Differential Effects of Tumor Necrosis Factor-α and Platelet-Activating Factor on Bovine Pulmonary Artery Endothelial Cells in Vitro

Abstract

The effects of tumor necrosis factor alpha (TNF alpha) and platelet-activating factor (PAF) on monolayer permeability, cytotoxicity, and release of prostacyclin (measured as the stable metabolite 6-ketoprostaglandin [PG]F1 alpha) and thromboxane (TX)B2 were investigated in bovine pulmonary artery endothelial cells (BPAEC). After 4 h of incubation, TNF alpha (2000 U/mL) induced an increase in steady-state 125I-albumin permeability across the BPAEC monolayer (2.9 +/- 0.3%/h vs. 1.8 +/- 0.3%/h in control monolayers; n = 7, p < .05), and induced release of 6-keto-PGF1 alpha (2581 +/- 226 pg/mL vs. 863 +/- 164 pg/mL in controls; n = 16, p < .05) and TXB2 (204 +/- 14 pg/mL vs. 105 +/- 23 pg/mL in controls; n = 10, p < .05). PAF-incubation was also associated with increased 6-keto-PGF1 alpha and TXB2 release (4157 +/- 471 pg/mL and 276 +/- 32 pg/mL, respectively), but did not markedly alter morphology or increase 125I-albumin permeability. Specific tritiated deoxyglucose release and specific LDH release were unaffected by both treatments. These results indicate that TNF alpha contributed directly to increased BPAEC permeability without cytotoxicity or requirement for other serum or cellular components. However, PAF did not directly alter endothelial barrier function despite increased release of 6-keto-PGF1 alpha and TXB2.

The inositol trisphosphate pathway mediates platelet-activating-factor-induced pulmonary oedema

“…One probable reason for the lack of mechanistic studies is the lack of effect of PAF on endothelial cell permeability in vitro [55][56][57], although such an effect has been reported once [58]. Therefore, nearly all of the studies on this subject have been performed in isolated organs or whole animals.…”

Differential Regulation of Lung Endothelial Permeability <b><i>in Vitro</i></b> and <b><i>in Situ</i></b>

“…This failure is not explained by lack of PAF-receptors that are present (Fig. 2) and functional as was shown by the PAF-dependent production of prostacyclin [35]. Some groups were successful in increasing vascular permeability in endothelial cell monolayers by PAF in HUVEC cells, in murine pulmonary artery and in murine pulmonary microvascular endothelial cells [29,36,37].…”

Tumor Necrosis Factor-&#945;-Mediated Pulmonary Endothelial Barrier Dysfunction

“…Although it may be tempting to ascribe the prolonged stimulus-to-response lag time of TNF--induced permeability to requisite de novo gene expression, inhibition of either new protein or mRNA synthesis with cycloheximide or actinomycin D, respectively, fails to block TNF--induced permeability changes [51,106]. Although TNF-reportedly induces endothelial injury under some circumstances, several studies have established that TNF--induced endothelial barrier dysfunction is not dependent on cell injury [60,[106][107][108][109]. That TNF--induced changes in endothelial barrier function occur independently of cell injury has been established by assays measuring membrane integrity (i.e., chromium 51 [106] and lactose dehydrogenase release [107,109]), mitochondrial activity [108], and direct determination of endothelial viability [106].…”

“…Although TNF-reportedly induces endothelial injury under some circumstances, several studies have established that TNF--induced endothelial barrier dysfunction is not dependent on cell injury [60,[106][107][108][109]. That TNF--induced changes in endothelial barrier function occur independently of cell injury has been established by assays measuring membrane integrity (i.e., chromium 51 [106] and lactose dehydrogenase release [107,109]), mitochondrial activity [108], and direct determination of endothelial viability [106]. Further, TNF--induced endothelial barrier dysfunction cannot be ascribed to apoptosis as human endothelial cells, which are resistant to direct TNF--induced apoptosis [57], are sensitive to the paracellular permeability-inducing effects of TNF- [110].…”

“…The TNF-effect is reversible and recovery of barrier function is observed within 24 -72h of TNF-removal [51,52,106,108]. Ovine Pulmonary Artery [102] Porcine Pulmonary Artery [8] Bovine Aortic [51,107,268] Bovine Pulmonary Artery [50,60,106,109,269,270] Bovine Pulmonary Microvascular [114,[271][272][273] Bovine Brain Microvascular [108] Rabbit Corneal [274] Human Umbilical Vein [52,227,239,243,[275][276][277][278][279] Human Brain Microvascular [280] Human Foreskin Microvascular [275] Human Pulmonary Artery [110,228] Human Pulmonary Microvascular [281,282] TNF--induced loss of endothelial barrier function occurs through opening of the paracellular pathway and appears to be dependent on the formation of intercellular gaps [50][51][52]. The formation of intercellular gaps correlates with the initial signs of increased paracellular permeability [50][51][52].…”