Uta Hillebrand

Increased inorganic phosphate induces human endothelial cell apoptosis in vitro

Chronic kidney disease with hyperphosphatemia is associated with accelerated atherosclerosis and endothelial dysfunction. However, the contribution of high serum phosphate levels to endothelial injury is incompletely understood. The aim of this work was to evaluate the responses of endothelial cells to elevated levels of extracellular phosphate in vitro. High phosphate in concentrations similar to those observed in uremia-associated hyperphosphatemia (>2.5 mM) induced apoptosis in two endothelial cell lines (EAhy926 cells and GM-7373 cells). This effect was enhanced when cells were incubated for 24 h in the presence of 2.8 mM calcium instead of 1.8 mM. By treating cells with 0.5 or 1.0 mM phosphonoformic acid, an inhibitor of the phosphate transporter, death was completely prevented. The process of phosphate-induced apoptosis was further characterized by increased oxidative stress, as detected by increased ROS generation and disruption of the mitochondrial membrane potential at approximately 2 h after treatment, followed by caspase activation. These findings show that hyperphosphatemia causes endothelial cell apoptosis, a process that impairs endothelial integrity. Endothelial cell injury induced by high phosphate concentrations may be an initial event leading to vascular complications in patients with chronic kidney disease.

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.

The Soluble VEGF Receptor sFlt1 Contributes to Endothelial Dysfunction in CKD

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.

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.

High phosphate directly affects endothelial function by downregulating annexin II

Hyperphosphatemia is associated with increased cardiovascular risk in patients with renal disease and in healthy individuals. Here we tested whether high phosphate has a role in the pathophysiology of cardiovascular events by interfering with endothelial function, thereby impairing microvascular function and angiogenesis. Protein expression analysis found downregulation of annexin II in human coronary artery endothelial cells, an effect associated with exacerbated shedding of annexin II-positive microparticles by the cells exposed to high phosphate media. EAhy926 endothelial cells exposed to sera from hyperphosphatemic patients also display decreased annexin II, suggesting a negative correlation between serum phosphate and annexin II expression. By using endothelial cell-based assays in vitro and the chicken chorioallantoic membrane assay in vivo, we found that angiogenesis, vessel wall morphology, endothelial cell migration, capillary tube formation, and endothelial survival were impaired in a hyperphosphatemic milieu. Blockade of membrane-bound extracellular annexin II with a specific antibody mimicked the effects of high phosphate. In addition, high phosphate stiffened endothelial cells in vitro and in rats in vivo. Thus, our results link phosphate and adverse clinical outcomes involving the endothelium in both healthy individuals and patients with renal disease.

Pathogenesis of Hypertension: Interactions Among Sodium, Potassium, and Aldosterone

Arterial hypertension is a major cause of disease-related morbidity and mortality worldwide. It is nearly absent in populations that consume natural foods low in sodium. However, in industrial countries, where the individual intake of sodium is at least 10 times higher, the prevalence of hypertension is approximately 40%. Major population-based studies link a high-sodium and low-potassium diet to an increase in blood pressure. A hallmark of arterial hypertension is endothelial dysfunction characterized by decreased synthesis of nitric oxide (NO). Plasma sodium and potassium are major determinants for the mechanical stiffness of endothelial cells. High plasma sodium levels stiffen endothelial cells and block NO synthesis. Aldosterone is a prerequisite for this action. However, high plasma potassium levels soften endothelial cells and activate NO release. There is increasing evidence that sodium can be stored transiently in considerable amounts and osmotically inactive in the interstitium. Taken together, it is recommended to maintain plasma sodium levels in the low physiologic range and potassium levels in the high physiologic range while suppressing plasma aldosterone as much as possible. A restriction in sodium intake that is accompanied by increased intake of potassium can profoundly improve the prevalence of hypertension and cardiovascular disease.

Blood pressure, antihypertensive treatment, and graft survival in kidney transplant patients.

Whether the use of angiotensin‐converting enzyme inhibitor or angiotensin receptor blocker inhibitor (ACEI/ARB) is beneficial in renal transplant recipients remains controversial. In this retrospective study on 505 renal transplant recipients, we analyzed blood pressure and graft survival according to antihypertensive treatment with ACE‐I/ARB and/or calcium channel blockers (CCB) over a period of 10 years. Patients were stratified according to their blood pressure 1 year after transplantation [controlled (≤130/80 mmHg; CTR, 181 patients) and noncontrolled (>130/80 mmHg; non‐CTR, 324 patients)] and according to antihypertensive treatment (ACE‐I/ARB and/or CCB taken for at least 2 years). One year after transplantation, 88.4% of CTR and 96.6% of non‐CTR received antihypertensive treatment (P < 0.05). Graft survival was longer in CTR than in non‐CTR (P < 0.05). Importantly, graft survival was longer in patients who received long‐term treatment with ACEI/ARB, CCB, or a combination of ACEI/ARB and CCB (P < 0.001). The beneficial effect of ACEI/ARB therapy was more pronounced in non‐CTR compared with that of CTR. We conclude that blood pressure control is a key target for long‐term graft survival in renal transplant patients. Long‐term ACEI/ARB and CCB therapy is beneficial for graft survival, especially in patients with diabetes and/or albuminuria.

Dose-dependent endothelial cell growth and stiffening by aldosterone: endothelial protection by eplerenone.

Background Aldosterone at high concentrations causes an expansion of apical surface area and volume of cultured endothelial cells. These morphological changes are associated with endothelial cell stiffening. Here, we tested the hypothesis that the aforementioned aldosterone actions are confined to aldosterone concentrations within the pathophysiological range. Moreover, we investigated whether endothelial cells of venous and arterial origin respond similarly to aldosterone and whether the new aldosterone antagonist eplerenone effectively prevents endothelial cell growth and stiffening. Methods We used an endothelial cell line of venous origin (EAHy 926) and primary cultures of human coronary artery endothelial cells (HCAEC). Cells were incubated for 72 h with aldosterone at concentrations of 0.1, 1, 10 and 100 nmol/l. Eplerenone was added at a concentration of 2 μmol/l. Applying atomic force microscopy, we scanned cell layers under fixed and living conditions, allowing measurement of endothelial cell apical surface, voIume and cellular stiffness. Results Aldosterone had comparable effects on EAHy 926 and HCAEC. In EAHy 926, the apical surface increased dose dependently by up to 72 ± 5% and cell volume by up to 36 ± 5%. In HCAEC, the maximum increase of apical surface was 78 ± 6% and maximum cell volume expansion was 58 ± 6%. Furthermore, aldosterone increased endothelial cell stiffness from 1.47 ± 0.08 kPa up to 3.95 ± 0.15 kPa in EAHy 926, and from 1.64 ± 0.13 kPa up to 4.31 ± 0.13 kPa in HCAEC. Physiological aldosterone concentrations had no effect, but starting at 1 nmol/l, corresponding to the low pathophysiological range, substantial cell alterations emerged. Eplerenone, at a therapeutic concentration, prevented the observed actions of aldosterone. Conclusions Aldosterone-induced endothelial cell growth and stiffening in vitro begins with concentrations in the low pathophysiological range. The preventive action of eplerenone indicates that the endothelium could be a major target of this drug in vivo.

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.

How steroid hormones act on the endothelium—insights by atomic force microscopy

Vascular actions of steroid hormones have gained increasing importance. Indeed, some steroid hormones favorably influence vascular structure and function, whereas others are detrimental. This review will focus on the endothelial effects of steroid hormones. In the first part, we summarize data from in vivo studies elucidating the regulation of endothelial function by steroid hormones. Accumulating data argue for an improvement of endothelium-derived relaxation and impaired vascular contraction by estradiol, whereas testosterone, progesterone, and aldosterone have contrary effects. In the second part, we present data from novel atomic force microscopy studies performed in living endothelial cells under the influence of steroid hormones. These studies provide insight into structural and functional alterations of endothelial cells characterized by changes in volume, apical surface, and stiffness. We summarize the available evidence that changes in shape of endothelial cells translate into changes of endothelial cell stiffness. Under the influence of estradiol, endothelial cells become spherical with consecutive improvement of elasticity, whereas aldosterone flattens endothelial cell-shape leading to increased stiffness. Both, endothelial cell shape and stiffness are major determinants of endothelial nitric oxide production. These studies emphasize the great potential of atomic force microscopy to investigate the function of living endothelial cells.