Panos Kouklis

An Essential Cytoskeletal Linker Protein Connecting Actin Microfilaments to Intermediate Filaments

Typified by rapid degeneration of sensory neurons, dystonia musculorum mice have a defective BPAG1 gene, known to be expressed in epidermis. We report a neuronal splice form, BPAG1n, which localizes to sensory axons. Both isoforms have a coiled-coil rod, followed by a carboxy domain that associates with intermediate filaments. However, the amino terminus of BPAG1n differs from BPAG1e in that it contains a functional actin-binding domain. In transfected cells, BPAG1n coaligns neurofilaments and microfilaments, establishing this as a cytoskeletal protein interconnecting actin and intermediate filament cytoskeletons. In BPAG1 null mice, axonal architecture is markedly perturbed, consistent with a failure to tether neurofilaments to the actin cytoskeleton and underscoring the physiological relevance of this protein.

Ca2+ signalling and PKCα activate increased endothelial permeability by disassembly of VE—cadherin junctions

The role of intracellular Ca2+ mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i in both cases was due to release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial 125I‐albumin permeability. Thapsigargin induced activation of PKCα and discontinuities in VE‐cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCα activation and similar alterations in VE‐cadherin junctions, but in association with actin stress fibre formation. Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC20), whereas thrombin induced MLC20 phosphorylation consistent with formation of actin stress fibres. Calphostin C pretreatment prevented the disruption of VE‐cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca2+]i elicited by thapsigargin and thrombin may activate a calphostin C‐sensitive PKC pathway that signals VE‐cadherin junctional disassembly and increased endothelial permeability. Results suggest a critical role for Ca2+ signalling and activation of PKCα in mediating the disruption of VE‐cadherin junctions, and thereby in the mechanism of increased endothelial permeability.

Cdc42 Regulates the Restoration of Endothelial Barrier Function

Abstract— The mechanisms involved in the restoration of endothelial cell junctions subsequent to barrier disruption remain unclear. It is known that formation of adherens junctions (AJs) affects cytoskeletal actin arrangement and that Rho GTPases regulate the state of actin polymerization. In the present study, we examined the role of the Rho GTPases, Rho, Rac, and Cdc42 in the reannealing of AJs. We studied the response to thrombin, which increases endothelial permeability through disassembly of AJs, followed by recovery of barrier function through junctional reannealing within 2 hours. Cdc42 was activated late, at ≈1 hour after thrombin exposure, concurrent with its translocation from the cytoplasm to the membrane. Activation and translocation of Cdc42 preceded the reformation of AJs. Expression of the dnCdc42 mutant (N17Cdc42) significantly delayed the reformation of the VE-cadherin-containing AJs and restoration of endothelial barrier function. We also studied the lung microcirculation to address the in vivo relevance of Cdc42 signaling in barrier restoration. N17Cdc42 expression in the mouse lung endothelium markedly attenuated the endothelial barrier recovery after the permeability increase induced by activation of the thrombin receptor protease-activated receptor-1. These findings demonstrate the critical function of Cdc42 in restoring AJ-dependent, endothelial cell homotypic adhesion and barrier function. The delayed activation of Cdc42 represents a negative-feedback mechanism that signals AJ reassembly after the increase in endothelial permeability induced by inflammatory mediators such as thrombin.

Cdc42 Regulates Adherens Junction Stability and Endothelial Permeability by Inducing α-Catenin Interaction With the Vascular Endothelial Cadherin Complex

The endothelial adherens junctions (AJs) consist of trans-oligomers of membrane spanning vascular endothelial (VE)-cadherin proteins, which bind β-catenin through their cytoplasmic domain. β-Catenin in turn binds α-catenin and connects the AJ complex with the actin cytoskeleton. We addressed the in vivo effects of loss of VE-cadherin interactions on lung vascular endothelial permeability and the role of specific Rho GTPase effectors in regulating the increase in permeability induced by AJ destabilization. We used cationic liposomes encapsulating the mutant of VE-cadherin lacking the extracellular domain (&Dgr;EXD) to interfere with AJ assembly in mouse lung endothelial cells. We observed that lung vascular permeability (quantified as microvessel filtration coefficient [Kf,c]) was increased 5-fold in lungs expressing &Dgr;EXD. This did not occur to the same degree on expression of the VE-cadherin mutant, &Dgr;EXD&Dgr;β, lacking the β-catenin–binding site. The increased vascular permeability was the result of destabilization of VE-cadherin homotypic interaction induced by a shift in the binding of β-catenin from wild-type VE-cadherin to the expressed &Dgr;EXD mutant. Because &Dgr;EXD expression in endothelial cells activated the Rho GTPase Cdc42, we addressed its role in the mechanism of increased endothelial permeability induced by AJ destabilization. Coexpression of dominant-negative Cdc42 (N17Cdc42) prevented the increase in Kf,c induced by &Dgr;EXD. This was attributed to inhibition of the association of α-catenin with the &Dgr;EXD–β-catenin complex. The results demonstrate that Cdc42 regulates AJ permeability by controlling the binding of α-catenin with β-catenin and the consequent interaction of the VE-cadherin/catenin complex with the actin cytoskeleton.

Critical role of Cdc42 in mediating endothelial barrier protection in vivo

Activation of the Rho GTPase Cdc42 has been shown in endothelial cell monolayers to prevent disassembly of interendothelial junctions and the increase in endothelial permeability. Here, we addressed the in vivo role of Cdc42 activity in mediating endothelial barrier protection in lungs by generating mice expressing the dominant active mutant V12Cdc42 protein in vascular endothelial cells targeted via the VE-cadherin promoter. These mice developed normally and exhibited constitutively active GTP-bound Cdc42. The increase in lung vascular permeability and gain in tissue water content in response to intraperitoneal lipopolysaccharide challenge (7 mg/kg) were markedly attenuated in the transgenic mice. To address the basis of the protective effect, we observed that expression of V12Cdc42 mutant in endothelial monolayers reduced the decrease in transendothelial electrical resistance, a measure of opening of interendothelial junctions, thus indicating that Cdc42 activity preserved junctional integrity. RhoA activity in V12Cdc42-expressing endothelial monolayers was reduced compared with untransfected cells, suggesting that activated Cdc42 functions by counteracting the canonical RhoA-mediated mechanism of endothelial hyperpermeability. Therefore, Cdc42 activity of microvessel endothelial cells is a critical determinant of junctional barrier restrictiveness and may represent a means of therapeutically modulating increased lung vascular permeability and edema formation.

Isolation of an ES-Derived Cardiovascular Multipotent Cell Population Based on VE-Cadherin Promoter Activity

Embryonic Stem (ES) or induced Pluripotent Stem (iPS) cells are important sources for cardiomyocyte generation, targeted for regenerative therapies. Several in vitro protocols are currently utilized for their differentiation, but the value of cell-based approaches remains unclear. Here, we characterized a cardiovascular progenitor population derived during ES differentiation, after selection based on VE-cadherin promoter (Pvec) activity. ESCs were genetically modified with an episomal vector, allowing the expression of puromycin resistance gene, under Pvec activity. Puromycin-surviving cells displayed cardiac and endothelial progenitor cells characteristics. Expansion and self-renewal of this cardiac and endothelial dual-progenitor population (CEDP) were achieved by Wnt/β-catenin pathway activation. CEDPs express early cardiac developmental stage-specific markers but not markers of differentiated cardiomyocytes. Similarly, CEDPs express endothelial markers. However, CEDPs can undergo differentiation predominantly to cTnT+ (~47%) and VE-cadherin+ (~28%) cells. Transplantation of CEDPs in the left heart ventricle of adult rats showed that CEDPs-derived cells survive and differentiate in vivo for at least 14 days after transplantation. A novel, dual-progenitor population was isolated during ESCs differentiation, based on Pvec activity. This lineage can self-renew, permitting its maintenance as a source of cardiovascular progenitor cells and constitutes a useful source for regenerative approaches.

Vascular endothelial-cadherin downregulation as a feature of endothelial transdifferentiation in monocrotaline-induced pulmonary hypertension

Increased pulmonary vascular resistance in pulmonary hypertension (PH) is caused by vasoconstriction and obstruction of small pulmonary arteries by proliferating vascular cells. In analogy to cancer, subsets of proliferating cells may be derived from endothelial cells transitioning into a mesenchymal phenotype. To understand phenotypic shifts transpiring within endothelial cells in PH, we injected rats with alkaloid monocrotaline to induce PH and measured lung tissue levels of endothelial-specific protein and critical differentiation marker vascular endothelial (VE)-cadherin. VE-cadherin expression by immonoblotting declined significantly 24 h and 15 days postinjection to rebound to baseline at 30 days. There was a concomitant increase in transcriptional repressors Snail and Slug, along with a reduction in VE-cadherin mRNA. Mesenchymal markers α-smooth muscle actin and vimentin were upregulated by immunohistochemistry and immunoblotting, and α-smooth muscle actin was colocalized with endothelial marker platelet endothelial cell adhesion molecule-1 by confocal microscopy. Apoptosis was limited in this model, especially in the 24-h time point. In addition, monocrotaline resulted in activation of protein kinase B/Akt, endothelial nitric oxide synthase (eNOS), nuclear factor (NF)-κB, and increased lung tissue nitrotyrosine staining. To understand the etiological relationship between nitrosative stress and VE-cadherin suppression, we incubated cultured rat lung endothelial cells with endothelin-1, a vasoconstrictor and pro-proliferative agent in pulmonary arterial hypertension. This resulted in activation of eNOS, NF-κB, and Akt, in addition to induction of Snail, downregulation of VE-cadherin, and synthesis of vimentin. These effects were blocked by eNOS inhibitor N(ω)-nitro-l-arginine methyl ester. We propose that transcriptional repression of VE-cadherin by nitrosative stress is involved in endothelial-mesenchymal transdifferentiation in experimental PH.