Marianne Wilhelmi

Microtubule-dependent matrix metalloproteinase-2/matrix metalloproteinase-9 exocytosis: Prerequisite in human melanoma cell invasion

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave and degrade a wide spectrum of extracellular matrix components. By enhancing turnover of extracellular matrix, MMP activity is also known to play a key role in tumor cell invasion. Because extracellular protease activity requires efficient release of these proteases to the cellular surface, we investigated storage, transport, and exocytosis of MMP-2 and MMP-9 in human melanoma cells using immunofluorescence, electrical, and biochemical techniques. Immunolabeling of melanoma cells with antibodies specific for MMP-2 and MMP-9 led to the identification of two distinct populations of small cytoplasmatic vesicles containing MMP-2 or MMP-9, respectively. In combination with α-tubulin–specific antibodies, both vesicle populations were found to be aligned along the microtubular network. Moreover, the molecular motor protein kinesin is shown to be localized on most of these vesicles, providing evidence that the identified vesicles are actively propelled along microtubules toward the plasma membrane. The functional relevance of these findings is demonstrated using low dosage (5.9 nmol/L) of paclitaxel to affect the microtubular function of melanoma cells. Although cell proliferation is not altered, paclitaxel treatment impairs secretion of MMP-2/MMP-9 and significantly reduces invasive activity in our new cell invasion assay. In conclusion, we demonstrate in melanoma cells that microtubule-dependent traffic of MMP-containing vesicles and exocytosis are critical steps for invasive behavior and therefore are potential targets for specific antitumor drugs.

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.

Nuclear pores collapse in response to CO2 imaged with atomic force microscopy.

Nuclear pore complexes (NPCs) are the rate-limiting barriers for the exchange of macromolecules (e.g. transcription factors or mRNA) between the nuclear and cytosolic compartments. NPC conformation determines movement of cargo in either direction and thus controls gene expression. ATP and calcium are known to induce an NPC shape change (increase in height and decrease in diameter) indicating pore contraction. Here we report a CO2-induced shape change which is different to the ATP/calcium response. Experiments were performed on the isolated nuclear envelope of Xenopus laevis oocytes. The nuclear envelope was spread on glass and the native cytoplasmic surface was imaged with atomic force microscopy (AFM). The preparation was scanned in a water-saturated 100% O2 atmosphere at room temperature. Exposure to 5% CO2 (95% O2) led over a time course of minutes to a dramatic NPC shape change (decrease in height and decrease in diameter) indicating pore closure. NPCs turned flat and central channel openings virtually disappeared. The CO2 response was only slowly reversible. We conclude that NPCs apparently collapse in response to CO2, a structural change that could lead to the functional isolation of the cell nucleus.

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.