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Shear detection and mechanotransduction by arterial endothelium requires junctional complexes containing PECAM-1 and VE-cadherin, as well as firm anchorage to the underlying basement membrane. While considerable information is available for junctional complexes in these processes, gained largely from in vitro studies, little is known about the contribution of the endothelial basement membrane. Using resistance artery explants, we show that the integral endothelial basement membrane component, laminin 511 (laminin α5), is central to shear detection and mechanotransduction and its elimination at this site results in ablation of dilation in response to increased shear stress. Loss of endothelial laminin 511 correlates with reduced cortical stiffness of arterial endothelium in vivo, smaller integrin β1-positive/vinculin-positive focal adhesions, and reduced junctional association of actin-myosin II In vitro assays reveal that β1 integrin-mediated interaction with laminin 511 results in high strengths of adhesion, which promotes p120 catenin association with VE-cadherin, stabilizing it at cell junctions and increasing cell-cell adhesion strength. This highlights the importance of endothelial laminin 511 in shear response in the physiologically relevant context of resistance arteries., (© 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Objectives: Recently, the nanomechanical properties (i.e. stiffness) of endothelial cells have been identified as crucial for appropriate endothelial function. One major determinant of endothelial stiffness is the endothelial sodium channel (EnNaC). EnNaC-dependent stiffening leads to reduced nitric oxide release, which is a hallmark for endothelial dysfunction. In the current study, we hypothesized that endothelial function is directly linked to the overall function of the arterial system., Methods: Sixty-four human ex-vivo arterial samples were collected from femoral bypass or vein-stripping procedures. Nanomechanical characteristics of ex-vivo endothelium from isolated arterial side branches were determined using atomic force microscopy. The endothelium's potential to respond to EnNaC inhibition by amiloride was defined as endothelial amiloride index. In addition, patients' arterial stiffness was determined by pulse wave velocity (PWV)., Results: Fifty-three percentage of the ex-vivo samples responded 'classically' to amiloride with endothelial softening, whereas 47% of the patients' samples did not. Interestingly, a lack of endothelial softening in the presence of amiloride in vitro was observed with higher frequency among samples obtained from individuals with elevated PWV. Further, an increased PWV was associated with impaired renal function and endothelial dysfunction (higher levels of von Willebrand factor)., Conclusions: Here, we report differential responses of human ex-vivo vessels to amiloride. Although the mechanism of differential amiloride response is still unknown, the data indicate that drug action on endothelial function could differ strongly among patients, especially in those with a vascular end-organ damage determined by PWV.

Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Previous studies show that polyphenol-rich compounds can induce a swelling of the endothelial glycocalyx (eGC). Our goal was to reveal the mechanism behind the eGC-swelling. As polyphenols are potent modulators of fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, the hypothesis was tested whether polyphenol-induced increase in CFTR activity is responsible for the eGC-swelling. The impact of the polyphenols resveratrol, (-)-epicatechin, and quercetin on nanomechanics of living endothelial GM7373 cells was monitored by AFM-nanoindentation. The tested polyphenols lead to eGC-swelling with a simultaneous decrease in cortical stiffness. EGC-swelling, but not the change in cortical stiffness, was prevented by the inhibition of CFTR. Polyphenol-induced eGC-swelling could be mimicked by cytochalasin D, an actin-depolymerizing agent. Thus, in the vascular endothelium, polyphenols induce eGC-swelling by softening cortical actin and activating CFTR. Our findings imply that CFTR plays an important role in the maintenance of vascular homeostasis and may explain the vasoprotective properties of polyphenols., From the Clinical Editor: Many vascular problems clinically can be attributed to a dysregulation of endothelial glycocalyx (eGC). The underlying mechanism however remains unclear. In this article, the authors used nanoindentation and showed that polyphenols could swell the endothelial glycocalyx and alter its function. This investigative method can lead to further mechanistic studies of other molecular pathways., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Smart mechanisms allow frictionless slipping of rather rigid erythrocytes (red blood cells, RBC) through narrow blood vessels. Nature solved this problem in an elegant way coating the moving object (RBC) and the tunnel wall (endothelium) by negative charges (glycocalyx). As long as these surfaces are intact, repulsive forces create a 'security zone' that keeps the respective surfaces separated from each other. However, damage of either one of these surfaces causes loss of negative charges, allowing an unfavorable physical interaction between the RBC and the endothelium. It has been recently shown that any alteration of the endothelial glycocalyx leaves nasty footprints on the RBC glycocalyx. In this scenario, sodium ions hold a prominent role. Plasma sodium is stored in the glycocalyx partially neutralizing the negative surface charges. A 'good' glycocalyx has a high sodium store capacity but still maintains sufficient surface negativity at normal plasma sodium. A 'bad' glycocalyx shows the opposite. This concept was used for the development of the so-called 'salt blood test' (SBT) that quantitatively measures RBC sodium store capacity of the glycocalyx and thus indirectly evaluates the quality of the inner vessel wall. In an initial step, the applicability of the SBT was tested in eight different medical facilities. The study shows that an increased salt sensitivity, as measured by the SBT, is more frequently found in individuals with a hypertensive history, despite antihypertensive medication. Taken together, preservation of the endothelial glycocalyx appears to be of utmost importance for maintaining a well-balanced function of the vascular system., (© 2015 S. Karger AG, Basel.)

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., (© FASEB.)

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., (© 2014 American Heart Association, Inc.)

Background and Objectives: The endothelial glycocalyx (eGC), a mesh of anionic biopolymers covering the luminal surface of endothelial cells, is considered as an intravascular compartment that protects the vessel wall against pathogenic insults in cardiovascular disease. We hypothesized that chronic kidney disease (CKD) is associated with reduced eGC integrity and subsequent endothelial dysfunction., Methods & Results: Shedding of two major components of the eGC, namely syndecan-1 (Syn-1) and hyaluronan (HA), was measured by ELISA in 95 patients with CKD (stages 3-5) and 31 apparently healthy controls. Plasma levels of Syn-1 and HA increased steadily across CKD stages (5- and 5.5-fold, respectively P < 0.001) and were independently associated with impaired renal function after multivariate adjustment. Furthermore, Syn-1 and HA correlated tightly with plasma markers of endothelial dysfunction such as soluble fms-like tyrosine kinase-1 (sFlt-1), soluble vascular adhesion molecule-1 (sVCAM-1), von-Willebrand-Factor (vWF) and angiopoietin-2 (P < 0.001). Experimentally, excessive shedding of the eGC, evidenced by 11-fold increased Syn-1 plasma levels, was also observed in an established rat model of CKD, the 5/6-nephrectomized rats. Consistently, an atomic force microscopy-based approach evidenced a significant decrease in eGC thickness (360 ± 79 vs. 157 ± 29 nm, P = 0.001) and stiffness (0.33 ± 0.02 vs. 0.22 ± 0.01 pN/nm, P < 0.001) of aorta endothelial cell explants isolated from CKD rats., Conclusion: Our findings provide evidence for damage of the atheroprotective eGC as a consequence of CKD and potentially open a new avenue to pathophysiology and treatment of cardiovascular disease in renal patients., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Once upon a time, the expression of the epithelial sodium channel (ENaC) was mainly assigned to the kidneys, colon and sweat glands where it was considered to be the main determinant of sodium homeostasis. Recent, though indirect, evidence for the possible existence of ENaC in a non-epithelial tissue was derived from the observation that the vascular endothelium is a target for aldosterone. Inhibitory actions of the intracellular aldosterone receptors by spironolactone and, more directly, by ENaC blockers such as amiloride supported this view. Shortly after, direct data on the expression of ENaC in vascular endothelium could be demonstrated. There, endothelial ENaC (EnNaC) could be defined as a major regulator of cellular mechanics which is a critical parameter in differentiating between vascular function and dysfunction. Foremost, the mechanical stiffness of the endothelial cell cortex, a layer 50-200 nm beneath the plasma membrane, has been shown to play a crucial role as it controls the production of the endothelium-derived vasodilator nitric oxide (NO) which directly affects the tone of the vascular smooth muscle cells. In contrast to soft endothelial cells, stiff endothelial cells release reduced amounts of NO, the hallmark of endothelial dysfunction. Thus, the combination of endothelial stiffness and myogenic tone might increase the peripheral vascular resistance. An elevation of arterial blood pressure is supposed to be the consequence of such functional changes. In this review, EnNaC is discussed as an aldosterone-regulated plasma membrane protein of the vascular endothelium that could significantly contribute to maintaining of an appropriate arterial blood pressure but, if overexpressed, could participate in the pathogenesis of arterial hypertension.

Sodium transport in the distal nephron is mediated by epithelial sodium channel activity. Proteolytic processing of external domains and inhibition with increased sodium concentrations are important regulatory features of epithelial sodium channel complexes expressed in the distal nephron. By contrast, sodium channels expressed in the vascular system are activated by increased external sodium concentrations, which results in changes in the mechanical properties and function of endothelial cells. Mechanosensitivity and shear stress affect both epithelial and vascular sodium channel activity. Guyton's hypothesis stated that blood pressure control is critically dependent on vascular tone and fluid handling by the kidney. The synergistic effects, and complementary regulation, of the epithelial and vascular systems are consistent with the Guytonian model of volume and blood pressure regulation, and probably reflect sequential evolution of the two systems. The integration of vascular tone, renal perfusion and regulation of renal sodium reabsorption is the central underpinning of the Guytonian model. In this Review, we focus on the expression and regulation of sodium channels, and we outline the emerging evidence that describes the central role of amiloride-sensitive sodium channels in the efferent (vascular) and afferent (epithelial) arms of this homeostatic system.

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.

In humans, when plasma sodium concentration rises slightly beyond 140 mM, vascular endothelium sharply stiffens and nitric oxide release declines. In search of a vascular sodium sensor, the endothelial glycocalyx was identified as being a negatively charged biopolymer capable of selectively buffering sodium ions. Sodium excess damages the glycocalyx and renders vascular endothelium increasingly permeable for sodium. In the long term, sodium accumulates in the interstitium and gradually damages the organism. It was discovered that circulating red blood cells (RBC) 'report' surface properties of the vascular endothelium. To some extent, the RBC glycocalyx mirrors the endothelial glycocalyx. A poor (charge-deprived) endothelial glycocalyx causes a poor RBC glycocalyx and vice versa. This observation led to the assumption that the current state of an individual's vascular endothelium in terms of electrical surface charges and sodium-buffering capabilities could be read simply from a blood sample. Recently, a so-called salt blood test was introduced that quantifies the RBC sodium buffer capacity and thus characterizes the endothelial function. The arguments are outlined in this article spanning a bridge from cellular nano-mechanics to clinical application.

Gliding of red blood cells (RBC) through blood vessels is mediated by the negatively charged glycocalyx located on the surfaces of both RBC and endothelial cells (EC). In various vasculopathies, EC gradually lose this protective surface layer. As a consequence, RBC come into close physical contact with the vascular endothelium. It is hypothesized that the RBC glycocalyx could be adversely affected by a poor EC glycocalyx. This hypothesis was tested by evaluating the RBC and EC surface layers with atomic force microscopy techniques. In the first series of experiments, EC monolayers grown in culture were exposed to rhythmic drag forces exerted from a blood overlay (drag force treatment), and thereafter, the EC surface was investigated in terms of thickness and adhesiveness. In the second series, the glycocalyx of the EC monolayers was disturbed by enzymatic cleavage of negatively charged heparan sulfates before drag force treatment, and thereafter, the RBC surface was evaluated. In the third series, the RBC glycocalyx of the blood overlay was enzymatically disturbed before drag force treatment, and thereafter, the EC surface was evaluated. A strong positive correlation between the RBC and EC surface properties was found (r (2) = 0.95). An enzymatically affected EC glycocalyx lead to the shedding of the RBC glycocalyx and vice versa. It is concluded that there is physical interaction between the blood and endothelium. Apparently, the RBC glycocalyx reflects properties of the EC glycocalyx. This observation could have a significant impact on diagnosis and treatment of cardiovascular diseases.

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 αENaC in endothelial cells evoked a reduced channel surface density and a lower cortical stiffness compared with the mock control. In turn, an increased αENaC 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.

About one third of the population worldwide is supposed to be salt sensitive which is a major cause for arterial hypertension later in life. For preventive actions it is thus desirable to identify salt-sensitive individuals before the appearance of clinical symptoms. Recent observations suggest that the vascular endothelium consists of two salt-sensitive barriers in series, the glycocalyx that buffers sodium and the endothelial cell membrane that contains sodium channels. Glycocalyx sodium buffer capacity and sodium channel activity are conversely related to each other. For proof of concept, a so-called salt provocation test (SPT) was developed that should unmask vascular salt sensitivity in humans at virtually any age. Nineteen healthy subjects, ranging from 25 to 63 years of age, underwent two series of 1-h blood pressure measurements after acute ingestion of a salt cocktail with or without addition of a sodium channel blocker effective in vascular endothelium. Differential analysis of the changes in diastolic blood pressure (net ∆DP) identified 12 individuals (63%) as being salt resistant (net ∆DP = -0.05 ± 0.62 mmHg) and seven individuals (37%) as being salt sensitive (net ∆DP = +6.98 ± 0.75 mmHg). Vascular salt sensitivity was not related to the age of the study participants. It is concluded that the SPT could be useful for identifying vascular salt sensitivity in humans already early in life.

Vascular endothelium plays a key role in blood pressure regulation. Recently, it has been shown that a 5% increase of plasma sodium concentration (sodium excess) stiffens endothelial cells by about 25%, leading to cellular dysfunction. Surface measurements demonstrated that the endothelial glycocalyx (eGC), an anionic biopolymer, deteriorates when sodium is elevated. In view of these results, a two-barrier model for sodium exiting the circulation across the endothelium is suggested. The first sodium barrier is the eGC which selectively buffers sodium ions with its negatively charged proteoglycans. The second sodium barrier is the endothelial plasma membrane which contains sodium channels. Sodium excess, in the presence of aldosterone, leads to eGC break-down and, in parallel, to an up-regulation of plasma membrane sodium channels. The following hypothesis is postulated: Sodium excess increases vascular sodium permeability. Under such conditions (e.g. high-sodium diet), day-by-day ingested sodium, instead of being readily buffered by the eGC and then rapidly excreted by the kidneys, is distributed in the whole body before being finally excreted. Gradually, the sodium overload damages the organism.

Plasma sodium, slightly above normal and in presence of aldosterone, stiffens vascular endothelium and reduces nitric oxide release with the consequence of endothelial dysfunction. This process is mediated by epithelial sodium channels (ENaC) and, most likely, the endothelial Na(+)/K(+)-ATPase. Both, ENaC and Na(+)/K(+)-ATPase, are located in the plasma membrane of endothelial cells and embedded in the endothelial glycocalyx (eGC). This negatively charged biopolymer is directly exposed to the blood stream and selectively buffers sodium ions. We hypothesize that the glycocalyx could interfere with endothelial sodium transport when extracellular sodium varies in the physiological range. Therefore, we modeled the endothelial cell as a pump-leak system measuring changes of intracellular sodium in cultured human endothelial cells. Experiments were performed under low/high extracellular sodium conditions before and after enzymatic eGC removal, and with inhibition of Na(+)/K(+)-ATPase and ENaC, respectively. Three major observations were made: (1) eGC removal by heparinase treatment facilitates sodium to enter/exit the endothelial cells. (2) The direction of net sodium movement across the endothelial plasma membrane depends on the concentration of extracellular sodium which regulates both the Na(+)/K(+)-ATPase and ENaC activity. (3) Removal of eGC and inhibition of sodium transport modify the electrical resistance of endothelial cells. We conclude that the eGC serves as a potential "firewall" preventing uncontrolled access of sodium to the pump-leak system of the endothelial cell. After eGC removal, sodium access to the system is facilitated. Thus the pump-leak system could be regulated by ambient sodium and control vascular permeability in pathophysiological conditions.

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 (К(cortex)) of living endothelial cells. It is hypothesized that cortical actin dynamics determines К(cortex) 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 К(cortex) and bulk cell stiffness (К(bulk)) and, additionally, the simultaneous analysis of submembranous actin web dynamics. We show that К(cortex) 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 К(bulk), regulate NO release and thus determines endothelial function.

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.

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.

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.

High blood pressure is the main cause of disease-related morbidity and mortality worldwide. It is virtually absent in populations that consume natural foods low in sodium. In other countries, however, where the individual intake of sodium is at least 10 times greater, the prevalence of arterial hypertension is about 40%. Vascular endothelium plays a central role in blood pressure regulation. In addition to the kidney, the vasculature is a major target for aldosterone where it controls nonexcitable sodium channels in the endothelium. High sodium channel expression/activity downregulates the release of nitric oxide (NO), and thus determines endothelial function. Mechanical cell stiffness is therefore the means whereby high sodium channel activity reduces NO release. We have found that small changes of plasma sodium, but only above 139 mM, and potassium above 4 mM regulate the stiffness of the submembrane actin web and thus control the shear stress-dependent activity of endothelial NO synthase, which lies directly beneath the cell membrane. Plasma sodium above 139 mM stiffens the actin web of the endothelial cell, and plasma potassium greater than 4 mM does the opposite. In conclusion, vascular endothelial cells are highly sensitive to changes in extracellular sodium and potassium but only within and above the normal range. This sensitivity may serve as a physiological feedback mechanism to regulate local blood flow. It becomes pathogenic, however, when the concentration of plasma sodium or potassium changes towards and beyond the upper limits of the normal range., (Copyright © 2011 S. Karger AG, Basel.)

Aldosterone, a mineralocorticoid hormone mainly synthesized in the adrenal cortex, has been recognized to be a regulator of cell mechanics. Recent data from a number of laboratories implicate that, besides kidney, the cardiovascular system is an important target for aldosterone. In the endothelium, it promotes the expression of epithelial sodium channels (ENaC) and modifies the morphology of cells in terms of mechanical stiffness, surface area and volume. Additionally, it renders the cells highly sensitive to small changes in extracellular sodium and potassium. In this context, the time course of aldosterone action is pivotal. In the fast (seconds to minutes), non-genomic signalling pathway vascular endothelial cells respond to aldosterone with transient swelling, softening and insertion of ENaC in the apical plasma membrane. In parallel, nitric oxide (NO) is released from the cells. In the long-term (hours), aldosterone has opposite effects: The mechanical stiffness increases, the cells shrink and NO production decreases. This leads to the conclusion that both the physiology and pathophysiology of aldosterone action in the vascular endothelium are closely related. Aldosterone, at concentrations in the physiological range and over limited time periods can stabilize blood pressure and regulate tissue perfusion while chronically high concentrations of this hormone over extended time periods impair sodium homeostasis promoting endothelial dysfunction and the development of tissue fibrosis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In the vascular endothelium, mechanical cell stiffness (К) and nitric oxide (NO) release are tightly coupled. "Soft" cells release more NO compared to "stiff" cells. Currently, however, it is not known whether NO itself is the primary factor that softens the cells or whether NO release is the result of cell softening. To address this question, a hybrid fluorescence/atomic force microscope was used in order to measure changes in К and NO release simultaneously in living vascular endothelial cells. Aldosterone was applied to soften the cells transiently and to trigger NO release. NO synthesis was then either blocked or stimulated and, simultaneously, К was measured. Cell indentation experiments were performed to evaluate К, while NO release was measured either by an intracellular NO-dependent fluorescence indicator (DAF-FM/DA) or by NO-selective electrodes located close to the cell surface. After the application of aldosterone, К decreases, within 10 min, to 80.5 ± 1.7% of control (100%). DAF-FM fluorescence intensity increases simultaneously to 132.9 ± 2.2%, which indicates a significant increase in the activity of endothelial NO synthase (eNOS). Inhibition of eNOS (by N (ω)-nitro-L: -arginine methyl ester) blocks the NO release, but does not affect the aldosterone-induced changes in К. Application of an eNOS-independent NO donor (NONOate/AM) raises intracellular NO concentration, but, again, does not affect К. Data analysis indicates that a decrease of К by about 10% is sufficient to induce a significant increase of eNOS activity. In conclusion, these nanomechanic properties of endothelial cells in vascular endothelium determine NO release, and not vice versa.

In the kidney the epithelial sodium channel (ENaC) is regulated by the mineralocorticoid hormone aldosterone, which is essential for long-term blood pressure control. Evidence has accumulated showing that ENaC is expressed in endothelial cells. Moreover, its activity modifies the biomechanical properties of the endothelium. Therefore, the vascular system is also an important target for aldosterone and responds to the hormone with an increase in cell volume, surface area, and mechanical stiffness. These changes occur in a concerted fashion from minutes to hours and can be prevented by the specific sodium channel blocker amiloride and the mineralocorticoid receptor (MR) blocker spironolactone. Aldosterone acts on cells of the vascular system via genomic and non-genomic pathways. There is evidence that the classical cytosolic MR could mediate both types of response. Using a nanosensor covalently linked to aldosterone, binding sites at the plasma membrane were identified by atomic force microscopy. The interaction of aldosterone and this newly identified surface receptor could precede the slow classic genomic aldosterone response resulting in fast activation of endothelial ENaC. Recent data suggest that aldosterone-induced ENaC activation initiates a sequence of cellular events leading to a reduced release of vasodilating nitric oxide. We propose a model in which ENaC is the key mediator of aldosterone-dependent blood pressure control in the vascular endothelium., (Copyright 2009 Elsevier Inc. All rights reserved.)

Endothelial dysfunction contributes to the increased cardiovascular risk that accompanies CKD. We hypothesized that the soluble VEGF receptor 1 (sFlt-1), a VEGF antagonist, plays a role in endothelial dysfunction and decreased angiogenesis in CKD. We enrolled 130 patients with CKD stages 3 to 5 and 56 age- and gender-matched control patients. Plasma sFlt-1 levels were higher in patients with CKD and, after multivariate regression analyses, exclusively associated with renal function and levels of vWF, a marker of endothelial dysfunction. Compared with serum from control patients, both recombinant sFlt-1 and serum from patients with CKD had antiangiogenic activity in the chick chorioallantoic membrane (CAM) assay, induced endothelial cell apoptosis in vitro, and decreased nitric oxide generation in two different endothelial cell lines. Pretreating the sera with an antibody against sFlt-1 abrogated all of these effects. Furthermore, we observed increased sFlt1 levels in 5/6-nephrectomized rats compared with sham-operated animals. Finally, using real-time PCR and ELISA, we identified monocytes as a possible source of increased sFlt-1 in patients with CKD. Our findings show that excess sFlt-1 associates with endothelial dysfunction in CKD and suggest that increased sFlt-1 may predict cardiovascular risk in CKD.

The mineralocorticoid hormone aldosterone acts on target cells of kidney, colon, and the cardiovascular system through genomic and nongenomic pathways. Although the classical intracellular mineralocorticoid receptor plays a key role in mediating both pathways, it is unclear whether there are specific aldosterone receptors located on the cell surface. To search for such sites in vascular endothelium, we used an atomic force microscope (AFM) which measures unbinding forces based on single molecular recognition between an aldosterone-loaded AFM tip and the cell membrane. Aldosterone was tethered covalently via linker molecules to an AFM tip. Human endothelial cells (EA.hy926) were grown in culture and studied in buffer at 37 degrees C. Using the aldosterone-functionalized AFM tip as a mechanical nanoscale indenter, unbinding forces could be measured at randomly chosen sites of the plasma membrane. Sites with strong interactions between AFM tip and cell surface could be identified exhibiting unbinding forces of about 65 pN. The binding probability between the aldosterone-loaded tip and the cell surface at selected membrane sites was 53 +/- 7.2%. Addition of an excess supply of aldosterone to the bath solution blocked the binding of the aldosterone-loaded tip to the cell surface. The binding probability was reduced to 8.0 +/- 1.8% when an excess supply of aldosterone was added to the bath. However, it was not influenced by the addition of spironolactone or dexamethasone. We conclude that aldosterone receptor sites exist on the cell surface of vascular endothelial cells distinct from the classical mineralocorticoid receptors and insensitive to glucocorticoids. Binding of aldosterone to these receptors initiates an intracellular signaling cascade that precedes the classical genomic response and most likely participates in the control of vascular resistance.

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.

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.

Fluid and electrolyte balance in the human organism is controlled by aldosterone, a mineralocorticoid hormone of the suprarenal glands. The major target cells are localized in the kidney where the hormone controls transepithelial salt transport. Over the past few years, evidence has been accumulated that cells of the cardiovascular system are also targeted by the hormone. As an example, endothelial cells resemble similar mechanisms triggered by aldosterone as shown for the kidney. Although the pathological alterations induced by aldosterone excess are obvious, the physiological changes are largely unknown. On the basis of recent experiments, using atomic force microscopy as an imaging tool and a mechanical sensor, I present a hypothesis on the physiological role of aldosterone in endothelial function and its potential implications in the control of blood pressure.

Reperfusion after ischemic conditions induces massive endothelial cell (EC) activation, an initial step of reperfusion injury. Reperfusion is characterized by reoxygenation, realkalinization and a localized increase of inflammatory stimuli. In this study, we focused on the influence of extracellular realkalinization on human umbilical vein endothelial cell (HUVEC) activation. We examined intracellular pH (pH(in)) and intracellular free calcium concentration ([Ca(2+)](in)), a second messenger known to mediate von Willebrand factor (VWF) exocytosis in endothelium, upon realkalinization. Furthermore, we measured the agonist-stimulated exocytosis of VWF, Interleukin-8 and soluble P-selectin (sP-Selectin) as markers of EC activation. To verify a morphological correlate of EC activation, we finally observed platelet-endothelial adherence during realkalinization using shear flow. Realkalinization of HUVEC was simulated by switching from bicarbonate buffered Ringer solution of an acidotic pH(ex) of 6.4 to a physiologic pH(ex) of 7.4. Extracellular realkalinization was accompanied by pH(in) recovery from 6.5 to 7.2 within 10 min. Application of cariporide, an inhibitor of the Na(+)/H(+) exchanger subtype 1 (NHE), during extracellular realkalinization significantly delayed the early kinetics of intracellular realkalinization. Histamine stimulated [Ca(2+)](in) was significantly increased upon realkalinization compared to control cells. Also agonist-stimulated release of VWF, Interleukin-8 and sP-Selectin was massively enhanced during pH(in) recovery in comparison to control. Furthermore, we observed an increased platelet binding to endothelium. Interestingly, each of these realkalinization-induced effects were significantly reduced by early application of cariporide. Therefore, delay of acute NHE-dependent pH(in) recovery may represent a promising mechanism for inhibition of EC activation upon reperfusion., ((c) 2007 Wiley-Liss, Inc.)

The signalling peptide bradykinin (BK) is implicated in inflammation and angiogenesis. It promotes fluid transport from blood vessels to interstitial space, and thus facilitates oedema formation. To clarify whether paracellular or transcellular pathways mediate this effect, we investigated the BK-stimulated fluid transport across endothelial monolayers in vitro by comparison of electrical and fluorescence methods. Electrical cell impedance sensing monitored a biphasic response of cell layers to BK with high time resolution: a short decrease (18%, 1 min) was followed by a more sustained increase in paracellular resistance (30%, 10 min). The two phases can be assigned to second messengers of the BK-signalling pathway: Ca(2+) for the decrease and cyclic adenosine monophosphate for the rise of resistance, respectively. Despite tightening of the intercellular clefts, BK increased the fluid permeability by 39%, indicating transcellular fluid transport. Additionally, BK stimulated both in- and outwardly directed membrane trafficking as assessed by vesicular fluid uptake (by 49%) and secretion of von Willebrandt factor (by 85%). In conclusion, the combination of electrical and fluorescence data suggests that BK induces a shift from para- to transcellular fluid transport across endothelium.

Aim: In response to aldosterone endothelial cells swell and stiffen. Although amiloride-sensitive sodium and water uptake is known to be involved, the underlying mechanisms are yet unclear. We tested the hypothesis whether the intracellular accumulation of water or organic matter is responsible for the structural and functional alterations., Methods: Atomic force microscopy was used as an imaging tool and a mechanical nanosensor. Cell water, organic cell matter and cell pressure was measured at single cell level in human umbilical vein endothelial cells (HUVEC). Furthermore, we tested by means of a miniature perfusion chamber in vitro the physical robustness to blood flow of the aldosterone-treated endothelium., Results: In response to a three-day treatment with 1 nM aldosterone HUVEC swell. To our surprise, cell water decreased from 82+/-6% to 71+/-5% while intracellular organic matter increased from 18+/-1.8% to 29+/-3.0%. These changes were paralleled by a rise in cell pressure of 114%, measured in living HUVEC in vitro. Blood flow across the endothelium was found significantly altered after aldosterone treatment. Imaging the endothelial monolayer after blood perfusion disclosed large gaps between cells treated with aldosterone. The mineralocorticoid receptor blockers, spironolactone and eplerenone could prevent the aldosterone actions., Conclusion: Mild aldosteronism causes intracellular accumulation of organic matter at the cost of cell water. This makes endothelium stiff and vulnerable to shear stress. The measurements could explain clinical observations that high blood pressure combined with high plasma aldosterone concentration may damage the endothelium of blood vessels.

Background: Aldosterone has long been known to control water and electrolyte balance by acting on mineralocorticoid receptors in kidney. However, recent studies identified these classic receptors also in the vascular system. Indeed, aldosterone alters structure and function of human endothelium. We applied nanotechniques to detect these changes., Methods: Experiments were performed in cultured monolayers of human umbilical vein endothelial cells (HUVECs). Ten nanomoles per liter aldosterone or solvent (control) was added to the culture medium for 72 hours. Atomic force microscopy (AFM) was used as a nanotool that physically interacts with the cell surface. In the monolayer we measured cell volume, apical cell surface, and cell stiffness., Results: Volume, apical surface area, and stiffness of individual cells increased in response to aldosterone by 18% (P < 0.05), 64% (P < 0.001), and 78% (P < 0.01), respectively. Imaging of the cell-to-cell contacts disclosed gaps in response to aldosterone. Furthermore, underneath the monolayer we detected an aldosterone-induced increase of protein deposition by 58% (P < 0.001)., Conclusion: Aldosterone remodels human endothelium in vitro. Cells increase in size and stiffness. Protein leakage through intercellular gaps could be caused by the increased apical membrane tension. The increase in cell rigidity could trigger endothelial dysfunction observed in hyperaldosteronism.

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 approximately 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 micromol/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.

Aldosterone controls electrolyte balance by acting on the renal epithelium. However, there is strong evidence that vascular endothelium is another target for mineralocorticoids. Endothelial cells gain sensitivity to diuretics when exposed to aldosterone. Atomic force microscopy detects such phenomena. It is speculated that endothelium and kidney join forces in the regulation of body fluids.

Weibel-Palade bodies (WPB) are endothelial vesicles that store von Willebrand factor (vWF), involved in the early phase of hemostasis. In the present study we investigated the morphodynamics of single WPB plasma membrane fusion events upon hypoxic stimulation by using atomic force microscopy (AFM). Simultaneously, we measured vWF release from endothelial cells to functionally confirm WPB exocytosis. Exposing human endothelial cells to hypoxia (pO2 = 5 mm Hg) we found an acute (within minutes) release of vWF. Despite acute vWF release, potential cellular modulators of secretion, such as intracellular pH and cell volume, remained unchanged. We only detected a slight instantaneous increase of cytosolic Ca2+ concentration. Although overall cell morphology remained virtually unchanged, high resolution AFM images of hypoxic endothelial cells disclosed secretion pores, most likely the loci of WPB exocytosis on luminal plasma membrane. We conclude that short-term hypoxia barely alters overall cell morphology and intracellular milieu. However, at nanometer scale, hypoxia instantaneously switches the smooth luminal plasma membrane to a rough activated cell surface, covered with secretion pores that release vWF to the luminal cell surface.

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 ohms cm2 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 alpha-, beta-, and gamma-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 beta-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.

To evaluate shear stress-induced effects on cultured cells we have extended the mechanical setup of a multichannel in vitro rheological system and developed software allowing entire processing control and image data analysis. The values of cell motility, degree of orientation (alignment), and cell elongation were correlated as a function of time (morphodynamics). Collective and individual endothelial cells within confluent cultures displayed a shear stress-dependent characteristic phase behavior of the following time course: resting conditions (phase I), change of motility (phase II), onset of alignment (phase III), and finally cell elongation (phase IV). Especially cell motility was characterized by a randomized zigzag movement around mean trajectories (fluctuations) together with mean cell locomotion. Onset of shear stress caused a down-regulation of fluctuations of 30% within <10 min and simultaneously increased locomotion velocities preferring the flow direction (phase II). After a lag period of 10 to 20 min cells orientated in the direction of flow (phase III) without significant cell elongation, which finally occurs within hours (phase IV). These data provide first evidence that cells within confluent endothelial monolayers respond to shear stress with a characteristic phase behavior.

Atomic force microscopy (AFM) is a useful technique for imaging the surface of living cells in three dimensions. The authors applied AFM to obtain morphological information of individual cultured endothelial cells of bovine aorta under stationary and strain conditions and to simultaneously measure changes in cell volume in response to aldosterone. This mineralocorticoid hormone is known to have acute, non-genomic effects on intracellular pH, intracellular electrolytes and inositol-1,4,5-triphosphate production. In this study whether endothelial cells under tension change their volume in response to aldosterone was tested. Such changes were already shown in human leukocytes measured by Coulter counter. In contrast to leukocytes that are more or less spherical and live in suspension, endothelial cells exhibit a complex morphology and adhere to a substrate. Thus, measurements of discrete cell volume changes in endothelial cells under physiological condition is only feasible with more sophisticated techniques. By using AFM we could precisely measure the absolute cell volume of individual living endothelial cells. Before the addition of aldosterone the cell volume of mechanically stressed endothelial cells mimicking arterial blood pressure was 1827 +/- 172 fl. Cell volume was found to increase by 28% 5 min after hormone exposure. Twenty-five minutes later cell volume was back to normal despite the continuous presence of aldosterone in the medium. Amiloride, a blocker of the plasma membrane Na+/H+ exchanger prevented the initial aldosterone-induced volume increase. Taken together, AFM disclosed a transient swelling of endothelial cells induced by the activation of an aldosterone sensitive plasma membrane Na+/H+ exchanger.