chain reaction, and western blot. Experimental tumors were generated by transplantation of the modified mouse mammary carcinoma cells into immunocompetent mice. Tumor growth was monitored, and tumor tissue was subjected to histological analysis.ResultsVE-cadherin and N-cadherin were largely co-expressed in invasive human breast cancers. Silencing of N-cadherin in mouse mammary carcinoma cells led to decreased VE-cadherin expression and induced changes indicative of mesenchymal-epithelial transition, as indicated by re-induction of E-cadherin, localization of β-catenin at the cell membrane, decreased expression of vimentin and SIP1, and gain of epithelial morphology. Suppression of N-cadherin expression also inhibited tumor growth in vivo, even when VE-cadherin expression was forced.ConclusionsOur results highlight the critical role of N-cadherin in breast cancer progression and show that N-cadherin is involved in maintaining the malignant tumor cell phenotype. The presence of N-cadherin prevents the re-expression of E-cadherin and localization of β-catenin at the plasma membrane of mesenchymal mammary carcinoma cells. N-cadherin is also required to maintain the expression of VE-cadherin in malignant tumor cells but not vice versa. Thus, N-cadherin acts in concert with VE-cadherin to promote tumor growth.

From basic mechanisms to clinical applications in heart protection, new players in cardiovascular diseases and cardiac theranostics: meeting report from the third international symposium on "New frontiers in cardiovascular research"

In this meeting report, particularly addressing the topic of protection of the cardiovascular system from ischemia/reperfusion injury, highlights are presented that relate to conditioning strategies of the heart with respect to molecular mechanisms and outcome in patients’ cohorts, the influence of co-morbidities and medications, as well as the contribution of innate immune reactions in cardioprotection. Moreover, developmental or systems biology approaches bear great potential in systematically uncovering unexpected components involved in ischemia–reperfusion injury or heart regeneration. Based on the characterization of particular platelet integrins, mitochondrial redox-linked proteins, or lipid-diol compounds in cardiovascular diseases, their targeting by newly developed theranostics and technologies opens new avenues for diagnosis and therapy of myocardial infarction to improve the patients’ outcome.

Meeting report from the 2nd International Symposium on New Frontiers in Cardiovascular Research. Protecting the cardiovascular system from ischemia: between bench and bedside

Recent advances in basic cardiovascular research as well as their translation into the clinical situation were the focus at the last “New Frontiers in Cardiovascular Research meeting”. Major topics included the characterization of new targets and procedures in cardioprotection, deciphering new players and inflammatory mechanisms in ischemic heart disease as well as uncovering microRNAs and other biomarkers as versatile and possibly causal factors in cardiovascular pathogenesis. Although a number of pathological situations such as ischemia–reperfusion injury or atherosclerosis can be simulated and manipulated in diverse animal models, also to challenge new drugs for intervention, patient studies are the ultimate litmus test to obtain unequivocal information about the validity of biomedical concepts and their application in the clinics. Thus, the open and bidirectional exchange between bench and bedside is crucial to advance the field of ischemic heart disease with a particular emphasis of understanding long-lasting approaches in cardioprotection.

Genetic manipulation of endothelial cells by viral vectors

The need for uncovering molecular mechanisms in endothelial cell biology has tremendously increased in the last decades as it became more and more clear that the endothelium is an important target in nearly all diseases and treatments (drug delivery) and plays a central role in regeneration processes. One of the critical methods generally applied in cell biology research to uncover structural and functional aspects is the modulation of protein expression by over-expression, expression of mutant variants or gene silencing. This strategy, however, requires genetic manipulation of the respective cells. The classical gene transfer by chemical transfection techniques works pretty well in a large variety of cultured cells but fails for most endothelial cell types. Insufficient transfection rates and gene expression levels as well as the sensitivity of the endothelium against chemical transfection reagents limits utilisation of this technique for endothelial cell biology research. This holds true not only for primary endothelial cell cultures and endothelial cells in vivo but also for endothelial cell lines, e.g. endothelioma cells. The development of viral vectors originally designed for gene therapy approaches has significantly improved the methodological spectrum in endothelial cell research. Two viral vector systems, based on retroviruses and adenoviruses, deliver transgenic information highly efficient into both cultured endothelial cells and in endothelial cells in vivo, respectively. This review aims to give a comprehensive overview of these two vector systems that appear to be reliable and efficient tools for gene delivery into endothelial cell types.

Imbalance of SMC1 and SMC3 Cohesins Causes Specific and Distinct Effects

SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.

Targeting of NADPH oxidase in vitro and in vivo suppresses fibroblast activation and experimental skin fibrosis.

Although there is increasing evidence that oxidative stress is involved in collagen synthesis and myofibroblast activation, the NADPH oxidase (Nox) system is incompletely investigated in the context of human dermal fibroblasts (HDFs) and skin fibrosis. Using the pan‐Nox inhibitor diphenyleneiodonium (DPI) as an initial tool, we show that gene expression of collagen type I, α‐smooth muscle actin (α‐SMA) and fibronectin 1 is suppressed in HDFs. Detailed expression analysis of all Nox isoforms and adaptors revealed expression of RNA and protein expression of Nox4, p22phox and Poldip2 but neither Nox1 nor Nox2. Nox4 could be immunolocalized to the endoplasmic reticulum. Importantly, TGF‐β1 had a dose‐ and time‐dependent upregulating effect on NADH activity and Nox4 gene expression in HDFs. Genetic silencing of Nox4 as demonstrated by siRNA in HDFs as well as in murine fibroblasts established from Nox4 knockout mice confirmed that TGF‐β1‐mediated collagen type I gene, α‐SMA and fibronectin 1 gene expressions were Nox4‐dependent. This TGF‐β1 effect was mediated by Smad3 as shown by in silico promoter analysis, pharmacological inhibition and gene silencing of Smad3. The relevance of these findings is highlighted in the bleomycin‐induced scleroderma mouse model. DPI treatment attenuated skin fibrosis and myofibroblast activation. Moreover, Nox4 knockdown by siRNA reduced skin collagen synthesis, α‐SMA and fibronectin 1 expression in vivo. Finally, analyses of HDFs from patients with systemic sclerosis confirmed the expression of Nox4 and its adaptors, whereas Nox1 and Nox2 were not detectable. Our findings indicate that Nox4 targeting is a promising future treatment for fibrotic skin diseases.

Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.The formation of new blood vessels requires both polarized cell migration and coordinated control of endothelial cell contacts. Here, Cao and colleagues describe at the sub-cellular level the cytoskeletal and cell junction dynamics regulating these processes upon VEGF-induced cell elongation.

Between microbial attack and defence: The endothelium as a vulnerable player in infectious diseases

Between microbial attack and defence: The endothelium as a vulnerable player in infectious diseases -