Sepsis is a leading cause of death worldwide. Increased vascular permeability is a major hallmark of sepsis. Dynamic alterations in actin fiber formation play an important role in the regulation of ...endothelial barrier functions and thus vascular permeability. Endothelial integrity requires a delicate balance between the formation of cortical actin filaments that maintain endothelial cell contact stability and the formation of actin stress fibers that generate pulling forces, and thus compromise endothelial cell contact stability. Current research has revealed multiple molecular pathways that regulate actin dynamics and endothelial barrier dysfunction during sepsis. These include intracellular signaling proteins of the small GTPases family (e.g., Rap1, RhoA and Rac1) as well as the molecules that are directly acting on the actomyosin cytoskeleton such as myosin light chain kinase and Rho kinases. Another hallmark of sepsis is an excessive recruitment of neutrophils that also involves changes in the actin cytoskeleton in both endothelial cells and neutrophils. This review focuses on the available evidence about molecules that control actin dynamics and regulate endothelial barrier functions and neutrophil recruitment. We also discuss treatment strategies using pharmaceutical enzyme inhibitors to target excessive vascular permeability and leukocyte recruitment in septic patients.
Changes in vascular permeability occur during inflammation and the actin cytoskeleton plays a crucial role in regulating endothelial cell contacts and permeability. We demonstrated recently that the ...actin-binding protein cortactin regulates vascular permeability via Rap1. However, it is unknown if the actin cytoskeleton contributes to increased vascular permeability without cortactin. As we consistently observed more actin fibres in cortactin-depleted endothelial cells, we hypothesised that cortactin depletion results in increased stress fibre contractility and endothelial barrier destabilisation. Analysing the contractile machinery, we found increased ROCK1 protein levels in cortactin-depleted endothelium. Concomitantly, myosin light chain phosphorylation was increased while cofilin, mDia and ERM were unaffected. Secretion of the barrier-stabilising hormone adrenomedullin, which activates Rap1 and counteracts actomyosin contractility, was reduced in plasma from cortactin-deficient mice and in supernatants of cortactin-depleted endothelium. Importantly, adrenomedullin administration and ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling actomyosin contractility with consequences for endothelial barrier integrity.
Vascular permeability is mediated by Cortactin (Cttn) and regulated by several molecules including cyclic-adenosine-monophosphate, small Rho family GTPases and the actin cytoskeleton. However, it is ...unclear whether Cttn directly interacts with any of the junctional components or if Cttn intervenes with signaling pathways affecting the intercellular contacts and the cytoskeleton. To address these questions, we employed immortalized microvascular myocardial endothelial cells derived from wild-type and Cttn-knock-out mice. We found that lack of Cttn compromised barrier integrity due to fragmented membrane distribution of different junctional proteins. Moreover, immunoprecipitations revealed that Cttn is within the VE-cadherin-based adherens junction complex. In addition, lack of Cttn slowed-down barrier recovery after Ca
repletion. The role of Cttn for cAMP-mediated endothelial barrier regulation was analyzed using Forskolin/Rolipram. In contrast to Cttn-KO, WT cells reacted with increased transendothelial electrical resistance. Absence of Cttn disturbed Rap1 and Rac1 activation in Cttn-depleted cells. Surprisingly, despite the absence of Cttn, direct activation of Rac1/Cdc42/RhoA by CN04 increased barrier resistance and induced well-defined cortical actin and intracellular actin bundles. In summary, our data show that Cttn is required for basal barrier integrity by allowing proper membrane distribution of junctional proteins and for cAMP-mediated activation of the Rap1/Rac1 signaling pathway.
Summary
The endothelial barrier of the vasculature is of utmost importance for separating the blood stream from underlying tissues. This barrier is formed by tight and adherens junctions (TJ and AJ) ...that form intercellular endothelial contacts. TJ and AJ are integral membrane structures that are connected to the actin cytoskeleton via various adaptor molecules. Consequently, the actin cytoskeleton plays a crucial role in regulating the stability of endothelial cell contacts and vascular permeability. While a circumferential cortical actin ring stabilises junctions, the formation of contractile stress fibres, e. g. under inflammatory conditions, can contribute to junction destabilisation. However, the role of actin-binding proteins (ABP) in the control of vascular permeability has long been underestimated. Naturally, ABP regulate permeability via regulation of actin remodelling but some actin-binding molecules can also act independently of actin and control vascular permeability via various signalling mechanisms such as activation of small GTPases. Several studies have recently been published highlighting the importance of actin-binding molecules such as cortactin, ezrin/ radixin/moesin, Arp2/3, VASP or WASP for the control of vascular permeability by various mechanisms. These proteins have been described to regulate vascular permeability under various pathophysiological conditions and are thus of clinical relevance as targets for the development of treatment strategies for disorders that are characterised by vascular hyperpermeability such as sepsis. This review highlights recent advances in determining the role of ABP in the control of endothelial cell contacts and vascular permeability.
Abstract
Adducin (Add) is an actin binding protein participating in the stabilization of actin/spectrin networks, epithelial junctional turnover and cardiovascular disorders such as hypertension. ...Recently, we demonstrated that Add is required for adherens junctions (AJ) integrity. Here we hypothesized that Add regulates tight junctions (TJ) as well and may play a role in cAMP-mediated barrier enhancement. We evaluated the role of Add in MyEnd cells isolated from WT and Add-Knock-Out (KO) mice. Our results indicate that the lack of Add drastically alters the junctional localization and protein levels of major AJ and TJ components, including VE-Cadherin and claudin-5. We also showed that cAMP signaling induced by treatment with forskolin and rolipram (F/R) enhances the barrier integrity of WT but not Add-KO cells. The latter showed no junctional reorganization upon cAMP increase. The absence of Add also led to higher protein levels of the small GTPases Rac1 and RhoA. In vehicle-treated cells the activation level of Rac1 did not differ significantly when WT and Add-KO cells were compared. However, the lack of Add led to increased activity of RhoA. Moreover, F/R treatment triggered Rac1 activation only in WT cells. The function of Rac1 and RhoA per se was unaffected by the total ablation of Add, since direct activation with CN04 was still possible in both cell lines and led to improved endothelial barrier function. In the current study, we demonstrate that Add is required for the maintenance of endothelial barrier by regulating both AJ and TJ. Our data show that Add may act upstream of Rac1 as it is necessary for its activation via cAMP.
Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial ...cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.
Neutrophil extravasation is a migratory event in response to inflammation that depends on cytoskeletal dynamics regulated by myosins. Myosin-1e (Myo1e) is a long-tailed class-I myosin that has not ...yet been studied in the context of neutrophil–endothelial interactions and neutrophil extravasation. Intravital microscopy of TNFα-inflamed cremaster muscles in Myo1e-deficient mice revealed that Myo1e is required for efficient neutrophil extravasation. Specifically,Myo1e deficiency caused increased rolling velocity, decreased firm adhesion, aberrant crawling, and strongly reduced transmigration. Interestingly, we observed a striking discontinuous rolling behavior termed “intermittent rolling,” during which Myo1e-deficient neutrophils showed alternating rolling and jumping movements. Surprisingly, chimeric mice revealed that these effects were due to Myo1e deficiency in leukocytes. Vascular permeability was not significantly altered in Myo1e KO mice. Myo1e-deficient neutrophils showed diminished arrest, spreading, uropod formation, and chemotaxis due to defective actin polymerization and integrin activation. In conclusion, Myo1e critically regulates adhesive interactions of neutrophils with the vascular endothelium and neutrophil extravasation. Myo1e may therefore be an interesting target in chronic inflammatory diseases characterized by excessive neutrophil recruitment.
Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial ...cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.
Sepsis remains an important health problem worldwide due to inefficient treatments often resulting in multi-organ failure. Neutrophil recruitment is critical during sepsis. While neutrophils are ...required to combat invading bacteria, excessive neutrophil recruitment contributes to tissue damage due to their arsenal of molecular weapons that do not distinguish between host and pathogen. Thus, neutrophil recruitment needs to be fine-tuned to ensure bacterial killing, while avoiding neutrophil-inflicted tissue damage. We recently showed that the actin-binding protein HS1 promotes neutrophil extravasation; and hypothesized that HS1 is also a critical regulator of sepsis progression. We evaluated the role of HS1 in a model of lethal sepsis induced by cecal-ligation and puncture. We found that septic HS1-deficient mice had a better survival rate compared to WT mice due to absence of lung damage. Lungs of septic HS1-deficient mice showed less inflammation, fibrosis, and vascular congestion. Importantly, systemic CLP-induced neutrophil recruitment was attenuated in the lungs, the peritoneum and the cremaster in the absence of HS1. Lungs of HS1-deficient mice produced significantly more interleukin-10. Compared to WT neutrophils, those HS1-deficient neutrophils that reached the lungs had increased surface levels of Gr-1, ICAM-1, and L-selectin. Interestingly, HS1-deficient neutrophils had similar F-actin content and phagocytic activity, but they failed to polymerize actin and deform in response to CXCL-1 likely explaining the reduced systemic neutrophil recruitment in HS1-deficient mice. Our data show that HS1 deficiency protects against sepsis by attenuating neutrophil recruitment to amounts sufficient to combat bacterial infection, but insufficient to induce tissue damage.
•HS1 deficiency increases survival during sepsis in mice.•Loss of HS1 decreases neutrophil recruitment.•HS1-deficient neutrophils do not respond to CXCL1 with actin remodeling and cell stiffening.•Lung damage is reduced in septic HS1 deficient mice.•Systemic inflammatory response is reduced without HS1.
The intestinal epithelium forms a stable barrier protecting underlying tissues from pathogens in the gut lumen. This is achieved by specialized integral membrane structures such as tight and adherens ...junctions that connect neighboring cells and provide stabilizing links to the cytoskeleton. Junctions are constantly remodeled to respond to extracellular stimuli. Assembly and disassembly of junctions is regulated by interplay of actin remodeling, endocytotic recycling of junctional proteins, and various signaling pathways. Accumulating evidence implicate small G proteins of the Ras superfamily as important signaling molecules for the regulation of epithelial junctions. They function as molecular switches circling between an inactive GDP-bound and an active GTP-bound state. Once activated, they bind different effector molecules to control cellular processes required for correct junction assembly, maintenance and remodelling. Here, we review recent advances in understanding how GTPases of the Rho, Ras, Rab and Arf families contribute to intestinal epithelial homeostasis.
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