Recharging red blood cell surface by hemodialysis.

BACKGROUND: Similar as in vascular endothelium the negatively charged glycocalyx of erythrocytes selectively buffers sodium. Loss of glycocalyx (i.e. loss of negative charges) leads to increased erythrocyte sodium sensitivity (ESS) quantified by a recently developed salt-blood-test (SBT). The hypothesis was tested whether a regular 4-hour hemodialysis (4h-HD) alters ESS. METHODS: In 38 patients with end stage renal disease (ESRD) ESS was measured before and after 4h-HD, together with standard laboratory and clinical parameters (electrolytes, acid-base status, urea, creatinine, hemoglobin, c-reactive protein and blood pressure). RESULTS: Before 4h-HD, 20 patients (out of 38) were classified as "salt sensitive" by SBT. After 4h-HD, this number decreased to 11. Erythrocyte sodium buffering power remained virtually constant in patients with already low ESS before dialysis, whereas in patients with high ESS, 4h-HD improved the initially poor sodium buffering power by about 20%. No significant correlations could be detected between standard blood parameters and the respective ESS values except for plasma sodium concentration which was found increased by 3.1 mM in patients with high salt sensitivity. CONCLUSIONS: 4h-HD apparently recharges "run-down" erythrocytes and thus restores erythrocyte sodium buffering capacity. Besides the advantage of efficient sodium buffering in blood, erythrocytes with sufficient amounts of free negative charges at the erythrocyte surface will cause less (mechanical) injury to the negatively charged endothelial surface due to efficient repulsive forces between blood and vessel wall. Hemodialysis improves erythrocyte surface properties and thus may prevent early vascular damage in patients suffering from ESRD.

Endothelial sodium channels trigger endothelial salt sensitivity with aging.

The epithelial sodium channel is also expressed in vascular endothelium (endothelial sodium channel [EnNaC]). Depending on ambient sodium concentration, EnNaC is associated with mechanical stiffening of the endothelial cell cortex, leading to endothelial dysfunction. Because the incidence of both salt sensitivity and endothelial dysfunction increases with age, we investigated the abundance of EnNaC in aging mice. To assess EnNaC functionality and endothelial salt sensitivity, stiffness was measured while ambient sodium was varied. Aortae of young (3 months) and old (15 months) C57BL/6J wild-type mice were kept ex vivo on a physiological concentration of aldosterone (0.45 nmol/L). Spironolactone (10 nmol/L) and amiloride (1 µmol/L) were applied for aldosterone antagonism and EnNaC blockage, respectively. EnNaC at the endothelial cell surface was quantified by immunofluorescence staining. Cortical stiffness was monitored by atomic force microscopy when ambient sodium was raised from 135 to 150 mmol/L. In ex vivo aortae of older mice, endothelial cells had significantly higher EnNaC numbers than those of younger mice (+23%). In parallel, cortical stiffness was found increased (+8.5%). Acute application of high sodium led to an immediate rise in stiffness in both groups but was pronounced in endothelium of older mice (+18% versus +26%). Spironolactone and amiloride lowered EnNaC abundance and prevented endothelial stiffening under all conditions. We conclude that EnNaC mediates endothelial salt sensitivity in the aging process. This mechanism might contribute to the development of age-related cardiovascular disease and suggests the usage of spironolactone and amiloride specifically in the elderly.

Long-term application of the aldosterone antagonist spironolactone prevents stiff endothelial cell syndrome.

Aldosterone triggers the stiff endothelial cell syndrome (SECS), characterized by an up-regulation of epithelial sodium channels (ENaCs) and mechanical stiffening of the endothelial cell cortex accompanied by endothelial dysfunction. In vivo, aldosterone antagonism exerts sustained protection on the cardiovascular system. To illuminate the molecular mechanisms of this time-dependent effect, a study on endothelial cells in vitro and ex vivo was designed to investigate SECS over time. Endothelia (from human umbilical veins, bovine aortae, and explants of human arteries) were cultured in aldosterone-supplemented medium with or without the mineralocorticoid receptor (MR) antagonist spironolactone. MR expression, ENaC expression, cortical stiffness, and shear-mediated nitric oxide (NO) release were determined after 3 d (short term) and up to 24 d (long term). Over time, MR expression increased by 129%. ENaC expression and surface abundance increased by 32% and 42% (13.8 to 19.6 molecules per cell surface), paralleled by a 49% rise in stiffness. Spironolactone prevented this development and, after 3 wk of treatment, increased NO release by 50%. Thus, spironolactone improves endothelial function long-lastingly by preventing a time-dependent manifestation of SECS. This emphasizes the key role of vascular endothelium as a therapeutical target in cardiovascular disorders and might explain blood pressure independent actions of MR antagonism.

Salt overload damages the glycocalyx sodium barrier of vascular endothelium.

Sodium overload stiffens vascular endothelial cells in vitro and promotes arterial hypertension in vivo. The hypothesis was tested that the endothelial glycocalyx (eGC), a mesh of anionic biopolymers covering the surface of the endothelium, participates in the stiffening process. By using a mechanical nanosensor, mounted on an atomic force microscope, height (∼400 nm) and stiffness (∼0.25 pN/nm) of the eGC on the luminal endothelial surface of split-open human umbilical arteries were quantified. In presence of aldosterone, the increase of extracellular sodium concentration from 135 to 150 mM over 5 days (sodium overload) led the eGC shrink by ∼50% and stiffening by ∼130%. Quantitative eGC analyses reveal that sodium overload caused a reduction of heparan sulphate residues by 68% which lead to destabilization and collapse of the eGC. Sodium overload transformed the endothelial cells from a sodium release into a sodium-absorbing state. Spironolactone, a specific aldosterone antagonist, prevented these changes. We conclude that the endothelial glycocalyx serves as an effective buffer barrier for sodium. Damaged eGC facilitates sodium entry into the endothelial cells. This could explain endothelial dysfunction and arterial hypertension observed in sodium abuse.

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.

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.

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.

Nebivolol decreases endothelial cell stiffness via the estrogen receptor beta: a nano-imaging study.

BACKGROUND: Nebivolol (NEB) is a [beta]1-receptor blocker with nitric oxide-dependent vasodilating properties. NEB-induced nitric oxide release is mediated through the estrogen receptor. METHOD: Here, we tested the hypothesis that NEB decreases endothelial cell stiffness and that these effects can be abolished by both endothelial nitric oxide synthase and estrogen receptor blockade. Human endothelial cells (EAHy-926) were incubated with vehicle, NEB 0.7 nmol/l, metoprolol 200 nmol/l, 17[beta]-estradiol (E2) 15 nmol/l, the estrogen receptor antagonists tamoxifen 100 nmol/l and ICI 182780 (ICI) 100 nmol/l, the nitric oxide synthase inhibitor N[omega]-nitro-L-arginine methyl ester 1 mmol/l and combinations of NEB and E2 with either tamoxifen, ICI or N[omega]-nitro-L-arginine methyl ester as well as metoprolol and ICI. Atomic force microscopy was performed to measure cellular stiffness, cell volume and apical surface. Presence of estrogen receptor protein in EAHy-926 was confirmed by western blot analysis; quantification of ER[alpha] and ER[beta] total RNA was performed by semiquantitative PCR. RESULTS: Both NEB as well as E2 decreased cellular stiffness to a similar extent (NEB: 0.83 +/- 0.03 pN/nm, E2: 0.87 +/- 0.03 pN/nm, vehicle: 2.19 +/- 0.07 pN/nm), whereas metoprolol had no effect on endothelial stiffness (2.07 +/- 0.04 pN/nm, all n = 60, P < 0.01). The decrease in stiffness occurred as soon as 5 min after starting NEB incubation. The effects are mediated through nongenomic ER[beta] pathways, as ER[alpha] is not translated into measurable protein levels in EAHy-926. Furthermore, NEB increased cell volume by 48 +/- 4% and apical surface by 34 +/- 3%. E2 had comparable effects. Tamoxifen, ICI and N[omega]-nitro-L-arginine methyl ester substantially diminished the effects of NEB and E2. CONCLUSION: NEB decreases cellular stiffness and causes endothelial cell growth. These effects are nitric oxide-dependent and mediated through nongenomic ER[beta] pathways. The morphological and functional alterations observed in endothelial cells may explain improved endothelial function with NEB treatment.

Direct aldosterone action on mouse cardiomyocytes detected with atomic force microscopy.

There is growing evidence that aldosterone acts on heart where it causes cellular remodeling and hypertrophy. Since it is still unclear whether aldosterone directly acts on cardiomyocytes or indirectly, by an altered electrolyte balance in the organism, we applied atomic force microscopy (AFM) in primary cultures of neonatal mouse cardiomyocytes to measure hormone-induced changes in cell volume and plasma membrane surface. AFM measures cell volume and, at the same time, provides quantitative information on cell surface properties. Neonatal mouse cardiomyocytes were cultured for 28 hours in absence or presence of 100 nM aldosterone. Spironolactone was applied as a selective aldosterone receptor antagonist. At the microscopic level, single cell volume and single cell surface were found unchanged by aldosterone. However, nanoscopy of the cell surface, i.e. analysis of the plasma membrane at the nanometer level, revealed a specific increase in plasma membrane nano-enfoldings (roughness). This aldosterone-mediated increase in cell surface roughness was completely prevented by spironolactone. We conclude: (i) Aldosterone directly acts upon cardiomyocytes. (ii) At the microscopic level, no changes of cell volume and cell surface are detectable. (iii) At the nanoscopic level, aldosterone increases plasma membrane roughness. These nanometer changes, detectable only with AFM in cells scanned in fluid after fixation under physiological conditions, indicate plasma membrane remodeling of cardiomyocytes by mineralocorticoids.