First trimester trophoblasts forming endothelial-like tubes in vitro emulate a ‘blood vessel development’ gene expression profile

Extravillous cytotrophoblasts isolated from first trimester placenta, and immortalised cell lines derived from them, have the intrinsic ability to form endothelial-like tubes when cultured on Matrigel™ extracellular matrix. This in vitro tube formation may model placental angiogenesis and/or endovascular differentiation by trophoblasts. To interpret the relevance of this phenomenon to placental development, we used a gene expression microarray approach to identify which genes and pathways are associated with the tube-forming phenotype of HTR8/SVneo first trimester trophoblasts (HTR8-M), compared with HTR8/SVneo not forming tubes on plastic culture surface (HTR8-P). Furthermore, we used weighted gene co-expression network analysis (WGCNA) of microarray data to identify modules of co-expressed genes underlying the biological processes. There were 481 genes differentially expressed between HTR8-M and HTR8-P and these were significantly enriched for blood vessel development and related gene ontologies. WGCNA clustered the genes into 9 co-expression modules. One module was significantly associated with HTR8-M (p = 1.15E-05) and contained genes involved in actin cytoskeleton organization, cell migration and blood vessel development, consistent with tube formation on Matrigel. Another module was significantly associated with HTR8-P (p = 1.94E-05) and was enriched for genes involved in mitosis, consistent with proliferation by cells on plastic which do not differentiate. Up-regulation of angiogenesis and vascular development pathways in endovascular trophoblasts in vivo could underpin spiral artery remodelling processes, which are defective in preeclamptic pregnancies.     

Ischemic Preconditioning Increases Endothelial Progenitor Cell Number to Attenuate Partial Nephrectomy-Induced Ischemia/Reperfusion Injury

Objectives

The objective of this study was to investigate the role of endothelial progenitor cells (EPCs) in the modulation of ischemia-reperfusion injury (IRI) in a partial nephrectomy (PN) rat model using early-phase ischemic preconditioning (IPC).

Materials and Methods

Ninety male Sprague-Dawley rats were randomly divided into three groups following right-side nephrectomy: Sham-operated rats (surgery without vascular clamping); PN rats (renal blood vessels were clamped for 40 min and PN was performed); and IPC rats (pretreated with 15 min ischemia and 10 min reperfusion). At 1, 3, 6, 12, 24 h, and 3 days after reperfusion, the pool of circulating EPCs and kidneys were harvested. The extent of renal injury was assessed, along with EPC number, cell proliferation, angiogenesis, and vascular growth factor expression.

Results

Pretreated rats exhibited significant improvements in renal function and morphology. EPC numbers in the kidneys were increased at 12 h following reperfusion in the IPC group as compared to the PN or Sham groups. Cell proliferation (including endothelial and tubular epithelial cells) and angiogenesis in peritubular capillaries were markedly increased in kidneys treated with IPC. In addition, vascular endothelial growth factor-A (VEGF-A) and stromal cell-derived factor-1α (SDF-1α) expression in the kidneys of pretreated rats was increased compared to rats subjected to PN.

Conclusions

Our investigation suggested that: (1) the early phase of IPC may attenuate renal IRI induced by PN; (2) EPCs play an important role in renal protection, involving promotion of cell proliferation and angiogenesis through release of several angiogenic factors.     

Ischemic preconditioning in the animal kidney, a systematic review and meta-analysis

Ischemic preconditioning (IPC) is a potent renoprotective strategy which has not yet been translated successfully into clinical practice, in spite of promising results in animal studies. We performed a unique systematic review and meta-analysis of animal studies to identify factors modifying IPC efficacy in renal ischemia/reperfusion injury (IRI), in order to enhance the design of future (clinical) studies. An electronic literature search for animal studies on IPC in renal IRI yielded fifty-eight studies which met our inclusion criteria. We extracted data for serum creatinine, blood urea nitrogen and histological renal damage, as well as study quality indicators. Meta-analysis showed that IPC reduces serum creatinine (SMD 1.54 [95%CI 1.16, 1.93]), blood urea nitrogen (SMD 1.42 [95% CI 0.97, 1.87]) and histological renal damage (SMD 1.12 [95% CI 0.89, 1.35]) after IRI as compared to controls. Factors influencing IPC efficacy were the window of protection (<24 h = early vs. ≥ 24 h = late) and animal species (rat vs. mouse). No difference in efficacy between local and remote IPC was observed. In conclusion, our findings show that IPC effectively reduces renal damage after IRI, with higher efficacy in the late window of protection. However, there is a large gap in study data concerning the optimal window of protection, and IPC efficacy may differ per animal species. Moreover, current clinical trials on RIPC may not be optimally designed, and our findings identify a need for further standardization of animal experiments.     

Endostatin associates with lipid rafts and induces reorganization of the actin cytoskeleton via down-regulation of RhoA activity

Endostatin, the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Observations that endostatin inhibits endothelial cell migration and induces disassembly of the actin cytoskeleton provide putative cellular mechanisms for this effect. To understand the mechanisms of endostatin-induced intracellular signaling, we analyzed the association of recombinant endostatin with endothelial cell lipid rafts and the roles of its heparin- and integrin-binding properties in this interaction. We observed that a fraction of cell surface-bound endostatin partitioned in low density membrane raft fractions together with caveolin-1. Heparinase treatment of cells prevented the recruitment of endostatin to the lipid rafts but did not affect the association of endostatin with the non-raft fraction, whereas preincubation of endostatin with soluble alpha5beta1 integrin prevented the association of endostatin with the endothelial cell membrane. Endostatin treatment induced recruitment of alpha5beta1 integrin into the raft fraction via a heparan sulfate proteoglycan-dependent mechanism. Subsequently, through alpha5beta1 integrin, heparan sulfate, and lipid raft-mediated interactions, endostatin induced Src-dependent activation of p190RhoGAP with concomitant decrease in RhoA activity and disassembly of actin stress fibers and focal adhesions. These observations provide a cell biological mechanism, which plausibly explains the anti-angiogenic mechanisms of endostatin in vivo.     

Relationship between organization of the actin cytoskeleton and the cell cycle in normal and adenovirus-infected rat cells.

Flow cytometry and staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin were used to investigate organization of the actin cytoskeleton in rat embryo cells at different stages of normal and adenovirus E1A-induced cell cycles. In uninfected cells in G0-G1 and S phases, actin was predominantly in the form of stress fibers. In G2, this organization changed to peripheral rings of thin filaments, while during mitosis, actin had a diffuse distribution. Infection of quiescent rat cells by adenovirus caused them to enter the cell cycle and replicate DNA and also caused disruption of stress fibers. Rapid disappearance of stress fibers and the appearance of peripheral rings of actin filaments began from 13 h after infection and closely followed synthesis of the E1A proteins. Infected cells began S phase at about 24 h after infection, and cells in G2 and mitosis were seen from 30 to 50 h. Thus, disruption of the actin cytoskeleton is an early effect of E1A and not an indirect consequence of the entry of infected cells into the cell cycle.     

The Actin Cytoskeleton and Signaling Network during Pollen Tube Tip Growth

The organization and dynamics of the actin cytoskeleton play key roles in many aspects of plant cell development. The actin cytoskeleton responds to internal developmental cues and en-vironmental signals and is involved in cell division, subcellular organelle movement, cell polarity and polar cell growth. The tip-growing pollen tubes provide an ideal model system to investigate fundamental mechanisms of underlying polarized cell growth. In this system, most signaling cascades required for tip growth, such as Ca~(2+)-, small GTPases- and lipid-mediated signaling have been found to be involved in transmitting signals to a large group of actin-binding proteins. These actin-binding proteins subsequently regulate the structure of the actin network, as well as the rapid turnover of actin filaments (F-actin), thereby eventually controlling tip growth. The actin cytoskeleton acts as an integrator in which multiple signaling pathways converge, providing a general growth and regulatory mechanism that applies not only for tip growth but also for polarized diffuse growth in plants.     

Rho小G蛋白相关激酶的结构与功能

Rho小G蛋白家族是Ras超家族成员之一,人类Rho小G蛋白包括20个成员,研究最清楚的有RhoA、Rac1和Cdc42。Rho小G蛋白参与了诸如细胞骨架调节、细胞移动、细胞增殖、细胞周期调控等重要的生物学过程。在这些生物学过程的调节中,Rho小G蛋白的下游效应蛋白质如蛋白激酶（p21-activated kinase,PAK）、ROCK（Rho-kinase）、PKN（protein kinase novel）和MRCK（myotonin-related Cdc42-binding kinase）发挥了不可或缺的作用。迄今研究发现,PAK可调节细胞骨架动力学和细胞运动,另外,PAK通过MAPK（mitogen-activated protein kinases）参与转录、细胞凋亡和幸存通路及细胞周期进程;ROCK与肌动蛋白应力纤维介导黏附复合物的形成及与细胞周期进程的调节有关;哺乳动物的PKN与RhoA/B/C相互作用介导细胞骨架调节;MRCK与细胞骨架重排、细胞核转动、微管组织中心再定位、细胞移动和癌细胞侵袭等有关。该文简要介绍Rho小G蛋白下游激酶PAK、ROCK、PKN和MRCK的结构及其在细胞骨架调节中的功能,重点总结它们在真核细胞周期调控中的作用,尤其是在癌细胞周期进程中所发挥的作用,为寻找癌症治疗的新靶点提供理论依据。     

Novel proteins linking the actin cytoskeleton to the endocytic machinery in Saccharomyces cerevisiae

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.     

Bioinformatics analysis of the biological changes involved in the osteogenic differentiation of human mesenchymal stem cells

The mechanisms underlying the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain unclear. In the present study, we aimed to identify the key biological processes during osteogenic differentiation. To this end, we downloaded three microarray data sets from the Gene Expression Omnibus (GEO) database: GSE12266, GSE18043 and GSE37558. Differentially expressed genes (DEGs) were screened using the limma package, and enrichment analysis was performed. Protein‐protein interaction network (PPI) analysis and visualization analysis were performed with STRING and Cytoscape. A total of 240 DEGs were identified, including 147 up‐regulated genes and 93 down‐regulated genes. Functional enrichment and pathways of the present DEGs include extracellular matrix organization, ossification, cell division, spindle and microtubule. Functional enrichment analysis of 10 hub genes showed that these genes are mainly enriched in microtubule‐related biological changes, that is sister chromatid segregation, microtubule cytoskeleton organization involved in mitosis, and spindle microtubule. Moreover, immunofluorescence and Western blotting revealed dramatic quantitative and morphological changes in the microtubules during the osteogenic differentiation of human adipose‐derived stem cells. In summary, the present results provide novel insights into the microtubule‐ and cytoskeleton‐related biological process changes, identifying candidates for the further study of osteogenic differentiation of the mesenchymal stem cells.     

Rosiglitazone inhibits endothelial proliferation and angiogenesis

Rosiglitazone, an insulin sensitizer, is known to offer beneficial effects in retarding atherosclerotic vascular diseases. Since proliferation and angiogenesis are involved in initiation and plaque instability, two critical steps in the cardiovascular events, this study was designed to evaluate the mechanisms of rosiglitazone on endothelial proliferation and angiogenesis. Rosiglitazone-treated human umbilical vein endothelial cells were analyzed for growth rate by use of cell number counting, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay as well as 3H-thymidine incorporation. Cell cycle analysis was detected by flow cytometry and cell cycle-related proteins were measured by Western blot. Effects of rosiglitazone on angiogenesis were assessed by vascular endothelial growth factor (VEGF)-induced tube formation and wound-healing migration. Furthermore, effects of rosiglitazone on actin stress fiber were observed under confocal microscopy. Our data showed that rosiglitazone inhibits endothelial proliferation in a dose-dependent manner. Rosiglitazone caused endothelial arrest at G1 phase via affecting several cell cycle-related proteins that led to attenuate phosphorylation of retinoblastoma protein. Rosiglitazone markedly decreased VEGF-induced tube formation and endothelial cell migration, which might be explained by a disorganization of the actin cytoskeleton. Our data suggest that both anti-proliferative and anti-angiogenic activities in endothelial cells might account for the greater than expected beneficial effects of rosiglitazone for the treatment and prevention of atherosclerosis.     

Reorganization of microtubule system in pulmonary endothelial cells in response to thrombin treatment

Thrombin induces rapid and reversible increase of endothelial (EC) barrier permeability associated with actin cytoskeleton remodeling and contraction. The role of microtubules (Mts) in EC barrier regulation compared with actin systems is poorly understood. In this work we studied pathways of Mt and actin regulation in response to thrombin treatment in cultured EC, and the involvement of trimeric G-proteins and in this process. Cells were treated with thrombin, and further analysed using immunofluorescent staining of actin and Mts, digital microscopy and morphometric analysis. In normal cells actin network consists of thin bundles basically located in the cell periphery, Mt density decreases from the cell center to the cell edge. Thrombin (25 nM) induced endothelial dysfunction associated with a rapid (within 5 min) decrease of peripheral Mt network and a slower actin stress fiber formation in the cytoplasm. Pretreatment with Pertussis toxin, which is Gi protein inhibitor, attenuated thrombin-induced stress fiber formation and Mt disassembly. Overexpression of activated G12, G13, Gi and Gq proteins, which are involved in thrombin receptor-mediated signaling, resulted in increasing stress fibers thickness and density and complete Mt disassembly. From the results obtained we suggest that thrombin regulates actin cytoskeleton of EC using local Mt depolymerization at the cell edge.     

Renoprotective Mechanism of Remote Ischemic Preconditioning Based on Transcriptomic Analysis in a Porcine Renal Ischemia Reperfusion Injury Model

Ischemic preconditioning (IPC) is a well-known phenomenon in which tissues are exposed to a brief period of ischemia prior to a longer ischemic event. This technique produces tissue tolerance to ischemia reperfusion injury (IRI). Currently, IPC’s mechanism of action is poorly understood. Using a porcine single kidney model, we performed remote IPC with renal IRI and evaluated the IPC mechanism of action. Following left nephrectomy, 15 female Yorkshire pigs were divided into three groups: no IPC and 90 minutes of warm ischemia (control), remote IPC immediately followed by 90 minutes of warm ischemia (rIPCe), and remote IPC with 90 minutes of warm ischemia performed 24 hours later (rIPCl). Differential gene expression analysis was performed using a porcine-specific microarray. The microarray analysis of porcine renal tissues identified 1,053 differentially expressed probes in preconditioned pigs. Among these, 179 genes had altered expression in both the rIPCe and rIPCl groups. The genes were largely related to oxidation reduction, apoptosis, and inflammatory response. In the rIPCl group, an additional 848 genes had altered expression levels. These genes were primarily related to immune response and inflammation, including those coding for cytokines and cytokine receptors and those that play roles in the complement system and coagulation cascade. In the complement system, the membrane attack complex was determined to be sublytic, because it colocalized with phosphorylated extracellular signal-regulated kinase. Furthermore, alpha 2 macroglobulin, tissue plasminogen activator, uterine plasmin trypsin inhibitor, and arginase-1 mRNA levels were elevated in the rIPCl group. These findings indicate that remote IPC produces renoprotective effects through multiple mechanisms, and these effects develop over a long timeframe rather than immediately following IPC.     

Actin cytoskeleton dynamics and the cell division cycle

The network of actin filaments is one of the crucial cytoskeletal structures contributing to the morphological framework of a cell and which participates in the dynamic regulation of cellular functions. In adherent cell types, cells adhere to the substratum during interphase and spread to assume their characteristic shape supported by the actin cytoskeleton. This actin cytoskeleton is reorganized during mitosis to form rounded cells with increased cortical rigidity. The actin cytoskeleton is re-established after mitosis, allowing cells to regain their extended shape and attachment to the substratum. The modulation of such drastic changes in cell shape in coordination with cell cycle progression suggests a tight regulatory interaction between cytoskeleton signalling, cell–cell/cell–matrix adhesions and mitotic events. Here, we review the contribution of the actin cytoskeleton to cell cycle progression with an emphasis on the effectors responsible for the regulation of the actin cytoskeleton and integration of their activities with the cell cycle machinery.     

Brefeldin A (BFA) disrupts the organization of the microtubule and the actin cytoskeletons

Previous inquiries into the effects of Brefeldin A (BFA) have largely concentrated on dynamics of ER-Golgi membrane traffic, predominantly after relatively short treatments with the drug. We have now analyzed the effects of long BFA treatment on overall cell morphology, behavior of resident and cycling Golgi proteins, and microtubular and actin cytoskeletons organization. Prolonged (15 h or 40 h) treatment of normal rat kidney (NRK) cells with BFA caused dramatic swelling of the Endoplasmic Reticulum (ER) and shifted its localization to the periphery of the cells. The Golgi complex was disassembled and Golgi proteins redistributed and persisted in partially distinct compartments. Prolonged BFA treatment resulted in marked disruption of the MT and actin cytoskeleton. Peripheral MT were absent and tubulin staining was concentrated in short astral MT emanating from the microtubule organizing center (MTOC). Actin stress fibers were largely absent and actin staining was concentrated within a perinuclear area. Within this region, actin localization overlapped that of the membrane transport factor p115. BFA effects on Golgi structure and on MT and actin organization showed the same threshold -- all could be partially reversed after 30 min and 15 h BFA treatment but were irreversible after 40h incubation with the drug. The observed effects were not induced by signaling pathways involved in apoptotic phenomena or in ER stress response pathways. These results suggest that BFA inhibits the activity of key molecules that regulate MT and actin cytoskeleton dynamics. The findings can be used as the basis for elucidating the molecular mechanism of BFA action on the cytoskeleton.     

Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton

Inhibition of angiogenesis could be a treatment strategy for diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy. PP2 is a pharmacological inhibitor of Src family kinases and was found to inhibit FGF-2 induced angiogenesis in vivo. Experiments in vitro showed that PP2 inhibited invasive growth and sprouting of both endothelial and vascular smooth muscle cells into a fibrin matrix. PP2 inhibited the formation of lamellopodia and expression of kinase inactive c-Src reduced phosphorylation of cortactin and paxillin, suggesting a model in which Src kinases are involved in organization of the actin cytoskeleton. Consequently, endothelial cells expressing kinase inactive c-Src failed to spread and form cord-like structures on a collagen matrix. These data suggest that pharmacological inactivation of Src family kinases inhibits FGF-2 stimulated angiogenesis by interference with organization of the actin cytoskeleton in both endothelial and vascular smooth muscle cells, which affects cell migration.