Cre/lox Studies Identify Resident Macrophages as the Major Source of Circulating Coagulation Factor XIII-A.

OBJECTIVE: To establish the cellular source of plasma factor (F)XIII-A. APPROACH AND RESULTS: A novel mouse floxed for the F13a1 gene, FXIII-Aflox/flox (Flox), was crossed with myeloid- and platelet-cre-expressing mice, and cellular FXIII-A mRNA expression and plasma and platelet FXIII-A levels were measured. The platelet factor 4-cre.Flox cross abolished platelet FXIII-A and reduced plasma FXIII-A to 23±3% (P<0.001). However, the effect of platelet factor 4-cre on plasma FXIII-A was exerted outside of the megakaryocyte lineage because plasma FXIII-A was not reduced in the Mpl-/- mouse, despite marked thrombocytopenia. In support of this, platelet factor 4-cre depleted FXIII-A mRNA in brain, aorta, and heart of floxed mice, where FXIII-Apos cells were identified as macrophages as they costained with CD163. In the integrin αM-cre.Flox and the double copy lysozyme 2-cre.cre.Flox crosses, plasma FXIII-A was reduced to, respectively, 75±5% (P=0.003) and 30±7% (P<0.001), with no change in FXIII-A content per platelet, further consistent with a macrophage origin of plasma FXIII-A. The change in plasma FXIII-A levels across the various mouse genotypes mirrored the change in FXIII-A mRNA expression in aorta. Bone marrow transplantation of FXIII-A+/+ bone marrow into FXIII-A-/- mice both restored plasma FXIII-A to normal levels and replaced aortic and cardiac FXIII-A mRNA, while its transplantation into FXIII-A+/+ mice did not increase plasma FXIII-A levels, suggesting that a limited population of niches exists that support FXIII-A-releasing cells. CONCLUSIONS: This work suggests that resident macrophages maintain plasma FXIII-A and exclude the platelet lineage as a major contributor.

Transglutaminase 2 moderates the expansion of mouse abdominal aortic aneurysms.

OBJECTIVE: Previously published work has indicated that transcripts encoding transglutaminase 2 (TG2) increase markedly in a rat model of abdominal aortic aneurysm. This study determines whether TG2 and the related TG, factor XIII-A (FXIII-A), protect against aortic aneurysm development in mice. METHODS: C57BL/6J wild-type, Tgm2 -/- knockout, F13a1 -/- knockout, and Tgm2 -/- /F13a1 -/- double knockout mice were subjected to laparotomy and periaortic application of CaCl2. RESULTS: Tgm2 -/- mice showed slightly greater aortic dilatation at 6 weeks after treatment when compared with wild type. However, vessels from Tgm2 -/- mice, but not wild-type mice, continued to dilate up to 6 months after injury and by 24 weeks, a greater number of Tgm2 -/- mice had developed aneurysms (16/17 vs 10/19; P = .008). Laparotomy resulted in a high death rate in F13a1 -/- knockout mice, more frequently from cardiac complications than from hemorrhage, but among F13a1 -/- mice that survived for 6 weeks after CaCl2 treatment, abdominal aortic aneurysm diameter was unaltered relative to wild-type mice. Laparotomy resulted in a higher death rate among Tgm2 -/- /F13a1 -/- double knockout mice, owing to an increased frequency of delayed bleeding. Surprisingly, Tgm2 -/- /F13a1 -/- double knockout mice showed a trend toward decreased dilatation of the aorta 6 weeks after injury, and this finding was replicated in Tgm2 -/- /F13a1 -/- mice subjected to carotid artery injury. Levels of transcripts encoding TG2 were not increased in the aortas of injured wild-type or F13a1 -/- knockout mice relative to uninjured mice, although changes in the levels of other transcripts accorded with previous descriptions of the CaCl2 aneurysm model in mice. CONCLUSIONS: Knockout of Tgm2, but not F13a1 exacerbates aortic dilatation, suggesting that TG2 confers protection. However, levels of TG2 messenger RNA are not acutely elevated after injury. FXIII-A plays a role in preventing postoperative damage after laparotomy, confirming previous reports that it prevents distal organ damage after trauma. TG2 promotes wound healing after surgery and, in its absence, the bleeding diathesis associated with FXIII-A deficiency is further exposed.

Transglutaminase 2 limits the extravasation and the resultant myocardial fibrosis associated with factor XIII-A deficiency.

BACKGROUND AND AIMS: Transglutaminase (TG) 2 and Factor (F) XIII-A have both been implicated in cardiovascular protection and repair. This study was designed to differentiate between two competing hypotheses: that TG2 and FXIII-A mediate these functions in mice by fulfilling separate roles, or that they act redundantly in this respect. METHODS: Atherosclerosis was assessed in brachiocephalic artery plaques of fat-fed mixed strain apolipoprotein (Apo)e deficient mice that lacked either or both transglutaminases. Cardiac fibrosis was assessed both in the mixed strain mice and also in C57BL/6J Apoe expressing mice lacking either or both transglutaminases. RESULTS: No difference was found in the density of buried fibrous caps within brachiocephalic plaques from mice expressing or lacking these transglutaminases. Cardiac fibrosis developed in both Apoe/F13a1 double knockout and F13a1 single knockout mice, but not in Tgm2 knockout mice. However, concomitant Tgm2 knockout markedly increased fibrosis, as apparent in both Apoe/Tgm2/F13a1 knockout and Tgm2/F13a1 knockout mice. Amongst F13a1 knockout and Tgm2/F13a1 knockout mice, the extent of fibrosis correlated with hemosiderin deposition, suggesting that TG2 limits the extravasation of blood in the myocardium, which in turn reduces the pro-fibrotic stimulus. The resulting fibrosis was interstitial in nature and caused only minor changes in cardiac function. CONCLUSIONS: These studies confirm that FXIII-A and TG2 fulfil different roles in the mouse myocardium. FXIII-A protects against vascular leakage while TG2 contributes to the stability or repair of the vasculature. The protective function of TG2 must be considered when designing clinical anti-fibrotic therapies based upon FXIII-A or TG2 inhibition.

RhoA-ROCK signaling is involved in contraction-mediated inhibition of SERCA2a expression in cardiomyocytes.

In neonatal ventricular cardiomyocytes (NVCM), decreased contractile activity stimulates sarco-endoplasmic reticulum Ca(2+)-ATPase2a (SERCA2a), analogous to reduced myocardial load in vivo. This study investigated in contracting NVCM the role of load-dependent RhoA-ROCK signaling in SERCA2a regulation. Contractile arrest of NVCM resulted in low peri-nuclear localized RhoA levels relative to contracting NVCM. In arrested NVCM, ROCK activity was decreased (59%) and paralleled a loss in F-actin levels. Y-27632-induced ROCK inhibition in contracting NVCM increased SERCA2a messenger RNA expression by 150%. This stimulation was transcriptional, as evident from transfections with the SERCA2a promoter. A reciprocal effect of Y-27632 treatment on the promoter activity of atrial natriuretic factor was observed. SERCA2a transcription was not altered by co-transfection of the RhoA-ROCK-dependent serum response factor (SRF) alone or in combination with myocardin. Furthermore, GATA4, another ROCK-dependent transcription factor, induced rather than repressed SERCA2a transcription. This study shows that contractile activity suppresses SERCA2a gene expression via RhoA-ROCK-dependent transcription modulation. This modulation is likely to be accomplished by a transcription factor other than SRF, myocardin, or GATA4.

Nuclear targeting of beta-catenin and p120ctn during thrombin-induced endothelial barrier dysfunction.

AIMS: Cytosolic and nuclear localization of beta-catenin was observed in leaky vessels and in tumours. Several lines of evidence indicate that nuclear beta-catenin facilitates angiogenesis. We hypothesized that nuclear beta-catenin liberated from endothelial junctional complexes marks the transition from hyperpermeability to angiogenesis. The aim of this study was, therefore, to investigate the fate of beta-catenin and the related catenin p120catenin (p120ctn), during disruption of the endothelial barrier function in human umbilical vein endothelial cells (ECs). METHODS AND RESULTS: The hyperpermeability-inducer thrombin caused a Rho kinase-dependent redistribution of beta-catenin from the membrane to the cytosol as evidenced by the western blot analysis of membrane and cytosol fractions and by immunohistochemistry. Glycogen synthase kinase 3beta, which phosphorylates cytosolic beta-catenin and thereby facilitates its proteasomal degradation, was inhibited by thrombin. The analysis of nuclear extracts demonstrated a thrombin-induced nuclear accumulation of beta-catenin as well as p120ctn. Thrombin stimulation activated beta-catenin-mediated transcriptional activity as evidenced by reporter assays. Finally, real-time-PCR revealed increased mRNA levels of several beta-catenin target genes. CONCLUSION: Thrombin induced a cytosolic stabilization of membrane-liberated beta-catenin, which, together with p120ctn, subsequently translocated to the nucleus where it induces several beta-catenin target genes. This supports the suggestion that membrane-liberated beta-catenin and p120ctn contribute to angiogenic responses of ECs following episodes of vascular leakage.

ROCK2 primes the endothelium for vascular hyperpermeability responses by raising baseline junctional tension.

Rho kinase mediates the effects of inflammatory permeability factors by increasing actomyosin-generated traction forces on endothelial adherens junctions, resulting in disassembly of intercellular junctions and increased vascular leakage. In vitro, this is accompanied by the Rho kinase-driven formation of prominent radial F-actin fibers, but the in vivo relevance of those F-actin fibers has been debated, suggesting other Rho kinase-mediated events to occur in vascular leak. Here, we delineated the contributions of the highly homologous isoforms of Rho kinase (ROCK1 and ROCK2) to vascular hyperpermeability responses. We show that ROCK2, rather than ROCK1 is the critical Rho kinase for regulation of thrombin receptor-mediated vascular permeability. Novel traction force mapping in endothelial monolayers, however, shows that ROCK2 is not required for the thrombin-induced force enhancements. Rather, ROCK2 is pivotal to baseline junctional tension as a novel mechanism by which Rho kinase primes the endothelium for hyperpermeability responses, independent from subsequent ROCK1-mediated contractile stress-fiber formation during the late phase of the permeability response.

Localized RhoA GTPase activity regulates dynamics of endothelial monolayer integrity.

AIMS: Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith connected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are associated with distinct localized RhoA activities at the subcellular level. METHODS AND RESULTS: Live fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility. Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for this protrusive activity and maintenance of barrier restoration. CONCLUSION: Spontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter-endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re-annealing of endothelial junctions.

Driving Rho GTPase activity in endothelial cells regulates barrier integrity.

In the past decade understanding of the role of the Rho GTPases RhoA, Rac1 and Cdc42 has been developed from regulatory proteins that regulate specific actin cytoskeletal structures - stress fibers, lamellipodia and filopodia - to complex integrators of cytoskeletal structures that can exert multiple functions depending on the cellular context. Fundamental to these functions are three-dimensional complexes between the individual Rho GTPases, their specific activators (GEFs) and inhibitors (GDIs and GAPs), which greatly outnumber the Rho GTPases themselves, and additional regulatory proteins. By this complexity of regulation different vasoactive mediators can induce various cytoskeletal structures that enable the endothelial cell (EC) to respond adequately. In this review we have focused on this complexity and the consequences of Rho GTPase regulation for endothelial barrier function. The permeability inducers thrombin and VEGF are presented as examples of G-protein coupled receptor- and tyrosine kinase receptor-mediated Rho GTPase activation, respectively. These mediators induce complex but markedly different networks of activators, inhibitors and effectors of Rho GTPases, which alter the endothelial barrier function. An interesting feature in this regulation is that Rho GTPases often have both barrier-protecting and barrier-disturbing functions. While Rac1 enforces the endothelial junctions, it becomes part of a barrier-disturbing mechanism as activator of reactive oxygen species generating NADPH oxidase. Similarly RhoA is protective under basal conditions, but becomes involved in barrier dysfunction after activation of ECs by thrombin. The challenge and promise lies in unfolding this complex regulation, as this will provide leads for new therapeutic opportunities.