Cdc42-driven podosome formation in endothelial cells

Ectopic expression of a constitutive active mutant of the GTPase Cdc42 (V12Cdc42) in vascular endothelial cells triggers the dissolution of stress fibres and focal adhesion contacts and causes the repolymerisation of actin into dots. Each punctate structure consists of an F-actin core surrounded by a vinculin ring, consistent with the definition of podosomes. We now report further analysis of these complexes and show the presence of established podosomal markers such as cortactin, gelsolin, dynamin, N-WASP, and Arp2/3 which are absent in focal adhesions. Endothelial podosomes appear as randomly distributed conical structures, distributed on, but restricted to, the ventral membrane and confined to contact sites between cells and their substratum. The nature of the extracellular matrix does not influence podosome formation nor their spatial organisation. Induction of podosomes in response to V12Cdc42 is not associated with a migratory nor with a proliferative phenotype. These results add endothelial cells to the list of cell types endowed with the ability to form podosomes in vitro and raise the possibility that endothelial cells could form such structures under certain physiological or pathological conditions.     

Regulatory signals for endothelial podosome formation

Podosomes are punctate actin-rich adhesion structures which spontaneously form in cells of the myelomonocytic lineage. Their formation is dependent on Src and RhoGTPases. Recently, podosomes have also been described in vascular cells. These podosomes differ from the former by the fact that they are inducible. In endothelial cells, such a signal can be provided by either constitutively active Cdc42, the PKC activator PMA or TGFbeta, depending on the model. Consequently, other regulatory pathways have been reported to contribute to podosome formation. To get more insight into the mechanisms by which podosomes form in endothelial cells, we have explored the respective contribution of signal transducers such as Cdc42-related GTPases, Smads and PKCs in three endothelial cell models. Results presented demonstrate that, in addition to Cdc42, TC10 and TCL GTPases can also promote podosome formation in endothelial cells. We also show that PKCalpha can be either necessary or entirely dispensable, depending on the cell model. In contrast, PKCdelta is essential for podosome formation in endothelial cells but not smooth muscle cells. Finally, although podosomes vary very little in their molecular composition, the signalling pathways involved in their assembly appear very diverse.     

Cortactin regulates podosome formation: roles of the protein interaction domains

Cortactin, a multi-domain scaffolding protein involved in actin polymerization, is enriched in podosomes induced by phorbol ester in vascular smooth muscle cells. We generated several functional and truncation mutants of cortactin to probe the roles of various protein interaction domains in the regulation of the dynamics of podosome formation. At the onset of podosome genesis, cortactin clustered near the ends of stress fibers that appeared to act as nucleation platforms onto which the actin polymerization machinery assembled. Translocation of cortactin to these pre-podosome clusters required the intact N-WASp-binding SH3 domain. Overexpression of the C-terminal third of cortactin containing the intact SH3 domain inhibited podosome formation presumably by sequestering of N-WASp and prevented cortactin clustering. Subsequent assembly of the actin-rich core of podosomes required translocation of additional cortactin to the actin core, a process that required the actin-binding repeats, but not the Arp2/3-binding N-terminal acidic region nor the SH3 domain. These results suggest that the SH3 domain and the actin-binding repeat region are involved, respectively, in the early and late stages of podosome formation process.     

The circle of life: Phases of podosome formation,turnover and reemergence

Podosomes are highly dynamic actin-rich structures in a variety of cell types, especially monocytic cells. They fulfill multiple functions such as adhesion, mechanosensing, or extracellular matrix degradation, thus allowing cells to detect and respond to a changing environment. These abilities are based on an intricate architecture that enables podosomes to sense mechanical properties of their substratum and to transduce them intracellularly in order to generate an appropriate cellular response. These processes are enabled through the tightly orchestrated interplay of more than 300 different components that are dynamically recruited during podosome formation and turnover. In this review, we discuss the different phases of the podosome life cycle and the current knowledge on regulatory factors that impact on the genesis, activity, dissolution and reemergence of podosomes. We also highlight mechanoregulatory processes that become important during these different stages, on the level of individual podosomes, and also at podosome sub- and superstructures.     

The inhibitory effects of endostatin on endothelial cells are modulated by extracellular matrix

We investigated the ability of extracellular matrix (ECM) proteins to modulate the response of endothelial cells to both promoters and inhibitors of angiogenesis. Using human dermal microvascular endothelial cells (HDMEC), we found that cells demonstrated different adhesive properties and proliferative responses to the growth factor VEGF depending upon which ECM protein with which they were in contact, with fibronectin having the most impact on VEGF-induced HDMEC proliferation and survival. More importantly, we observed that ECM could modulate the ability of the angiogenic inhibitor endostatin to prevent endothelial cell proliferation, survival and migration. We observed that growth on vitronectin or fibronectin impaired the ability of endostatin to inhibit VEGF-induced HDMEC proliferation to the greatest extent as determined by BrdU incorporation. We found that, following growth on collagen I or collagen IV, endostatin only inhibited VEGF-induced HDMEC proliferation at the highest dose tested (2500 ng/ml). In a similar manner, we observed that growth on ECM proteins modulated the ability of endostatin to induce endothelial cell apoptosis, with growth on collagen I, fibronectin and collagen IV impairing endostatin-induced apoptosis. Interestingly, endostatin inhibited VEGF-induced HDMEC migration following culture on collagen I, collagen IV and laminin, while migration was not inhibited by endostatin following HDMEC culture on other matrices including vitronectin, fibronectin and tenascin-C. These results suggest that different matrix proteins may affect different mechanisms of endostatin inhibition of angiogenesis. Taken together, our results suggest that the ECM may have a profound impact on the ability of angiostatic molecules such as endostatin to inhibit angiogenesis and thus may have impact on the clinical efficacy of such inhibitors.     

Lipopolysaccharide-stimulated responses in rat aortic endothelial cells by a systems biology approach

The vascular endothelium plays an important role in regulating immune and inflammatory responses to resist pathogens infection. Although it has been known that lipopolysaccharide (LPS) is a critical inducer of sepsis or endotoxemia, the systematic responses of LPS-stimulation in endothelial cells (ECs) are still unclear. The present study aims to analyze the late-phase responses of LPS-induced rat aortic ECs by using systematic biology approaches, including rat cDNA microarray, 2-DE and MALDI-TOF MS/MS, and cytokine protein array. Furthermore, to improve the efficiency of analysis of the bulk systematic data of rat, we designed a set of bioinformatic tools to convert and integrate these rat data into the corresponding human genes or proteins IDs based on BioCarta, KEGG, and Gene Ontology databases. Using the systematic analysis, it was shown that LPS could promote some signaling or metabolic pathways as well as pathophysiologic phenomena of proliferation, atherogenesis, inflammation, and apoptosis through activated nuclear factor-kappaB pathway in ECs. Interestingly, ECs also activated the mediators of anti-inflammation, antiapoptosis, and antioxidation to protect themselves. Moreover, the expressions of altered genes, proteins, and their involvement in the hypothetical signaling pathway can provide further understanding of inflammation associated responses in ECs.     

Caution regarding the combined effects of extracellular matrices and nutrient media on cultured endothelial cells

To study the influence of smooth muscle cells (SMC) on endothelial cells (EC), different co-culture designs are available, including EC seeding on SMC extracellular matrix (ECM). We explored human umbilical vein endothelial cell (HUVEC) adhesion and proliferation on either in situ or coated ECM, elaborated by HUVECs or human arterial smooth muscle cells (HUASMCs), in the presence of different nutrient media containing varying amounts of fetal calf serum. Coating wells with HUVEC or HUASMC ECMs did not improve HUVEC adhesion 1 h after cell seeding, compared with uncoated wells. HUVEC adhesion on in situ HUVEC-ECM and HUASMC-ECM was significantly increased compared with uncoated wells. The substratum upon which cells are maintained was found to play a crucial role, in conjunction with the medium to which HUVECs are exposed for their proliferative response. These results stress the importance of selecting media in relation to the particular substratum, in order to avoid misinterpretation of data.     

The synthesis of subendothelial matrix by bovine aortic endothelial cells in culture

Bovine aortic endothelial cells cultured on collagenous or plastic substrata continuously synthesize and deposit a subendothelial matrix, independently of whether the cells are in the logarithmic or the stationary phase of growth. This subendothelial matrix contains fibrillar and amorphous elements comparable with those observed in the subendothelium in vivo. Deposition of subendothelial matrix on a collagen gel substratum both started earlier and progressed at approximately double the rate than that on denatured collagen. The relative composition of the subendothelial matrix was assessed by sequential incubation with trypsin, elastase and collagenase (Jones et al., 1979). The subendothelial matrix deposited on collagen gels by early confluent cultures and late post-confluent cultures differed in their enzyme sensitivity. These age-related changes in the enzyme sensitivity of the subendothelial matrix were characteristic for each cloned cell population examined. Comparable variations in the composition of the subendothelial matrix were not observed when the cells were cultured on plastic or gelatin-coated dishes; the subendothelial matrix deposited on these two substrata contained considerably more trypsin-sensitive material and less elastase and collagenase-sensitive material than the matrix deposited on native collagen gels. Age-related changes in the enzyme sensitivity of the subendothelial matrix deposited on collagen gels was found to be a function of the time elapsed since confluence and it was not related to the time elapsed since plating or to the number of cells present.     

Enhanced assay of endothelial exocytosis using extracellular matrix components

Vascular inflammation plays a key role in the pathogenesis of atherosclerosis. The first step in vascular inflammation is endothelial exocytosis, in which endothelial granules fuse with the plasma membrane, releasing prothrombotic and proinflammatory messenger molecules. The development of cell culture models to study endothelial exocytosis has been challenging because the factors that modulate exocytosis in vitro are not well understood. Here we report a method for studying endothelial exocytosis that optimizes extracellular matrix components, cell density, and duration of culture. Human umbilical vein endothelial cells plated on collagen I-coated plates and cultured in the confluent state for 7–12 days in low-serum medium showed robust secretion of von Willebrand factor when stimulated with various agonists. This exocytosis assay is rapid and applicable to high-throughput screening.     

Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells

Extracellular Ca(2+) concentration ([Ca(2+)](o)) regulates the functions of many cell types through a G protein-coupled [Ca(2+)](o)-sensing receptor (CaR). Whether the receptor is functionally expressed in vascular endothelial cells is largely unknown. In cultured human aortic endothelial cells (HAEC), RT-PCR yielded the expected 555-bp product corresponding to the CaR, and CaR protein was demonstrated by fluorescence immunostaining and Western blot. RT-PCR also demonstrated the expression in HAEC of alternatively spliced variants of the CaR lacking exon 5. Although stimulation of fura 2-loaded HAEC by several CaR agonists (high [Ca(2+)](o), neomycin, and gadolinium) failed to increase intracellular Ca(2+) concentration ([Ca(2+)](i)), the CaR agonist spermine stimulated an increase in [Ca(2+)](i) that was diminished in buffer without Ca(2+) and was abolished after depletion of an intracellular Ca(2+) pool with thapsigargin or after blocking IP(3)- and ryanodine receptor-mediated Ca(2+) release with xestospongin C and with high concentration ryanodine, respectively. Spermine stimulated an increase in DAF-FM fluorescence in HAEC, consistent with NO production. Both the increase in [Ca(2+)](i) and in NO production were reduced or absent in HAEC transfected with siRNA specifically targeted to the CaR. HAEC express a functional CaR that responds to the endogenous polyamine spermine with an increase in [Ca(2+)](i), primarily due to release of IP(3)- and ryanodine-sensitive intracellular Ca(2+) stores, leading to the production of NO. Expression of alternatively spliced variants of the CaR may result in the absence of a functional response to other known CaR agonists in HAEC.     

Atorvastatin induces tissue transglutaminase in human endothelial cells

Tissue transglutaminase (tTgase) contributes to the organisation of the basement membrane and is therefore thought to be important for the integrity and stability of the vessel wall. In the present study, we hypothesised that the HMG-CoA reductase inhibitor atorvastatin may up-regulate the tTgase expression in endothelial cells and thereby exert beneficial effects on endothelial function. Treatment of human umbilical vein endothelial cells (HUVEC) with atorvastatin (1-10 microM) caused a clear increased expression of tTgase in both permeabilised and non-permeabilised HUVEC. In contrast, stimulation of HUVEC with TNFalpha had no substantial effect on tTgase expression or localisation but inhibited the atorvastatin-induced up-regulation and externalisation of tTgase. Propidium iodide staining revealed that statin-induced apoptosis is not responsible for the enhanced expression. By inducing the expression of tTgase, statins may promote tTgase-mediated stabilisation of the basement membrane. This effect of atorvastatin may contribute to the beneficial role of statins on endothelial function.     

Regulation of endothelin-1 release from human endothelial cells by sex steroids and angiotensin-II

It is well documented that there are gender differences in the incidence and patterns of cardiovascular disease; males have a higher incidence of cardiovascular disease than premenopausal women. We have therefore investigated whether the sex hormones, estradiol and testosterone, could directly influence the secretion of vascular peptides from human aortic endothelial cells (HAEC). Previously we have shown that testosterone can increase the number of HAECs that secrete adrenomedullin. In this study we investigated sex hormone regulation of endothelin-1 in HAEC. Several studies have observed a reduction in endothelin-1 secretion from endothelial cells in the presence of estradiol, the effect being more marked for stimulated cells. Studies on the actions of testosterone are much fewer and inconclusive. In this study we observed that estradiol did not change the number of cells secreting endothelin-1 during 4 h incubation under basal conditions but decreased the number of secreting cells stimulated with angiotensin-II. Testosterone induced an increase in the number of cells secreting endothelin-1 (p = 0.03). Complementary incubations revealed that testosterone up-regulated endothelin-1 mRNA at 1–3 h (p < 0.05). These results, together with our previous observations, indicate that angiotensin-II, testosterone and estradiol have parallel effects on the production of endothelin-1 as on adrenomedullin in HAEC. We conclude that there is potential for coordinated modulation by sex steroids and angiotensin-II of vasoactive peptide production in human endothelial cells.     

VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

Actin dynamics at sites of extracellular matrix degradation

The degradation of extracellular matrix (ECM) by proteases is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Here we present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia highlights the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover. The results offer new insight into the tight coordination between signalling, actin remodelling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. In conclusion, invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that also represent a valuable experimental paradigm.     

Lysophosphatidylglycerol stimulates chemotactic migration and tube formation in human umbilical vein endothelial cells

In this study, we observed that lysophosphatidylglycerol (LPG) stimulates extracellular signal-regulated kinase (ERK) in human umbilical vein endothelial cells (HUVECs). LPG-stimulated ERK activity was not inhibited by pertussis toxin (PTX), indicating PTX-sensitive G-proteins-independent manner. In terms of functional aspect, LPG induced chemotactic migration of HUVECs in a PTX-insensitive manner. Preincubation of HUVECs with an ERK inhibitor (PD98059) completely inhibited LPG-induced chemotactic migration, suggesting the crucial role of ERK in the process. LPG-induced ERK activation and chemotactic migration in HUVECs were not affected by an lysophosphatidic acid receptor-selective antagonist (Ki16425), indicating lysophosphatidic acid receptors-independency. We also found that LPG stimulated tube formation in HUVECs. Taken together we suggest that LPG stimulates HUVECs and result in chemotactic migration and tube formation, suggesting a new aspect of LPG as a modulator of endothelial cell functioning.     

Tyrosine phosphatase epsilonM stimulates migration and survival of porcine aortic endothelial cells by activating c-Src

The cell growth, survival, and migration of vascular endothelial cells (ECs) are positively regulated by several protein tyrosine kinase receptors. Therefore, protein tyrosine phosphatases (PTPs) must also be important for these processes. The present study found that transmembranal PTPepsilonM, but not cytoplasmic PTPepsilonC, is expressed in porcine ECs and in rat smooth muscle cells, both of which were prepared from the aorta. The overexpression of wild-type PTPepsilonM promoted cell survival and migration in porcine aortic ECs even in medium without and with 1% serum, respectively. A catalytically inactive, substrate-trapping mutant of PTPepsilonM, respectively, did not affect and conversely suppressed cell survival and migration. Interestingly, the forced expression of wild-type PTPepsilonC reduced cell viability in contrast to PTPepsilonM in ECs lacking endogenous PTPepsilonC, indicating the biological significance of selective expression of PTPepsilon isoforms in the vasculature. PTPepsilonM activated c-Src kinase probably by directly dephosphorylating phospho-Tyr527, a negative regulatory site of c-Src. The increases in cell survival and migration induced by overexpressed PTPepsilonM were suppressed by the c-Src inhibitor SU6656. Considering the behaviors of vascular ECs in the pathogenesis of atherosclerosis, these data suggest that PTPepsilonM negatively regulates the development of this disease by activating c-Src.     

Sodium fluoride induces podosome formation in endothelial cells

Background information. Fluoride is a well‐known G‐protein activator. Exposure of cultured cells to its derivatives results in actin cytoskeleton remodelling. Podosomes are actin‐based structures endowed with adhesion and matrix‐degradation functions. This study investigates actin cytoskeleton reorganization induced by fluoride in endothelial cells. Results. Treatment of cultured endothelial cells with sodium fluoride (NaF) results in a rapid and potent stimulation of podosome formation. Furthermore, we show that Cdc42 (cell‐division cycle 42), Rac1 and RhoA activities are stimulated in NaF‐treated cells. However, podosome assembly is dependent on Cdc42 and Rac1, but not RhoA. Although the sole activation of Cdc42 is sufficient to induce individual podosomes, a balance between RhoGTPase activities regulates podosome formation in response to NaF, which in this case are often found in groups or rosettes. As in other models, podosome formation in endothelial cells exposed to NaF also involves Src. Finally, we demonstrate that NaF‐induced podosomes are fully competent for matrix protein degradation. Conclusions. Taken together, our findings establish NaF as a novel inducer of podosomes in endothelial cells in vitro.     

Subconfluent endothelial cells form podosomes downstream of cytokine and RhoGTPase signaling

Adhesion, migration and invasion of endothelial cells are prerequisites for the formation of blood vessels and have to be controlled on a subcellular level. We report that subconfluent human umbilical vein endothelial cells (HUVEC) are able to constitutively form podosomal adhesions that are sites of matrix metalloprotease concentration and matrix degradation. Importantly, incubation of serum-starved cells with VEGF or TNFalpha revealed the dependence of podosomes on cytokine signaling. Podosome formation was also stimulated by addition of monocytes to HUVEC. Microinjection/application of specific inhibitors or active/inactive mutants showed that regulatory pathways include Src kinase and RhoGTPase signaling, N-WASP activation and Arp2/3 complex-dependent actin nucleation. In sum, our data show that HUVEC displaying a migratory phenotype constitutively form f-actin-rich adhesions with podosomal characteristics downstream of cytokine signaling. We propose that HUVEC podosomes play an important role in endothelial cell migration and invasion.