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Endothelial barrier disruption and vascular leak importantly contribute to organ dysfunction and mortality during inflammatory conditions like sepsis and acute respiratory distress syndrome. We identified the kinase Arg/Abl2 as a mediator of endothelial barrier disruption, but the role of Arg in endothelial monolayer regulation and its relevance in vivo remain poorly understood. Here we show that depletion of Arg in endothelial cells results in the activation of both RhoA and Rac1, increased cell spreading and elongation, redistribution of integrin-dependent cell-matrix adhesions to the cell periphery, and improved adhesion to the extracellular matrix. We further show that Arg is activated in the endothelium during inflammation, both in murine lungs exposed to barrier-disruptive agents, and in pulmonary microvessels of septic patients. Importantly, Arg-depleted endothelial cells were less sensitive to barrier-disruptive agents. Despite the formation of F-actin stress fibers and myosin light chain phosphorylation, Arg depletion diminished adherens junction disruption and intercellular gap formation, by reducing the disassembly of cell-matrix adhesions and cell retraction. In vivo, genetic deletion of Arg diminished vascular leak in the skin and lungs, in the presence of a normal immune response. Together, our data indicate that Arg is a central and non-redundant regulator of endothelial barrier integrity, which contributes to cell retraction and gap formation by increasing the dynamics of adherens junctions and cell-matrix adhesions in a Rho GTPase-dependent fashion. Therapeutic inhibition of Arg may provide a suitable strategy for the treatment of a variety of clinical conditions characterized by vascular leak. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-021-09781-x.

Aims: Endothelial barrier dysfunction leads to edema and vascular leak, carrying high morbidity and mortality. Previously, Abl kinase inhibition was shown to protect against vascular leak. Using the distinct inhibitory profiles of clinically available Abl kinase inhibitors, we aimed to provide a mechanistic basis for novel treatment strategies against vascular leakage syndromes. Methods & Results: Bosutinib most potently protected against inflammation-induced endothelial barrier disruption. In vivo, bosutinib prevented LPS-induced alveolar protein extravasation in an acute lung injury mice model. Mechanistically, Mitogen-activated Protein 4 Kinase 4 (MAP4K4) was identified as important novel mediator of endothelial permeability, which signals via ezrin, radixin and moesin proteins to increase turnover of integrin-based focal adhesions. The combined inhibition of MAP4K4 and Arg by bosutinib preserved adherens junction integrity and reduced turnover of focal adhesions, which synergistically act to stabilize the endothelial barrier during inflammation. Conclusion: MAP4K4 was identified as important regulator of endothelial barrier integrity, increasing focal adhesion turnover and disruption of cell-cell junctions during inflammation. Inhibiting both Arg and MAP4K4, the clinically available drug bosutinib may form a viable strategy against vascular leakage syndromes.

Endothelial cells line the vasculature and act as gatekeepers that control the passage of plasma, macromolecules and cells from the circulation to the interstitial space. Dysfunction of the endothelial barrier can lead to uncontrolled leak or edema. Vascular leakage is a hallmark of a range of diseases and despite its large impact no specialized therapies are available to prevent or reduce it. RhoGTPases are known key regulators of cellular behavior that are directly involved in the regulation of the endothelial barrier. We recently performed a comprehensive analysis of the effect of all RhoGTPases and their regulators on basal endothelial integrity. In addition to novel positive regulators of endothelial barrier function, we also identified novel negative regulators, of which the ArhGAP45 (also known as HMHA1) was the most significant. We now demonstrate that ArhGAP45 acts as a Rac-GAP (GTPase-Activating Protein) in endothelial cells, which explains its negative effect on endothelial barrier function. Silencing ArhGAP45 not only promotes basal endothelial barrier function, but also increases cellular surface area and induces sprout formation in a 3D-fibrin matrix. Our data further shows that loss of ArhGAP45 promotes migration and shear stress adaptation. In conclusion, we identify ArhGAP45 (HMHA1) as a novel regulator, which contributes to the fine-tuning of the regulation of basal endothelial integrity.

RhoGTPases are known regulators of intracellular actin dynamics that are important for maintaining endothelial barrier function. RhoA is most extensively studied as a key regulator of endothelial barrier function, however the function of the 2 highly homologous family-members (> 88%) RhoB and RhoC in endothelial barrier function is still poorly understood. This study aimed to determine whether RhoA, RhoB and RhoC have overlapping or distinct roles in barrier function and permeability in resting and activated endothelium. By using primary endothelial cells in combination with siRNA transfection to establish individual, double or triple knockdown of the RhoA/B/C RhoGTPases, we found that RhoB, but not RhoA or RhoC, is in resting endothelium a negative regulator of permeability. Loss of RhoB accounted for an accumulation of VE-cadherin at cell-cell contacts. Thrombin-induced loss of endothelial integrity is mediated primarily by RhoA and RhoB. Combined loss of RhoA/B showed decreased phosphorylation of Myosin Light Chain and increased expression of VE-cadherin at cell-cell contacts after thrombin stimulation. RhoC contributes to the Rac1-dependent restoration of endothelial barrier function. In summary, this study shows that these highly homologous RhoGTPases differentially control the dynamics of endothelial barrier function.

Indoxyl sulfate (IS) is an accumulative protein-bound uremic toxin found in patients with kidney disease. It is reported that IS impairs the vascular endothelium, but a comprehensive overview of all mechanisms active in IS-injury currently remains lacking. Here we performed RNA sequencing in human umbilical vein endothelial cells (HUVECs) after IS or control medium treatment and identified 1293 genes that were affected in a IS-induced response. Gene enrichment analysis highlighted pathways involved in altered vascular formation and cell metabolism. We confirmed these transcriptome profiles at the functional level by demonstrating decreased viability and increased cell senescence in response to IS treatment. In line with the additional pathways highlighted by the transcriptome analysis, we further could demonstrate that IS exposure of HUVECs promoted tubule formation as shown by the increase in total tubule length in a 3D HUVECs/pericytes co-culture assay. Notably, the pro-angiogenic response of IS and increased ROS production were abolished when CYP1B1, one of the main target genes that was highly upregulated by IS, was silenced. This observation indicates IS-induced ROS in endothelial cells is CYP1B1-dependent. Taken together, our findings demonstrate that IS promotes angiogenesis and CYP1B1 is an important factor in IS-activated angiogenic response.

Vascular homeostasis and regeneration in ischemic tissue relies on intrinsic competence of the tissue to rapidly recruit endothelial cells for vascularization. The mononuclear cell (MNC) fraction of blood contains circulating progenitors committed to endothelial lineage. These progenitors give rise to endothelial colony-forming cells (ECFCs) that actively participate in neovascularization of ischemic tissue. To evaluate if the initial clonal outgrowth of ECFCs from cord (CB) and peripheral blood (PB) was stimulated by hypoxic conditions, MNCs obtained from CB and PB were subjected at 20% and 1% O2 cell culture conditions. Clonal outgrowth was followed during a 30 days incubation period. Hypoxia impaired the initial outgrowth of ECFC colonies from CB and also reduced their number that were developing from PB MNCs. Three days of oxygenation (20% O2) prior to hypoxia could overcome the initial CB-ECFC outgrowth. Once proliferating and subcultured the CB-ECFCs growth was only modestly affected by hypoxia (18±2% reduction); proliferation of PB-ECFCs was reduced to a similar extent. Early passages of subcultured CB- and PB-ECFCs contained only viable cells and few if any senescent cells, but the clonal ability of subcultured PB-ECFCs was reduced by 30% for PB-ECFCs. Tube formation by subcultured PB-ECFCs was also markedly inhibited by continuous exposure to 1% O2. Gene expression profiles point to regulation of the cell cycle and metabolism as major altered gene clusters. Finally we discuss our counterintuitive observations in the context of the important role that hypoxia has in promoting neovascularization.

In many pathological conditions the endothelium becomes activated and dysfunctional, resulting in hyperpermeability and plasma leakage. No specific therapies are available yet to control endothelial barrier function, which is regulated by inter-endothelial junctions and the generation of acto-myosin-based contractile forces in the context of cell-cell and cell-matrix interactions. However, the spatiotemporal distribution and stimulus-induced reorganization of these integral forces remain largely unknown. Traction force microscopy of human endothelial monolayers was used to visualize contractile forces in resting cells and during thrombin-induced hyperpermeability. Simultaneously, information about endothelial monolayer integrity, adherens junctions and cytoskeletal proteins (F-actin) were captured. This revealed a heterogeneous distribution of traction forces, with nuclear areas showing lower and cell-cell junctions higher traction forces than the whole-monolayer average. Moreover, junctional forces were asymmetrically distributed among neighboring cells. Force vector orientation analysis showed a good correlation with the alignment of F-actin and revealed contractile forces in newly formed filopodia and lamellipodia-like protrusions within the monolayer. Finally, unstable areas, showing high force fluctuations within the monolayer were prone to form inter-endothelial gaps upon stimulation with thrombin. To conclude, contractile traction forces are heterogeneously distributed within endothelial monolayers and force instability, rather than force magnitude, predicts the stimulus-induced formation of intercellular gaps.

This editorial refers to ‘Loss of osteoglycin promotes angiogenesis in limb ischaemia mouse models via modulation of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 signalling pathway’ by Gao et al. , pp. 70–80. The circulatory system regulates adequate distribution of blood and delivery of oxygen and nutrients to the tissues. When arterial blood flow is interrupted a rapid neovascularization response occurs by expansion of collateral blood vessels and angiogenic sprouting in the hypoxic tissue. VEGF-A plays a central role in hypoxia-induced angiogenesis.1 According to insight obtained from developing tissues, VEGF-A binds to VEGF receptor-2 dimers on the protruding filopodia of sprouting endothelial tips. The receptor is subsequently internalized, after which intracellular signalling evokes a proper sprouting response.2 However, clinical studies in which VEGF was used to induce angiogenesis in chronically ischemic heart or limb have been unsuccessful.3,4 Apparently these tissues are refractory to VEGF stimulation. It is generally assumed that the extracellular matrix harbours information that regulates and guides endothelial sprouting. Matrix association of VEGF-A via its heparin-binding domain can direct the invading sprout, while angiogenesis inhibitors such as thrombospondin and matrikines (proteolytically generated matrix fragments that modulate angiogenesis) do the opposite. In this issue of Cardiovascular Research , Wu et al .5 report on a potentially important role of osteoglycin in limiting the cellular response to VEGF. Osteoglycin inhibits the VEGF-VEGFR2 interaction in healthy tissue, while its absence in the ischemic mouse hindleg enhances both sprouting angiogenesis and collateralization. Osteoglycin (gene symbol: OGN), also called mimecan (UniProt entry name: MIME), is a member of the small leucine-rich proteoglycans (SLRPs) and is expressed in most cells of the body.6 Many SLRPs including osteoglycin can bind to matrix proteins, can expose a binding site to collagen fibrils, and can act …

Thrombin and other inflammatory mediators may induce vascular permeability through the disruption of adherens junctions between adjacent endothelial cells. If uncontrolled, hyperpermeability leads to an impaired barrier, fluid leakage and edema, which can contribute to multi-organ failure and death. RhoGTPases control cytoskeletal dynamics, adhesion and migration and are known regulators of endothelial integrity. Knowledge of the precise role of each RhoGTPase, and their associated regulatory and effector genes, in endothelial integrity is incomplete. Using a combination of a RNAi screen with electrical impedance measurements, we quantified the effect of individually silencing 270 Rho-associated genes on the barrier function of thrombin-activated, primary endothelial cells. Known and novel RhoGTPase-associated regulators that modulate the response to thrombin were identified (RTKN, TIAM2, MLC1, ARPC1B, SEPT2, SLC9A3R1, RACGAP1, RAPGEF2, RHOD, PREX1, ARHGEF7, PLXNB2, ARHGAP45, SRGAP2, ARHGEF5). In conclusion, with this siRNA screen, we confirmed the roles of known regulators of endothelial integrity but also identified new, potential key players in thrombin-induced endothelial signaling.

Endothelial barrier function is carefully controlled to protect tissues from edema and damage inflicted by extravasated leukocytes. RhoGTPases, in conjunction with myriad regulatory proteins, exert both positive and negative effects on the endothelial barrier integrity. Precise knowledge about the relevant mechanisms is currently fragmented and we therefore performed a comprehensive analysis of endothelial barrier regulation by RhoGTPases and their regulators. Combining RNAi with electrical impedance measurements we quantified the relevance of 270 Rho-associated genes for endothelial barrier function. Statistical analysis identified 10 targets of which six promoted- and four reduced endothelial barrier function upon downregulation. We analyzed in more detail two of these which were not previously identified as regulators of endothelial integrity. We found that the Rac1-GEF (Guanine nucleotide Exchange Factor) TIAM2 is a positive regulator and the Cdc42(Rac1)-GAP (GTPase-Activating Protein) SYDE1 is a negative regulator of the endothelial barrier function. Finally, we found that the GAP SYDE1 is part of a Cdc42-centered signaling unit, also comprising the Cdc42-GEF FARP1 and the Cdc42 effector PAK7 which controls the integrity of the endothelial barrier. In conclusion, using a siRNA-based screen, we identified new regulators of barrier function and found that Cdc42 is a dominant positive regulator of endothelial integrity.

Purpose of review Neovascularization is a prominent feature in advanced human atherosclerotic plaques. This review surveys recent evidence for and remaining uncertainties regarding a role of neovascularization in atherosclerotic plaque progression. Specific emphasis is given to hypoxia, angiogenesis inhibition, and perivascular adipose tissue (PVAT). Recent findings Immunohistochemical and imaging studies showed a strong association between hypoxia, inflammation and neovascularization, and the progression of the atherosclerotic plaque both in humans and mice. Whereas in humans, a profound invasion of microvessels from the adventitia into the plaque occurs, neovascularization in mice is found mainly (peri)adventitially. Influencing neovascularization in mice affected plaque progression, possibly by improving vessel perfusion, but supportive clinical data are not available. Whereas plaque neovascularization contributes to monocyte/macrophage accumulation in the plaque, lymphangiogenesis may facilitate egress of cells and waste products. A specific role for PVAT and its secreted factors is anticipated and wait further clinical evaluation. Summary Hypoxia, inflammation, and plaque neovascularization are associated with plaque progression as underpinned by recent imaging data in humans. Recent studies provide new insights into modulation of adventitia-associated angiogenesis, PVAT, and plaque development in mice, but there is still a need for detailed information on modulating human plaque vascularization in patients.

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.

During prolonged hypoxic conditions, endothelial cells change their gene expression to adjust to the low oxygen environment. This process is mainly regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Although endothelial cells do not form sprouts during prolonged hypoxic culturing, silencing of HIF-2α partially restores sprout formation. The present study identifies novel HIF-2α-target genes that may regulate endothelial sprouting during prolonged hypoxia. The gene expression profile of primary human microvascular endothelial cells (hMVECs) that were cultured at 20 % oxygen was compared to hMVECs that were cultured at 1 % oxygen for 14 days by using genome-wide RNA-sequencing. The differentially regulated genes in hypoxia were compared to the genes that were differentially regulated upon silencing of HIF-2α in hypoxia. Surprisingly, KEGG pathway analysis showed that metabolic pathways were enriched within genes upregulated in response to hypoxia and enriched within genes downregulated upon HIF-2α silencing. Moreover, 51 HIF-2α-regulated genes were screened for their role in endothelial sprouting in hypoxia, of which four genes ARRDC3, MME, PPARG and RALGPS2 directly influenced endothelial sprouting during prolonged hypoxic culturing. The manipulation of specific downstream targets of HIF-2α provides a new, but to be further evaluated, perspective for restoring reduced neovascularization in several pathological conditions, such as diabetic ulcers or other chronic wounds, for improvement of vascularization of implanted tissue-engineered scaffolds. Electronic supplementary material The online version of this article (doi:10.1007/s10456-016-9527-4) contains supplementary material, which is available to authorized users.

Summary Endothelial progenitor cell (EPC) nomenclature remains ambiguous and there is a general lack of concordance in the stem cell field with many distinct cell subtypes continually grouped under the term “EPC.” It would be highly advantageous to agree on standards to confirm an endothelial progenitor phenotype and this should include detailed immunophenotyping, potency assays, and clear separation from hematopoietic angiogenic cells which are not endothelial progenitors. In this review, we seek to discourage the indiscriminate use of “EPCs,” and instead propose precise terminology based on defining cellular phenotype and function. Endothelial colony forming cells and myeloid angiogenic cells are examples of two distinct and well-defined cell types that have been considered EPCs because they both promote vascular repair, albeit by completely different mechanisms of action. It is acknowledged that scientific nomenclature should be a dynamic process driven by technological and conceptual advances; ergo the ongoing “EPC” nomenclature ought not to be permanent and should become more precise in the light of strong scientific evidence. This is especially important as these cells become recognized for their role in vascular repair in health and disease and, in some cases, progress toward use in cell therapy.

During short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis.We investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling.Human microvascular endothelial cells (hMVECs), cultured at 20% oxygen for 14 days and seeded on top of 3D fibrin matrices, formed sprouts when stimulated with VEGF-A/TNFα. In contrast, hMVECs precultured at 1% oxygen for 14 days were viable and proliferative, but did not form sprouts into fibrin upon VEGF-A/TNFα stimulation at 1% oxygen. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting, whereas HIF-1α or HIF-3α by si-RNA had no effect. No involvement of Dll4/Notch pathway in the inhibitory effect on endothelial sprouting by prolonged hypoxia was found. In addition, hypoxia decreased the production of urokinase-type plasminogen activator (uPA), needed for migration and invasion, without a significant effect on its inhibitor PAI-1. This was independent of HIF-2α, as si-HIF-2α did not counteract uPA reduction.Prolonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia.

Despite considerable progress in the understanding of endothelial barrier regulation and the identification of approaches that have the potential to improve endothelial barrier function, no drug- or stem cell-based therapy is presently available to reverse the widespread vascular leak that is observed in acute respiratory distress syndrome (ARDS) and sepsis. The translational gap suggests a need to develop experimental approaches and tools that better mimic the complex environment of the microcirculation in which the vascular leak develops. Recent studies have identified several elements of this microenvironment. Among these are composition and stiffness of the extracellular matrix, fluid shear stress, interaction of endothelial cells (ECs) with pericytes, oxygen tension, and the combination of toxic and mechanic injurious stimuli. Development of novel cell culture techniques that integrate these elements would allow in-depth analysis of EC biology that closely approaches the (patho)physiological conditions in situ. In parallel, techniques to isolate organ-specific ECs, to define EC heterogeneity in its full complexity, and to culture patient-derived ECs from inducible pluripotent stem cells or endothelial progenitor cells are likely to advance the understanding of ARDS and lead to development of therapeutics. This review 1) summarizes the advantages and pitfalls of EC cultures to study vascular leak in ARDS, 2) provides an overview of elements of the microvascular environment that can directly affect endothelial barrier function, and 3) discusses alternative methods to bridge the gap between basic research and clinical application with the intent of improving the translational value of present EC culture approaches.

Successful stem cell therapy after acute myocardial infarction (AMI) is hindered by lack of engraftment of sufficient stem cells at the site of injury. We designed a novel technique to overcome this problem by assembling stem cell-microbubble complexes, named ‘StemBells’.StemBells were assembled through binding of dual-targeted microbubbles (~3μm) to adipose-derived stem cells (ASCs) via a CD90 antibody. StemBells were targeted to the infarct area via an ICAM-1 antibody on the microbubbles. StemBells were characterized microscopically and by flow cytometry. The effect of ultrasound on directing StemBells towards the vessel wall was demonstrated in an in vitro flow model. In a rat AMI-reperfusion model, StemBells or ASCs were injected one week post-infarction. A pilot study demonstrated feasibility of intravenous StemBell injection, resulting in localization in ICAM-1-positive infarct area three hours post-injection. In a functional study five weeks after injection of StemBells cardiac function was significantly improved compared with controls, as monitored by 2D-echocardiography. This functional improvement neither coincided with a reduction in infarct size as determined by histochemical analysis, nor with a change in anti- and pro-inflammatory macrophages.In conclusion, the StemBell technique is a novel and feasible method, able to improve cardiac function post-AMI in rats.

Endothelial colony-forming cells (ECFC) are grown from circulating CD34+ progenitors present in adult peripheral blood, but during in vitro expansion part of the cells lose CD34. To evaluate whether the regulation of CD34 characterizes the angiogenic phenotypical features of PB-ECFCs, we investigated the properties of CD34+ and CD34− ECFCs with respect to their ability to form capillary-like tubes in 3D fibrin matrices, tip-cell gene expression, and barrier integrity. Selection of CD34+ and CD34− ECFCs from subcultured ECFCs was accomplished by magnetic sorting (FACS: CD34+: 95 % pos; CD34−: 99 % neg). Both fractions proliferated at same rate, while CD34+ ECFCs exhibited higher tube-forming capacity and tip-cell gene expression than CD34− cells. However, during cell culture CD34− cells re-expressed CD34. Cell-seeding density, cell–cell contact formation, and serum supplements modulated CD34 expression. CD34 expression in ECFCs was strongly suppressed by newborn calf serum. Stimulation with FGF-2, VEGF, or HGF prepared in medium supplemented with 3 % albumin did not change CD34 mRNA or surface expression. Silencing of CD34 with siRNA resulted in strengthening of cell–cell contacts and increased barrier function of ECFC monolayers as measured by ECIS. Furthermore, CD34 siRNA reduced tube formation by ECFC, but did not affect tip-cell gene expression. These findings demonstrate that CD34+ and CD34− cells are different phenotypes of similar cells and that CD34 (1) can be regulated in ECFC; (2) is positively involved in capillary-like sprout formation; (3) is associated but not causally related to tip-cell gene expression; and (4) can affect endothelial barrier function. Electronic supplementary material The online version of this article (doi:10.1007/s10456-016-9506-9) contains supplementary material, which is available to authorized users.

Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

Upon implantation of tissue-engineered scaffolds, hypoxia will occur until neovascularization takes place. In vivo, the temporary fibrin matrix forms a suitable matrix for this process and fibrin variants can influence the extent of neovascularization. In this study, the influence of oxygen tension and naturally occurring fibrinogen variants on adipose tissue-derived mesenchymal stem cell (ASC) expansion and differentiation were determined. ASC proliferated 1.7-fold faster in 1% oxygen and showed reduced cell aging, and their stemness was preserved. The stem cell surface marker expression was similar in 1% and 20% oxygen. The various fibrinogen coatings did not influence ASC expansion and differentiation. Differentiation of ASC toward adipogenic and osteogenic lineages was improved in 20% oxygen, whereas 1% oxygen improved chondrogenic differentiation. In conclusion, optimal oxygen concentrations vary for the intended ASC application, and fibrinogen variants, which can be used to influence neovascularization, do not alter ASC behavior. These data emphasize the importance of oxygen concentrations during stem cell growth and differentiation.

Subsequent to myocardial infarction, cardiomyocytes within the infarcted areas and border zones expose phosphatidylserine (PS) in the outer plasma membrane leaflet (flip-flop). We showed earlier that in addition to apoptosis, this flip-flop can be reversible in cardiomyocytes. We now investigated a possible role for Rho and downstream effector Rho-associated kinase (ROCK) in the process of (reversible) PS exposure and apoptosis in cardiomyocytes. In rat cardiomyoblasts (H9c2 cells) and isolated adult ventricular rat cardiomyocytes clostridium difficile Toxin B (TcdB), a Rho GTPase family inhibitor, C3 transferase (C3), a Rho(A,B,C) inhibitor and the ROCK inhibitors Y27632 and H1152 were used to inhibit Rho - ROCK signaling. PS exposure was assessed via flow cytometry and fluorescent digital imaging microscopy using annexin-V. Akt expression and phosphorylation were analyzed via Western blot, and Akt activity was inhibited by wortmannin. The cellular concentration activated caspase 3 was determined as a measure of apoptosis, and flippase activity was assessed via flow cytometry using NBD labeled PS. TcdB, C3, Y27632 and H1152 all significantly increased PS exposure. TcdB, Y27632 and H1152 all significantly inhibited phosphorylation of the anti-apoptotic protein Akt and Akt inhibition by wortmannin lead to increased PS exposure. However, only TcdB and C3, but not ROCK- or Akt inhibition led to caspase 3 activation and thus apoptosis. Notably, pancaspase inhibitor zVAD only partially inhibited TcdB induced PS exposure indicating the existence of apoptotic and non-apoptotic PS exposure. The induced PS exposure coincided with decreased flippase activity as measured with NBD-labeled PS flip-flop. In this study, we show a regulatory role for a novel signaling route, Rho - ROCK - flippase signaling, in maintaining asymmetrical membrane phospholipid distribution in cardiomyocytes

In anti-cancer therapy, current investigations explore the possibility of two different strategies to target tumor vasculature: one aims at interfering with angiogenesis, the process involving the outgrowth of new blood vessels from pre-existing vessels, while the other directs at affecting the already established tumor vasculature. However, the majority of in vitro model systems currently available examine the process of angiogenesis, while the current focus in anti-vascular therapies moves towards exploring the benefit of targeting established vasculature as well. This urges the need for in vitro systems that are able to differentiate between the effects of compounds on angiogenesis as well as on established vasculature. To achieve this, we developed an in vitro model in which effects of compounds on different vascular targets can be studied specifically. Using this model, we examined the actions of the fumagillin derivate TNP-470, the MMP-inhibitor marimastat and the recently developed tubulin-binding agent Ang-510. We show that TNP-470 and marimastat solely inhibited angiogenesis, whereas Ang-510 potently inhibited angiogenesis and caused massive disruption of newly established vasculature. We show that the use of this in vitro model allows for specific and efficient screening of the effects of compounds on different vascular targets, which may facilitate the identification of agents with potential clinical benefit. The indicated differences in the mode of action between marimastat, TNP-470 and Ang-510 to target vasculature are illustrative for this approach. (C) 2009 Elsevier Inc. All rights reserved.

Aims Cytosolic and nuclear localization of β-catenin was observed in leaky vessels and in tumours. Several lines of evidence indicate that nuclear β-catenin facilitates angiogenesis. We hypothesized that nuclear β-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 β-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 β-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 3β, which phosphorylates cytosolic β-catenin and thereby facilitates its proteasomal degradation, was inhibited by thrombin. The analysis of nuclear extracts demonstrated a thrombin-induced nuclear accumulation of β-catenin as well as p120ctn. Thrombin stimulation activated β-catenin-mediated transcriptional activity as evidenced by reporter assays. Finally, real-time-PCR revealed increased mRNA levels of several β-catenin target genes. Conclusion Thrombin induced a cytosolic stabilization of membrane-liberated β-catenin, which, together with p120ctn, subsequently translocated to the nucleus where it induces several β-catenin target genes. This supports the suggestion that membrane-liberated β-catenin and p120ctn contribute to angiogenic responses of ECs following episodes of vascular leakage.

Expression of the transcription factor hypoxia-inducible factor 1 (HIF-1), which plays a key role in cellular adaptation to hypoxia, was investigated in normal colorectal mucosa (ten), adenomas (61), and carcinomas (23). Tissue samples were analyzed for HIF-1 alpha, its upstream regulators, von Hippel-Lindau factor, AKT, and mammalian target of rapamycin (mTOR) and its downstream targets glucose transporter 1 (GLUT1), carbonic anhydrase IX, stromal-cell-derived factor 1 (SDF-1) by immunohistochemistry. In normal colorectal mucosa, HIF-1 alpha was observed in almost all nuclei of surface epithelial cells, probably secondary to a gradient of oxygenation, as indicated by pimonidazole staining. The same staining pattern was present in 87% of adenomas. In carcinomas, HIF-1 alpha was present predominantly around areas of necrosis (78%). Active AKT and mTOR, were present in all adenomas, carcinomas, and in normal colorectal mucosa. GLUT1 and SDF-1 were present in the normal surface epithelium of all adenoma cases, whereas in the carcinoma GLUT1 was located around necrotic regions and SDF-1 was present in all epithelial cells. In conclusion, HIF-1 alpha appears to be physiologically expressed in the upper part of the colorectal mucosa. The present observations support that upregulation of HIF-1 alpha and its downstream targets GLUT1 and SDF-1 in colorectal adenomas and carcinomas may be due to hypoxia, in close interaction with an active phosphatidylinositol 3-kinases-AKT-mTOR pathway.

Proteolysis is essential for decidual development during embryonic implantation, but little is known regarding the expression and functions of membrane-type matrix metalloproteinases (MT-MMPs) and urokinase-type plasminogen activator (uPA) and its receptor uPAR in decidua. Therefore, their protein and mRNA levels were analysed in three first trimester decidual tissues, decidual secretory endometrium (DSE), decidua parietalis (DP) and basalis (DB). Decidua was obtained during first trimester pregnancy termination. uPA, uPAR, and MT1/2/3/5-MMP expression were studied by RT-PCR and immunohistochemistry, and CD56-positive uNK cells and CD68-positive macrophages were quantified in serial sections. The mRNAs and antigens of all proteases and uPAR were detectable in the decidual tissues and extravillous trophoblasts (EVT). mRNA levels of all proteases and uPAR, except MT5-MMP, were elevated in both DB and DP compared to DSE, being significant for MT1-MMP and uPAR in DP. MT2- and MT3-MMP mRNAs in DB were 24- and 10-fold higher than in DSE, and 19- and 7-fold increased compared to DP. At the protein level uPA and uPAR were particularly elevated in DB, while pro-angiogenic MT1- and MT3-MMPs were elevated in both DB and DP compared to DSE. MT2-MMP was prominently present in all conditions. The number of uNK cells was increased in DB and DP versus DSE, while a comparable increase in macrophages did not reach statistical significance. These data are consistent with a differential regulation of pericellular proteases in decidua by pregnancy-induced hormones, immune cells and EVT. © The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Objective— Rho kinase mediates vascular leakage caused by many vasoactive agents including thrombin. Enhanced Rho kinase activity induces endothelial barrier dysfunction by a contractile mechanism via inactivation of Myosin Phosphatase (MP). Here, we investigated the contribution of basal Rho kinase activity to the regulation of endothelial barrier integrity. Methods and Results— Using a phospho-specific antibody against the myosin phosphatase targeting subunit (Thr 696− MYPT1) as a marker for Rho kinase activity, basal endothelial Rho kinase activity was observed at cell-cell contact sites, in vitro and in situ. Thrombin enhanced MYPT phosphorylation at F-actin stress fibers. Inhibition of basal Rho kinase activity for 24 hours or depletion of Rho kinase (ROCK-I and -II) by siRNA disrupted endothelial barrier integrity, opposite to the previously observed protection from the thrombin-enhanced endothelial permeability. This barrier dysfunction could not be explained by changes in RhoA, Rac1, eNOS, or apoptosis. Remarkably, basal Rho kinase activity was essential for proper expression of the adhesion molecule VE-cadherin. Conclusions— Rho kinase has opposing activities in regulation of endothelial barrier function: (1) an intrinsic barrier-protective activity at the cell margins, and (2) an induced barrier-disruptive activity at contractile F-actin stress fibers. These findings may have implications for long-term antivascular leak therapy.

Objective— Besides having a key role in fibrinolysis, the plasminogen system has been implicated in cell migration and angiogenesis. A common mechanism is the binding of plasminogen to carboxy-terminal lysine residues in partially degraded fibrin or on cellular surfaces. Here we examined the involvement of thrombin activatable fibrinolysis inhibitor (TAFI) and pancreatic carboxypeptidase B (CPB) in an in vitro capillary tube formation system, which is largely plasminogen-dependent. Methods and Results— Human microvascular endothelial cells (hMVECs) were seeded on a 3D plasma clot matrix and subsequently stimulated with bFGF/tumor necrosis factor (TNF)-α. Tube formation was analyzed and fibrin degradation products (FbDP) were determined in the medium. Supplementation of the matrix with additional TAFI or CPB produced a reduction in tube formation. Pretreatment of hMVECs with CPB before seeding resulted in a similar effect. FbDP-levels indicated a concomitant reduction in matrix proteolysis. A TAFIa inhibitor increased tube formation and FbDP release into the medium. In separate assays, CPB impaired the migration of hMVECs in a dose-dependent manner, whereas proliferation and adhesion remained unaffected. Conclusions— Overall, these results demonstrate that TAFI and CPB in these systems modulate the plasminogen system both in the matrix and on the cell surface, thus leading to the inhibition of endothelial cell movement and tube formation.

In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d-glucose produced approximately 2-fold higher levels of methylglyoxal but not 3-deoxyglucosone and glyoxal, as compared to 5 mM d-glucose. Under hyperglycaemic conditions, the methylglyoxal-arginine adduct argpyrimidine as detected with a specific antibody, but not N(e)-(carboxymethyl)lysine and N(e)-(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARgamma ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine-modified protein. With in-gel digestion and mass spectrometric analysis, we identified this major protein as heat-shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.

Background: During wound repair, fibrin acts both as a barrier to prevent blood loss and as a temporary matrix for the invasion and ingrowth of endothelial and tissue cells. A well-controlled angiogenesis process in the fibrinous exudate matrix is crucial for optimal wound healing. The composition and structure of the fibrin matrix are important determinants of the invasion of endothelial cells and capillary-tube formation into the matrix. Objective: Fibrinogen circulates in a high and low molecular weight form (HMW and LMW, respectively) and the purpose of this study was to investigate how fibrin matrices from these naturally occurring fibrinogen variants influence angiogenesis. Angiogenesis was studied using an in vitro model in which human microvascular endothelial cells (hMVEC) were cultured on three-dimensional fibrin matrices from different fibrinogen forms, and using two in vivo mouse models. Results: The in vitro angiogenesis in an HMW-fibrin matrix shows increased cell and tubular structure ingrowth compared with unfractionated fibrin matrix (median increase 58%, range 46-234%). The ingrowth of tubular structures in an LMW-fibrin matrices is decreased when compared with unfractionated fibrin (median decrease 70%, range 67-100%). Similar results were observed for in vivo angiogenesis. Conclusions: The naturally occurring fibrinogen variants HMW- and LMW-fibrin modulate the angiogenic capacity of endothelial cells in fibrin matrices. The different effects of the molecular weight fibrinogen variants provide further insight in the matrix characteristics in angiogenesis and could possibly be applied in the context of tissue engineering and wound healing. © 2006 International Society on Thrombosis and Haemostasis.

Objective— Emerging evidence suggests that human blood contains bone marrow (BM)-derived endothelial progenitor cells that contribute to postnatal neovascularization. Clinical trials demonstrated that administration of BM-cells can enhance neovascularization. Most studies, however, used crude cell populations. Identifying the role of different cell populations is important for developing improved cellular therapies. Methods and Results— Effects of the hematopoietic stem cell–containing CD34 + cell population on migration, proliferation, differentiation, stimulation of, and participation in capillary-like tubule formation were assessed in an in vitro 3-dimensional matrix model using human microvascular endothelial cells. During movement over the endothelial monolayer, CD34 + cells remained stuck at sites of capillary tube formation and time- and dose-dependently formed cell clusters. Immunohistochemistry confirmed homing and proliferation of CD34 + cells in and around capillary sprouts. CD34 + cells were transduced with the LNGFR marker gene to allow tracing. LNGFR gene–transduced CD34 + cells integrated in the tubular structures and stained positive for CD31 and UEA-1. CD34 + cells alone stimulated neovascularization by 17%. Coculture with CD34 − cells led to 68% enhancement of neovascularization, whereas CD34 − cells displayed a variable response by themselves. Cell–cell contact between CD34 + and CD34 − cells facilitated endothelial differentiation of CD34 + cells. Conclusions— Our data suggest that administration of CD34 + -enriched cell populations may significantly improve neovascularization and point at an important supportive role for (endogenous or exogenous) CD34 − cells.

The endothelium plays a central role in the logistics of the immune system by allowing the selective transmigration of leukocytes, as well as the maintenance of the circulation and coagulation homeostasis. Evidence is increasing that the carbohydrate composition of the endothelial cell surface is critical for the cells to exert their physiological function. The major aim of this study is to unravel the mechanisms underlying the expression of carbohydrate structures by endothelial cells, which are involved in leukocyte adhesion and migration. Using quantitative real-time PCR, the expression profile of a selected group of 74 glycosylation-related genes has been determined in human umbilical vein endothelial cells (HUVEC) and human foreskin microvascular endothelial cells (FMVEC) under control and TNFα-induced conditions. The set of genes comprised 59 glycosyltransferases, 6 mannosidases and 9 sulfotransferases. In parallel, the overall cell surface glycan profile has been assessed by the use of glycan-specific lectins and monoclonal antibodies. The results demonstrate that HUVEC and FMVEC differ substantially in the expression of glycosylation-related genes and, accordingly, also in the presence of different glycan epitopes on the cell membrane. Induction of an inflamed phenotype of the cells by treatment with TNFα differentially modulates a set of these genes in HUVEC and FMVEC resulting in a change in the cell membrane associated glycans that are of importance in inflammation-related endothelial cell-surface processes. © 2005 Wiley-Liss, Inc.

Since the recognition of basicfibroblast growth factor (bFGF, FGF-2) and acidic FGF (aFGF, FGF-1) as proliferation and maintenance factors for endothelial cells in culture,1,2 much interest has arisen regarding their role as potential angiogenic factors in tumors and tissue healing in vivo. The FGFs, of which presently at least 22 members have been recognized,3 interact with FGF receptors (FGFRs). After FGF binding, the FGFRs dimerize and become activated displaying protein tyrosine kinase activity and subsequent signaling in the cell.4,5 Four functional FGFRs have been recognized, FGFR1–4, most of which can be encountered in various alternative spliced isoforms. FGFR1 plays an essential role during early stages of development of the mesoderm and at several levels during organogenesis.3,6 FGF/FGFR1 is expressed almost exclusively in the mesenchyme, including endothelial cells, smooth muscle cells, and hemopoietic progenitor cells. FGFR1 is involved in the stimulation and proliferation of endothelial cells and vascularization by FGFs, particularly bFGF and aFGF. FGFs also promote the formation of hematopoietic progenitor cells in early development, which requires the FGFR1 as well.7,8 In line with this, chromosomal translocation involving FGFR1 has been observed in patients with stem-cell myeloproliferative disease, probably caused by constitutive activation of the FGFR1 gene.9 See page 944 It has long been anticipated that hematopoietic progenitor cells and endothelial progenitor cells derive from a common cell type, the hemangioblast.10 Studies on mouse embryos have shown that such hemangioblast cells indeed exist. These cells develop in the primitive streak of the embryo and migrate to the yolk sac, where they form blood islands.11 The outer cells of these blood islands mature into endothelial cells and form a vascular plexus, while the inner cells develop into primitive and definitive hematopoietic progenitor cells and their subsequent lineages of blood cells. In addition, in …

Endostatin is a cleavage product of collagen XVIII that has shown to inhibit tumor-angiogenesis in experimental tumor models. At present, the exact molecular mechanism of action of endostatin is not completely elucidated. In this study, we wanted to identify specific target genes of endostatin. For this purpose, the human renal cell carcinoma RC-9 was subcutaneously implanted in nude mice and treated with endostatin. Tumor growth was inhibited by endostatin after 4 days of treatment. Using immunohistochemistry and the hypoxia marker pimonidazole, we demonstrate disintegration of blood vessels and hypoxia and anoxia as a result of the treatment. Hereafter, we applied the polymerase chain reaction (PCR)-based subtractive suppression hybridization (SSH) method, together with the mirror orientation selection (MOS) technique to identify specifically induced and suppressed genes after endostatin-treatment. We found eight genes to be specifically induced and 11 to be suppressed by the endostatin-treatment. Among other genes, core binding factor a-1/osteoblast-specific factor-2 (cbfa1/osf2) was found to be specifically suppressed by endostatin. Unexpectedly, cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor, not only in the experimental RC-9 tumor model, but in sections of human breast cancer as well. Since an effect of antiangiogenic therapy on granulocytes has been reported before, this might lead to new insights in the role of granulocytes in antiangiogenic therapy in general. In conclusion, the SSH-PCR implemented with the MOS-technique is a powerful tool to identify differentially expressed genes. Using these techniques, we have identified several target genes of endostatin, of which cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor.

Objective - Growth factor-induced angiogenesis involves migration of endothelial cells (ECs) into perivascular areas and requires active remodeling of the endothelial F-actin cytoskeleton. The small GTPase RhoA previously has been implicated in vascular endothelial growth factor (VEGF)-induced signaling pathways, but its role has not been clarified. Methods and Results - VEGF induced the activation of RhoA and recruited RhoA to the cell membrane of human ECs. This increase in RhoA activity is necessary for the VEGF-induced reorganization of the F-actin cytoskeleton, as demonstrated by adenoviral transfection of dominant-negative RhoA. Rho kinase mediated this effect of RhoA, as was demonstrated by the use of Y-27632, a specific inhibitor of Rho kinase. Inhibition of Rho kinase prevented the VEGF-enhanced EC migration in response to mechanical wounding but had no effect on basal EC migration. Furthermore, in an in vitro model for angiogenesis, inhibition of either RhoA or Rho kinase attenuated the VEGF-mediated ingrowth of ECs in a 3-dimensional fibrin matrix. Conclusions - VEGF-induced cytoskeletal changes in ECs require RhoA and Rho kinase, and activation of RhoA/Rho kinase signaling is involved in the VEGF-induced in vitro EC migration and angiogenesis. Chemicals/CAS: 4 (1 aminoethyl) n (4 pyridyl)cyclohexanecarboxamide, 146986-50-7; F actin, 39409-31-9; fibrin, 9001-31-4; guanosine triphosphatase, 9059-32-9; vasculotropin, 127464-60-2; Amides; Endothelial Growth Factors; Enzyme Inhibitors; Fibrin, 9001-31-4; Intercellular Signaling Peptides and Proteins; Lymphokines; Pyridines; rho GTP-Binding Proteins, EC 3.6.5.2; rhoA GTP-Binding Protein, EC 3.6.5.2; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Y 27632, 138381-45-0

SummaryThe aminopeptidase inhibitor bestatin has been shown to have anti-angiogenic effects in a number of model systems. These effects are thought to result from inhibition of CD13 activity. Because tumor angiogenesis can evolve in a fibrin-rich stroma matrix we have studied for the first time the effects of bestatin on microvascular endothelial capillary-like tube formation in a fibrin matrix. Bestatin enhanced the formation of capillary-like tubes dose-dependently. Its effects were apparent at 8 µM; the increase was 3.7-fold at 125 µM; while high concentrations (>250 µM), that were shown to have anti-angiogenic effects in other systems, caused extensive matrix degradation. Specific CD13-blocking antibodies WM15 and MY-7, and the aminopeptidase inhibitors amastatin and actinonin also enhanced capillary-like tube formation (maximally 1.5-fold), but these effects did not reach statistical significance. The effect of bestatin was not due to a change in uPAR availability because the relative involvement of the u-PA/u-PAR activity was not altered by bestatin. In view of the present findings we hypothesize that aminopeptidases other than CD13 predominantly contribute to the observed pro-angiogenic effect of bestatin in a fibrin matrix. The identification of this novel effect of bestatin is important in the light of the proposed use of bestatin as anti-angiogenic and/or anti-tumor agent.

Many diseases share the common feature of vascular leakage, and endothelial barrier dysfunction is often the underlying cause. The subsequent stages of endothelial barrier dysfunction contribute to endothelial hyperpermeability. Vasoactive agents induce loss of junctional integrity, a process that involves actin-myosin interaction. Subsequently, the interaction of leukocytes amplifies leakage by the leukocyte-derived mediators. The processes mainly occur at the postcapillary venules. The whole microvascular bed, including the capillaries, becomes involved in vascular leakage by the induction of angiogenesis. Plasma leakage results from gaps between endothelial cells as well as by the induction of transcellular transport pathways. Several mechanisms can improve endothelial barrier function, depending on the tissue affected and the cause of hyperpermeability. They include blockade of specific receptors and elevation of cyclic AMP (cAMP) by agents such as beta(2)-adrenergic agents. However, current therapies based on these principles often fail. Recent research has identified several new promising targets for pharmacological therapy. Endogenous compounds were also found with barrier-improving characteristics. Important insights were obtained in the different pathways involved in barrier dysfunction. Such insights regard the regulation of endothelial contraction and endothelial junction integrity: inhibitors of RhoA activation and Rho kinase represent a potentially valuable group of agents with endothelial hyperpermeability reducing properties, and strategies to target vascular endothelial growth factor (VEGF)-mediated edema are under current investigation. In clinical practice, not only tools to improve an impaired endothelial barrier function are necessary. Sometimes, a controlled, temporal, and local increase in permeability can also be desired, for example, with the aim to enhance drug delivery. Therefore, vessel leakiness is also being exploited to enable tissue access of liposomes, viral vectors, and other therapeutic agents that do not readily cross healthy endothelium. This review discusses strategies for targeting signaling molecules in therapies for diseases involving altered endothelial permeability.

The endothelium dynamically regulates the extravasation of hormones, macromolecules and other solutes. In pathological conditions, endothelial hyperpermeability can be induced by vasoactive agents, which induce tiny leakage sites between the cells, and by cytokines, in particular vascular endothelial growth factor, which increase the exchange of plasma proteins by vesicles and intracellular pores. It is generally believed that the interaction of actin and non-muscle myosin in the periphery of the endothelial cell, and the destabilization of endothelial junctions, are required for endothelial hyperpermeability induced by vasoactive agents. Transient short-term hyperpermeability induced by histamine involves Ca2+/calmodulin-dependent activation of the myosin light chain (MLC) kinase. Prolonged elevated permeability induced by thrombin in addition involves activation of the small GTPase RhoA and Rho kinase, which inhibits dephosphorylation of MLC. It also involves the action of other protein kinases. Several mechanisms can increase endothelial barrier function, depending on the tissue affected and the cause of hyperpermeability. They include blockage of specific receptors, and elevation of cyclic AMP by agents such as beta2-adrenergic agents. Depending on the vascular bed, nitric oxide and cyclic GMP can counteract or aggravate endothelial hyperpermeability. Finally, inhibitors of RhoA activation and Rho kinase represent a potentially valuable group of agents with endothelial hyperpermeability-reducing properties.

Angiogenesis, or the formation of new microvessels, is often encountered in pathological situations. A fibrinous exudate can often act as a temporary matrix for the ingrowth of these new microvessels. This matrix consists mainly of fibrin, but is mingled with other plasma components and interstitial collagen fibres. In vitro, capillary-like tube formation can be mimicked by exposing human microvascular endothelial cells (hMVECs), seeded on top of a three-dimensional fibrin matrix, to an angiogenic growth factor (e.g. fibroblast growth factor (FGF)-2) and the cytokine tumour necrosis factor (TNF)-α. Plasmin activity is required in this process. We investigated whether the angiogenic potential of hMVECs was altered by the presence of collagen. The addition of type I collagen to fibrin matrices dose-dependently inhibited tube-formation. Tube-formation in these fibrin/collagen matrices by hMVECs required matrix metalloprotease (MMP) activity, as well as plasmin activity. On a pure collagen type I matrix, hMVECs were not able to form tube-like structures in the matrix but formed sprouts. This sprouting required MMP activity and was, in contrast to the tube-like structures in a fibrin matrix, not influenced by hypoxia. These data indicate that the interaction between endothelial cells and different matrix components is of importance for the angiogenic potential of these cells.

Within many general functions the endothelium is equipped with a number of mechanisms that prevent thrombus formation in the circulatory system. It harbours factors that interrupt the coagulation cascade, such as antithrombin III, the protein C receptor thrombomodulin, and tissue factor pathway inhibitor. It prevents platelet activation by the production of nitric oxide and prostacyclin, exonucleotidases and surface heparan sulphates. Furthermore, it can trigger and control fibrinolysis by the synthesis and release of tissue-type plasminogen activator and its inhibitor PAI-1. The general properties of the endothelium are subject to adaptation by environmental factors, such as inflammatory mediators and shear forces. Interleukin-1 and tumour necrosis factor-alpha reduce the antithrombotic properties of the endothelium. Furthermore, local variation exists between different vascular beds and vessel types, such as in the endometrium. While the endothelium controls blood fluidity on its apical side, adaptation of the endothelium also prepares its involvement in tissue repair upon inflammation or damage. The fibrin matrix, which is formed after damage of the vascular system, not only acts as a sealing of the wound, but also facilitates the repair process by providing a scaffolding for cell invasion and angiogenesis.

SummaryAmong other proteolytic enzymes, the urokinase-type plasminogen activator (u-PA)/plasmin cascade contributes to cell migration and the formation of capillary-like structures in a fibrinous exudate. The u-PA receptor (u-PAR) focuses proteolytical activity on the cell surface of the endothelial cell and hereby accelerates the pericellular matrix degradation. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 enhance u-PA receptor expression in human endothelial cells. In this paper we show that the protein kinase C (PKC) inhibitors Ro31-8220 and GF109203X inhibit VEGF165-induced u-PAR antigen expression in human endothelial cells, whereas PKC inhibition had no effect on FGF-2-induced u-PAR antigen enhancement. In addition, inhibition of PKC activity had no effect on VEGF165-or FGF-2-induced proliferation in human endothelial cells. We conclude that VEGF165 induces u-PAR via a PKC-dependent pathway, whereas proliferation is induced via a different pathway probably involving tyrosine phosphorylation of proteins downstream of the VEGF receptors.

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix, hMVECs stimulated with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor-α (TNF-α) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (20% oxygen) conditions. In both conditions the in-growth of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-α was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the α(v)-integrin subunit was enhanced by hypoxia, blocking antibodies against α(v)β3 and α(v)β5-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process. (C) 2000 by The American Society of Hematology. Chemicals/CAS: fibrin, 9001-31-4; phorbol 13 acetate 12 myristate, 16561-29-8; plasmin, 9001-90-5, 9004-09-5; plasminogen activator, 9039-53-6; tissue plasminogen activator, 105913-11-9; urokinase, 139639-24-0; vasculotropin, 127464-60-2; Antigens, CD; Culture Media; Culture Media, Conditioned; DNA, Complementary; Endothelial Growth Factors; Fibrin, 9001-31-4; Fibroblast Growth Factor 2, 103107-01-3; Integrin alphaV; integrin alphaVbeta5; Integrins; Lymphokines; Oxygen, 7782-44-7; Plasmin, EC 3.4.21.7; plasminogen activator, urokinase receptors; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases, EC 2.7.1.112; Receptors, Cell Surface; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor, EC 2.7.1.112; Receptors, Vitronectin; RNA, Messenger; Tumor Necrosis Factor; Urinary Plasminogen Activator, EC 3.4.21.73; Vascular Endothelial Growth Factor Receptor-1, EC 2.7.1.112; vascular endothelial growth factor

The hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children. The role of a verocytotoxin (VT)-producing Escherichia coli has been strongly implicated in the epidemic form of HUS. Although direct toxicity of VT on glomerular endothelial cells has been demonstrated, it remained still unclear how the VT is transported from the intestine to the target organs. In this study we demonstrate that VT, when incubated in whole blood, binds rapidly and completely to human polymorphonuclear leukocytes (PMNs) and not to other components of blood. Binding studies with 125I-VT-1 showed a single class of binding sites on freshly isolated, non-stimulated human PMNs. The K(d) of VT-binding to PMNs was 10-8 mol/L, 100-fold less than that of the VT-receptor globotriaosylceramide. On incubation of VT-preloaded PMNs with human glomerular microvascular endothelial cells (GMVECs), transfer of VT-1 to the endothelial cells occurred. Incubation of non-stimulated GMVECs with VT- preloaded PMNs, but not with PMNs or VT-1 alone, caused inhibition of protein synthesis and cell death. Our data are in concert with a role of PMNs in the transfer of VT from the intestine to the kidney endothelium. This transfer occurs by selective binding to a specific receptor on PMNs and subsequent passing of the ligand VT to the VT-receptor on GMVECs, which causes cell damage. This new mechanism further underpins the important role of PMNs in HUS. (C) 2000 by The American Society of Hematology.

SummarySmooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

The retinal pigment epithelium (RPE) maintains the choriocapillaris (CC) in the normal eye and is involved in the pathogenesis of choroidal neovascularization in age-related macular degeneration. Vascular endothelial growth factor-A (VEGF) is produced by differentiated human RPE cells in vitro and in vivo and may be involved in paracrine signaling between the RPE and the CC. We investigated whether there is a polarized secretion of VEGF by RPE cells in vitro. Also, the localization of VEGF receptors in the human retina was investigated. We observed that highly differentiated human RPE cells, cultured on transwell filters in normoxic conditions, produced two- to sevenfold more VEGF toward their basolateral side as compared to the apical side. In hypoxic conditions, VEGF-A secretion increased to the basal side only, resulting in a three- to 10-fold higher basolateral secretion. By immunohistochemistry in 30 human eyes and in two cynomolgus monkey eyes, KDR (VEGFR-2) and flt-4 (VEGFR-3) were preferentially localized at the side of the CC endothelium facing the RPE cell layer, whereas flt-1 (VEGFR-1) was found on the inner CC and on other choroidal vessels. Our results indicate that RPE secretes VEGF toward its basal side where its receptor KDR is located on the adjacent CC endothelium, suggesting a role of VEGF in a paracrine relation, possibly in cooperation with flt-4 and its ligand. This can explain the known trophic function of the RPE in the maintenance of the CC and its fenestrated permeable phenotype and points to a role for VEGF in normal eye functioning. Up-regulated basolateral VEGF secretion by RPE in hypoxia or loss of polarity of VEGF production may play a role in the pathogenesis of choroidal neovascularization.

Abstract —In the present study, we differentiated between short- and long-term effects of vasoactive compounds on human endothelial permeability in an in vitro model. Histamine induced a rapid and transient (22 Na ions. This increase in permeability was inhibited completely by chelation of intracellular calcium ions by BAPTA-AM and inhibition of calmodulin activity and myosin light chain (MLC) phosphorylation. The presence of serum factors prolonged the barrier dysfunction induced by histamine. Thrombin by itself induced a prolonged barrier dysfunction (>30 minutes) as evidenced by an increased passage of peroxidase and 40 kDa dextran. It was dependent only partially on calcium ions and calmodulin. The protein tyrosine kinase inhibitors genistein and herbimycin A, but not the inactive analogue daidzein, inhibited to a large extent the increase in permeability induced by thrombin. Genistein and BAPTA-AM inhibited the thrombin-induced permeability in an additive way, causing together an almost complete prevention of the thrombin-induced increase in permeability. Inhibition of protein tyrosine kinase was accompanied by a decrease in MLC phosphorylation and a reduction in the extent of F-actin fiber and focal attachment formation. Inhibition of Rho A by C3 transferase toxin reduced both the thrombin-induced barrier dysfunction and MLC phosphorylation. Genistein and C3 transferase toxin did not elevate the cellular cAMP levels. No evidence was found for a significant role of protein kinase C in the thrombin-induced increase in permeability or in the accompanying MLC phosphorylation. These data indicate that in endothelial cell monolayers that respond to histamine in a physiological way, thrombin induces a prolonged increase in permeability by “calcium sensitization,” which involves protein tyrosine phosphorylation and Rho A activation.

See related article, pages 50–58 Vascular leakage is a hallmark of many, often life-threatening, inflammatory diseases, and contributes to disease severity in disorders such as sepsis, cancer, diabetes, and atherosclerosis.1 Despite the tremendous medical importance of vascular leakage, few specific therapies are available today to counteract it, and current therapies often fail to do so.2 This is in part because the in vivo molecular targets are incompletely identified, although a wealth of data obtained from in vitro studies is available on signal transduction pathways that regulate vascular permeability.3,4 Interestingly, among various new agents that potentially reduce endothelial hyperpermeability, the cholesterol-lowering statin drugs have been proposed to reduce vascular leakage by means of their inhibitory effects on RhoA proteins.5,6 In the current issue of Circulation Research Gorovoy et al provide an elegant proof-of-principle that increased RhoA activity results in vascular hyperpermeability in vivo.7 They show that elevation of RhoA activity by deletion of one of its inhibitory proteins, RhoGDI, causes a loss of endothelial junctional integrity and decrease in vascular barrier function. Rho GTPases, particularly RhoA, Rac-1 and cdc42 have received much attention as key regulators of cell shape, movement and proliferation. In vitro studies have shown that the balance of activities of these small G proteins determines the status of endothelial barrier8: Cdc42 enhancing recovery of a disturbed barrier,9 Rac-1 being required for establishing a tight barrier,8 and RhoA being involved in inducing endothelial hyperpermeability by various stimuli, such as thrombin, VEGF, angiopoietin-2 and LPA (see Figure).10–12 Similarly, these Rho GTPases are acting …

Angiogenesis, the formation of new capillary blood vessels, is a feature of a variety of pathological processes. To study the effects of a specific group of hormones (all ligands of the steroid/retinoid/thyroid hormone receptor superfamily) on the angiogenic process in humans, we have used a model system in which human microvascular endothelial cells from foreskin (hMVEC) are cultured on top of a human fibrin matrix in the presence of basic fibroblast growth factor and tumor necrosis factor-α. This model mimics the in vivo situation where fibrin appears to be a common component of the matrix present at sites of chronic inflammation and tumor stroma. Our results show that testosterone and dexamethasone are strong inhibitors and all-trans retinoic acid (at-RA) and 9-cis retinoic acid (9-cis RA) are potent stimulators of the formation of capillary-like tubular structures. These effects are mediated by their respective nuclear hormone receptors as demonstrated by the use of specific synthetic receptor agonists and antagonists. 17β-estradiol, progesterone, and 1,25-dihydroxyvitamin D3 did not affect or only weakly affected in vitro angiogenesis, which may be related to the lack of significant nuclear receptor expression. Although hMVEC express both thyroid hormone receptors α and β, no effect of thyroid hormone on tube formation was found. The effects of testosterone, dexamethasone,at-RA, and 9-cis RA on tube formation were accompanied by parallel changes in urokinase-type plasminogen activator (u-PA) expression, at both mRNA and antigen levels. Exogenous suppletion of the medium with single chain u-PA enhances tube formation in our in vitro model, whereas quenching of u-PA activity (but not of tissue-type plasminogen activator activity) or of u-PA binding to u-PA receptor by specific antibodies suppressed basal and retinoid-stimulated tube formation. Moreover, addition of scu-PA to testosterone- or dexamethasone-treated hMVEC restored the suppressed angiogenic activity for a substantial part. Aprotinin, an inhibitor of plasmin activity, completely inhibited tube formation, indicating that the proteolytic properties of the u-PA/u-PA receptor complex are crucial in this process. Our results show that steroid hormones (testosterone and dexamethasone) and retinoids have strong, but opposite effects on tube formation in a human in vitro model reflecting pathological angiogenesis in the presence of fibrin and inflammatory mediators. These effects can be explained by hormone-receptor–mediated changes in u-PA expression, resulting in enhanced local proteolytic capacity of the u-PA/u-PA receptor complex.© 1998 by The American Society of Hematology.

Neutrophil collagenase (matrix metalloproteinase-8 or MMP-8) is regarded as being synthesized exclusively by polymorphonuclear neutrophils (PMN). However, in vivo MMP-8 expression was observed in mononuclear fibroblast- like cells in the rheumatoid synovial membrane. In addition, we detected MMP- 8 mRNA expression in cultured rheumatoid synovial fibroblasts and human endothelial cells. Up-regulation of MMP-8 was observed after treatment of the cells with either tumor necrosis factor-α (10 ng/ml) or phorbol 12-myristate 13-acetate (10 nM). Western analysis showed a similar regulation at the protein level. The size of secreted MMP-8 was 50 kDa, which is about 30 kDa smaller than MMP-8 from PMN. Conditioned media from rheumatoid synovial fibroblasts contained both type I and II collagen degrading activity. However, degradation of type II collagen, but not that of type I collagen, was completely inhibited by 50 μM doxycycline, suggesting specific MMP-8 activity. In addition, doxycycline down-regulated MMP-8 induction, at both the mRNA and protein levels. Thus MMP-8 exerts markedly wider expression in human cells than had been thought previously, implying that PMN are not the only source of cartilage degrading activity at arthritic sites. The inhibition of both MMP-8 activity and synthesis by doxycycline provides an incentive for further studies on the clinical effects of doxycycline in the treatment of rheumatoid arthritis.

IL-6 −/− mice showed impaired leukocyte accumulation in subcutaneous air pouches. Defective leukocyte accumulation was not due to a reduced migratory capacity of IL-6 −/− leukocytes and was associated with a reduced in situ production of chemokines. These observations led to a reexamination of the interaction of IL-6 with endothelial cells (EC). EC express only the gp130 signal transducing chain and not the subunit-specific IL-6R and are therefore unresponsive to IL-6. However, EC are responsive to a combination of IL-6 and soluble IL-6R as measured by the activation of STAT3, chemokine expression, and augmentation of ICAM-1. Activation by IL-6–IL-6R complexes was inhibited by an IL-6 receptor antagonist and potentiated by a superagonist. Hence, in vivo and in vitro evidence supports the concept that the IL-6 system plays an unexpected positive role in local inflammatory reactions by amplifying leukocyte recruitment.

Proteases play an important role in various aspects of angiogenesis. First, pericellular proteolysis facilitates matrix degradation as well as migration and invasion of capillary sprouts into its surrounding matrix. (Membrane-type) matrix metalloproteases [(MT-)MMPs], ADAM(TS) metalloproteases, serine proteases, their inhibitors, and occasionally cysteine proteases are important players in pericellular proteolysis. The specific location and short exposure of protease activity can be explained by a tread-milling model with internalization of protease–receptor complexes as was proposed for MT1-MMP/MMP2 and urokinase/urokinase receptor complexes. Second, proteolytic events play a role in regulating VEGF-induced sprouting, such as proteasomal degradation of hypoxia inducible factors (HIFs), Notch-mediated cell signaling, and turnover of VEGF receptors. Third, proteases can activate and modify growth factors and receptors and thus generate split products with new biological functions and contribute to cell recruitment. Proteases likely also contribute to formation of anastomoses, which is required to restore circulation. Finally, new biological functions are also acquired by generation of split products from and modifications of matrix proteins. They include angiogenesis-inhibiting matrikines, and modified fibrin forms with altered angiogenic properties, which may bear perspective for tissue engineering applications.