RESUMEN
We investigated the influence of heparin on the composition of the subendothelial matrix and on endothelial permeability to elucidate the structure-function relationship of matrix composition and permeability. Albumin flux across the confluent endothelial monolayers was used to assess the macromolecular permeability. Lowest values were obtained when 100% serum was used as medium for permeability studies. The endothelial matrix components, fibronectin and basement membrane-associated heparan sulfate proteoglycan (HSPG), were measured by enzyme immunoassay. Treatment of proliferating endothelial cells with heparin (0-900 micrograms/ml) induced a dose-dependent decrease in endothelial HSPG content, whereas the fibronectin content was unaltered. This structural change was accompanied by an increase in albumin permeability. Both heparin effects exhibited similar dose-response curves with half-maximal effects at approximately 5 micrograms/ml heparin. Acute addition of 300 micrograms/ml heparin had no effect on permeability or HSPG content. When endothelial cells were preincubated with an HSPG antiserum, the endothelial permeability increased nearly threefold. Our results indicate that heparin-induced loss of HSPG may cause the increase in endothelial permeability. The data underline the importance of HSPG for the integrity of the endothelial barrier.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Heparina/farmacología , Animales , Sangre , División Celular , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Fibronectinas/biosíntesis , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/biosíntesis , Concentración Osmolar , Proteoglicanos/biosíntesis , Porcinos , Factores de TiempoRESUMEN
In the present study we investigated the influence of elevated low density lipoprotein (LDL) concentration on endothelial permeability. Endothelial cells were cultured on microporous membranes until confluence and albumin, dextran and LDL transfer across endothelial monolayers was determined to assess macromolecular permeability. Exposure of proliferating aortic endothelial cells to LDL levels of more than 1 mg/ml LDL-cholesterol induced a concentration-dependent exponential increase in the permeability of confluent endothelial monolayers. Acute addition of high LDL concentration did not alter macromolecular permeability. Once elevated permeability was induced, it persisted. It was not readily reversible after addition of normal LDL levels. Change in permeability was accompanied by a selective decrease in basement membrane associated heparan sulfate proteoglycan (HSPG) content. The apparent parallel between the loss in endothelial barrier function and HSPG decrease implicates a connection between the two events. Prolonged, but not acute, incubation with antiserum directed against the core-protein of HSPG also led to increased permeability, suggesting a causal role of HSPG for the proper function of endothelium. The fact that non-atherogenic LDL-cholesterol levels had no effect indicates that a 'threshold' concentration for LDL-cholesterol may exist, leading to nondenuding injury in the endothelial barrier as an early event in development of atherosclerosis.
Asunto(s)
Arteriosclerosis/etiología , Permeabilidad de la Membrana Celular/fisiología , Endotelio Vascular/fisiología , Lipoproteínas LDL/fisiología , Animales , Aorta/patología , Aorta/fisiología , Arteriosclerosis/patología , Células Cultivadas , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Fibronectinas/metabolismo , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/metabolismo , Heparitina Sulfato/farmacología , Lipoproteínas LDL/farmacología , Microscopía Fluorescente , Proteoglicanos/metabolismo , Proteoglicanos/farmacología , PorcinosRESUMEN
The effect of increasing low density lipoprotein (LDL) concentrations on the synthesis of basement membrane components was investigated in proliferating porcine aortic endothelial cells (PAEC) in culture. Basement membrane-associated heparan sulfate proteoglycan (HSPG) and fibronectin were determined by enzyme immunoassay. Low extracellular LDL-levels increase, high extracellular LDL-levels decrease the HSPG content of PAEC. Fibronectin synthesis was only slightly affected while proliferation and metabolic activity as assessed by lactate production were constant. Insulin or high extracellular glucose did not influence the effect of LDL on basement membrane components.