In our studies, we have found that ET decreases IL-8-driven TEM of PMNs across human lung microvascular endothelia. We asked whether the observed ET effect could be attributed to action on either the PMN and/or endothelium. We found that ET blocked TEM even when PMNs were not directly exposed to ET (Figure ) and required the presence of both EF and PA (Figure ). At the same concentrations, ET did not inhibit PMN chemotaxis in an EC-free system (Figure ). In contrast, we found that ET decreased 14
C-albumin flux across preconfluent endothelia (Figure ). Further, ET attenuated the increase in 14
C-albumin flux provoked by both endogenous (TNF-α) and exogenous (LPS) mediators of barrier disruption (Figure ). Prior inhibition of PKA with H-89 or KT-5720 did not reverse the ET effect on TEM (Figure ), and agents demonstrated to elevate intracellular levels of cAMP in HMVEC-Ls (Figure , Additional File 1
: Figure S1A, B) could not reconstitute the ET effect (Figure , and Additional File 1
: Figure S1C). These combined data indicate that ET diminishes TEM of PMNs at the level of the endothelial paracellular pathway and does so independent of via cAMP/PKA activity.
Several studies have examined the direct effect of ET on in vitro
PMN function. O'Brien et al found that ET inhibited PMN phagocytosis of opsonized B. anthracis
]. Pretreatment of PMNs with ET profoundly reduced superoxide production in response to either LPS or muramyl dipeptide. Crawford et al demonstrated that ET impaired PMN NADPH oxidase activation and downstream N-formyl-methionine-leucine-phenylalanine (fMLP)-induced superoxide production [37
]. Taken together, these studies indicate that ET down-regulates PMN phagocytic and oxidative functions. Other studies have focused on the impact of ET on PMN chemotaxis and migration [9
]. In the current studies, ET did not alter the PMN chemotactic response to IL-8 in an EC-free system (Figure ). To address concerns that calcein is a Ca2+
-binder and would interfere with any Ca2+
-mediated ET effect, these experiments were performed in the absence of the fluoroprobe. Even in the absence of calcein, ET had no effect on IL-8 chemotaxis of PMNs (Figure ). Chemotaxis was not as vigorous in the latter experiment, and this may be secondary to differences in methodology; mainly the use of a modified Boyden chambers, a shorter incubation time, as well as a different means of measuring PMN migration.
Wade et al found that ET stimulated directed neutrophil migration without having any effect on unstimulated random migration [22
]. They also found that although ET increased cAMP in PMNs, the absolute level of that increase was < 1% of that caused by the Bordetella pertussis
toxin. In contrast, Szarowicz et al found that ET reduces chemoattractant-stimulated PMN actin assembly, chemokinesis, chemotaxis and polarization [9
]. In PMNs, ET provoked a > 50-fold increase in cAMP and a 4-fold increase in PKA phosphorylation. The differences between our findings and these other reports may be attributed to dissimilar techniques. For instance, Wade et al measured chemotaxis of PMNs preincubated for 1 h with ET in an agarose-gel based system, both of which were EC-free [22
], whereas Szarowicz's group utilized video microscopy to study adherence of PMNs preincubated for 2 h with ET to a fibronectin-coated surface [9
]. To our knowledge, none of these previous reports studied PMN migration in the context of the endothelial paracellular pathway. Another potential explanation for these disparities may be due to differences in potency of various EF preparations and their abilities to generate cAMP. Of note, the EF preparation offered by List Biologics is the least potent (personal communication, Dr. Erik Hewlett, University of Virginia, Charlottesville).
Far less is known about the direct effect of ET on ECs. Hong et al demonstrated that ET reorganizes the cytoskeleton and inhibits chemotaxis of human microvascular ECs [7
]. Tessier's group found that ET induces a gradual increase in transendothelial electrical resistance (TEER) across human umbilical vein EC monolayers cultured on collagen-coated inserts. They concluded that ET-induced edema could not be accounted for by the direct effect of ET on the endothelium [38
]. Of interest, in our experimental systems for both TEM of PMNs and transendothelial 14
C-albumin flux, the ECs were similarly cultured on collagen-impregnated filters. Although Tessier et al studied TEER, their experiments did not include transendothelial flux of a permeability tracer or TEM of PMNs.
ET is an intrinsic adenyl cyclase that increases cAMP [1
]. Data exists to support a cAMP-mediated mechanism underlying the ET effect on TEM of PMNs. Moy et al found that cAMP agonists attenuated the ability of thrombin to increase permeability [27
]. Similarly, Fukuhara et al found that cAMP agonists decreased cell permeability and enhanced vascular EC-EC adhesion [11
]. In ECs, cAMP targets multiple downstream signaling molecules that might promote endothelial barrier integrity, including PKA [39
] and EPAC1 [40
One key effector of cAMP is PKA [10
]. PKA has been shown to inhibit myosin-based contractility through phosphorylation of myosin-light-chain-kinase, thereby decreasing its activity [10
]. PKA also inhibits RhoA activity, stabilizes microtubules, reorganizes cortical actin and strengthens tight junctions through phosphorylation of vasodilator stimulated protein (VASP) [10
]. In our studies, we found that ET activates PKA in HMVEC-Ls in a dose- and time- dependent manner (Figure ). Although ET increases EC PKA activity, its inhibitory effect on TEM could not be ascribed to PKA activity. Two structurally dissimilar pharmacologic inhibitors of PKA, H-89 and KT-5720, each failed to attenuate the ET-induced decrease in IL-8-driven TEM of PMNs (Figure ). Further, we were unable to reproduce the ET effect on TEM with either of two structurally and functionally distinct pharmacologic agents each known to increase cAMP, FSK or IBMX (Figure ). Taken together, these data indicate that the mechanism through which ET counter-regulates IL-8-driven TEM of PMNs cannot be explained solely through cAMP/PKA activation.
Another downstream target for cAMP is EPAC1, which is a GEF for the ras GTPase, RAP1 [10
]. Like PKA activity, the EPAC1-RAP1 pathway also enhances endothelial barrier function [11
]. The EPAC1-specific analog 8CPT-2'O-Me-cAMP, which directly activates EPAC1 while bypassing PKA, has been shown to decrease permeability of endothelial cell monolayers, an effect which is ablated by prior siRNA-induced EPAC1 knockdown [12
]. Birukova et al [44
] and Fukuhara et al [11
] both demonstrated that activation of EPAC1 attenuated thrombin-induced increases in permeability. As in the case of PKA, the mechanism(s) by which EPAC1 improves barrier function is still being elucidated. Potential EPAC1 targets include activation of VASP, as well as activation of ARAP3, which in turn is a GEF for RhoA, and vinculin, which supports EC-EC adherens junctions through association with α-catenin [10
]. As PKA inhibition did not impair the ET effect on TEM (Figure ), one potential pathway is through EPAC1-RAP1 and its effectors.
Since ET evokes biological responses in both PMNs and ECs, it was unclear as to whether the ability of ET to regulate TEM of PMNs could be ascribed to its impact on PMNs, ECs, or both. Although prior studies had demonstrated that ET directly influenced PMN chemotaxis, in our experiments, it did not (Figure ). Further, ET diminished TEM of PMNs never exposed to ET (Figure ). Finally, not only did ET decrease the paracellular movement of PMNs (Figure ), but of a permeability tracer as well (Figure ). These combined data indicate that ET counter-regulates PMN diapedesis exclusively through its effects on the endothelium. Further support of this concept is offered by Wittchen et al, who reported direct activation of RAP1 in EC monolayers decreased both their permeability as well as TEM of leukocytes [43