Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

Quantification of random motility and chemotaxis bacterial transport coefficients using individual-cell and population-scale assays

A number of individual-cell and population-scale assays have been introduced to quantify bacterial motility and chemotaxis. The transport coefficients reported in the literature, however, span several orders of magnitude, making it difficult to ascertain to what degree variations in bacterial species/strain, growth medium, growth and experimental conditions, and experiment type contribute to the reported differences in coefficient values. We quantified the random motility of Escherichia coli AW405 using the capillary assay, stopped-flow diffusion chamber (SFDC), and tracking microscope. We obtained good agreement for the random motility coefficient between these assays when using the same bacterial strain and consistent growth and experimental conditions. Chemotaxis of E. coli toward the attractant alpha-methylaspartate was quantified using the SFDC and capillary assay. Good agreement for the chemotactic sensitivity coefficient between the SFDC and the capillary assay was obtained across a limited attractant concentration range. Three different mathematical models were considered for analyzing capillary assay data to obtain a chemotactic sensitivity coefficient. These models differed by their treatment of the bacterial concentration in the chamber and the attractant concentration at the mouth. Results from our study indicate that the capillary assay, the most commonly used bacterial random motility and chemotaxis assay, can be used to accurately quantify bacterial transport coefficients over a limited range of attractant concentrations, provided experiments are performed carefully and appropriate mathematical models are used to interpret the experimental data.     

Quantification of bacterial chemotaxis by measurement of model parameters using the capillary assay

The capillary assay for quantitative characterization of bacterial motility and chemotaxis is analyzed in terms of a mathematical model for cell population migration, in order to determine values for the cell random motility coefficient, mu and the cell chemotaxis coefficient, chi. The analysis involves both analytical perturbation methods and numerical finite-difference techniques. Transient cell density profiles within the capillary tube are determined as they depend upon mu and chi, providing a means for estimating mu and chi from the common protocol measurements of cell accumulation in the tube at specified observation times. The effects of extraneous factors such as assay geometry, stimulus diffusivity, bacterial density, and observation time are thus separated from the intrinsic cell-stimulus interaction and response. This allows independent population measurements of cell chemosensory movement properties to be extrapolated to situations involving growth and competition of populations, for purposes of better understanding microbial population dynamics in systems of biotechnological and microbial ecological importance.     

Traveling bands of chemotactic bacteria in the context of population growth

A mathematical model for traveling bands of motile and chemotactic bacteria in the presence of cell growth and death is examined. It is found that asymptotic traveling wave solutions exist in the absence of chemotaxis, due to the balance of growth, death and random motility. Thus random motility confers the ecological advantage of population propagation through migration into nutrient-rich regions. The presence of chemotaxis amplifies this advantage by moving more cells into higher nutrient concentration regions, resulting in larger and faster bands. Therefore there seem to be two types of traveling bands that can be attained by chemotactic bacteria in the presence of growth and death: (1) these growth/death/motility bands; and (2) pure chemotactic ‘Keller-Segel'-type bands. Comparison to experimental observations by Chapman in 1973 indicate that the latter seem to be formed. The relationship between these two types of solution is at present uncertain. The growth/death/motility bands may have relevance on longer time or distance scales characteristic of microbial ecological systems.     

Focal adhesion disassembly is an essential early event in hepatic stellate cell chemotaxis

Chemotaxis (i.e., directed migration) of hepatic stellate cells to areas of inflammation is a requisite event in the liver's response to injury. Previous studies of signaling pathways that regulate stellate cell migration suggest a key role for focal adhesions, but the exact function of these protein complexes in motility remains unclear. Focal adhesions attach a cell to its substrate and therefore must be regulated in a highly coordinated manner during migration. To test the hypothesis that focal adhesion turnover is an essential early event for chemotaxis in stellate cells, we employed a live-cell imaging technique in which chemotaxis was induced by locally stimulating the tips of rat stellate cell protrusions with platelet-derived growth factor-BB (PDGF). Focal adhesions were visualized with an antibody directed against vinculin, a structural component of the focal adhesion complex. PDGF triggered rapid disassembly of adhesions within 6.25 min, subsequent reassembly by 12.5 min, and continued adhesion assembly in concert with the spreading protrusion until the completion of chemotaxis. Blockade of adhesion disassembly by growing cells on fibronectin or treatment with nocodazole prevented a chemotactic response to PDGF. Augmentation of adhesion disassembly with ML-7 enhanced the chemotactic response to PDGF. These data suggest that focal adhesion disassembly is an essential early event in stellate cell chemotaxis in response to PDGF.     

Growth rate effects on fundamental transport properties of bacterial populations

In many natural environments, bacterial populations experience suboptimal growth due to the competition with other microorganisms for limited resources. The chemotactic response provides a mechanism by which bacterial populations can improve their situation by migrating toward more favorable growth conditions. For bacteria cultured under suboptimal growth conditions, evidence for an enhanced chemotactic response has been observed previously. In this article, for the first time, we have quantitatively characterized this behavior in terms of two macroscopic transport coefficients, the random motility and chemotactic sensitivity coefficients, measured in the stopped-flow diffusion chamber assay. Escherichia coli cultured over a range of growth rates in a chemostat exhibits a dramatic increase in the chemotactic sensitivity coefficient for D-fucose at low growth rates, while the random motility coefficient remains relatively constant by comparison. The change in the chemotactic sensitivity coefficient is accounted for by an independently measured increase in the number of galactose-binding proteins which mediate the chemotactic signal. This result is consistent with the relationship between macroscopic and microscopic parameters for chemotaxis, which was proposed in the mathematical model of Rivero and co-workers. (c) 1993 John Wiley & Sons, Inc.     

Stimulation of Locomotion of Peripheral Blood Monocytes by Human Plasma Fibronectin

The motility of human peripheral blood granulocytes and monocytes in response to human plasma fibronectin was quantified by an in vitro assay using blind-well chemotaxis chambers. Purified fibronectin under nondenaturing conditions produced increased migration of granulocytes only at concentrations higher than 100 nm, and induced increased chemotactic and random locomotion of monocytes at concentrations higher than 0.1 nm. The monocyte migration-inducing activity of fibronectin was concentration dependent, and was strongly inhibited by low concentrations of colchicine (100 nm–100 μm). These findings suggest the possibility that plasma fibronectin serves as a chemotactic stimulus for monocytes in vivo and attracts these cells to sites of microscopic tissue injury where plasma fibronectin is deposited.     

The mathematical modelling of tumour angiogenesis and invasion

In order to accomplish the transition from avascular to vascular growth, solid tumours secrete a diffusible substance known as tumour angiogenesis factor (TAF) into the surrounding tissue. Endothelial cells which form the lining of neighbouring blood vessels respond to this chemotactic stimulus in a well-ordered sequence of events comprising, at minimum, of a degradation of their basement membrane, migration and proliferation. Capillary sprouts are formed which migrate towards the tumour eventually penetrating it and permitting vascular growth to take place. It is during this stage of growth that the insidious process of invasion of surrounding tissues can and does take place. A model mechanism for angiogenesis is presented which includes the diffusion of the TAF into the surrounding host tissue and the response of the endothelial cells to the chemotactic stimulus. Numerical simulations of the model are shown to compare very well with experimental observations. The subsequent vascular growth of the tumour is discussed with regard to a classical reaction-diffusion pre-pattern model.     

Localization of submembranous cations to the leading end of human neutrophils during chemotaxis

Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.     

Migration of connective tissue-derived cells is mediated by ultra-low concentration gradient fields of EGF

The directed migration of cells towards chemical stimuli incorporates simultaneous changes in both the concentration of a chemotactic agent and its concentration gradient, each of which may influence cell migratory response. In this study, we utilized a microfluidic system to examine the interactions between epidermal growth factor (EGF) concentration and EGF gradient in stimulating the chemotaxis of connective tissue-derived fibroblast cells. Cells seeded within microfluidic devices were exposed to concentration gradients established by EGF concentrations that matched or exceeded those required for maximum chemotactic responses seen in transfilter migration assays. The migration of individual cells within the device was measured optically after steady-state gradients had been experimentally established. Results illustrate that motility was maximal at EGF concentration gradients between .01- and 0.1-ng/(mL.mm) for all concentrations used. In contrast, the number of motile cells continually increased with increasing gradient steepness for all concentrations examined. Microfluidics-based experiments exposed cells to minute changes in EGF concentration and gradient that were in line with the acute EGFR phosphorylation measured. Correlation of experimental data with established mathematical models illustrated that the fibroblasts studied exhibit an unreported chemosensitivity to minute changes in EGF concentration, similar to that reported for highly motile cells, such as macrophages. Our results demonstrate that shallow chemotactic gradients, while previously unexplored, are necessary to induce the rate of directed cellular migration and the number of motile cells in the connective tissue-derived cells examined.     

Effects of leukocyte random motility and chemotaxis in tissue inflammatory response.

A rapidly growing body of experimental evidence indicates that defects in leukocyte motility and chemotactic response correlate with increased susceptibility to and severity of bacterial infection in tissue. While this is understandable in qualitative terms, the sensitivity of the correlation is remarkable.In the present study, a theoretical analysis has been developed to relate the dynamics of bacterial growth to the growth and transport parameters of bacteria and leukocytes in tissue. The model considers a local tissue region in the vicinity of a venule and applies continuum unsteady state species conservation equations to the bacterial population, the phagocytic leukocytes, and a chemotactically active chemical mediator assumed to be produced by the bacteria. The analysis quantifies the effects of key parameters, such as leukocyte random motility and chemotactic coefficients, phagocytic and growth rate constants, and leukocyte vessel wall permeability, upon host ability to eliminate the bacteria.As an example, the model's predictions are compared to experimental results correlating inhibition of leukocyte chemotaxis by hemoglobin with its adjuvant action in experimental peritoneal infection by E. coli.     

Quantitative analysis of experiments on bacterial chemotaxis to naphthalene

A mathematical model was developed to quantify chemotaxis to naphthalene by Pseudomonas putida G7 (PpG7) and its influence on naphthalene degradation. The model was first used to estimate the three transport parameters (coefficients for naphthalene diffusion, random motility, and chemotactic sensitivity) by fitting it to experimental data on naphthalene removal from a discrete source in an aqueous system. The best-fit value of naphthalene diffusivity was close to the value estimated from molecular properties with the Wilke-Chang equation. Simulations applied to a non-chemotactic mutant strain only fit the experimental data well if random motility was negligible, suggesting that motility may be lost rapidly in the absence of substrate or that gravity may influence net random motion in a vertically oriented experimental system. For the chemotactic wild-type strain, random motility and gravity were predicted to have a negligible impact on naphthalene removal relative to the impact of chemotaxis. Based on simulations using the best-fit value of the chemotactic sensitivity coefficient, initial cell concentrations for a non-chemotactic strain would have to be several orders of magnitude higher than for a chemotactic strain to achieve similar rates of naphthalene removal under the experimental conditions we evaluated. The model was also applied to an experimental system representing an adaptation of the conventional capillary assay to evaluate chemotaxis in porous media. Our analysis suggests that it may be possible to quantify chemotaxis in porous media systems by simply adjusting the model's transport parameters to account for tortuosity, as has been suggested by others.     

Three-dimensional simulation of obstacle-mediated chemotaxis

Amoeboid cells exhibit a highly dynamic motion that can be directed by external chemical signals, through the process of chemotaxis. Here, we propose a three-dimensional model for chemotactic motion of amoeboid cells. We account for the interactions between the extracellular substances, the membrane-bound proteins, and the cytosolic components involved in the signaling pathway that originates cell motility. We show two- and three-dimensional simulations of cell migration on planar substrates, flat surfaces with obstacles, and fibrous networks. The results show that our model reproduces the main features of chemotactic amoeboid motion. Our simulations unveil a complicated interplay between the geometry of the cell’s environment and the chemoattractant dynamics that tightly regulates cell motion. The model opens new opportunities to simulate the interactions between extra- and intra-cellular compounds mediated by the matrix geometry.     

Regulation of vascular endothelial cell polarization and migration by Hsp70/Hsp90-organizing protein

Hsp70/Hsp90-organizing protein (HOP) is a member of the co-chaperone family, which directly binds to chaperones to regulate their activities. The participation of HOP in cell motility and endothelial cell functions remains largely unknown. In this study, we demonstrate that HOP is critically involved in endothelial cell migration and angiogenesis. Tube formation and capillary sprouting experiments reveal that depletion of HOP expression significantly inhibits vessel formation from endothelial cells. Wound healing and transwell migration assays show that HOP is important for endothelial cell migration. By examination of centrosome reorientation and membrane ruffle dynamics, we find that HOP plays a crucial role in the establishment of cell polarity in response to migratory stimulus. Furthermore, our data show that HOP interacts with tubulin and colocalizes with microtubules in endothelial cells. These findings indicate HOP as a novel regulator of angiogenesis that functions through promoting vascular endothelial cell polarization and migration.     

Effects of p-Fluorophenylalanine and Chloramphenicol on Chemotaxis in Escherichia coli

The effects of chloramphenicol and p-fluorophenylalanine (p-FPA) on growth, proportion of motile cells, average rate of motility, and the chemotactic response of a methionine auxotroph of Escherichia coli K-12 were studied. Kinetic studies revealed that the inhibition of chemotaxis by p-FPA can be explained by the effect on growth, proportion of motile cells, and average rate of motility rather than a selective inhibition of chemotaxis per se. The effect of chloramphenicol on chemotaxis could not be explained in terms of these characteristics. It is concluded that low concentrations of chloramphenicol, unlike p-FPA, selectively inhibit chemotaxis.     

Thrombospondin-induced tumor cell migration: haptotaxis and chemotaxis are mediated by different molecular domains

Thrombospondin induces the migration of human melanoma and carcinoma cells. Using a modified Boyden chamber assay, tumor cells migrated to a gradient of soluble thrombospondin (chemotaxis). Checkerboard analysis indicated that directional migration was induced 27-fold greater than stimulation of random motility. Tumor cells also migrated in a dose-dependent manner to a gradient of substratum-bound thrombospondin (haptotaxis). A series of human melanoma and carcinoma cells were compared for their relative motility stimulation by thrombospondin haptotaxis vs. chemotaxis. Some cell lines exhibited a stronger haptotactic response compared to their chemotactic response while other lines exhibited little or no migration response to thrombospondin. Human A2058 melanoma cells which exhibit a strong haptotactic and chemotactic response to thrombospondin were used to study the structural domains of thrombospondin required for the response. Monoclonal antibody C6.7, which binds to the COOH-terminal region of thrombospondin, inhibited haptotaxis in a dose-dependent optimal manner. C6.7 had no significant effect on thrombospondin chemotaxis. In contrast, monoclonal antibody A2.5, heparin, and fucoidan, which bind to the NH2-terminal heparin-binding domain of thrombospondin, inhibited thrombospondin chemotaxis but not haptotaxis. Monoclonal antibody A6.1 directed against the internal core region of thrombospondin had no significant effect on haptotaxis or chemotaxis. Synthetic peptides GRGDS (50 micrograms/ml), but not GRGES, blocked tumor cell haptotaxis on fibronectin, but had minimal effect on thrombospondin or laminin haptotaxis. The 140-kD fragment of thrombospondin lacking the heparin-binding amino-terminal region retained the property to fully mediate haptotaxis but not chemotaxis. When the COOH region of the 140-kD fragment, containing the C6.7-binding site, was cleaved off, the resulting 120-kD fragment (which retains the RGDA sequence) failed to induce haptotaxis. Separate structural domains of thrombospondin are therefore required for tumor cell haptotaxis vs. chemotaxis. This may have implications during hematogenous cancer metastases formation.     

Independent stimulation of motility and the oxidative metabolic burst of human polymorphonuclear leukocytes.

It has recently been suggested that a single stimulus to the membrane of the polymorphonuclear neutrophil leukocyte (PMN) produces a sequential, stereotyped response involving motility, degranulation, and the oxidative metabolic burst, and conversely, that the chemotactic response is dependent upon the stimulation of the hexose monophosphate shunt (HMPS). We have used small, synthetic substances, known to cause either increased motility or the metabolic burst, to examine whether these events can be stimulated independently. Phorbol myristate acetate (PMA) is a surface active agent that causes marked stimulation of iodination, superoxide production, chemiluminescence, and the HMPS. Such stimulation by PMA did not alter random or directional motility of PMN in the chemotaxis-under-agarose assay. Also, preincubation of PMN with PMA did not deplete their energy source for chemotaxis as demonstrated by a normal chemotactic response to zymosan activated serum. N-formylmethionyl peptides (f-met-phe, f-met-leu-phe) caused a dose-related stimulation of random and directional motility of PMN, but only a very slight stimulation of the HMPS, protein iodination, superoxide production, or chemiluminescence, and this minimal response occurred at more than 1000 times the concentration needed for stimulation of motility. These results indicate that stimulation of motility in the metabolic burst may involve separate events at the membrane of the PMN and that the events are not necessarily interdependent.     

Factor associated with neutral sphingomyelinase activity mediates navigational capacity of leukocytes responding to wounds and infection: live imaging studies in zebrafish larvae

Factor associated with neutral sphingomyelinase activity (FAN) is an adaptor protein that specifically binds to the p55 receptor for TNF (TNF-RI). Our previous investigations demonstrated that FAN plays a role in TNF-induced actin reorganization by connecting the plasma membrane with actin cytoskeleton, suggesting that FAN may impact on cellular motility in response to TNF and in the context of immune inflammatory conditions. In this study, we used the translucent zebrafish larvae for in vivo analysis of leukocyte migration after morpholino knockdown of FAN. FAN-deficient zebrafish leukocytes were impaired in their migration toward tail fin wounds, leading to a reduced number of cells reaching the wound. Furthermore, FAN-deficient leukocytes show an impaired response to bacterial infections, suggesting that FAN is generally required for the directed chemotactic response of immune cells independent of the nature of the stimulus. Cell-tracking analysis up to 3 h after injury revealed that the reduced number of leukocytes is not due to a reduction in random motility or speed of movement. Leukocytes from FAN-deficient embryos protrude pseudopodia in all directions instead of having one clear leading edge. Our results suggest that FAN-deficient leukocytes exhibit an impaired navigational capacity, leading to a disrupted chemotactic response.     

VEGF is a chemoattractant for FGF-2-stimulated neural progenitors

Migration of undifferentiated neural progenitors is critical for the development and repair of the nervous system. However, the mechanisms and factors that regulate migration are not well understood. Here, we show that vascular endothelial growth factor (VEGF)-A, a major angiogenic factor, guides the directed migration of neural progenitors that do not display antigenic markers for neuron- or glia-restricted precursor cells. We demonstrate that progenitor cells express both VEGF receptor (VEGFR) 1 and VEGFR2, but signaling through VEGFR2 specifically mediates the chemotactic effect of VEGF. The expression of VEGFRs and the chemotaxis of progenitors in response to VEGF require the presence of fibroblast growth factor 2. These results demonstrate that VEGF is an attractive guidance cue for the migration of undifferentiated neural progenitors and offer a mechanistic link between neurogenesis and angiogenesis in the nervous system.