Co-operative Cdc42 and Rho signalling mediates ephrinB-triggered endothelial cell retraction

Cell repulsion responses to Eph receptor activation are linked to rapid actin cytoskeletal reorganizations, which in turn are partially mediated by Rho-ROCK (Rho kinase) signalling, driving actomyosin contractility. In the present study, we show that Rho alone is not sufficient for this repulsion response. Rather, Cdc42 (cell division cycle 42) and its effector MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) are also critical for ephrinB-induced cell retraction. Stimulation of endothelial cells with ephrinB2 triggers rapid, but transient, cell retraction. We show that, although membrane retraction is fully blocked by blebbistatin (a myosin-II ATPase inhibitor), it is only partially blocked by inhibiting Rho-ROCK signalling, suggesting that there is ROCK-independent signalling to actomyosin contractility downstream of EphBs. We find that a combination of either Cdc42 or MRCK inhibition with ROCK inhibition completely abolishes the repulsion response. Additionally, endocytosis of ephrin-Eph complexes is not required for initial cell retraction, but is essential for subsequent Rac-mediated re-spreading of cells. Our data reveal a complex interplay of Rho, Rac and Cdc42 in the process of EphB-mediated cell retraction-recovery responses.     

Rac1 mediates laminar shear stress-induced vascular endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. ECs are constantly subjected to shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular behaviors and functions. The aim of this study is to elucidate the effects of Rac1, which is the member of small G protein family, on EC migration under different laminar shear stress (5.56, 10.02, and 15.27 dyn/cm²). The cell migration distance under laminar shear stress increased significantly than that under the static culture condition. Especially, under relative high shear stress (15.27 dyn/cm²) there was a higher difference at 8 h (P < 0.01) and 2 h (P < 0.05) compared with static controls. RT-PCR results further showed increasing mRNA expression of Rac1 in ECs exposed to laminar shear stress than that exposed to static culture. Using plasmids encoding the wild-type (WT), an activated mutant (Q61L), and a dominant-negative mutant (T17N), plasmids encoding Rac1 were transfected into EA.hy 926 cells. The average net migration distance of Rac1Q61L group increased significantly, while Rac1T17N group decreased significantly in comparison with the static controls. These results indicated that Rac1 mediated shear stress-induced EC migration. Our findings conduce to elucidate the molecular mechanisms of EC migration induced by shear stress, which is expected to understand the pathophysiological basis of wound healing in health and diseases.     

Notch signaling mediates melanoma–endothelial cell communication and melanoma cell migration

Stromal and cellular components within the tumor microenvironment significantly influence molecular signals mediating tumor growth and progression. We recently performed a screen to evaluate critical mediators of melanoma–endothelial communication and identified several molecular pathways associated with these cellular networks, including Notch3. Here, we evaluate the nature of melanoma–endothelial communication mediated by Notch3 and its functional significance. We find that Notch3 is specifically upregulated in melanoma–endothelial cell cocultures and is functionally associated with increased Notch signaling in melanoma cells. Furthermore, induced Notch3 signaling in melanoma cell lines leads to enhanced tumor cell migration without associated increases in tumor cell growth. Additionally, Notch3 expression is specifically associated with malignant patient samples and is not evident in benign nevi. We conclude that Notch3 mediates melanoma–endothelial cell communication and tumor cell migration and may serve as a meaningful therapeutic target for this aggressive malignancy.     

Role of Rho kinase in sphingosine 1-phosphate-mediated endothelial and smooth muscle cell migration and differentiation

The role of sphingosine 1-phosphate (S1P)-induced Rho kinase (ROCK) activation in the angiogenic responses of pulmonary artery-derived endothelial cells (PAEC) and smooth muscle cells (PASMC) was examined. S1P, a biologically active phospholipid that regulates angiogenesis, promoted PAEC chemotaxis and capillary morphogenesis; furthermore, this activity was unaltered by pretreatment with the pharmacological inhibitor of ROCK, H1152. In contrast, S1P (500 nM) significantly inhibited spontaneous PASMC chemotaxis and differentiation; however, this inhibition was eradicated upon H1152 pretreatment. Similarly, PASMCs transfected with ROCK II siRNA diminished S1P-induced inhibition of the development of multi-cellular structures. Analysis by RT-PCR identified the presence of S1P₁ and S1P₃ receptors on both PAECs and PASMCs, while S1P₂ receptor expression was confined to only PASMCs. Consistent with this observation, the S1P₁ and S1P₃ receptor antagonist, VPC23019, virtually abolished the S1P-initiated PAEC differentiation but did not impede the S1P-induced inhibition of PASMC differentiation. However, the S1P₂ receptor antagonist, JTE013, had no effect on S1P-mediated differentiation of PAECs but abolished the S1P-induced inhibition of PASMC function. Co-cultured endothelial and smooth muscle cells differentiated into “neovascular-like” networks, which were significantly inhibited by S1P. The inhibition of co-culture differentiation in both PAECs and PASMCs was negated by H1152 pretreatment. However, when smooth muscle cells were added to S1P-initiated endothelial cell networks, additional S1P treatment did not inhibit the cellular networks generated by these cells. In conclusion, S1P-induced PAEC angiogenic responses are regulated by S1P₁ and/or S1P₃ receptors independent of Rho kinase activation, whereas S1P₂ receptor-mediated curtailment of PASMC function by S1P.     

Friction-controlled traction force in cell adhesion

The force balance between the extracellular microenvironment and the intracellular cytoskeleton controls the cell fate. We report a new (to our knowledge) mechanism of receptor force control in cell adhesion originating from friction between cell adhesion ligands and the supporting substrate. Adherent human endothelial cells have been studied experimentally on polymer substrates noncovalently coated with fluorescent-labeled fibronectin (FN). The cellular traction force correlated with the mobility of FN during cell-driven FN fibrillogenesis. The experimental findings have been explained within a mechanistic two-dimensional model of the load transfer at focal adhesion sites. Myosin motor activity in conjunction with sliding of FN ligands noncovalently coupled to the surface of the polymer substrates is shown to result in a controlled traction force of adherent cells. We conclude that the friction of adhesion ligands on the supporting substrate is important for mechanotransduction and cell development of adherent cells in vitro and in vivo.     

Mechanisms of force generation and force transmission during interstitial leukocyte migration

For innate and adaptive immune responses it is essential that inflammatory cells use quick and flexible locomotion strategies. Accordingly, most leukocytes can efficiently infiltrate and traverse almost every physiological or artificial environment. Here, we review how leukocytes might achieve this task mechanistically, and summarize recent findings on the principles of cytoskeletal force generation and transduction at the leading edge of leukocytes. We propose a model in which the cells switch between adhesion‐receptor‐mediated force transmission and locomotion modes that are based on cellular deformations, but independent of adhesion receptors. This plasticity in migration strategies allows leukocytes to adapt to the geometry and molecular composition of their environment.     

Raf-1 regulates Rho signaling and cell migration

Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.     

Enhancement of endothelial progenitor cell numbers and migration by H1152, a Rho kinase specific inhibitor

Endothelial progenitor cells (EPCs) are applied in the treatment of ischemic diseases. In ex vivo culture of human cord-blood derived EPCs, H1152, (S)-(+)-2-methyl-1-[(4-methyl-5-iso-quinolinyl) sulfonyl]-homopiperazine, markedly increased the number of EPCs. It also induced EPC migration, stimulated the phosphorylation of AKT, and reduced the expression of p27 in the EPCs. Thus H1152 can be used effectively in ex vivo expansion of EPCs.     

Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway

The goals of this study were 2-fold: 1) to determine whether stimulation of Eph B4 receptors promotes microvascular endothelial cell migration and/or proliferation, and 2) to elucidate signaling pathways involved in these responses. The human endothelial cells used possessed abundant Eph B4 receptors with no endogenous ephrin B2 expression. Stimulation of these receptors with ephrin B2/Fc chimera resulted in dose- and time-dependent phosphorylation of Akt. These responses were inhibited by LY294002 and ML-9, blockers of phosphatidylinositol 3-kinase (PI3K) and Akt, respectively. Eph B4 receptor activation increased proliferation by 38%, which was prevented by prior blockade with LY294002, ML-9, and inhibitors of protein kinase G (KT5823) and MEK (PD98059). Nitrite levels increased over 170% after Eph B4 stimulation, indicating increased nitric oxide production. Signaling of endothelial cell proliferation appears to be mediated by a PI3K/Akt/endothelial nitric-oxide synthase/protein kinase G/mitogen-activated protein kinase cascade. Stimulation with ephrin B2 also increased migration by 63% versus controls. This effect was inhibited by blockade with PP2 (Src inhibitor), LY294002 or ML-9 but was unaffected by the PKG and MEK blockers. Eph B4 receptor stimulation increased activation of both matrix metalloproteinase-2 and -9. The results from these studies indicate that Eph B4 stimulates migration and proliferation and may play a role in angiogenesis.     

RhoA/rho-associated kinase mediates fibroblast contractile force generation

The intracellular signals governing contractile force generation by non-muscle cells remain uncertain. Our aim was to test the hypothesis that the rhoA/rho-associated kinase signaling pathway is a principal mediator of contractile force generation in non-muscle cells. We measured myosin II regulatory light chain (MLC) phosphorylation and directly quantitated force generation by chicken embryo fibroblasts in the absence and presence of selective inhibitors of rhoA, and its downstream effector, rho-associated kinase. Inactivation of rhoA, with C3 transferase, inhibited serum-stimulated MLC phosphorylation and contractile force generation. Y-27632, an inhibitor of rho-associated kinase, reduced basal contractile tension, and inhibited both serum and endothelin-1 stimulated MLC phosphorylation and contractile force generation. The results of this study provide novel evidence indicating that the rhoA/rho-associated kinase signaling pathway is a principal mediator of MLC phosphorylation and consequent contractile force generation by non-muscle cells.     

Roles of mechanical force and CXCR1/CXCR2 in shear-stress-induced endothelial cell migration

We previously demonstrated that CXCR1 and CXCR2 are novel mechanosensors mediating laminar shear-stress-induced endothelial cell (EC) migration (Zeng et al. in Cytokine 53:42–51, 2011). In the present study, an analytical model was proposed to further analyze the underlying mechanisms, assuming the mechanical force (MF) and mechanosensor-mediated biochemical reactions induce cell migration together. Shear stress can regulate both mechanosensor-mediated migration in the flow direction (Ms–M_FD) and mechanosensor-mediated migration toward a wound (Ms–M_W). Next, the migration distance, the roles of MF-induced cell migration (MF–M), and the mobilization mechanisms of mechanosensors were analyzed. The results demonstrated that MF–M plays an important role in 15.27 dyn/cm² shear-stress-induced EC migration but is far weaker than Ms–M_W at 5.56 dyn/cm². Our findings also indicated that CXCR2 played a primary role, in synergy with CXCR1. The Ms–M_FD was primarily mediated by the synergistic effect of CXCR1 and CXCR2. In Ms–M_W, when shear stress was beyond a certain threshold, the synergistic effect of CXCR1 and CXCR2 was enhanced, and the effect of CXCR1 was inhibited. Therefore, the retarding of EC migration and wound closure capacity under low shear flow was related to the low magnitude of shear stress, which may contribute to atherogenesis and many other vascular diseases.     

Alpha-smooth muscle actin expression enhances cell traction force

Using an established corneal stromal cell differentiation model, we manipulated alpha-smooth muscle actin (alpha-SMA) protein expression levels in fibroblasts by treating them with TGF-beta1, bFGF, TGF-beta type I receptor inhibitor (SB-431542), and siRNA against alpha-SMA. The corresponding cell traction forces (CTFs) were determined by cell traction force microscopy. With all these treatments, we found that alpha-SMA is not required for CTF induction, but its expression upregulates CTF. This upregulation involves the modification of stress fibers but does not appear to relate to non-muscle myosin II expression or beta-actin expression. Moreover, there exists a linear relationship between alpha-SMA protein expression level and CTF magnitude. Finally, CTFs were found to vary among a population of myofibroblasts, suggesting that alpha-SMA protein expression levels of individual cells also vary.     

Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates CD151-induced endothelial cell proliferation and cell migration

We previously reported that CD151 promotes neovascularization and improves blood perfusion in rat hind-limb ischemia model, but the precise mechanism is still unclear. Endothelial cell proliferation and cell migration play critical roles in angiogenesis. Many growth factors and hormones have been shown to regulate cell proliferation, cell migration and angiogenesis, including the activation of eNOS activity, via the PI3K/Akt signaling pathway. Whether CD151 induces cell proliferation and cell migration via PI3K/Akt signaling pathway is not known. Here we showed that CD151 promotes human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation in vitro, accompanied by increased phosphorylation of Akt and eNOS, leading to increased eNOS activity and nitric oxide (NO) levels after rAAV-CD151 infection, whereas infection with rAAV-anti-CD151 attenuated the effects of CD151, which suggested that CD151 can activate PI3K/Akt pathway. Moreover, inhibitors of PI3K (LY294002) and eNOS (l-NAME) can attenuate CD151-induced cell proliferation and cell migration. The results suggested that activation of PI3K/Akt signaling pathway mediates CD151-induced cell proliferation and migration.     

Rho family GTPases regulate VEGF-stimulated endothelial cell motility

Migration of endothelial cells induced by vascular endothelial growth factor (VEGF) is a critical step in angiogenesis. Stimulation of motility by growth factors such as VEGF requires interaction with the signal transduction pathways activated by the extracellular matrix (ECM). Here we demonstrate that the Rac GTPase is the critical intersection activated by type 1 collagen ECM and VEGF during stimulation of endothelial cell motility. To analyze the role of the Rho family GTPases in VEGF-stimulated endothelial cell chemotaxis and ECM-stimulated haptotaxis, we transduced the respective fusion proteins in human foreskin dermal endothelial cells using a Tat peptide from the human immunodeficiency virus Tat protein. VEGF signaling required Rac activation during chemotaxis, and Rac and Cdc42 were activated during haptotaxis on type I collagen. Similar to VEGF, Rac activation induced an increase in endothelial cell stress fiber and focal adhesion. Surprisingly, Rho activation was not present in collagen-induced haptotaxis or stimulation of chemotaxis by VEGF, although Rho induced stress fibers and focal adhesions similar to Rac activation. The result of constitutive Rho activation was an inhibition of haptotaxis. Thus, Rac is required and sufficient for the activation of endothelial cell haptotaxis and VEGF-stimulated chemotaxis.     

Physical limits of cell migration: Control by ECM space and nuclear deformation and tuning by proteolysis and traction force

Cell migration through 3D tissue depends on a physicochemical balance between cell deformability and physical tissue constraints. Migration rates are further governed by the capacity to degrade ECM by proteolytic enzymes, particularly matrix metalloproteinases (MMPs), and integrin- and actomyosin-mediated mechanocoupling. Yet, how these parameters cooperate when space is confined remains unclear. Using MMP-degradable collagen lattices or nondegradable substrates of varying porosity, we quantitatively identify the limits of cell migration by physical arrest. MMP-independent migration declined as linear function of pore size and with deformation of the nucleus, with arrest reached at 10% of the nuclear cross section (tumor cells, 7 µm²; T cells, 4 µm²; neutrophils, 2 µm²). Residual migration under space restriction strongly depended upon MMP-dependent ECM cleavage by enlarging matrix pore diameters, and integrin- and actomyosin-dependent force generation, which jointly propelled the nucleus. The limits of interstitial cell migration thus depend upon scaffold porosity and deformation of the nucleus, with pericellular collagenolysis and mechanocoupling as modulators.     

The scaffolding adapter Gab1 mediates vascular endothelial growth factor signaling and is required for endothelial cell migration and capillary formation

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.     

Mechanotransduction in endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. EC migration can be regulated by different mechanisms such as chemotaxis, haptotaxis, and mechanotaxis. This review will focus on fluid shear stress-induced mechanotransduction during EC migration. EC migration and mechanotransduction can be modulated by cytoskeleton, cell surface receptors such as integrins and proteoglycans, the chemical and physical properties of extracellular matrix (ECM) and cell-cell adhesions. The shear stress applied on the luminal surface of ECs can be sensed by cell membrane and associated receptor and transmitted throughout the cell to cell-ECM adhesions and cell-cell adhesions. As a result, shear stress induces directional migration of ECs by promoting lamellipodial protrusion and the formation of focal adhesions (FAs) at the front in the flow direction and the disassembly of FAs at the rear. Persistent EC migration in the flow direction can be driven by polarized activation of signaling molecules and the positive feedback loops constituted by Rho GTPases, cytoskeleton, and FAs at the leading edge. Furthermore, shear stress-induced EC migration can overcome the haptotaxis of ECs. Given the hemodynamic environment of the vascular system, mechanotransduction during EC migration has a significant impact on vascular development, angiogenesis, and vascular wound healing.     

Tissue engineering science: Consequences of cell traction force

Blood and tissue cells mechanically interact with soft tissues and tissue-equivalent reconstituted collagen gels in a variety of situations relevant to biomedicine and biotechnology. A key phenomenon in these interactions is the exertion of traction force by cells on local collagen fibers which typically constitute the solid network of these tissues and gels and impart gross mechanical integrity. Two important consequences of cells exerting traction on such collagen networks are first, when the cells co-ordinate their traction, resulting in cell migration, and second, when their traction is sufficient to deform the network. Such cell-collagen network interactions are coupled in a number of ways. Network deformation, for example, can result in net alignment of collagen fibers, eliciting contact guidance, wherein cells move with bidirectional bias along an axis of fiber alignment, potentially leading to a nonuniform cell distribution. This may govern cell accumulation in wounds and be exploited to control cell infiltration of bioartificial tissues and organs. Another consequence of cell traction is the resultant stress and strain in the network which modulate cell protein and DNA synthesis and differentiation. We summarize, here, relevant mathematical theories which we have used to describe the inherent coupling of cell dynamics and tissue mechanics in cell-populated collagen gels via traction. The development of appropriate models based on these theories, in an effort to understand how events in wound healing govern the rate and extent of wound contraction, and to measure cell traction forces in vitro, are described. Relevant observations and speculation from cell biology and medicine that motivate or serve to critique the assumptions made in the theories and models are also summarized.Abbreviations ECM Extracellular Matrix - FPCL Fibroblast-Populated Collagen Lattice - FPCM Fibroblast-Populated Collagen Microsphere     

Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis

To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-L-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.