Development of vascular smooth muscle contractility by endothelium-derived transforming growth factor ß proteins.

It is well established that the release of vasodilators and vasoconstrictors from vascular endothelium regulates vascular smooth muscle contraction. In this report, we investigate the role of the endothelium in the development and maintenance of constitutive vascular contractility. For that purpose, contractile activity of cultured bovine aortic smooth muscle cells (BASMCs) embedded in collagen gels was monitored by changes in gel diameter. After culturing for 5 days, ATP- and high KCl solution-induced contractions were significantly enhanced in the gels that were overlaid with bovine aortic endothelial cells (BAECs) or were cultured with conditioned medium of cultured BAECs. ATP-induced Ca(2+) transients, recorded in BASMCs cultured with conditioned medium of BAECs, were markedly augmented, but high KCl-induced Ca(2+) transients were not affected. BASMCs in control gels were spindle shaped, and those in endothelium-treated gels were more elongated and interconnected. The endothelial conditioned medium also strongly affected the intracellular distribution of actin fibers. Conditioned medium of BAECs contained TGFß1 and TGFß2. The TGFß receptor antagonist SB431542 as well as simultaneous treatment with TGFß1 and TGFß2 neutralizing antibodies completely reversed the above effects of endothelial conditioned medium on BASMCs. BAECs medium induced phosphorylation of Smad2 and increased ATP-induced phosphorylation of myosin light chain in BASMCs. The present results indicate that the release of TGFß1 and TGFß2 from vascular endothelium affects the contractility of vascular smooth muscle cells by altering their morphology and agonist-induced Ca(2+) mobilization.

Endothelium-dependent epithelial-mesenchymal transition of tumor cells: exclusive roles of transforming growth factor ß1 and ß2.

BACKGROUND: Induction of epithelial-mesenchymal transition (EMT) is essential for the metastasis of tumor cells and maintaining their stemness. This study aimed to examine whether endothelial cells, which are most closely located to tumor cells in vivo, play a role in inducing EMT in tumor cells or not. METHODS: Concentrated culture medium of bovine aortic endothelial cells (BAECs) was applied to tumor cell lines (A549 and PANC-1) and epithelial cell line (NMuMg). Cadherin conversion, expressions of α-smooth muscle actin and ZO-1, actin fiber formation and cell migration were examined as hallmarks of the induction of EMT in these cell lines. Transforming growth factor ß (TGFß) antibodies were used to neutralize TGFß1, TGFß2 and TGFß3. Expression and release of TGFß proteins in BAECs as well as in porcine and human endothelial cells were assessed by Western blotting and ELISA, respectively. RESULTS: Conditioned medium of BAEC induced EMT in the examined cell lines. All endothelial cells from various species and locations expressed TGFß1 and TGFß2 proteins and much lower level of TGFß3 protein. Conditioned medium from these endothelial cells contained TGFß1 and TGFß2, but TGFß3 could not be detected. Neutralizing antibody against each of TGFß1 or TGFß2 did not reverse endothelium-dependent EMT, but simultaneous neutralization of both TGFß1 and TGFß2 completely abolished it. CONCLUSIONS: Endothelial cells may play a role in the induction and maintenance of EMT in tumor cells by constitutively releasing TGFß1 and TGFß2. GENERAL SIGNIFICANCE: The present results provide a novel strategy of the inhibition of tumor metastasis by targeting vascular endothelium.

Transforming growth factor beta1 alters calcium mobilizing properties and endogenous ATP release in A549 cells: possible implications for cell migration.

We examined the effects of transforming growth factor beta(1) (TGFbeta(1)) on cellular functions in human lung cancer cell line A549. Treatment of A549 cells with 1 ng/ml TGFbeta(1) for more than 3 days altered their morphology from an epithelial cobblestone-like appearance to a fibroblast-like one, reduced the expression of E-cadherin mRNA and protein, and induced the formation of F-actin fibers. These hallmarks indicate that TGFbeta(1) induced the epithelial-mesenchymal transition in A549 cells. Migration of TGFbeta(1)-treated A549 cells, which was quantified by the wound-healing assay, was markedly accelerated by 3 microM ATPgammaS, a non-hydrolyzable ATP analogue. ATPgammaS-induced migration of TGFbeta(1)-treated A549 cells was reversed by the P2 antagonist suramin. In contrast, migration of control A549 cells was not altered by ATPgammaS. TGFbeta(1)-treated A549 cells showed an augmentation of ATP-induced Ca(2+) transients, thapsigargin-induced Ca(2+) transients, and store-operated Ca(2+) entry compared with those in control cells. Basal level of the extracellular ATP concentration was significantly lower in TGFbeta(1)-treated A549 cells than in control cells. We conclude from these results that TGFbeta(1) augments ATP-induced Ca(2+) mobilization, which leads to the acceleration of migration, in A549 cells but, it markedly reduces endogenous ATP release. This implies that the actions of ATP would become a novel therapeutic target for inhibiting cancer cell migration.

Hypergravity induces ATP release and actin reorganization via tyrosine phosphorylation and RhoA activation in bovine endothelial cells.

Mechanical stresses regulate physiological and pathological functions of vascular endothelial cells. We examined, in this study, the effects of hypergravity on endothelial functions. Hypergravity (3 G) applied by low speed centrifuge immediately induced a membrane translocation of small G-protein RhoA and tyrosine phosphorylation of 125 kDa FAK in bovine aortic endothelial cells (BAECs). Hypergravity also induced a transient reorganization of actin fibers in 3 min, which was inhibited by Rho-kinase inhibitor (Y27632) and tyrosine kinase inhibitors (herbimycin A and tyrphostin 46). Furthermore, the extracellular ATP concentration ([ATP]o) was increased by 2 G and 3 G hypergravity in 5 min, and the inhibitors of Rho-kinase, tyrosine kinase, and volume-regulated anion channels (VRAC; verapamil, tamoxifen and fluoxetine) significantly suppressed [ATP]o elevation. Application of 3 G hypergravity for 1 h increased the nuclear uptake of BrdU, which was inhibited by Rho-kinase inhibitor and VARC inhibitors. Furthermore, intermittent application of 3 G hypergravity for 1 or 2 h/day stimulated endothelial migration in 5 days, and this was inhibited by suramin, a P2 antagonist. Collectively, these results indicate that hypergravity induces ATP release and actin reorganization via RhoA activation and FAK phosphorylation, thereby activating cell proliferation and migration in BAECs. These also suggest that gravity can be regarded as an extracorporeal signal that could significantly affect endothelial functions.

Heparan sulfate proteoglycan is essential to thrombin-induced calcium transients and nitric oxide production in aortic endothelial cells.

Thrombin induces Ca(2+) transients and subsequent nitric oxide (NO) production in vascular endothelial cells. Thrombin cleaves protease-activated receptors, resulting in activation of intracellular signals, but it is not clarified how the extracellular thrombin stays around the cells to exert its enzyme activities. This study aimed to investigate the possible involvement of heparin sulfate proteoglycan (HSPG) in the effects of thrombin on vascular endothelium. Heparinase III completely removed the polysaccharide chain of HSPG in bovine aortic endothelial cells (BAECs). Thrombin induced Ca(2+) transients in control BAECs, but not in heparinase III-treated BAECs. In contrast, ATP induced Ca(2+) transients both in control and heparinase III-treated BAECs. Thrombin that was pre-incubated with heparin also failed to induced Ca(2+) transients in BAECs. Furthermore, thrombin-induced NO production, as assessed with DAF-2 fluorescence, was suppressed in heparinase III-treated BAECs and by the pre-incubation of thrombin with heparin. ATP-induced NO production was, however, not affected in heparinase III-treated BAECs. These results indicate that it is essential for thrombin to bind to the polysaccharide chain of HSPG for inducing Ca(2+) transients and NO production in BAECs.

Involvement of heparan sulfate proteoglycan in sensing hypotonic stress in bovine aortic endothelial cells.

Hypotonic stress (HTS) induces various responses in vascular endothelium, but the molecules involved in sensing HTS are not known. To investigate a possible role of heparan sulfate proteoglycan (HSPG) in sensing HTS, we compared the responses of control bovine aortic endothelial cells (BAECs) with those of cells treated with heparinase III, which exclusively degrades HSPG. Tyrosine phosphorylation of 125 kDa FAK induced by HTS (-30%) in control cells was abolished in heparinase III-treated BAECs. The amplitude of the volume-regulated anion channel (VRAC) current, whose activation is regulated by tyrosine kinase, was significantly reduced by the treatment with heparinase III. Also, HTS-induced ATP release through the VRAC pore and the concomitant Ca(2+) transients were significantly reduced in the heparinase III-treated BAECs. In contrast, exogenously applied ATP evoked similar Ca(2+) transients in both control and heparinase III-treated BAECs. The transient formation of actin stress fibers induced by HTS in control cells was absent in heparinase III-treated BAECs. Lysophosphatidic acid (LPA) also induced FAK phosphorylation, actin reorganization and ATP release in control BAECs, but heparinase III did not affect these LPA-induced responses. We conclude from these observations that HSPG is one of the sensory molecules of hypotonic cell swelling in BAECs.

Protein kinase A inhibits lysophosphatidic acid-induced migration of airway smooth muscle cells.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that is released from activated platelets and affects contractile properties of airway smooth muscle cells. However, possible roles of LPA on cell migration, one of the initial events of airway remodeling, are not clarified. This study aimed to examine the effects of LPA on migration and actin fiber formation in bovine tracheal smooth muscle cells (BTSMCs). Random and oriented cell migrations were examined with wound assay and Boyden chamber assay, respectively. Cytosolic actin fibers were stained with rhodamine-phalloidin. Membrane translocation of RhoA, a hallmark of RhoA activation, was assessed by Western blotting. LPA augmented the migration of BTSMCs from wounded confluent monolayer but did not accelerate the chemotactic migration toward LPA. LPA also induced a transient actin reorganization and RhoA activation. Dense actin fibers were observed mainly in the wound edge but not in migrated cells, thereby suggesting the role of actin reorganization in the initiation of cell migration. LPA-induced actin fiber formation was blocked by Y27632 [R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide], an inhibitor of Rho kinase. Effects of LPA on migration and actin fiber formation were also inhibited by cAMP-elevating agents, i.e., dibutyryl cAMP, forskolin, isoproterenol, and theophylline. KT5720 (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester], a protein kinase A (PKA) inhibitor, reversed the inhibitory actins of cAMP on LPA-induced responses. These results indicate that LPA induces cAMP/PKA-sensitive, RhoA-mediated random migration of BTSMCs. Regulation of this mechanism would be beneficial for the control of airway remodeling.

Proapoptotic nitric oxide production in amyloid beta protein-treated cerebral microvascular endothelial cells.

OBJECTIVE: The objective of this study was to investigate the effects of amyloid beta protein (Abeta) on cerebral microvascular endothelium, and their possible involvement in Abeta-induced apoptosis in the neighboring cells. METHODS: Cultured bovine brain microvascular endothelial cells (BBECs) were incubated with Abeta for 24 h. Production of nitric oxide (NO) was assessed by nitric oxide-sensitive fluorescent dye, DAF-2, and the expression of NO synthase (NOS) proteins was examined by Western blotting. Effects of Abeta-treated microvascular endothelium on the DNA damage of the neighboring cells were assessed by single-cell gel electrophoresis. RESULTS: Abeta increased the expression of iNOS protein, but did not affect eNOS and nNOS expressions in BBECs. Abeta-treated BBECs showed spontaneous NO production in the presence of L-arginine. The neural cell line PC12 showed marked apoptosis after being co-cultured with Abeta-treated BBECs for 48 h, and the apoptosis was as potent as that induced by the inflammatory stimuli lipopolysaccharide and interferon-gamma. The DNA damage of PC12 cells evoked by co-culture with Abeta-treated BBECs was prevented by L-NG-nitroarginine methyl ester, an inhibitor of NOS. CONCLUSIONS: These results indicate that Abeta induces the expression of iNOS in BBECs, and that microvascular endothelium-derived NO may induce apoptosis in neighboring neural cells.

Constitutive nitric oxide production in bovine aortic and brain microvascular endothelial cells: a comparative study.

Vascular endothelium constitutively generates nitric oxide (NO) in large vessels and induces a relaxation of smooth muscle cells. However, little is known about the production of NO in microvessels, where smooth muscle layers are thin or absent. In this study, we have compared the constitutive production of NO in bovine brain microvascular endothelial cells (BBECs) with that in bovine aortic endothelial cells (BAECs). ATP, acetylcholine (ACh) and A23187 induced Ca(2+) transients both in BBECs and BAECs. In contrast, although ATP and A23187 evoked a similar degree of [Ca(2+)](i) increase in both types of cell, they failed to induce NO production in BBECs, as measured with an NO-sensitive fluorescent dye DAF-2, whereas in BAECs there was an increase in DAF-2 fluorescence. Hypotonic stress induced ATP release and subsequent NO production in BAECs, but not in BBECs. We have developed an in vitro model vessel system that consists of aortic smooth muscle cells embedded in a collagen gel lattice and overlaid with endothelial cells. Precontracted gels showed relaxation in response to ACh, when BAECs were overlaid. However, ACh-induced relaxation was not observed in BBEC-overlaid gels. Expression of eNOS protein as well as cellular uptake of l-[(3)H]arginine were significantly lower in BBECs than in BAECs. These results indicate that Ca(2+)-dependent NO production is at an undetectable level in BBEC, for which at least two factors, i.e. low levels of eNOS expression and l-arginine uptake, are responsible.

Functional implications of Ca2+ mobilizing properties for nitric oxide production in aortic endothelium.

We have investigated the relationship between Ca2+ mobilization and the cellular production of nitric oxide (NO) by using fura-2 and diaminofluorescein-2 (DAF-2), an NO-sensitive dye, in bovine aortic endothelial cells (BAEC). High concentrations of ATP (100 microM) or thapsigargin (1 micro M) depleted intracellular Ca2+ store sites with a single Ca2+ transient, and induced an increase in DAF-2 fluorescence even in Ca2+-free solution, thereby indicating that store depletion leads to NO production. The same level of increase in DAF-2 fluorescence was elicited by low concentrations of ATP (1 micro M), which induced Ca2+ oscillations but did not deplete store sites, only in the presence of extracellular Ca2+. Furthermore, inhibition of ATP (1 micro M)-induced Ca2+ entry with La3+ suppressed DAF-2 fluorescence. ATP (0.3 micro M), applied in Ca2+-free, Mn2+-containing solution induced Mn2+ entry-coupled fura-2 quenching, repeating shortly after each oscillation peak. These results indicate that NO is produced preferentially by entered Ca2+, and that Ca2+ oscillations, which are induced by low levels of stimulation, play a significant role in NO production by strongly modulating Ca2+ entry.

Superoxide anion impairs contractility in cultured aortic smooth muscle cells.

We examined the effects of superoxide anion (O) generated by xanthine plus xanthine oxidase (X/XO) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and muscle contractility in cultured bovine aortic smooth muscle cells (BASMC). Cells were grown on collagen-coated dish for the measurement of [Ca(2+)](i). Pretreatment with X/XO inhibited ATP-induced Ca(2+) transient and Ca(2+) release-activated Ca(2+) entry (CRAC) after thapsigargin-induced store depletion, both of which were reversed by superoxide dismutase (SOD). In contrast, Ca(2+) transients induced by high-K(+) solution and Ca(2+) ionophore A-23187 were not affected by X/XO. BASMC-embedded collagen gel lattice, which was pretreated with xanthine alone, showed contraction in response to ATP, thapsigargin, high-K(+) solution, and A-23187. Pretreatment of the gel with X/XO impaired gel contraction not only by ATP and thapsigargin, but also by high-K(+) solution and A-23187. The X/XO-treated gel showed normal contraction; however, when SOD was present during the pretreatment period. These results indicate that O(2)(-) attenuates smooth muscle contraction by impairing CRAC, ATP-induced Ca(2+) transient, and Ca(2+) sensitivity in BASMC.

Volume-regulated anion channels serve as an auto/paracrine nucleotide release pathway in aortic endothelial cells.

Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca(2+) oscillations and NO production. They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a "permeating blocker model," in which ATP binds with an affinity of 1.0 +/- 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl(-) hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.