Basal vascular endothelial permeability is normally kept low in part by the restrictiveness of interendothelial junctions (IEJs). We investigated the possible role of nitric oxide (NO) in controlling IEJ integrity and thereby regulating basal vascular permeability. We determined the permeability of continuous endothelia in multiple murine vascular beds, including lung vasculature, of wild-type mice, endothelial nitric oxide synthase (eNOS) null mice, and mice treated with NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME). Light and electron microscopic studies revealed that L-NAME treatment resulted in IEJs opening within a few minutes with a widespread response within 30 min. We observed a 35% increase in transendothelial transport of albumin, using as tracer dinitrophenylated albumin in mouse lungs and other organs studied. To rule out the involvement of blood cells in the mechanism of increased endothelial permeability, vascular beds were flushed free of blood, treated with L-NAME, and perfused with the tracer. The open IEJs observed in these studies indicated a direct role for NO in preserving the normal structure of endothelial junctions. We also used the electron-opaque tracer lanthanum chloride to assess vascular permeability. Lanthanum chloride was presented by perfusion to various vascular beds of mice lacking NO. Open IEJs were seen only in capillary and venular endothelial segments of mice lacking NO, and there was a concomitant increase in vascular permeability to the tracer. Together, these data demonstrate that constitutive eNOS-derived NO is a crucial determinant of IEJ integrity and thus serves to maintain the low basal permeability of continuous endothelia.

Caveolin-1, the principal integral membrane protein of caveolae, has been implicated in regulating the structural integrity of caveolae, vesicular trafficking, and signal transduction. Although the functions of caveolin-1 are beginning to be explored in caveolin-1-/- mice, these results are confounded by unknown compensatory mechanisms and the development of pulmonary hypertension, cardiomyopathy, and lung fibrosis. To address the role of caveolin-1 in regulating lung vascular permeability, in the present study we used small interfering RNA (siRNA) to knock down caveolin-1 expression in mouse lung endothelia in vivo. Intravenous injection of siRNA against caveolin-1 mRNA incorporated in liposomes selectively reduced the expression of caveolin-1 by approximately 90% within 96 h of injection compared with wild-type mice. We observed the concomitant disappearance of caveolae in lung vessel endothelia and dilated interendothelial junctions (IEJs) as well as increased lung vascular permeability to albumin via IEJs. The reduced caveolin-1 expression also resulted in increased plasma nitric oxide concentration. The nitric oxide synthase inhibitor L-NAME, in part, blocked the increased vascular albumin permeability. These morphological and functional effects of caveolin-1 knockdown were reversible within 168 h after siRNA injection, corresponding to the restoration of caveolin-1 expression. Thus our results demonstrate the essential requirement of caveolin-1 in mediating the formation of caveolae in endothelial cells in vivo and in negatively regulating IEJ permeability.

The interaction between bacteria and endothelial cell plasma membrane is mediated by components of the bacterial wall outer membrane, the most important being LPS.3 LPS binds to CD14 (1-3) and Toll-like receptor 4 (TLR4) (1-4) expressed in the membrane. NF-B, the transcription factor activated by LPS-CD14-TLR4 signaling (5), results in the transcriptional induction of cytokines (interleukin-1 (IL-1), IL-6, IL-8), tissue factor, and adhesion molecules (E-and P-selectins, VCAM-1 (vascular cell adhesion molecule), and ICAM-1) (6). Cav-1, the structural protein of caveolae in endothelial cells and other cell types, regulates the formation of caveolae, the vesicle carriers involved in the transcytosis of albumin across the endothelial barrier (7). Studies showed that caveolae-mediated transcytosis contributes to the regulation of microvascular permeability (7) secondary to the activation of Src kinase (8). Cav-1-null mice, lacking caveolae (9), showed defective albumin transcytosis (10). In an experimental model of diabetes, increased Cav-1 expression in endothelial cells was associated with increased transcytosis of albumin (11). LPS was shown to induce the expression of Cav-1 in endothelial cells (12) and murine macrophages (13, 14); however, the mechanisms of the response and its consequences in regulating endothelial barrier function are not clear.NF-B is composed of dimers of five different proteins (p50, p52, p65/RelA, RelB, c-Rel) (15). These dimers exist in the cytoplasm in inactive forms bound to the inhibitory protein I-B (IB) (15). A variety of agonists activate IB kinases ␣ and ␤ (15), which in turn phosphorylate serines 32 and 36 of IB␣ and serines 19 and 23 of IB␤, respectively (15). Phosphorylation of IB␣ and IB␤ leads to the proteolytic degradation of IB and dissociation of NF-B, and NF-B translocates to the nucleus to induce gene transcription (15). The IB kinase complex consists of two catalytic IKK␣ and IKK␤, and a regulatory subunit, IKK␥ (or NF-B essential modulator (NEMO)) (16). NEMO interaction with IKK␣ and IKK␤ is required for IB kinase catalytic activity. Based on our observation that the intronic region of Cav-1 contains NF-B consensus sites, we addressed the possibility that LPS mediates Cav-1 expression by an NF-B-dependent mechanism. We surmised that this pathway thereby contributes to the mechanism of increased transendothelial albumin permeability seen with LPS. We demonstrate here that LPS activation of endothelial cells increased Cav-1 protein expression as well as caveolae number and that both were dependent on activation of NF-B. Moreover, inhibiting NF-B activation pharmacologically, knockdown of p65/RelA expression and knockdown of Cav-1 expression each interfered with the increase in transendothelial albumin permeability induced