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Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated
R R Schleef1, D J Loskutoff, T J Podor
1Research Institute of Scripps Clinic, Committee on Vascular Biology, La Jolla, California 92037.
Insights
Activation of endothelial cells with TNF alpha or LPS significantly increases surface plasminogen activator inhibitor-1 (PAI-1). This PAI-1 redistribution on the cell surface may impair fibrinolysis and promote thrombosis during inflammation.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Endothelial cells play a crucial role in regulating blood coagulation and fibrinolysis.
- Plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system.
- The surface distribution of PAI-1 on endothelial cells is not fully understood, particularly under inflammatory conditions.
Purpose of the Study:
- To investigate the topographical distribution of PAI-1 on the surface of activated human umbilical vein endothelial cells (HUVECs).
- To compare PAI-1 distribution on activated versus unactivated HUVECs.
- To explore the potential implications of altered PAI-1 surface expression on endothelial cell function.
Main Methods:
- Immunogold electron microscopy (EM) was used to visualize PAI-1 on HUVEC surfaces.
- Cells were activated with tumor necrosis factor alpha (TNF alpha) or lipopolysaccharide (LPS).
- High-resolution surface replication was combined with immunogold staining for detailed topographical analysis.
Main Results:
- Unactivated HUVECs showed minimal PAI-1 on the apical surface, with most found in the extracellular matrix.
- Activation with TNF alpha or LPS led to a 10-fold increase in PAI-1 on the apical cell surface and extracellular matrix.
- Specific immunoabsorption or treatment with tissue-type plasminogen activator significantly reduced PAI-1 staining.
- Similar increases in surface PAI-1 were observed in activated bovine aortic endothelial cells.
Conclusions:
- Inflammatory stimuli induce a significant redistribution of PAI-1 to the apical surface of endothelial cells.
- This increased surface PAI-1 may contribute to reduced endothelial fibrinolytic activity.
- The findings suggest a mechanism by which inflammation-induced PAI-1 expression could promote localized intravascular thrombosis.
Abstract:
Immunogold EM was employed to compare the distribution of type 1 plasminogen activator inhibitor (PAI-1) on the surface of agonist-activated human umbilical vein endothelial cells (HUVECs) with that of control, unactivated cells. As previously observed, (Schleef, R.R., T.J. Podor, E. Dunne, J. Mimuro, and D.J. Loskutoff. J. Cell Biol. 110:155-163), analysis of cross-sections of nonpermeabilized control HUVEC monolayers stained first with affinity-purified rabbit antibodies to PAI-1 and then with gold-conjugated goat anti-rabbit IgG, revealed the presence of relatively few gold particles (less than 1-2% of the total) on the apical cell surface. The majority of gold particles were detected primarily in the extracellular matrix between the culture substratum and the cell membrane. In contrast, treatment of HUVECs with tumor necrosis factor alpha (TNF alpha; 200 U/ml, 24 h) or with lipopolysaccharide (LPS; 10 micrograms/ml, 24 h) resulted in an increased staining of PAI-1 not only in the extracellular matrix, but also on the apical cell surface (10-fold increase). Immunoabsorption of the rabbit anti-PAI-1 with purified PAI-1, or treatment of HUVECs with tissue-type plasminogen activator (2.5 micrograms/ml, 2 h, 4 degrees C) reduced the amount of staining both on the apical surface and in the extracellular matrix of agonist-activated HUVECs by 80-95%. The topographical location of PAI-1 on the cell surface was examined further by coupling immunogold staining with high resolution surface replication. Transmission EM of surface replicas from TNF alpha- or LPS-activated HUVECs revealed a general increase in PAI-1 staining both on planar regions and within indentations of the apical cell surface. Nonactivated HUVECs revealed PAI-1-specific immunogold particles only in areas of exposed extracellular matrix between the cells and occasionally at regions of cell-cell contacts. Analysis of activated bovine aortic endothelial cells by immuno-electron microscopy, immunologic assays, and flow cytometry revealed similar increases in surface PAI-1. These increases in surface PAI-1 could be detected by 3 h and continued over a 24-h period. The expression of PAI-1 on the luminal surface of endothelial cells during immune or inflammatory reactions could reduce endothelial fibrinolytic activity, thus, promoting the localized, pathologic formation of intravascular thrombi.