Changes in dynamics of tumor/endothelial cell adhesive interactions depending on endothelial cell growth state and elastic properties.

Cancer cell adhesion to the endothelium is a crucial process in hematogenous metastasis, but how the integrity of the endothelial barrier and endothelial cell (EC) mechanical properties influence the adhesion between metastatic cancer cells and the endothelium remain unclear. In the present study, we have measured the adhesion between single cancer cells and two types of ECs at various growth states and their mechanical properties (elasticity) using atomic force microscopy single cell force spectroscopy. We demonstrated that the EC stiffness increased and adhesion with cancer cells decreased, as ECs grew from a single cell to a confluent state and developed cell-cell contacts, but this was reversed when confluent cells returned to a single state in a scratch assay. Our results suggest that the integrity of the endothelial barrier is an important factor in reducing the ability of the metastatic tumor cells to adhere to the vascular endothelium, extravasate and lodge in the vasculature of a distant organ where secondary metastatic tumors would develop.

Temporal and molecular dynamics of human metastatic breast carcinoma cell adhesive interactions with human bone marrow endothelium analyzed by single-cell force spectroscopy.

Bone is a common site of metastasis for breast cancer and the mechanisms of metastasis are not fully elucidated. The purpose of our study was to characterize temporal and molecular dynamics of adhesive interactions between human breast cancer cells (HBCC) and human bone marrow endothelium (HBME) with piconewton resolution using atomic force microscopy (AFM). In adhesion experiments, a single breast cancer cell, MDA-MB-231 (MB231) or MDA-MB-435 (MB435) was attached to the AFM cantilever and brought into contact with a confluent HBME monolayer for different time periods (0.5 to 300 sec). The forces required to rupture individual molecular interactions and completely separate interacting cells were analyzed as measures of cell-cell adhesion. Adhesive interactions between HBME and either MB231 or MB435 cells increased progressively as cell-cell contact time was prolonged from 0.5 to 300 sec due to the time-dependent increase in the number and frequency of individual adhesive events, as well as to the involvement of stronger ligand-receptor interactions over time. Studies of the individual molecule involvement revealed that Thomsen-Friedenreich antigen (TF-Ag), galectin-3, integrin-ß1, and integrin-α3 are all contributing to HBCC/HBME adhesion to various degrees in a temporally defined fashion. In conclusion, cell-cell contact time enhances adhesion of HBCC to HBME and the adhesion is mediated, in part, by TF-Ag, galectin-3, integrin-α3, and integrin-ß1.

Applications of Atomic Force Microscopy for Adhesion Force Measurements in Mechanotransduction.

Adhesive interactions between living cells or ligand-receptor interactions can be studied at the molecular level using atomic force microscopy (AFM). Adhesion force measurements are performed with functionalized AFM probes. In order to measure single ligand-receptor interactions, a cantilever with a pyramidal tip is functionalized with a bio-recognized ligand (e.g., extracellular matrix protein). The ligand-functionalized probe is then brought into contact with a cell in culture to investigate adhesion between the respective probe-bound ligand and endogenously expressed cell surface receptors (e.g., integrins or other adhesion receptor). For experiments designed to examine cell-cell adhesions, a single cell is attached to a tipless cantilever which is then brought into contact with other cultured cells. Force curves are recorded to determine the forces necessary to rupture discrete adhesions between the probe-bound ligand and receptor, or to determine total adhesion force at cell-cell contacts. Here, we describe the procedures for measuring adhesions between (a) fibronectin and α5ß1 integrin, and (b) breast cancer cells and bone marrow endothelial cells.

Dipeptidyl Peptidase-4 Inhibition With Saxagliptin Ameliorates Angiotensin II-Induced Cardiac Diastolic Dysfunction in Male Mice.

Activation of the renin-angiotensin-aldosterone system is common in hypertension and obesity and contributes to cardiac diastolic dysfunction, a condition for which no treatment currently exists. In light of recent reports that antihyperglycemia incretin enhancing dipeptidyl peptidase (DPP)-4 inhibitors exert cardioprotective effects, we examined the hypothesis that DPP-4 inhibition with saxagliptin (Saxa) attenuates angiotensin II (Ang II)-induced cardiac diastolic dysfunction. Male C57BL/6J mice were infused with either Ang II (500 ng/kg/min) or vehicle for 3 weeks receiving either Saxa (10 mg/kg/d) or placebo during the final 2 weeks. Echocardiography revealed Ang II-induced diastolic dysfunction, evidenced by impaired septal wall motion and prolonged isovolumic relaxation, coincident with aortic stiffening. Ang II induced cardiac hypertrophy, coronary periarterial fibrosis, TRAF3-interacting protein 2 (TRAF3IP2)-dependent proinflammatory signaling [p-p65, p-c-Jun, interleukin (IL)-17, IL-18] associated with increased cardiac macrophage, but not T cell, gene expression. Flow cytometry revealed Ang II-induced increases of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and CD45+CD4+ lymphocytes. Treatment with Saxa reduced plasma DPP-4 activity and abrogated Ang II-induced cardiac diastolic dysfunction independent of aortic stiffening or blood pressure. Furthermore, Saxa attenuated Ang II-induced periarterial fibrosis and cardiac inflammation, but not hypertrophy or cardiac macrophage infiltration. Analysis of Saxa-induced changes in cardiac leukocytes revealed Saxa-dependent reduction of the Ang II-mediated increase of cardiac CD11c messenger RNA and increased cardiac CD8 gene expression and memory CD45+CD8+CD44+ lymphocytes. In summary, these results demonstrate that DPP-4 inhibition with Saxa prevents Ang II-induced cardiac diastolic dysfunction, fibrosis, and inflammation associated with unique shifts in CD11c-expressing leukocytes and CD8+ lymphocytes.

Uric acid promotes vascular stiffness, maladaptive inflammatory responses and proteinuria in western diet fed mice.

OBJECTIVE: Aortic vascular stiffness has been implicated in the development of cardiovascular disease (CVD) and chronic kidney disease (CKD) in obese individuals. However, the mechanism promoting these adverse effects are unclear. In this context, promotion of obesity through consumption of a western diet (WD) high in fat and fructose leads to excess circulating uric acid. There is accumulating data implicating elevated uric acid in the promotion of CVD and CKD. Accordingly, we hypothesized that xanthine oxidase(XO) inhibition with allopurinol would prevent a rise in vascular stiffness and proteinuria in a translationally relevant model of WD-induced obesity. MATERIALS/METHODS: Four-week-old C57BL6/J male mice were fed a WD with excess fat (46%) and fructose (17.5%) with or without allopurinol (125mg/L in drinking water) for 16weeks. Aortic endothelial and extracellular matrix/vascular smooth muscle stiffness was evaluated by atomic force microscopy. Aortic XO activity, 3-nitrotyrosine (3-NT) and aortic endothelial sodium channel (EnNaC) expression were evaluated along with aortic expression of inflammatory markers. In the kidney, expression of toll like receptor 4 (TLR4) and fibronectin were assessed along with evaluation of proteinuria. RESULTS: XO inhibition significantly attenuated WD-induced increases in plasma uric acid, vascular XO activity and oxidative stress, in concert with reductions in proteinuria. Further, XO inhibition prevented WD-induced increases in aortic EnNaC expression and associated endothelial and subendothelial stiffness. XO inhibition also reduced vascular pro-inflammatory and maladaptive immune responses induced by consumption of a WD. XO inhibition also decreased WD-induced increases in renal TLR4 and fibronectin that associated proteinuria. CONCLUSIONS: Consumption of a WD leads to elevations in plasma uric acid, increased vascular XO activity, oxidative stress, vascular stiffness, and proteinuria all of which are attenuated with allopurinol administration.

Fascin2 regulates cisplatin-induced apoptosis in NRK-52E cells.

Previous studies have shown that the aging kidney has a marked loss of α(E)-catenin in proximal tubular epithelium. α-Catenin, a key regulator of the actin cytoskeleton, interacts with a variety of actin-binding proteins. Cisplatin-induced loss of fascin2, an actin bundling protein, was observed in cells with a stable knockdown of α(E)-catenin (C2 cells), as well as in aging (24 mon), but not young (4 mon), kidney. Fascin2 co-localized with α-catenin and the actin cytoskeleton in NRK-52E cells. Knockdown of fascin2 increased the susceptibility of tubular epithelial cells to cisplatin-induced injury. Overexpression of fascin2 in C2 cells restored actin stress fibers and attenuated the increased sensitivity of C2 cells to cisplatin-induced apoptosis. Interestingly, fascin2 overexpression attenuated cisplatin-induced mitochondrial dysfunction and oxidative stress in C2 cells. These data demonstrate that fascin2, a putative target of α(E)-catenin, may play important role in preventing cisplatin-induced acute kidney injury.

Brief serotonin exposure initiates arteriolar inward remodeling processes in vivo that involve transglutaminase activation and actin cytoskeleton reorganization.

Inward remodeling of the resistance vasculature is strongly associated with life-threatening cardiovascular events. Previous studies have demonstrated that both actin polymerization and the activation of transglutaminases mediate early stages of the transition from a structurally normal vessel to an inwardly remodeled one. Ex vivo studies further suggest that a few hours of exposure to vasoconstrictor agonists induces inward remodeling in the absence of changes in intraluminal pressure. Here we report that a short, 10-min, topical exposure to serotonin (5-HT) + N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME) was sufficient to initiate inward remodeling processes in rat cremasteric feed arterioles (100-200 µm lumen diameter), in vivo. Addition of the transglutaminase inhibitor, cystamine, blocked the in vivo remodeling. We further demonstrate that, in isolated arterioles, 5-HT + l-NAME activates transglutaminases and modulates the phosphorylation state of cofilin, a regulator of actin depolymerization. The 5-HT + l-NAME-induced remodeling process in isolated arterioles was also inhibited by an inhibitor of Lim Kinase, the kinase that phosphorylates and inactivates cofilin. Therefore, our results indicate that a brief vasoconstriction induced by 5-HT + l-NAME is able to reduce the passive structural diameter of arterioles through processes that are dependent on the activation of transglutaminases and Lim kinase, and the subsequent phosphorylation of cofilin.

Vascular smooth muscle cell stiffness and adhesion to collagen I modified by vasoactive agonists.

In vascular smooth muscle cells (VSMCs) integrin-mediated adhesion to extracellular matrix (ECM) proteins play important roles in sustaining vascular tone and resistance. The main goal of this study was to determine whether VSMCs adhesion to type I collagen (COL-I) was altered in parallel with the changes in the VSMCs contractile state induced by vasoconstrictors and vasodilators. VSMCs were isolated from rat cremaster skeletal muscle arterioles and maintained in primary culture without passage. Cell adhesion and cell E-modulus were assessed using atomic force microscopy (AFM) by repetitive nano-indentation of the AFM probe on the cell surface at 0.1 Hz sampling frequency and 3200 nm Z-piezo travelling distance (approach and retraction). AFM probes were tipped with a 5 µm diameter microbead functionalized with COL-I (1 mg\ml). Results showed that the vasoconstrictor angiotensin II (ANG-II; 10-6) significantly increased (p<0.05) VSMC E-modulus and adhesion probability to COL-I by approximately 35% and 33%, respectively. In contrast, the vasodilator adenosine (ADO; 10-4) significantly decreased (p<0.05) VSMC E-modulus and adhesion probability by approximately -33% and -17%, respectively. Similarly, the NO donor (PANOate, 10-6 M), a potent vasodilator, also significantly decreased (p<0.05) the VSMC E-modulus and COL-I adhesion probability by -38% and -35%, respectively. These observations support the hypothesis that integrin-mediated VSMC adhesion to the ECM protein COL-I is dynamically regulated in parallel with VSMC contractile activation. These data suggest that the signal transduction pathways modulating VSMC contractile activation and relaxation, in addition to ECM adhesion, interact during regulation of contractile state.

Vasoactive agonists exert dynamic and coordinated effects on vascular smooth muscle cell elasticity, cytoskeletal remodelling and adhesion.

In this study, we examined the ability of vasoactive agonists to induce dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion, and tested the hypothesis that these events are coordinated with rapid remodelling of the cortical cytoskeleton. Real-time measurement of cell elasticity was performed with atomic force microscopy (AFM) and adhesion was assessed with AFM probes coated with fibronectin (FN). Temporal data were analysed using an Eigen-decomposition method. Elasticity in VSMCs displayed temporal oscillations with three components at approximately 0.001, 0.004 and 0.07 Hz, respectively. Similarly, adhesion displayed a similar oscillatory pattern. Angiotensin II (ANG II, 10(-6) M) increased (+100%) the amplitude of the oscillations, whereas the vasodilator adenosine (ADO, 10(-4) M) reduced oscillation amplitude (-30%). To test whether the oscillatory changes were related to the architectural alterations in cortical cytoskeleton, the topography of the submembranous actin cytoskeleton (100-300 nm depth) was acquired with AFM. These data were analysed to compare cortical actin fibre distribution and orientation before and after treatment with vasoactive agonists. The results showed that ANG II increased the density of stress fibres by 23%, while ADO decreased the density of the stress fibres by 45%. AFM data were supported by Western blot and confocal microscopy. Collectively, these observations indicate that VSMC cytoskeletal structure and adhesion to the extracellular matrix are dynamically altered in response to agonist stimulation. Thus, vasoactive agonists probably invoke unique mechanisms that dynamically alter the behaviour and structure of both the VSMC cytoskeleton and focal adhesions to efficiently support the normal contractile behaviour of VSMCs.

Atomic force microscopy to characterize binding properties of α7-containing nicotinic acetylcholine receptors on neurokinin-1 receptor-expressing medullary respiratory neurons.

In the present study, we used atomic force microscopy (AFM) to examine the ligand-binding properties of α7-containing nicotinic acetylcholine receptors (nAChRs) expressed on neurons from the ventral respiratory group. We also determined the effect of acute and prolonged exposure to nicotine on the binding probability of nAChRs. Neurons from neonatal (postnatal day 5-10) and juvenile rats (3-4 weeks old) were cultured. Internalization of Alexa Fluor 488-conjugated substance P was used to identify respiratory neurons that expressed the neurokinin-1 receptor (NK1-R), a recognized marker of ventral respiratory group neurons. To assess functional changes in nAChRs, AFM probes conjugated with anti-α7 subunit nAChR antibody were used to interact cyclically with the surface of the soma of NK1-R-positive neurons. Measurements were made of the frequency of antibody adhesion to the α7 receptor subunit and of the detachment forces between the membrane-attached receptor and the AFM probe tip. Addition of α-bungarotoxin (a specific antagonist of α7 subunit-containing nAChRs) to the cell bath produced a 69% reduction in binding to the α7 subunit (P < 0.05, n = 10), supporting specificity of binding. Acute exposure to nicotine (1 µM added to culture media) produced an 80% reduction in nAChR antibody binding to the α7 subunit (P < 0.05, n = 9). Prolonged incubation (72 h) of the cell culture in nicotine significantly reduced α7 binding in a concentration-dependent manner. Collectively, these findings demonstrate that AFM is a sensitive tool for assessment of functional changes in nAChRs expressed on the surface of living NK1-R-expressing medullary neurons. Moreover, these data demonstrate that nicotine exposure decreases the binding probability of α7 subunit-containing nAChRs.

Selective measurement and manipulation of adhesion forces between cancer cells and bone marrow endothelial cells using atomic force microscopy.

AIMS: The lack of understanding of the biology of bone cancer metastasis has limited the development of effective treatment strategies. The aim of this study was to characterize tumor cell adhesion molecules and determine active tumor cell interactions with human bone marrow endothelial (BME) cells using atomic force microscopy. MATERIALS & METHODS: A single prostate (PC3) cancer cell was coupled (concanavalin A) to the atomic force microscopy cantilever then placed in contact with BME cells for cell force spectroscopy measurements. RESULTS & DISCUSSION: Strong adhesive interactions between PC3 and BME cells were significantly (p < 0.05) reduced by anti-ICAM-1, anti-ß1 and anti-P-selectin, but not anti-VCAM-1. The combined blocking antibodies or the therapeutic agent zoledronic acid significantly (p < 0.005) reduced the adhesive interactions by 65 and 63%, respectively, which was confirmed using a functional in vitro assay. CONCLUSION: Atomic force microscopy provides a highly sensitive screening assay to determine and quantify nanoscale adhesion events between different cell types important in the metastatic cascade.

Coordination of fibronectin adhesion with contraction and relaxation in microvascular smooth muscle.

AIMS: The regulation of vascular diameter by vasoconstrictors and vasodilators requires that vascular smooth muscle cells (VSMCs) be physically coupled to extracellular matrix (ECM) and neighbouring cells in order for a vessel to mechanically function and transfer force. The hypothesis was tested that integrin-mediated adhesion to the ECM is dynamically up-regulated in VSMCs during contractile activation in response to a vasoconstrictor and likewise down-regulated during relaxation in response to a vasodilator. METHODS AND RESULTS: VSMCs were isolated from the Sprague-Dawley rat cremaster muscles. Atomic force microscopy (AFM) with fibronectin (FN)-functionalized probes was employed to investigate the biomechanical responses and adhesion of VSMCs. Responses to angiotensin II (Ang II; 10(-6) M) and adenosine (Ado; 10(-4) M) were recorded by measurements of cell cortical elasticity and cell adhesion. The results showed that Ang II caused an immediate increase in adhesion (+27%) between the probe and cell. Cell stiffness increased (+70%) in parallel with the adhesion change. Ado decreased adhesion (-15%) to FN and reduced (-30%) stiffness. CONCLUSION: Changes in the receptor-mediated activation of the contractile apparatus cause parallel alterations in cell adhesion and cell cortical elasticity. These studies support the hypothesis that the regulation of cell adhesion is coordinated with contraction and demonstrate the dynamic nature of cell adhesion to the ECM. It is proposed that coordination of adhesion and VSMC contraction is an important mechanism that allows for an efficient transfer of force between the contractile apparatus of the cell and the extracellular environment.

Cardiomyocyte contractile status is associated with differences in fibronectin and integrin interactions.

Integrins link the extracellular matrix (ECM) with the intracellular cytoskeleton and other cell adhesion-associated signaling proteins to function as mechanotransducers. However, direct quantitative measurements of the cardiomyocyte mechanical state and its relationship to the interactions between specific ECM proteins and integrins are lacking. The purpose of this study was to characterize the interactions between the ECM protein fibronectin (FN) and integrins in cardiomyocytes and to test the hypothesis that these interactions would vary during contraction and relaxation states in cardiomyocytes. Using atomic force microscopy, we quantified the unbinding force (adhesion force) and adhesion probability between integrins and FN and correlated these measurements with the contractile state as indexed by cell stiffness on freshly isolated mouse cardiomyocytes. Experiments were performed in normal physiological (control), high-K(+) (tonically contracted), or low-Ca(2+) (fully relaxed) solutions. Under control conditions, the initial peak of adhesion force between FN and myocyte alpha(3)beta(1)- and/or alpha(5)beta(1)-integrins was 39.6 +/- 1.3 pN. The binding specificity between FN and alpha(3)beta(1)- and alpha(5)beta(1)-integrins was verified by using monoclonal antibodies against alpha(3)-, alpha(5)-, alpha(3) + alpha(5)-, or beta(1)-integrin subunits, which inhibited binding by 48%, 65%, 70%, or 75%, respectively. Cytochalasin D or 2,3-butanedione monoxime (BDM), to disrupt the actin cytoskeleton or block myofilament function, respectively, significantly decreased the cell stiffness; however, the adhesion force and binding probability were not altered. Tonic contraction with high-K(+) solution increased total cell adhesion (1.2-fold) and cell stiffness (27.5-fold) compared with fully relaxed cells with low-Ca(2+) solution. However, it could be partially prevented by high-K(+) bath solution containing BDM, which suppresses contraction by inhibiting the actin-myosin interactions. Thus, our results demonstrate that integrin binding to FN is modulated by the contractile state of cardiac myocytes.

Cell-based and direct gene transfer-induced angiogenesis via a secreted chimeric fibroblast growth factor-1 (sp-FGF-1) in the chick chorioallantoic membrane (CAM).

Fibroblast growth factor-1 (FGF-1) is a potent angiogenic factor; its structure lacks a signal peptide for secretion. We previously reported that the overexpression of a secreted version of FGF-1 (sp-FGF-1) in microvascular endothelial cells (ECs) enhances cell migration [Partridge et al. J Cell Biochem 2000; 78(3): 487]. In the current study, we have examined the angiogenic effects of sp-FGF-1 in chicken chorioallantoic membranes (CAMs). Two methods of examining the effects of sp-FGF-1 in CAMs were used: cell-mediated transfection via bovine ECs and direct gene transfection. In the cell-mediated gene transfection, those eggs that were implanted with a gelatin sponge seeded with ECs stably transfected to over-express sp-FGF-1 protein showed a significant increase in angiogenesis inside the sponge when compared to eggs treated with vector control-transfected ECs. In the direct gene transfer, eggs received sp-FGF-1 showed a significant increase in vascularization when compared to eggs received vector alone plasmids. These CAM models are useful both for studying molecular mechanisms of angiogenesis and for developing better gene therapy strategies.

Delayed arteriolar relaxation after prolonged agonist exposure: functional remodeling involving tyrosine phosphorylation.

Although arteriolar contraction is dependent on Ca2+-induced myosin phosphorylation, other mechanisms including Ca2+ sensitization and time-dependent phenomena such as cytoskeletal and cellular reorganization may contribute to contractile events. We hypothesized that if arteriolar smooth muscle exhibits time-dependent behavior this may be manifested in differences in relaxation after short- and long-term exposure to contractile agonists. Studies were conducted in isolated arterioles pressurized to 70 mmHg. In initial experiments (n = 10), rate of relaxation was measured after acute (5 min) or prolonged (4 h) exposure to 5 microM norepinephrine (NE). Prolonged exposure to NE resulted in significantly (P < 0.05) increased time for relaxation in physiological salt solution. Rapid relaxation of vessels exposed to NE for 4 h was observed after superfusion with 0 mM Ca2+ buffer, indicating that the alteration in relaxation was reversible and Ca2+ dependent. A similarly impaired dilation was not observed with 4-h exposure to KCl (75 mM). To determine mechanisms contributing to the effects of prolonged NE exposure, studies were performed in the presence of the microtubule depolymerizing agent demecolcine (10 microM) or a series of tyrosine phosphorylation inhibitors. Although demecolcine caused significant vasoconstriction (P < 0.05) and potentiated NE vasoconstriction, it did not prevent the effect of long-term NE exposure on relaxation. Genistein, although having no effect on acute NE-induced contraction, concentration-dependently inhibited prolonged NE constriction. Similarly, Src (PP1) and p42/44 MAP kinase (PD-98059) inhibitors prevented maintenance of long-term NE contraction. The data indicate that prolonged exposure to NE induces biochemical alterations that impair relaxation after removal of the agonist. The contractile effects are Ca2+ dependent and involve tyrosine phosphorylation but do not appear to involve the polymerization state of the microtubule network.

Microtubule-dependent regulation of vasomotor tone requires Rho-kinase.

Changes in microtubule polymerization state have been shown to affect many cellular events, including the contractile properties of smooth muscle. We have previously shown that depolymerization of microtubules causes significant vasoconstriction in arterioles. This vasoconstriction does not require the endothelium or an increase in vascular smooth muscle Ca2+. Consequently, we hypothesized that a Ca2+-sensitizing mechanism may be involved in the constrictor response. The purpose of these experiments was to further elucidate cell signaling pathways responsible for vasoconstriction following microtubule disruption. Rat skeletal muscle arterioles were isolated, cannulated and pressurized without intraluminal flow. All arterioles used for experiments developed spontaneous, myogenic tone (54% of passive diameter). Microtubule depolymerization with colcemid or vinblastine caused arterioles to constrict by an additional 20% from resting basal diameter. In addition, arterioles treated with colcemid showed significantly enhanced responsiveness to norepinephrine and reduced responsiveness to adenosine. To investigate a role for Rho-kinase, vessels were incubated with inhibitors of the Rho-kinase pathway - Y-27632 or C3 exoenzyme. Inhibition of Rho-kinase significantly inhibited the constriction associated with colcemid-induced microtubule depolymerization. Inhibition of Rho-kinase also abolished the increased responsiveness to norepinephrine whereas adenosine responsiveness continued to be reduced. By comparison, inhibition of the tyrosine kinase, Src, with PP2 did not have any effect on the colcemid-induced changes in vascular tone or reactivity. These data indicate that the vasoconstriction and enhanced norepinephrine reactivity associated with microtubule disruption involves a Ca2+-sensitization process that is mediated by the Rho-kinase pathway.

Shear stress-induced release of basic fibroblast growth factor from endothelial cells is mediated by matrix interaction via integrin alpha(v)beta3.

Considering that chronic elevation of shear stress results in remodeling of the vasculature, we analyzed whether mechanical load could mediate basic fibroblast growth factor (bFGF) release and whether bFGF would act as mediator of shear stress-induced endothelial proliferation and differentiation. Supernatant media of shear stress-exposed endothelial cells (EC) contained significantly higher amounts of bFGF than medium from static cells. Released bFGF was fully intact with regard to its function as an inductor of proliferation and differentiation. Shear stress-conditioned media induced capillary-like structure formation, whereas static control medium did not. Likewise, only shear stress-conditioned medium induced proliferation of serum starved EC. Both capillary-like structure formation and proliferation could be inhibited by neutralization of bFGF or its receptor. The release of bFGF was subject to specific, integrin-mediated control, since inhibition of alpha(v)beta(3) integrin prevented it, whereas inhibition of alpha(5)beta(1) integrin had no effect. We conclude that shear stress induces the release of bFGF from EC in a tightly controlled manner. The release is dependent on specific cell-matrix interactions via alpha(v)beta(3) integrins. The effects on cell proliferation and differentiation suggest that release of bFGF is functionally significant and may represent a necessary initial step in adaptive remodeling processes induced by shear stress.