Pia Jeggle

Epithelial sodium channel stiffens the vascular endothelium in vitro and in Liddle mice.

Liddle syndrome, an inherited form of hypertension, is caused by gain-of-function mutations in the epithelial Na+ channel (ENaC), the principal mediator of Na+ reabsorption in the kidney. Accordingly, the disease pathology was ascribed to a primary renal mechanism. Whether this is the sole responsible mechanism, however, remains uncertain as dysregulation of ENaC in other tissues may also be involved. Previous work indicates that ENaC in the vascular endothelium is crucial for the regulation of cellular mechanics and thus vascular function. The hormone aldosterone has been shown to concomitantly increase ENaC surface expression and stiffness of the cell cortex in vascular endothelial cells. The latter entails a reduced release of the vasodilator nitric oxide, which eventually leads to an increase in vascular tone and blood pressure. Using atomic force microscopy, we have found a direct correlation between ENaC surface expression and the formation of cortical stiffness in endothelial cells. Stable knockdown of &agr;ENaC in endothelial cells evoked a reduced channel surface density and a lower cortical stiffness compared with the mock control. In turn, an increased &agr;ENaC expression induced an elevated cortical stiffness. More importantly, using ex vivo preparations from a mouse model for Liddle syndrome, we show that this disorder evokes enhanced ENaC expression and increased cortical stiffness in vascular endothelial cells in situ. We conclude that ENaC in the vascular endothelium determines cellular mechanics and hence might participate in the control of vascular function.

Role of cellular mechanics in the function and life span of vascular endothelium

The vascular endothelium plays a crucial role in vessel homeostasis and is implicated in the pathogenesis of cardiovascular disease. The function and life span of endothelial cells, therefore, have a large impact upon the quality and expectancy of an individual’s life. Exposure to haemodynamic forces determines the phenotype of endothelial cells. Turbulent blood flow, disturbed shear stress and a rising tension of the vessel wall result in endothelial dysfunction and an enhanced endothelial cell turnover. In this scenario, the role of endothelial mechanics is yet poorly described. The streaming blood exerts shear forces transmitted to the soft cortical actin mesh immediately underneath the plasma membrane. The mechanical properties of this actin cortex seem to be an important regulator of endothelial function. Aldosterone and high plasma sodium stiffen the endothelial cell cortex which is accompanied by a decrease in NO release. If endothelial stiffening is only transient, it may be a useful mechanism to compensate for any decrease in arterial blood pressure. Long-term stiffening of the cell, however, may lead to endothelial dysfunction and may contribute to cardiovascular disorders, as observed in disturbed aldosterone/sodium homeostasis. In this case, the mineralocorticoid receptor antagonist spironolactone maintains the endothelial cell cortex soft and thereby preserves normal endothelial function and longevity. This may explain the recently observed beneficial effects of spironolactone on the cardiovascular system. Taken together, the review highlights the importance of elasticity for normal endothelial function.

Direct evidence for functional TRPV1/TRPA1 heteromers

Transient receptor potential cation channel, subfamily V, member 1 (TRPV1) plays a key role in sensing environmental hazards and in enhanced pain sensation following inflammation. A considerable proportion of TRPV1-expressing cells also express transient receptor potential cation channel, subfamily A, member 1 (TRPA1). There is evidence for a TRPV1-TRPA1 interaction that is predominantly calcium-dependent, and it has been suggested that the two proteins might form a heteromeric channel. Here, we constructed subunit concatemers to search for direct evidence for such an interaction. We found that a TRPV1::TRPV1 concatemer and TRPV1 formed channels with similar properties. A TRPV1::TRPA1 concatemer was responsive to TRPV1 agonists capsaicin, acidic pH and ethanol, but not to TRPA1 agonists. Isolated TRPV1 and TRPV1::TRPA1 imaged by atomic force microscopy (AFM) both had molecular volumes consistent with the formation of tetrameric channels. Antibodies decorated epitope tags on TRPV1 with a four-fold symmetry, as expected for a homotetramer. In contrast, pairs of antibodies decorated tags on TRPV1::TRPA1 predominantly at 180°, indicating the formation of a channel consisting of two TRPV1::TRPA1 concatemers arranged face to face. TRPV1::TRPA1 was sensitized by PKC activation and could be inhibited by a TRPV1 antagonist. TRPV1::TRPA1 was activated by heat and displayed a threshold and temperature coefficient similar to TRPV1. However, the channel formed by TRPV1::TRPA1 has only two binding sites for capsaicin and shows less total current and a smaller capsaicin-induced shift in voltage-dependent gating than TRPV1::TRPV1 or TRPV1. We conclude that the presence of TRPA1 exerts a functional inhibition on TRPV1.

Membrane potential depolarization decreases the stiffness of vascular endothelial cells.

The stiffness of vascular endothelial cells is crucial to mechanically withstand blood flow and, at the same time, to control deformation-dependent nitric oxide release. However, the regulation of mechanical stiffness is not yet understood. There is evidence that a possible regulator is the electrical plasma membrane potential difference. Using a novel technique that combines fluorescence-based membrane potential recordings with atomic force microscopy (AFM)-based stiffness measurements, the present study shows that membrane depolarization is associated with a decrease in the stiffness of endothelial cells. Three different depolarization protocols were applied, all of which led to a similar and significant decrease in cell stiffness, independently of changes in cell volume. Moreover, experiments using the actin-destabilizing agent cytochalasin D indicated that depolarization acts by affecting the cortical actin cytoskeleton. A model is proposed whereby a change of the electrical field across the plasma membrane is directly sensed by the submembranous actin network, regulating the actin polymerization:depolymerization ratio and thus cell stiffness. This depolarization-induced decrease in the stiffness of endothelial cells could play a role in flow-mediated nitric-oxide-dependent vasodilation.

Nanomechanics of vascular endothelium

The mechanical characteristics of endothelial cells reveal four distinct compartments, namely glycocalyx, cell cortex, cytoplasm and nucleus. There is accumulating evidence that endothelial nanomechanics of these individual compartments control vascular physiology. Depending on protein composition, filament formation and interaction with cross-linker proteins, these four compartments determine endothelial stiffness. Structural organization and mechanical properties directly influence physiological processes such as endothelial barrier function, nitric oxide release and gene expression. This review will focus on endothelial nanomechanics and its impact on vascular function.

Exocytotic fusion pores are composed of both lipids and proteins

During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE proteins catalyze fusion, the structure and composition of fusion pores remain unknown. Here, we exploited the rigid framework and defined size of nanodiscs to interrogate the properties of reconstituted fusion pores, using the neurotransmitter glutamate as a content-mixing marker. Efficient Ca2+-stimulated bilayer fusion, and glutamate release, occurred with approximately two molecules of mouse synaptobrevin 2 reconstituted into ∼6-nm nanodiscs. The transmembrane domains of SNARE proteins assumed distinct roles in lipid mixing versus content release and were exposed to polar solvent during fusion. Additionally, tryptophan substitutions at specific positions in these transmembrane domains decreased glutamate flux. Together, these findings indicate that the fusion pore is a hybrid structure composed of both lipids and proteins.

Cortical actin nanodynamics determines nitric oxide release in vascular endothelium.

The release of the main vasodilator nitric oxide (NO) by the endothelial NO synthase (eNOS) is a hallmark of endothelial function. We aim at elucidating the underlying mechanism how eNOS activity depends on cortical stiffness (Кcortex) of living endothelial cells. It is hypothesized that cortical actin dynamics determines Кcortex and directly influences eNOS activity. By combined atomic force microscopy and fluorescence imaging we generated mechanical and optical sections of single living cells. This approach allows the discrimination between Кcortex and bulk cell stiffness (Кbulk) and, additionally, the simultaneous analysis of submembranous actin web dynamics. We show that Кcortex softens when cortical F-actin depolymerizes and that this shift from a gel-like stiff cortex to a soft G-actin rich layer, triggers the stiffness-sensitive eNOS activity. The results implicate that stiffness changes in the ∼100 nm phase of the submembranous actin web, without affecting Кbulk, regulate NO release and thus determines endothelial function.

Feedforward activation of endothelial ENaC by high sodium

Kidney epithelial sodium channels (ENaCs) are known to be inactivated by high sodium concentrations (feedback inhibition). Recently, the endothelial sodium channel (EnNaC) was identified to control the nanomechanical properties of the endothelium. EnNaC‐dependent endothelial stiffening reduces the release of nitric oxide, the hallmark of endothelial dysfunction. To study the regulatory impact of sodium on EnNaC, endothelial cells (EA.hy926 and ex vivo mouse endothelium) were incubated in aldosterone‐free solutions containing either low (130 mM) or high (150 mM) sodium concentrations. By applying atomic force microscopy‐based nanoindentation, an unexpected positive correlation between increasing sodium concentrations and cortical endothelial stiffness was observed, which can be attributed to functional EnNaC. In particular, an acute rise in sodium concentration (+20 mM) was sufficient to increase EnNaC membrane abundance by 90% and stiffening of the endothelial cortex by 18%. Despite the absence of exogenous aldosterone, these effects were prevented by the aldosterone synthase inhibitor FAD286 (100 nM) or the mineralocorticoid receptor (MR)‐antagonist spironolactone (100 nM), indicating endogenous aldosterone synthesis and MR‐dependent signaling. Interestingly, in the presence of high‐sodium concentrations, FAD286 increased the transcription of the MR by 69%. Taken together, a novel feedforward activation of EnNaC by sodium is proposed that contrasts ENaC feedback inhibition in kidney.—Korte, S., Sträter, A. S., Drüppel, V., Oberleithner, H., Jeggle, P., Grossmann, C., Fobker, M., Nofer, J.‐R., Brand, E., Kusche‐Vihrog, K. Feedforward activation of endothelial ENaC by high sodium. FASEB J. 28, 4015‐4025 (2014). www.fasebj.org

Cartilage-like electrostatic stiffening of responsive cryogel scaffolds

Cartilage is a structural tissue with unique mechanical properties deriving from its electrically-charged porous structure. Traditional three-dimensional environments for the culture of cells fail to display the complex physical response displayed by the natural tissue. In this work, the reproduction of the charged environment found in cartilage is achieved using polyelectrolyte hydrogels based on polyvinyl alcohol and polyacrylic acid. The mechanical response and morphology of microporous physically-crosslinked cryogels are compared to those of heat-treated chemical gels made from the same polymers, as a result of pH-dependent swelling. In contrast to the heat-treated chemically-crosslinked gels, the elastic modulus of the physical cryogels was found to increase with charge activation and swelling, explained by the occurrence of electrostatic stiffening of the polymer chains at large charge densities. At the same time, the permeability of both materials to fluid flow was impaired by the presence of electric charges. This cartilage-like mechanical behavior displayed by responsive cryogels can be reproduced in other polyelectrolyte hydrogel systems to fabricate biomimetic cellular scaffolds for the repair of the tissue.

Atomic force microscopy imaging reveals the formation of ASIC/ENaC cross-clade ion channels

ASIC and ENaC are co-expressed in various cell types, and there is evidence for a close association between them. Here, we used atomic force microscopy (AFM) to determine whether ASIC1a and ENaC subunits are able to form cross-clade hybrid ion channels. ASIC1a and ENaC could be co-isolated from detergent extracts of tsA 201 cells co-expressing the two subunits. Isolated proteins were incubated with antibodies against ENaC and Fab fragments against ASIC1a. AFM imaging revealed proteins that were decorated by both an antibody and a Fab fragment with an angle of ∼120° between them, indicating the formation of ASIC1a/ENaC heterotrimers.