Role of NO in Regulating Vascular Permeability

One of the initial events in an acute inflammatory response is increased extravasation of fluid and protein from postcapillary venules at sites of tissue injury. Under physiological conditions, microvascular fluid exchange is primarily determined by blood flow, Starling forces, and the perfused surface area. The perm-selectivity barrier to solute flux is determined by multiple factors including the identity of the endothelium (tissue bed), composition of tight versus adherens junctions, and extracellular matrix.

Using cannulated, venular microvessels in the mesentery, a variety of inflammatory agents such as ATP, ionomycin, bradykinin, histamine, and VEGF stimulated increases in [Ca2+]j slightly preceding the initial peak in vascular leakage. The delay of the onset of changes in permeability suggests that in addition to the initial increase of [Ca2+]P calcium-dependent signaling cascades (i.e., eNOS) may contribute to vascular permeability. Furthermore, NOS inhibitors can reduce ATP-induced increases in single vessel permeability, while 8-Br-cGMP, a membrane-permeable cGMP analog, can potentiate ATP driven increases in permeability. These agents, however, did not affect the ATP induced rise in [Ca2+]p which further suggests that the increase in [Ca2+] was necessary for an agonist-induced increase in permeability but not entirely sufficient.

Over the years, studies on the action of NO in regulating vascular permeability have yielded conflicting observations (Figure 1). Early studies using NO donors and NOS

inhibitors demonstrated the negative regulatory effect of NO in microvascular permeability. For example, administration of a NOS inhibitor, ^-nitro-L-arginine-methyl ester (L-NAME), increased transvascular fluid and protein flux, whereas subsequent NO donors such as sodium nitroprus-side (SNP) reversed this effect in the intestinal circulation. The increase in permeability was not due to hemodynamic alteration, a phenomenon in which L-NAME has been suggested to play a role. Many other studies using NOS inhibitors or NO donors also demonstrated that NO negatively regulates permeability in response to a host of inflammatory agents including bradykinin, carrageenan, substance P, and mustard oil. In addition, increases in intracellular levels of cGMP by NO donors or activators of guanylate cyclase (sGC) have also been associated with a decrease in endothelial permeability.

On the other hand, the NO-sGC-cGMP pathway can positively regulate microvascular permeability. Upon receptor activation and subsequent stimulation of phospholipase C (PLC), increase in cytosolic calcium can lead to activation of eNOS and NO production. NO activates guanylate cyclase (GC) and increases cGMP production, resulting in protein kinase G (PKG) activation and phosphorylation of proteins that regulate the contractile apparatus or cytoskele-ton. In cannulated porcine coronary venules, VEGF and his-tamine can induce increases in permeability, as measured by the rate of FITC-albumin transfer into the extravascular space in a cGMP-PKG dependent manner. Administration of the NO donor SNP increased permeability similar to that induced by histamine, and this increase can be blocked completely by an inhibitor of guanylate cyclase.

Recent studies have examined the effect of eNOS in modulating vascular permeability using eNOS (-/-) mice or using a synthetic peptide that inhibits eNOS activity. A pivotal study by Jain's laboratory [9] demonstrated that eNOS (-/-) mice, but not iNOS (-/-) mice showed impairment in VEGF-induced increases in permeability, further confirming the importance of eNOS as the predominant NOS isoforms in the regulation of VEGF induced vascular permeability. To further examine the role of eNOS in regulating vascular permeability, our laboratory has generated a synthetic fusion protein containing the eNOS inhibitory domain of caveolin-1 and a cell permeable peptide that facilitates the uptake of cargo proteins/oligonucleotides [10]. This peptide, which we termed cavtratin, can inhibit acetylcholine-induced, endothelium-dependent relaxation, thereby reducing the amount of NO generated block eNOS and not iNOS activity. Furthermore, it can specifically. Administration of this peptide in vivo can block both carrageenan- and mustard oil-induced inflammation and edema formation. Furthermore, this peptide, which did not have an antiangiogenic or cytostatic effect, can selectively block tumor microvascular permeability, leading to decreased tumor progression. Recently, cavtratin has been shown to block PAF-mediated increases in hydraulic conductivity in venules, thus validating the antileak actions of the peptide [11]. Combined with our observations that eNOS (-/-) mice exhibited decreased tumor permeability and progression, these studies may pro vide novel insights into development of new antipermeabil-ity drugs to target tumor growth.

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