Endothelial nitric oxide synthase regulates microvascular hyperpermeability in vivo

Abstract

Nitric oxide (NO) is an important regulator of blood flow, but its role in permeability is still challenged. We tested in vivo the hypotheses that: (a) endothelial nitric oxide synthase (eNOS) is not essential for regulation of baseline permeability; (b) eNOS is essential for hyperpermeability responses in inflammation; and (c) molecular inhibition of eNOS with caveolin-1 scaffolding domain (AP-Cav) reduces eNOS-regulated hyperpermeability. We used eNOS-deficient (eNOS-/-) mice and their wild-type control as experimental animals, platelet-activating factor (PAF) at 10(-7) m as the test pro-inflammatory agent, and integrated optical intensity (IOI) as an index of microvascular permeability. PAF increased permeability in wild-type cremaster muscle from a baseline of 2.4 +/- 2.2 to a peak net value of 84.4 +/- 2.7 units, while the corresponding values in cremaster muscle of eNOS-/- mice were 1.0 +/- 0.3 and 15.6 +/- 7.7 units (P < 0.05). Similarly, PAF increased IOI in the mesentery of wild-type mice but much less in the mesentery of eNOS-/- mice. PAF increased IOI to comparable values in the mesenteries of wild-type mice and those lacking the gene for inducible NOS (iNOS). Administration of AP-Cav blocked the microvascular hyperpermeability responses to 10(-7) m PAF. We conclude that: (1) baseline permeability does not depend on eNOS; (2) eNOS and NO are integral elements of the signalling pathway for the hyperpermeability response to PAF; (3) iNOS does not affect either baseline permeability or hyperpermeability responses to PAF; and (4) caveolin-1 inhibits eNOS regulation of microvascular permeability in vivo. Our results establish eNOS as an important regulator of microvascular permeability in inflammation.

Figure 1. Microvascular permeability in mouse cremaster muscle

Time course of changes in net integrated optical intensity (IOI; an index of permeability) at baseline and in response to topically applied 10⁻⁷ m PAF (arrow) in wild-type and eNOS−/− mice. The data are means ± s.e.m. (P < 0.05). The lower panels show typical images of the extravasation of FITC-dextran 77 in wild-type and eNOS−/− mice during baseline and after application of 10⁻⁷ m PAF. Note the large increase in interstitial fluorescence (IOI) after application of PAF in wild-type mice. In contrast, PAF elicited a small and short-lived change in interstitial fluorescence in eNOS−/− mice. n = 8 for both eNOS+/+ and eNOS−/− mice.

Figure 2. Microvascular permeability in mouse mesentery

Time course of changes in net IOI at baseline and in response to topically applied 10⁻⁷ m PAF (arrow) in wild-type and eNOS−/− mice. The data are means ± s.e.m. (P < 0.05). n = 7 for both eNOS+/+ and eNOS−/− mice.

Figure 3. Time course of microvascular permeability in cremaster muscle

Administration of AP-Cav significantly reduces the hyperpermeability response to 10⁻⁷ m PAF in the mouse cremaster muscle (PAF applied at arrow). The data are means ± s.e.m. (P < 0.05). PAF group, n = 7; PAF + AP-Cav, n = 6.

Figure 4. Time course of microvascular permeability in mesentery

Administration of AP-Cav significantly reduces the hyperpermeability response to 10⁻⁷ m PAF in the mouse mesentery (PAF applied at arrow). The data are means ± s.e.m. (P < 0.05). PAF group, n = 7; PAF + AP-Cav, n = 6.