Alma Siflinger-Birnboim

Culture and characterization of pulmonary microvascular endothelial cells.

SummarySurface proteins were compared in endothelial cells (EC) obtained from bovine peripheral lung, pulmonary artery and vein, and dorsal aorta using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Galactose-containing glycoproteins [molecular weight (Mr) 160–220 and 40 kDa] binding to theRicinus communis agglutinin (RCA) and peanut agglutinin (PNA) were selectively observed on pulmonary microvessel EC as compared to EC from pulmonary artery, pulmonary vein, and dorsal aorta. The unique RCA- and PNA-binding profiles of EC from the pulmonary artery and microvessels may be important in characterizing EC from different sites in the pulmonary circulation. The pulmonary microvessel EC monolayer was also 15-fold more restrictive to transendothelial flux of [14C]sucrose (Mr=342 Da) than the pulmonary artery EC monolayer. In contrast, the microvessel EC were only six- and twofold more restrictive to the flux of larger tracer molecules, ovalbumin (Mr 43 kDa) and albumin (Mr=69 kDa) than pulmonary artery EC. The greater restrictiveness of pulmonary microvessel EC monolayer indicates a major phenotypic difference in the cultured pulmonary microvessel EC barrier function.

Serum albumin decreases transendothelial permeability to macromolecules

We examined the effects of serum albumin and other serum proteins on the fluxes of tracer 125I-albumin (MW 69 kDa) and 125I-haptoglobin (MW 100 kDa) across the pulmonary artery endothelial monolayer in vitro to test the role of serum proteins in modulating the endothelial barrier function. Replacement of control complete culture medium (20% fetal calf serum in DMEM) with DMEM alone increased the transendothelial 125I-albumin clearance rate (a measure of 125I-albumin permeability) by 83% of the control value. Repletion with 50% calf serum or with 2.0 g% albumin (i.e., the albumin concentration in 50% serum) decreased 125I-albumin permeability to the control value. This effect of serum or albumin was concentration-dependent since neither 12.5% serum nor 0.5 g% albumin (i.e., albumin concentration in 12.5% serum) altered 125I-albumin permeability from control values. The ammonium sulfate-precipitated serum protein fraction rich in albumin decreased 125I-albumin permeability from the control DMEM value, whereas serum fractions containing predominantly gamma-globulin or depleted of protein did not significantly alter 125I-albumin permeability. Other serum proteins that have been proposed to reduce endothelial permeability, alpha 1-acid glycoprotein (0.035-0.14 g/100 ml) and fibronectin (5 mg/100 ml), did not decrease 125I-albumin permeability from DMEM values. The endothelial permeability of 125I-haptoglobin of 4.63 +/- 0.53 x 10(-6) cm/sec in the presence of DMEM was 30% of the 125I-albumin permeability value. The addition of 2.0 g% albumin or 50% serum decreased 125I-haptoglobin permeability to 57 and 31%, respectively, of the DMEM value. These results indicate the critical role of serum albumin in regulating the restrictiveness of the endothelial barrier to macromolecules.

Selectivity of the endothelial monolayer: effects of increased permeability.

We investigated the mechanism of thrombin-induced increases in endothelial monolayer permeability by examining the effect of thrombin on the molecular sieving characteristics of the endothelial monolayer and comparing the responses of arterial- and venous-derived endothelial cell lines. Bovine pulmonary artery (BPA) and pulmonary vein (BPV) endothelial cells were similarly harvested and cultured. The endothelial cells were grown to confluence on gelatinized polycarbonate filters and the permeabilities to sucrose, albumin, and IgG were measured and corrected for effects of unstirred layers. The control permeabilities of BPA and BPV were similar with both monolayers, demonstrating selectivity to different sized tracer molecules. alpha-Thrombin (10(-6) M) increased the permeability of both BPA and BPV to albumin and sucrose. The permeability of BPA was increased to a greater extent than BPV, perhaps due to phenotypic differences. In both cell lines, the permeability increase was most pronounced for albumin, which by pore theory is best described by an increase in the radius of the small pore pathway for diffusion.

Vascular Endothelial Barrier Function and Its Regulation

The vascular endothelium has a variety of functions [hemostasis, defense reaction (inflammatory response), angiogenesis], among which the control of the exchange of substances between blood and tissues is of prime importance. Capillary permeability to plasma proteins is a critical factor in regulating tissue-fluid balance. The endothelial cell monolayer lining the vessel wall is a porous (semipermeable) membrane through which fluid and solutes are transported. The substances transported include water, respiratory gases and other small lipid-soluble molecules, ions, small lipid-insoluble organic molecules, large hydrophilic and lipophilic proteins. The transport of molecules from the plasma to the vascular endothelium is governed by several factors (Renkin, 1977; Simionescu and Simionescu, 1984): (1) vascular driving forces (i.e., hydrostatic and oncotic pressure gradients), (2) physiochemical properties of the permeant molecule, and (3) the surface properties of the endothelial membrane (Table I). Transport to tissues also depends to a significant extent on the physiochemical properties of the components underlying the endothelium (basement membrane, extracellular matrix, interstitial fluid). This review discusses some critical and novel aspects of transport of solutes and water across the endothelial barrier. Although we have discussed much current literature in the field,it is highly likely that there have been oversights.