In this study, β3 KO mice developed striking increases in mortality and systemic vascular leak after intraperitoneal LPS and CLP, and increased lung vascular leak after endotracheal LPS-induced ALI. Bone marrow reconstitution experiments ruled out significant effects caused by β3 deficiency in hematopoietic cells, including platelet activation defects, which may have led to bleeding diathesis and hemorrhage; changes in secretion of vasoactive mediators from hematopoietic cells (including S1P); and alterations in inflammatory cell function (e.g., abnormal neutrophil trafficking) (45
). We hypothesized, therefore, that increased susceptibility of β3 KO mice to sepsis mortality and ALI was a manifestation of a vascular endothelial permeability defect.
Previous studies have described increased VEGF-induced dermal vascular permeability in β3 KO mice and attributed this effect to increased VEGF receptor 2 (Flk-1) expression in endothelial cells (18
). In our study, in vivo
blockade of VEGF signaling did not alter mortality after intraperitoneal LPS, nor was Flk-1 expression increased in newly isolated β3 KO endothelial cells. Furthermore, αvβ3 antibodies produced large increases in endothelial permeability in response to edemagenic agonists including TGF-β and thrombin, and VEGF. These data suggest that αvβ3 modulates endothelial permeability through mechanisms downstream to multiple distinct signaling pathways.
S1P is an agonist generated at sites of increased vascular leak and is an important regulator of endothelial permeability and homeostasis in vivo
). We found that αvβ3 antibodies disrupted S1P-induced barrier enhancement and cortical actin formation in human endothelial cell monolayers. Furthermore, S1P induced unique αvβ3 (compared with αvβ5) translocation to peripheral vinculin-containing structures resolvable by TIRF microscopy, suggesting that αvβ3 is induced by S1P to form cortical focal adhesions. αvβ3 antibodies inhibited formation of these cortical focal adhesions, suggesting that stabilization of these structures requires αvβ3 ligation, and that redistribution of αvβ3-containing focal adhesions is required for S1P-induced cortical actin formation.
S1P-induced αvβ3 translocation occurred through Gi- and Rac1-mediated pathways, which are known to be involved in S1P-induced barrier enhancement and cortical actin formation (21
). In this study, we addressed whether αvβ3 effects on the S1P-Rac1 axis could be occurring upstream of or downstream to Rac1 activation. Previous investigations have shown that the activation state of Rac1 and other Rho GTPases can be regulated by integrin ligation state (46
). These studies also suggest that downstream effects of Rac1 activation, such as activation of Rac1 effectors, may also require effects governed by integrins (47
). Therefore, it is possible for integrins to regulate both activation state and downstream effects of Rho GTPases. This paradigm likely varies with different cell types and states (altered integrin ligation) and with different signaling pathways. The current study shows that for human endothelial cell monolayers grown on a stable extracellular matrix, S1P-mediated induction of Rac1 activity is not affected by αvβ3 antibodies, whereas αvβ3 translocation, cortical focal adhesion formation, and cortical actin formation are, suggesting that relevant αvβ3 effects occur downstream of S1P-mediated Rac1 activation.
Several other integrins are expressed in endothelial cells, including αvβ5, α6β4, and multiple β1-contatining integrins (49
), so it is surprising that there does not seem to be compensatory support of S1P-induced barrier resistance and cortical actin formation. Furthermore, the apparent specificity of our findings to αvβ3 is of interest. Integrins do not possess intrinsic enzymatic or actin-binding activity; therefore, specificity of their regulatory functions depends largely on interactions with additional cytoplasmic or transmembrane partners. Identification of αvβ3-interacting proteins that facilitate SIP-signaling, promote αvβ3 translocation and stabilization, form and stabilize cortical actin, and function as Rac1 effector targets would provide valuable clues to the mechanisms underlying these processes and would help explain how functional specificity is conferred to αvβ3.
Vinculin, which was found to colocalize with cortical αvβ3 in response to S1P, does not directly bind to integrins, but is thought to support focal adhesion assembly by indirectly coupling talin and α-actinin to the actin cytoskeleton and by recruiting additional proteins, such as paxillin and vinexin (50
). Therefore, vinculin could theoretically facilitate formation of cortical αvβ3-containing focal adhesions and participate in stabilization of cortical actin. However, because activated vinculin binds to talin (51
), which promiscuously binds to multiple integrin β subunit cytoplasmic domains (53
), it seems unlikely that vinculin itself could confer specific of the observed effects to αvβ3.
In conclusion, we have identified αvβ3 as a unique integrin regulator of barrier resistance in the vascular endothelium. αvβ3 is not thought to regulate normal blood vessel development and function (18
) (male β3 KO mice have abnormal development of coronary capillaries [56
]; our experiments used female mice exclusively); however, our study suggests that loss or functional blockade of αvβ3 results in uncompensated vascular leak in inflammatory states. Novel mechanisms that may be associated with this function include S1P-induced translocation of αvβ3 to cortical focal adhesion sites and stabilization of these cortical focal adhesions through ligation of αvβ3. Elucidation of underlying mechanistic details would provide valuable insights into how αvβ3 and perhaps other integrins modulate endothelial barrier function in response to inflammation, and thus identify novel therapeutic targets to treat pathologic vascular endothelial permeability.
To the extent that endotracheal and intraperitoneal LPS and CLP can adequately model human ALI and sepsis, our results suggest that functional blockade of αvβ3 in humans may produce increased susceptibility to vascular permeability and its associated consequences in these disease states. Drugs designed to block αvβ3 are currently in various stages of clinical trials as treatments for diseases including postmenopausal osteoporosis, rheumatoid arthritis, and cancer. Our results, therefore, suggest that increased intensity of vascular endothelial leak in the setting of sepsis and ALI may be an important undesirable consequence of otherwise promising αvβ3-targeted therapies.