Christlieb Haller

Cell-cell contacts in the human cell line ECV304 exhibit both endothelial and epithelial characteristics.

Abstract Endothelial cells separate the intra- and extravascular space and regulate transport processes between these compartments. Since intercellular junctions are required for these specific cell functions, the cell-cell contacts in the permanent cell line ECV304 were systematically analyzed and compared with human umbilical vein endothelial cells (HUVECs) in primary culture and with the epithelial Madin Darby Canine Kidney (MDCK) cell line. Filter-grown ECV304 cells generate a distinct electrical resistance and a permeability barrier between cell culture compartments. Electron microscopy of ECV304 cells revealed lateral membrane interdigitations, typically found in endothelial cells in vivo, with direct membrane contact sites, which prevented the diffusion of lanthanum. By immunoblot and immunofluorescence analysis, the expression and cellular localization of the tight junction and adherens-type junction proteins occludin, ZO-1, symplekin, β-catenin, and plakoglobin were analyzed. ECV304 cells display further characteristics of endothelial cells, including the expresssion of thrombomodulin and of the vitronectin receptor CD51, as well as the secretion of plasminogen activator inhibitor 1 (PAI-1) and endothelin. However, ECV304 cells also express proteins characteristically found in epithelial cells, including E-cadherin and the desmosomal proteins desmoplakin, desmocollin, and desmoglein; occasionally desmosomal structures can be identified by electron microscopy. In conclusion, ECV304 cells express many endothelial markers and form specialized intercellular junctions that display some epithelial features. Thus this reportedly endothelial-derived permanent human cell line may be dedifferentiated toward an epithelial phenotype.

Cytotoxicity of radiocontrast agents on polarized renal epithelial cell monolayers

OBJECTIVE Radiocontrast-induced nephropathy is a clinically important complication of coronary angiography. The cellular mechanisms of radiocontrast-induced renal dysfunction are not clear. Since tubular transport functions depend on the polarity of renal epithelial cells, we investigated the effects of radiocontrast agents on polarized tubular cells in vitro. METHODS We studied the effects of iso-iodine concentrations (37 and 74 mg iodine/ml) of an ionic (diatrizoate) and a non-ionic (iopamidol) monomeric radiocontrast agent and of hyperosmolal mannitol control solutions on filter-grown renal epithelial cell (MDCK, LLCPK) monolayers in vitro. The cytotoxicity was assayed by measurement of cell viability, transepithelial resistance, inulin permeability and (polarized) cellular enzyme release. The polarized MDCK cell phenotype was assessed by transmission electron microscopy and indirect immunofluorescence microscopy using monoclonal antibodies against specific apical (gp135) and basal (gp60, uvomorulin) MDCK surface markers. RESULTS The radiocontrast agents reduced cell viability to a greater extent than hyperosmolal mannitol solutions in both cell lines; diatrizoate was more toxic than iopamidol. LLCPK cells were more susceptible to radiocontrast cytotoxicity than MDCK cells. This cytotoxicity was associated with an alteration of MDCK cell polarity as assessed by the redistribution of surface marker proteins. CONCLUSIONS Diatrizoate is more toxic than iopamidol, which is partly related to its higher osmolality. The cytotoxicity of radiocontrast agents induces a redistribution of polarized membrane proteins which could contribute to the pathophysiology of radiocontrast-induced nephropathy.

Heterologous Expression of Human VEGF165 in Rat Brain: Dose-Dependent, Heterogeneous Effects on CBF in Relation to Vascular Density and Cross-Sectional Area

Vascular endothelial growth factor (VEGF) induces increased vessel permeability and formation of abnormal vessels. To investigate cerebral blood flow (CBF) during local overexpression of VEGF recombinant adenoviruses carrying the human VEGF165 complementary DNA (2.3 to 23 · 108 pfu/mL) were injected stereotactically into the caudate nucleus of anesthetized rats. Saline and adenoviruses carrying the β-galactosidase gene served as controls. Eleven days later (1) size and density of vessels were assessed in hematoxylin–eosin–stained sections, (2) vascular permeability was measured by intravenous Evans blue injections, and (3) local CBF (lCBF) was quantified using the iodo-[14C]antipyrine technique. Dose-dependent increases were found in (1) vessel density and size (only vessels >43 μm could be quantified morphologically), (2) Evans blue extravasation and brain edema formation, and (3) lCBF (up to eightfold). At medium doses, hyperemic areas and smaller areas of decreased lCBF were found. In low flow areas, vascular cross-sectional areas were increased 223-fold and vessel density up to 10-fold. In high flow areas, these parameters were increased 32-fold and up to 15-fold, respectively. Adenovirus mediated VEGF overexpression results in (1) increased vessel size and density, (2) areas of increased and of decreased flow, and (3) more and smaller vessels in high flow than in low flow areas. These results indicate a diverging flow pattern of newly formed vessels.

Current issues in the diagnosis and management of patients with renal artery stenosis: a cardiologic perspective

Renal artery stenosis most often is caused by atherosclerosis. Although patients with renal artery stenosis can be managed conservatively, renal revascularization may be indicated, particularly in patients with refractory hypertension on a multidrug regimen and patients with declining renal function. Duplex ultrasonography of the renal arteries and magnetic resonance angiography are currently the most efficient noninvasive methods for the evaluation of renal artery stenosis. Selective digital subtraction renal arteriography remains the gold standard for the definitive diagnosis. In selected patients undergoing coronary studies and angiography immediately after the coronary procedure can be efficient. Atherosclerotic renal artery lesions, which commonly affect the renal artery ostium, can be treated safely and effectively with balloon-expandable stents. Successful angioplasty commonly results in improved control of hypertension, but an overall benefit on renal function and/or patient survival has not been shown. Generally the risk/benefit ratio of renal artery stenting seems favorable, but further randomized studies are needed for evidence-based decision making. All patients with atherosclerotic renal artery stenosis should receive rigorous secondary prevention measures including platelet inhibitors, statins, and beta-blockers.

POLARIZED EXPRESSION OF HETEROLOGOUS MEMBRANE PROTEINS TRANSFECTED IN A HUMAN ENDOTHELIAL-DERIVED CELL LINE

The generation and maintenance of cell polarity in endothelial cells is poorly understood, partly because of a lack of a permanent endothelial in vitro model system. Here we evaluated the spontaneously immortalized human endothelial-derived cell line ECV304 as an in vitro model system for the study of the polarized expression of heterologous membrane proteins. Several stable ECV304 clones were generated by calcium phosphate transfection/G418 selection with cDNAs encoding membrane proteins of known cell surface distribution in the epithelial Madin Darby canine kidney (MDCK) cell line: influenza hemagglutinin and uvomorulin/E-cadherin were used as markers for the apical, respectively lateral cell membrane, the human lymphocyte surface marker CD7 served as an example of a circumferentially distributed membrane protein. Analysis of the transfected ECV304 clones using conventional and confocal immunofluorescence microscopy and immunoelectron microscopy revealed the same membrane distribution of the heterologous proteins in ECV304 cells as in MDCK cells. This polarized expression of heterologous membrane proteins in the endothelial-derived ECV304 cell line indicates efficient protein sorting/membrane trafficking mechanisms. The apical, lateral and basal cell membrane domains could be distinguished in ECV304 cells by confocal immunofluorescence microscopy. The permanent endothelial-derived ECV304 cell line may be a useful in vitro model system for the study of endothelial cell polarity.

Ionic Radiocontrast Media Disrupt Intercellular Contacts via an Extracellular Calcium-Independent Mechanism

Direct cytotoxic effects of radiocontrast (RC) agents have been implicated in radiocontrast nephropathy (RCIN). The interaction between extracellular calcium, which plays a central role in intercellular contacts, and the in vitro toxicity of RC was tested in Madin-Darby canine kidney (MDCK) cell monolayers grown on permeable supports. Cell viability was determined by trypan blue exclusion. The function of intercellular junctions was assessed by measuring the electrical transmonolayer resistance (TMR). The cell contacts were examined with indirect immunofluorescence microscopy using antibodies against the junctional proteins E-cadherin, ZO-1 and occludin. The ionic RC agents diatrizoate and ioxaglate (74 mg iodine/ml), but not the nonionic compounds iohexol or iodixanol, decreased ionized calcium (Ca2+) in the incubation media from 1.48 ± 0.04 mM (control) to 0.89 ± 0.06 mM (diatrizoate), respectively to 1.05 ± 0.08 mM (ioxaglate). Diatrizoate, and to a lesser extent ioxaglate, reduced the number of viable MDCK cells and showed a redistribution of the E-cadherin, ZO-1 and occludin immunofluorescence signal with a parallel decrease of the TMR indicating an impaired monolayer integrity. A similar reduction of extracellular Ca2+ through EGTA failed to reproduce these effects. Conversely, raising Ca2+ in diatrizoate-containing media to control levels did not abrogate its toxicity. In conclusion, the ionic RC agents diatrizoate and ioxaglate, but not the nonionic compounds iohexol or iodixanol, reduce extracellular Ca2+ in vitro. However, this reduction of Ca2+ does not explain their cytotoxic effects which could contribute to the pathogenesis of RCIN in vivo by opening intercellular junctions.

Refractory oedema in congestive heart failure: a contributory role of loop diuretics?

Abstract. We report a patient with congestive heart failure (CHF) who presented with massive oedema resistant to therapy with maximal doses of loop diuretics, despite an adequate renal function. After a diuretic pause and dietary salt restriction, a conventional dose of furosemide in combination with distally active diuretics induced a prompt weight loss exceeding 30 kg with stable renal function. We suggest that the ‘refractory’ oedema in this patient was due to a combination of CHF and inappropriate (loop) diuretic therapy in conjunction with a high dietary sodium intake. We conclude that in the absence of hyponatraemia and renal failure, even severe oedema may not represent a negative prognostic indicator. The recognition of diuretic‐associated mechanisms complicating cardiac oedema is essential to avoid the vicious circle of worsening oedema whilst escalating therapy with loop diuretics.

Influence of intercellular junctions on endothelin secretion of human umbilical vein endothelial cells in vitro.

Abstract The endothelium plays a pivotal role in the theological regulation of blood flow by the secretion of vasoactive factors. The interaction between shear forces and the endothelium is determined by the mechanical properties of the endothelial cell layer which are associated with intercellular junctions. Cell-cell contacts could therefore modulate the secretion of vasocative factors in response to theological stimuli. We investigated the relationship between intercellular junctions and the secretion of the vasoconstrictor peptide endothelin and the coagulation co-factor von Willebrand factor (vWF). Human umbilical vein endothelial cells (HUVECs) were used as in vitro endothelial model system. Intercellular junctions were reversibly disrupted by calcium chelation or hypertonic stress; alternatively, the formation of intercellular junctions was inhibited by culturing the cells in suspension or by plating them in the presence of an inhibitory anti-VE-cadherin antibody. The opening of intercellular junctions was verified by assessing transmonolayer electrical resistance (TMR) and immunofluorescence morphology. The concentration of endothelin and vWF was measured in the cell culture supernatants using specific ELISAs. The secretion of endothelin was inhibited by EGTA (5 mM) and stimulated by incubation with tumor necrosis factor α (TNFα, 40 ng/ml). Treatment with hypertonic medium (glycerol, 1200 mosmol/l) for 10 minutes opened intercellular junctions and markedly reduced the secretion of endothelin. HUVECs in suspension culture did not secrete endothelin and failed to respond to TNFα, but readily resumed these functions upon forming a new monolayer on plastic. The reconstitution of intercellular junctions after suspension culture could be inhibited using a specific anti-VE-cadherin antibody. This antibody, but not a non-specific anti-humanIgG antibody reduced endothelin secretion. The secretion of von Willebrand Factor was less dependent on intercellular junctions. The opening of intercellular junctions did not induce cell death, since the cells continued to exclude trypan blue. The results of this study suggest a novel and potentially pathophysiologically/clinically relevant correlation between intercellular junctions and the secretion of endothelin in endothelial cells.

Coronary Artery Ectasia and Systolic Flow Cessation in Hypertrophic Cardiomyopathy

A 24-year-old woman was evaluated for exertional dyspnea and chest pain. Echocardiography showed marked apical, septal (14 mm), and anterolateral (21 mm) hypertrophy with normal inferior and posterior wall thickness (Figure 1⇓). The patient underwent right and left heart catheterization, with coronary and biventricular angiography. The cardiac index was 2.2 L · min−1 · m−2. The left ventricular pressure was 100/0 to 10 mm Hg, without evidence of an intracavitary gradient. Right anterior oblique ventriculography demonstrated a subtotal obliteration of the left ventricular cavity during systole (Figure 2⇓). Simultaneous right and left ventricular angiography revealed a massively thickened interventricular septum (Figure 3⇓). Coronary angiography showed no hemodynamically relevant fixed stenosis. The striking finding was the dilation and pronounced tortuosity of the coronary arteries, particularly the left anterior descending arterial (LAD) system (Figure 4⇓, bottom), without signs of a …

Dr Haller's reply

1 Smith DK. Neal IJ, Holmberg SD. and the Centers for Disease Control Idiopathic CD4 + T-lymphocytopenia Task Force. Unexplained opportunistic infections and CD4 + T-lymphocytopenia without HIV Infection. An investigation of cases in the United States. N Engl / Mrd 1993: 238: 373-9. 2 Fauci AS. CD4 + T-lymphocytopenia without HIV infection no lights. no camera, just facts (Editorial). N E n g l ] Mrd 1993 :