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.

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.

Pivotal role of integrin alpha5beta1 in hypotonic stress-induced responses of human endothelium.

We have previously reported that both hypotonic stress (HTS) and lysophosphatidic acid (LPA) induce ATP release and a transient reorganization of actin through sequential activation of RhoA/Rho-kinase and focal adhesion kinase F-actin (FAK)/paxillin in human umbilical cord vein endothelial cells (HUVECs). LPA is known to induce the activation of RhoA via its specific receptors, but the mechanisms by which HTS initiates these intracellular signals are not known. The present study aimed to identify the molecule(s) that are unique to the sensing and/or transducing the mechanical stress. Reverse transcriptase-polymerase chain reaction revealed the expression of several integrin subunits in HUVECs. Anti-integrin alpha5beta1 antibody (Ab), but not anti-integrin alpha2, alpha6, alpha v, or beta4 antibodies, inhibited HTS-induced RhoA translocation, tyrosine phosphorylation of FAK and paxillin, ATP release, and actin reorganization. However, the LPA-induced ATP release and actin reorganization were not inhibited by any of these anti-integrin antibodies, indicating that integrin alpha5beta1 plays a pivotal role in the HTS-induced but not in the LPA-induced responses. It is therefore reasonable to assume that this particular subtype of integrin is involved in the initiation of the responses induced by mechanical stimuli in HUVECs.

A rare case of idiopathic hypereosinophilic syndrome involving the oral cavity associated with the esophagus and gastrointestinal tract.

We report the rare case of HES involving oral cavity associated with esophagus, and gastrointestinal tract, which we succeeded in diagnosing precisely through a biopsy specimen obtained from the lip. A 64-year-old man had dysphagia, swelling of the oral mucosa and the posterior cervical muscles, accompanied by an abdominal pain and diarrhea. Peripheral blood cell count showed marked eosinophilia. Computed tomography showed thickening of posterior wall of the pharynx, esophagus, and gastrointestinal tract. Histologic specimen obtained from the lower lip demonstrated a moderate infiltration of eosinophils. His clinical condition was improved by oral prednisolone therapy.