Hermann Schillers

Plasma sodium stiffens vascular endothelium and reduces nitric oxide release

Dietary salt plays a major role in the regulation of blood pressure, and the mineralocorticoid hormone aldosterone controls salt homeostasis and extracellular volume. Recent observations suggest that a small increase in plasma sodium concentration may contribute to the pressor response of dietary salt. Because endothelial cells are (i) sensitive to aldosterone, (ii) in physical contact with plasma sodium, and (iii) crucial regulators of vascular tone, we tested whether acute changes in plasma sodium concentration, within the physiological range, can alter the physical properties of endothelial cells. The tip of an atomic force microscope was used as a nanosensor to measure stiffness of living endothelial cells incubated for 3 days in a culture medium containing aldosterone at a physiological concentration (0.45 nM). Endothelial cell stiffness was unaffected by acute changes in sodium concentration <135 mM but rose steeply between 135 and 145 mM. The increase in stiffness occurred within minutes. Lack of aldosterone in the culture medium or treatment with the epithelial sodium channel inhibitor amiloride prevented this response. Nitric oxide formation was found down-regulated in cells cultured in aldosterone-containing high sodium medium. The results suggest that changes in plasma sodium concentration per se may affect endothelial function and thus control vascular tone.

Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli.

Many mucosal pathogens use type III secretion systems for the injection of effector proteins into target cells. The type III‐secreted proteins EspB and EspD of enteropathogenic Escherichia coli (EPEC) are inserted into the target cell membrane. Together with EspA, these proteins are supposed to constitute a molecular syringe, channelling other effector proteins into the host cell. In this model, EspB and EspD would represent the tip of the needle forming a pore into target cell membranes. Although contact‐dependent and Esp‐mediated haemolytic activity by EPEC has already been described, the formation of a putative pore resulting in haemolysis has not been demonstrated so far. Here, we show that (i) diffusely adhering (DA)‐EPEC strains exhibit a type III‐dependent haemolytic activity too; (ii) this activity resides in the secreted proteins and, for DA‐EPEC strains, in contrast to EPEC strains, does not require bacterial contact; and (iii) pores are introduced into the target cell membrane. Osmoprotection revealed a minimal pore size of 3–5 nm. The pores induced by type III‐secreted proteins of DA‐EPEC were characterized by electron microscopy techniques. Analysis by atomic force microscopy demonstrated the pores to be composed of six to eight subunits with a lateral extension of 55–65 nm and to be raised 15–20 nm above the membrane plane. We could also demonstrate an association of EspB and EspD with erythrocyte membranes and an interaction of both proteins with each other in vitro. These results, together with the homologies of EspB and EspD to proposed functional domains of other pore‐forming proteins (Yop/Ipa), strongly support the idea that both proteins are directly involved in pore formation, which might represent the type III secretion system translocon.

Potassium softens vascular endothelium and increases nitric oxide release.

In the presence of aldosterone, plasma sodium in the high physiological range stiffens endothelial cells and reduces the release of nitric oxide. We now demonstrate effects of extracellular potassium on stiffness of individual cultured bovine aortic endothelial cells by using the tip of an atomic force microscope as a mechanical nanosensor. An acute increase of potassium in the physiological range swells and softens the endothelial cell and increases the release of nitric oxide. A high physiological sodium concentration, in the presence of aldosterone, prevents these changes. We propose that the potassium effects are caused by submembranous cortical fluidization because cortical actin depolymerization induced by cytochalasin D mimics the effect of high potassium. In contrast, a low dose of trypsin, known to activate sodium influx through epithelial sodium channels, stiffens the submembranous cell cortex. Obviously, the cortical actin cytoskeleton switches from gelation to solation depending on the ambient sodium and potassium concentrations, whereas the center of the cell is not involved. Such a mechanism would control endothelial deformability and nitric oxide release, and thus influence systemic blood pressure.

Human Endothelium: Target for Aldosterone

Abstract—Aldosterone has long been known to control water and electrolyte balance by acting on mineralocorticoid receptors in kidney. However, recent studies demonstrated the presence of these receptors in nonclassical locations, including the cardiovascular system. We tested the hypothesis whether endothelial cells respond to aldosterone with changes in cell volume, a measure for ion-mediated water movement across the cell membrane. By means of atomic force microscopy in fluid, we measured volume of adherent human umbilical venous endothelial cells exposed for 72 hours to 10 nmol/L aldosterone. Over this period of time, cells swell by ≈ 18%. Aldosterone-induced swelling is prevented by 100 nmol/L of the mineralocorticoid receptor antagonist spironolactone, added to the primary endothelial cell culture. Aldosterone-treated cells dramatically shrink when 1 μmol/L of the diuretic amiloride is applied. Cells deprived of aldosterone do not respond to amiloride. Our conclusions are: (1) aldosterone leads to sustained cell swelling inhibited by administration of spironolactone or the sodium channel blocker amiloride; (2) cells respond to amiloride after aldosterone exposure; (3) renal diuretics act on endothelial cells; and (4) both amiloride and spironolactone could be useful for medical applications to prevent aldosterone-mediated endothelial dysfunction.

Endothelial barrier function under laminar fluid shear stress

It has been suggested that increasing levels of shear stress could modify endothelial permeability. This might be critical in venous grafting and in the pathogenesis of certain vascular diseases. We present a novel setup based on impedance spectroscopy that allows online investigation of the transendothelial electrical resistance (TER) under pure laminar shear stress. Shear stress–induced change in TER was associated with changes in cell motility and cell shape as a function of time (morphodynamics) and accompanied by a reorganization of catenins that regulate endothelial adherens junctions. Confluent cultures of porcine pulmonary trunk endothelial cells typically displayed a TER between 6 and 15 Ωcm2 under both resting conditions and low shear stress levels (0.5 dyn/cm2). Raising shear stress to the range of 2 to 50 dyn/cm2 caused a transient 2% to 15% increase in TER within 15 minutes that was accompanied by a reduction in cell motility. Subsequently, TER slowly decreased to a minimum of 20% below the starting value. During this period, acceleration of shape change occurred. In the ensuing period, TER values recovered, reaching control levels within hours and associated with an entire deceleration of shape change. A heterogeneous distribution of α-, β-, and γ-catenin, main components of the endothelial adherens type junctions, was also observed, indicating a differentiated regulation of shear stress–induced junction rearrangement. Additionally, catenins were partly colocalized with β-actin at the plasma membrane, indicating migration activity of these subcellular parts. Shear stress, even at peak levels of 50 dyn/cm2, did not cause intercellular gap formation. These data show that endothelial monolayers exposed to increased levels of laminar shear stress respond with a shear stress–dependent regulation of permeability and a reorganization of junction-associated proteins, whereas monolayer integrity remains unaffected.

Endothelial Cell Swelling by Aldosterone

There is accumulating evidence that mineralocorticoids not only act on kidney but also on the cardiovascular system. We investigated the response of human umbilical venous endothelial cells (HUVECs) to aldosterone at a time scale of 20 minutes in absence and presence of the aldosterone antagonist spironolactone or other transport inhibitors. We applied atomic force microscopy (AFM), which measures cell volume and volume shifts between cytosol and cell nucleus. We observed an immediate cell volume increase (about 10%) approximately 1 min after addition of aldosterone (0.1 µmol/l), approaching a maximum (about 18%) 10 min after aldosterone treatment. Cell volume returned to normal 20 min after hormone exposure. Spironolactone (1 µmol/l) or amiloride (1 µmol/l) prevented the late aldosterone-induced volume changes but not the immediate change observed 1 min after hormone exposure. AFM revealed nuclear swelling 5 min after aldosterone addition, followed by nuclear shrinkage 15 min later. The Na+/H+ exchange blocker cariporide (10 µmol/l) was ineffective. We conclude: (i) Aldosterone induces immediate (1 min) swelling independently of plasma membrane Na+ channels and intracellular mineralocorticoid receptors followed by late mineralocorticoid receptor- and Na+-channel-dependent swelling. (ii) Intracellular macromolecule shifts cause the changes in cell volume. (iii) Both amiloride and spironolactone may be useful for medical applications to prevent aldosterone-induced vasculopathies.

Steroids dilate nuclear pores imaged with atomic force microscopy

Macromolecules that act in the cell nucleus must overcome the nuclear envelope (NE). This barrier between cytosol and the nucleus is perforated by nuclear pore complexes (NPCs) that serve as translocation machineries. We visualized the translocation process at the NE surface, applying a nanotechnical approach using atomic force microscopy (AFM). In order to initiate protein targeting to NPCs, dexamethasone (dex) was injected into Xenopus laevis oocytes. Dex is a synthetic steroid of great therapeutic relevance that specifically binds to glucocorticoid receptors and thus triggers an intracellular signal cascade involving the cell nucleus. Ninety and 180 sec after dex injection cell nuclei were isolated, the NEs spread on glass and scanned with AFM. With single molecule resolution we observed that dex initiated proteins (DIPs) first bind to NPC‐free areas of the outer nuclear membrane. This causes NPCs to dilate. Then, in a second step, DIPs attach directly to NPCs and enter the dilated central channels. DIPs accumulation and NPC conformational changes were blocked by RU486, a specific glucocorticoid receptor antagonist. In conclusion, dex exposure induces NPC dilation. NPCs change conformation already prior to transport. The NPC dilation signal is most likely transmitted through NPC associated filaments or yet unknown structures in the NE outer membrane. NPC dilation could have significant impact on nuclear targeting of therapeutic macromolecules.

Aldosterone and amiloride alter ENaC abundance in vascular endothelium

The amiloride-sensitive epithelial sodium channel (ENaC) is usually found in the apical membrane of epithelial cells but has also recently been described in vascular endothelium. Because little is known about the regulation and cell surface density of ENaC, we studied the influence of aldosterone, spironolactone, and amiloride on its abundance in the plasma membrane of human endothelial cells. Three different methods were applied, single ENaC molecule detection in the plasma membrane, quantification by Western blotting, and cell surface imaging using atomic force microscopy. We found that aldosterone increases the surface expression of ENaC molecules by 36% and the total cellular amount by 91%. The aldosterone receptor antagonist spironolactone prevents these effects completely. Acute application of amiloride to aldosterone-pretreated cells led to a decline of intracellular ENaC by 84%. We conclude that, in vascular endothelium, aldosterone induces ENaC expression and insertion into the plasma membrane. Upon functional blocking with amiloride, the channel disappears from the cell surface and from intracellular pools, indicating either rapid degradation and/or membrane pinch-off. This opens new perspectives in the regulation of ENaC expressed in the vascular endothelium.

C-Reactive Protein Makes Human Endothelium Stiff and Tight

Elevation of C-reactive protein (CRP) in human blood accompanies inflammatory processes, including cardiovascular diseases. There is increasing evidence that the acute-phase reactant CRP is not only a passive marker protein for systemic inflammation but also affects the vascular system. Further, CRP is an independent risk factor for atherosclerosis and the development of hypertension. Another crucial player in atherosclerotic processes is the mineralocorticoid hormone aldosterone. Even in low physiological concentrations, it stimulates the expression and membrane insertion of the epithelial sodium channel, thereby increasing the mechanical stiffness of endothelial cells. This contributes to the progression of endothelial dysfunction. In the present study, the hypothesis was tested that the acute application of CRP (25 mg/L), in presence of aldosterone (0.5 nmol/L; 24 hour incubation), modifies the mechanical stiffness and permeability of the endothelium. We found that endothelial cells stiffen in response to CRP. In parallel, endothelial epithelial sodium channel is inserted into the plasma membrane, while, surprisingly, the endothelial permeability decreases. CRP actions are prevented either by the inhibition of the intracellular aldosterone receptors using spironolactone (5 nmol/L) or by the inactivation of epithelial sodium channel using specific blockers. In contrast, inhibition of the release of the vasodilating gas nitric oxide via blockade of the phosphoinositide 3-kinase/Akt pathway has no effect on the CRP-induced stiffening of endothelial cells. The data indicate that CRP enhances the effects of aldosterone on the mechanical properties of the endothelium. Thus, CRP could counteract any decrease in arterial blood pressure that accompanies severe acute inflammatory processes.

Real-Time Monitoring of Cell Elasticity Reveals Oscillating Myosin Activity

The cytoskeleton is the physical and biochemical interface for a large variety of cellular processes. Its complex regulation machinery is involved upstream and downstream in various signaling pathways. The cytoskeleton determines the mechanical properties of a cell. Thus, cell elasticity could serve as a parameter reflecting the behavior of the system rather than reflecting the specific properties of isolated components. In this study, we used atomic force microscopy to perform real-time monitoring of cell elasticity unveiling cytoskeletal dynamics of living bronchial epithelial cells. In resting cells, we found a periodic activity of the cytoskeleton. Amplitude and frequency of this spontaneous oscillation were strongly affected by intracellular calcium. Experiments reveal that basal cell elasticity and superimposed elasticity oscillations are caused by the collective action of myosin motor proteins. We characterized the cell as a mechanically multilayered structure, and followed cytoskeletal dynamics in the different layers with high time resolution. In conclusion, the collective activities of the myosin motor proteins define overall mechanical cell dynamics, reflecting specific changes of the chemical and mechanical environment.