A three-dimensional model of myxobacterial fruiting-body formation

Myxobacterial cells are social; they swarm by gliding on surfaces as they feed cooperatively. When they sense starvation, tens of thousands of cells change their movement pattern from outward spreading to inward concentration and form aggregates that become fruiting bodies. Cells inside fruiting bodies differentiate into round, nonmotile, environmentally resistant spores. Traditionally, cell aggregation has been considered to imply chemotaxis, a long-range cell interaction that shares many features of chemical reaction-diffusion dynamics. The biological evidence, however, suggests that Myxococcus xanthus aggregation is the consequence of direct cell-contact interactions that are different from chemotaxis. To test whether local interactions suffice to explain the formation of fruiting bodies and the differentiation of spores within them, we have simulated the process. In this article, we present a unified 3D model that reproduces in one continuous simulation all the stages of fruiting-body formation that have been experimentally observed: nonsymmetric initial aggregates (traffic jams), streams, formation of toroidal aggregates, hemispherical 3D mounds, and finally sporulation within the fruiting body.     

A novel three-dimensional computer-assisted method for a quantitative study of microvascular networks of the human cerebral cortex

OBJECTIVE: Detailed information on microvascular network anatomy is a requirement for understanding several aspects of microcirculation, including oxygen transport, distributions of pressure, and wall shear stress in microvessels, regulation of blood flow, and interpretation of hemodynamically based functional imaging methods, but very few quantitative data on the human brain microcirculation are available. The main objective of this study is to propose a new method to analyze this microcirculation. METHODS: From thick sections of india ink-injected human brain, using confocal laser microscopy, the authors developed algorithms adapted to very large data sets to automatically extract and analyze center lines together with diameters of thousands of brain microvessels within a large cortex area. RESULTS: Direct comparison between the original data and the processed vascular skeletons demonstrated the high reliability of this method and its capability to manage a large amount of data, from which morphometry and topology of the cerebral microcirculation could be derived. CONCLUSIONS: Among the many parameters that can be analyzed by this method, the capillary size, the frequency distributions of diameters and lengths, the fractal nature of these networks, and the depth-related density of vessels are all vital features for an adequate model of cerebral microcirculation.     

Suppression of the proliferation and migration of oncogenicras-dependent cell lines,cultured in a three-dimensional collagen matrix,by flavonoid-structured molecules

The effects of 7-hydroxycoumarin, genistein and quercetin on tworas-oncogene-driven tumour cells (rat breast adenocarcinoma and human bladder carcinoma) were investigated using cellular (proliferation and migration) and molecular targets (p21 ^ras GTPase activity and intracellular amount of p21 ^ras protein). All three compounds inhibited the growth of both cell lines. Genistein was the most effective substance. Further-more, 7-hydroxycoumarin and genistein affected the motile machinery of both cell lines because major fractions of the cells were slowed down or stopped locomotion. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), a well-known tumour promoter, increased the locomotion behaviour of the cells; the time of migration, the velocity and the distance of migration increased under the control of PMA. 7-Hydroxycoumarin decreased the relative amount of intracellular p21 ^ras , and concomitantly a PMA-induced decrease of p21 ^ras , GTPase activity could be partially antagonized by 7-hydroxycoumarin. Because of the low toxicity and the mode of action evaluated, it is likely that the best role for these substances may be adjuvant therapy of some malignancies following surgery. Profiles directed to migration and proliferation inhibition make these drugs exceptional candidates for chemopreventive strategies in tumours diagnosed as having increasedras oncogene levels.Abbreviations PMA phorbol 12-myristate 13-acetate - PMS phenazine methosulphate - RBA rat breast adenocarcinoma - XTT 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide inner salt, sodium salt     

TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration

Transmembrane-4-L-six-family-1 (TM4SF1) is a tetraspanin-like membrane protein that is highly and selectively expressed by cultured endothelial cells (EC) and, in vivo, by EC lining angiogenic tumor blood vessels. TM4SF1 is necessary for the formation of unusually long (up to a 50 μm), thin (~100–300 nm wide), F-actin-poor EC cell projections that we term ‘nanopodia’. Immunostaining of nanopodia at both the light and electron microsopic levels localized TM4SF1 in a regularly spaced, banded pattern, forming TM4FS1-enriched domains. Live cell imaging of GFP-transduced HUVEC demonstrated that EC project nanopodia as they migrate and interact with neighboring cells. When TM4SF1 mRNA levels in EC were increased from the normal ~90 mRNA copies/cell to ~400 copies/cell through adenoviral transduction, EC projected more and longer nanopodia from the entire cell circumference but were unable to polarize or migrate effectively. When fibroblasts, which normally express TM4SF1 at ~5 copies/cell, were transduced to express TM4SF1 at EC-like levels, they formed typical TM4SF1-banded nanopodia, and broadened, EC-like lamellipodia. Mass-spectrometry demonstrated that TM4SF1 interacted with myosin-10 and β-actin, proteins involved in filopodia formation and cell migration. In summary, TM4SF1, like genuine tetraspanins, serves as a molecular organizer that interacts with membrane and cytoskeleton-associated proteins and uniquely initiates the formation of nanopodia and facilitates cell polarization and migration.     

Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials

Co-cultures of endothelial cells (EC) and mesenchymal stem cells (MSC) in three-dimensional (3D) protein hydrogels can be used to recapitulate aspects of vasculogenesis in vitro. MSC provide paracrine signals that stimulate EC to form vessel-like structures, which mature as the MSC transition to the role of mural cells. In this study, vessel-like network formation was studied using 3D collagen/fibrin (COL/FIB) matrices seeded with embedded EC and MSC and cultured for 7 days. The EC:MSC ratio was varied from 5:1, 3:2, 1:1, 2:3 and 1:5. The matrix composition was varied at COL/FIB compositions of 100/0 (pure COL), 60/40, 50/50, 40/60 and 0/100 (pure FIB). Vasculogenesis was markedly decreased in the highest EC:MSC ratio, relative to the other cell ratios. Network formation increased with increasing fibrin content in composite materials, although the 40/60 COL/FIB and pure fibrin materials exhibited the same degree of vasculogenesis. EC and MSC were co-localized in vessel-like structures after 7 days and total cell number increased by approximately 70%. Mechanical property measurements showed an inverse correlation between matrix stiffness and network formation. The effect of matrix stiffness was further investigated using gels made with varying total protein content and by crosslinking the matrix using the dialdehyde glyoxal. This systematic series of studies demonstrates that matrix composition regulates vasculogenesis in 3D protein hydrogels, and further suggests that this effect may be caused by matrix mechanical properties. These findings have relevance to the study of neovessel formation and the development of strategies to promote vascularization in transplanted tissues.     

CXCR1 and CXCR2 silencing modulates CXCL8-dependent endothelial cell proliferation, migration and capillary-like structure formation

CXCR1 and CXCR2 are receptors for angiogenic ELR + CXC chemokines and are differentially expressed on endothelial cells; however, their functional significance in angiogenesis remains unclear. In this study, we determined the functional significance of these receptors in modulating endothelial cell phenotype by knocking-down the expression of CXCR1 and/or CXCR2 in human microvascular endothelial cells (HMEC-1) using short-hairpin RNA (shRNA). Cell proliferation, migration, invasion and capillary-like structure (CLS) formation were analyzed. Our data demonstrate that knock-down of CXCR1 and/or CXCR2 expression inhibited endothelial cell proliferation, survival, migration, invasion and CLS formation. Additionally, we examined the mechanism of CXCL8-dependent CXCR1 and/or CXCR2 mediated phenotypic changes by evaluating ERK phosphorylation and cytoskeletal rearrangement and observed inhibition of ERK phosphorylation and cytoskeletal rearrangement in HMEC-1-shCXCR1, HMEC-1-shCXCR2 and HMEC-1-shCXCR1/2 cells. Together, these data demonstrate that CXCR1 and CXCR2 expression plays a critical role in regulating multiple biological activities in human microvascular endothelial cells.     

Thromboxane A2 receptor signaling inhibits vascular endothelial growth factor-induced endothelial cell differentiation and migration

Vascular endothelial growth factor (VEGF) is an important patho-physiological mediator of angiogenesis. VEGF-induced endothelial cell (EC) migration and angiogenesis often occur in complicated environments containing multiple agents capable of modifying the response. Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of pathogenesis of many vascular diseases. Human EC express both TXA2 receptor (TP) isoforms; however, the effects of individual TP isoforms on VEGF-induced EC migration and angogenesis are unknown. We report here that the TXA2 mimetic [1S-(1alpha, 2beta(5Z), 3alpha(1E, 3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5'-heptenoic acid (IBOP) (100 nmol/L) is a potent antagonist (IC50 30 nmol/L) of VEGF-induced EC migration and differentiation. TPbeta, but not TPalpha, expression is required for the inhibition of VEGF-induced migration and angiogenesis. IBOP costimulation suppressed nitric oxide (NO) release from VEGF-treated EC through decreased activation of Akt, eNOS, and PDK1. TPbeta costimulation also ablated the increase in focal adhesion formation in response to VEGF. This mechanism was characterized by decreased recruitment of focal adhesion kinase (FAK) and vinculin to the alpha(v)beta3 integrin and reduced FAK and Src activation in response to VEGF. Addition of NO donors together with transfection of a constitutively active Src construct could circumvent the blockade of VEGF-induced migration by TP; however, neither intervention alone was sufficient. Thus, TP stimulation appears to limit angiogenesis, at least in part, by inhibiting the pro-angiogenic cytokine VEGF. These data further support a role for antagonism of TP activation in enhancing the angiogenic response in tissues exposed to elevated TXA2 levels in which revascularization is important.     

Roles of microtubule dynamics and small GTPase Rac in endothelial cell migration and lamellipodium formation under flow

Endothelial cell (EC) migration is required for vascular development and wound healing. We investigated the roles of microtubule (MT) dynamics and the small GTPase Rac in the fluid shear stress-induced protrusion of lamellipodia and enhancement of migration of bovine aortic ECs (BAECs). Shear stress increased lamellipodial protrusion and cell migration. Treating BAECs with paclitaxel (Taxol), an MT-stabilizing agent, inhibited lamellipodial protrusion and reduced migration speed in both the static and sheared groups. After Taxol washout, both lamellipodial protrusion and cell migration increased in the flow direction. Taxol treatment also decreased the shear-induced Rac activation. Transfection of BAECs with a dominant negative mutant of Rac1 inhibited lamellipodial protrusion and cell migration under static and shear conditions. Transfection with an activated mutant of Rac1 induced lamellipodia in all directions and attenuated the shear-induced migration, suggesting that an appropriate level of Rac activity and a polarized lamellipodial protrusion are important for cell migration under static and shear conditions. Our findings suggest that MT dynamics and optimum Rac activation are required for the polarized protrusion of lamellipodia that drives the directional EC migration under flow.     

Adenoviruses increase endothelial cell proliferation, migration, and tube formation: partial reversal by the focal adhesion kinase inhibitor, FRNK

During the course of examining the feasibility of using an adenoviral vector to deliver a potential anti-angiogenic agent to endothelial cells, we discovered that adenoviruses, themselves, have pro-angiogenic activities. Thus, an adenoviral vector containing a green fluorescent protein transgene (Ad-GFP) stimulated the growth, migration, tube formation, and phosphorylation of focal adhesion kinase (FAK) of human lung microvascular endothelial cells. However, adenovirus-mediated endothelial cell mitogenesis, tube formation, and FAK phosphorylation were completely reduced and migration was partially reversed by the addition of a Fak-Related Non-Kinase (FRNK) transgene to the vector. Because FRNK inhibits focal adhesion kinase (FAK) activity, this suggests that the adenoviral effects on endothelial cells are in part mediated through FAK. These data, as well as data obtained in other laboratories, suggest that adenoviruses should be used with caution in cancer gene therapy due to potential pro-angiogenic effects. However, some of these untoward effects may be modulated by concurrent use of a FAK inhibitor.     

Slits and Roundabouts in cancer,tumour angiogenesis and endothelial cell migration

Angiogenesis describes the development of new blood vessels from pre-existing vessels. The hijacking of this physiological process by tumours allows them to develop their own supplies of nutrients and oxygen, enabling their growth and metastasis. A large body of literature has accumulated over the last 20 years relating to angiogenesis, including signalling pathways involved in this process. One such pathway uses Slit–Roundabout proteins that are implicated in the development of cancers and tumour angiogenesis. The Roundabout family of receptors are large, single-pass transmembrane cell surface receptors involved in directing cell migration in response to their cognate Slit ligands. Although best known for their role in neuronal development, Slits and Roundabouts have now been implicated in myogenesis, leukocyte chemotaxis and tumour angiogenesis, confirming that the Robo signalling pathway functions across multiple cell types. We review here the evidence for a role for Slits and Roundabouts in cancer. In particular, we focus on the role of Robo1 and Robo4 in tumour angiogenesis and discuss the signalling pathways downstream of these proteins mediating endothelial cell migration.     

Identification of VLDLR as a novel endothelial cell receptor for fibrin that modulates fibrin-dependent transendothelial migration of leukocytes

While testing the effect of the (β15-66)(2) fragment, which mimics a pair of fibrin βN-domains, on the morphology of endothelial cells, we found that this fragment induces redistribution of vascular endothelial-cadherin in a process that is inhibited by the receptor-associated protein (RAP). Based on this finding, we hypothesized that fibrin may interact with members of RAP-dependent low-density lipoprotein (LDL) receptor family. To test this hypothesis, we examined the interaction of (β15-66)(2), fibrin, and several fibrin-derived fragments with 2 members of this family by ELISA and surface plasmon resonance. The experiments showed that very LDL (VLDL) receptor (VLDLR) interacts with high affinity with fibrin through its βN-domains, and this interaction is inhibited by RAP and (β15-66)(2). Furthermore, RAP inhibited transendothelial migration of neutrophils induced by fibrin-derived NDSK-II fragment containing βN-domains, suggesting the involvement of VLDLR in fibrin-dependent leukocyte transmigration. Our experiments with VLDLR-deficient mice confirmed this suggestion by showing that, in contrast to wild-type mice, fibrin-dependent leukocyte transmigration does not occur in such mice. Altogether, the present study identified VLDLR as a novel endothelial cell receptor for fibrin that promotes fibrin-dependent leukocyte transmigration and thereby inflammation. Establishing the molecular mechanism underlying this interaction may result in the development of novel inhibitors of fibrin-dependent inflammation.     

A technique for quantitative three-dimensional analysis of microvascular structure

Research into angiogenesis and vascular microarchitecture has contributed to progress in a wide variety of biomedical fields, but increased understanding is limited, in part, by the available assays and imaging modalities. Techniques that allow quantitation of vascular microarchitecture are needed. A comprehensive method is presented that uses 6-microm-thick serial sections of frozen tissue samples, immunostaining for CD31, brightfield microscopy, automated alignment of two-dimensional serial sections, and volume rendering to produce high-resolution, three-dimensional, quantifiable images of microvascular structure. Application of the technique is shown by characterizing vascularization into a fibrin gel implanted against the skeletal muscle of rats and explanted after 7 days. Comparing measurements from automated and manually aligned MRI and fibrin samples verified quantitation. Automation removes concerns of observer bias or variation inherent in manual alignment and increases the speed of analysis. Analysis of the fibrin gel reveals a dense (4.3 +/- 1.1% endothelial cell density) network of tortuous (1.37 +/- 0.05 tortuosity) capillaries that replaces the gel as it degrades. There is a high level of void space (22.8 +/- 3.6%) in the gel, and average capillary length in the fibrovascular tissue was 93.0 +/- 7.4 microm. Data obtained from these automatically aligned images agreed with those obtained using manual analysis (no statistical difference), and the results are consistent with data from traditional methods.     

Collagen triple helix repeat containing 1, a novel secreted protein in injured and diseased arteries, inhibits collagen expression and promotes cell migration

Collagen triple helix repeat containing 1 (Cthrc1) was identified in a screen for differentially expressed sequences in balloon-injured versus normal arteries. Cthrc1 expression was not detectable in normal arteries. However, on injury it was transiently expressed by fibroblasts of the remodeling adventitia and by smooth muscle cells of the neointima. It was also found in the matrix of calcifying human atherosclerotic plaques. CTHRC1 is a secreted 28-kDa protein that is glycosylated and highly conserved from lower chordates to mammals. A short collagen motif with 12 Gly-X-Y repeats appears to be responsible for trimerization of the protein and this renders the molecule susceptible to cleavage by collagenase. Cthrc1 mRNA expression levels are increased in response to transforming growth factor-beta and bone morphogenetic protein-4. Cell migration assays performed with CTHRC1-overexpressing fibroblasts and smooth muscle cells demonstrate that increased CTHRC1 levels are associated with enhanced migratory ability. Furthermore, CTHRC1 overexpression caused a dramatic reduction in collagen type I mRNA and protein levels. Our data indicate that the novel molecule CTHRC1 is transiently expressed in the arterial wall in response to injury where it may contribute to vascular remodeling by limiting collagen matrix deposition and promoting cell migration.     

细胞因子及内皮细胞游走与川崎病发病的关系

目的 :探讨川崎病的发病与细胞因子、内皮细胞游走的关系。方法 :用 EL ISA双抗夹心法和改良Boyden法 ,检测 2 8例川崎病急性期及丙种球蛋白治疗后患儿的血浆细胞因子含量变化及其对内皮细胞游走的影响 ,并与 1 0例健康儿童对照。结果 :在川崎病急性期时 ,白细胞介素 1 β(IL- 1 β)、IL- 6、肿瘤坏死因子 α(TNF- α)、γ干扰素 (IFN-γ)等蛋白含量明显增高 ,用丙种球蛋白治疗后下降 ;试管内加入上述细胞因子使得内皮细胞的游走明显增加。结论 :川崎病急性期时 ,细胞因子的产生具有加速内皮细胞游走 ,破坏动脉内膜 ,诱发动脉炎的作用 ,而丙种球蛋白的治疗效应与其能抑制内皮细胞游走有密切关系