Primary Human Lung Alveolus-on-a-chip Model of Intravascular Thrombosis for Assessment of Therapeutics.

Pulmonary thrombosis is a significant cause of patient mortality; however, there are no effective in vitro models of thrombi formation in human lung microvessels that could also assess therapeutics and toxicology of antithrombotic drugs. Here, we show that a microfluidic lung alveolus-on-a-chip lined by human primary alveolar epithelium interfaced with endothelium and cultured under flowing whole blood can be used to perform quantitative analysis of organ-level contributions to inflammation-induced thrombosis. This microfluidic chip recapitulates in vivo responses, including platelet-endothelial dynamics and revealed that lipopolysaccharide (LPS) endotoxin indirectly stimulates intravascular thrombosis by activating the alveolar epithelium, rather than acting directly on endothelium. This model is also used to analyze inhibition of endothelial activation and thrombosis due to a protease activated receptor-1 (PAR-1) antagonist, demonstrating its ability to dissect complex responses and identify antithrombotic therapeutics. Thus, this methodology offers a new approach to study human pathophysiology of pulmonary thrombosis and advance drug development.

Rho small G-protein-dependent binding of mDia to an Src homology 3 domain-containing IRSp53/BAIAP2.

mDia1 is a downstream effector of Rho small G protein that is implicated in stress fiber formation and cytokinesis. We isolated an mDia1-binding protein and identified it to be IRSp53/BAIAP2. IRSp53 and BAIAP2 have independently been isolated as a 58/53-kDa protein tyrosine phosphorylated in response to insulin and a BAI1-binding protein, respectively. BAI1 is a brain-specific seven-span transmembrane protein capable of inhibiting angiogenesis. The proline-rich formin homology 1 domain of mDia1 bound the Src homology 3 domain of IRSp53/BAIAP2 in a GTP-Rho-dependent manner. The results suggest that IRSp53/BAIAP2 is a downstream effector of mDia1.

Physical and functional interaction of rabphilin-11 with mammalian Sec13 protein. Implication in vesicle trafficking.

Rab11a small G protein (Rab11p) is implicated in vesicle trafficking, especially vesicle recycling. We have previously isolated a downstream effector of Rab11p, named rabphilin-11. We found here that rabphilin-11 directly bound the mammalian counterpart of yeast Sec13 protein (mSec13p) in cell-free and intact cell systems. Yeast Sec13p is involved as a component of coat proteins II in the Sar1p-induced vesicle formation from the endoplasmic reticulum, but the precise role of mSec13p is unknown. The interaction of rabphilin-11 with mSec13p was enhanced by GTP-Rab11p. Rabphilin-11 localized on the vesicles in perinuclear regions and along microtubules oriented toward the plasma membrane, whereas mSec13p partly colocalized with rabphilin-11 in the perinuclear regions, most presumably the Golgi complex. Disruption of the rabphilin-11-mSec13p interaction by overexpression of the mSec13p-binding region of rabphilin-11 impaired vesicle trafficking. These results indicate that the rabphilin-11-mSec13p interaction is implicated in vesicle trafficking.

Distinct actions and cooperative roles of ROCK and mDia in Rho small G protein-induced reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells.

Rho, a member of the Rho small G protein family, regulates the formation of stress fibers and focal adhesions in various types of cultured cells. We investigated here the actions of ROCK and mDia, both of which have been identified to be putative downstream target molecules of Rho, in Madin-Darby canine kidney cells. The dominant active mutant of RhoA induced the formation of parallel stress fibers and focal adhesions, whereas the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, and the dominant active mutant of mDia induced the weak formation of parallel stress fibers without affecting the formation of focal adhesions. In the presence of C3 ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, whereas the dominant active mutant of mDia induced only the diffuse localization of actin filaments. These results indicate that ROCK and mDia show distinct actions in reorganization of the actin cytoskeleton. The dominant negative mutant of either ROCK or mDia inhibited the formation of stress fibers and focal adhesions, indicating that both ROCK and mDia are necessary for the formation of stress fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK and mDia were necessary for the 12-O-tetradecanoylphorbol-13-acetate-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. The morphologies of stress fibers and focal adhesions in the cells expressing both the dominant active mutants of ROCK and mDia were not identical to those induced by the dominant active mutant of Rho. These results indicate that at least ROCK and mDia cooperatively act as downstream target molecules of Rho in the Rho-induced reorganization of the actin cytoskeleton.

Rab11BP/Rabphilin-11, a downstream target of rab11 small G protein implicated in vesicle recycling.

Rab11 small G protein has been implicated in vesicle recycling, but its upstream regulators or downstream targets have not yet been identified. We isolated here a downstream target of Rab11, named rabphilin-11, from bovine brain. Moreover, we isolated from a rat brain cDNA library its cDNA, which encoded a protein with a M(r) of 100,946 and 908 amino acids (aa). Rabphilin-11 bound GTP-Rab11 more preferentially than GDP-Rab11 at the N-terminal region and was specific for Rab11 and inactive for other Rab and Rho small G proteins. Both GTP-Rab11 and rabphilin-11 were colocalized at perinuclear regions, presumably the Golgi complex and recycling endosomes, in Madin-Darby canine kidney cells. In HeLa cells cultured on fibronectin, both the proteins were localized not only at perinuclear regions but also along microtubules, which were oriented toward membrane lamellipodia. Treatment of HeLa cells with nocodazole caused disruption of microtubules and dispersion of GTP-Rab11 and rabphilin-11. Overexpression of the C-terminal fragment of rabphilin-11 (aa 607-730), lacking the GTP-Rab11 binding domain, in HeLa cells reduced accumulation of transferrin at perinuclear regions and cell migration. Rabphilin-11 turned out to be a rat counterpart of recently reported bovine Rab11BP. These results indicate that rabphilin-11 is a downstream target of Rab11 which is involved in vesicle recycling.

Interaction of radixin with Rho small G protein GDP/GTP exchange protein Dbl.

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various actin cytoskeleton-dependent cell functions. The Rho subfamily members regulate ERM (ezrin, radixin and moesin)-dependent association of the actin cytoskeleton with the plasma membrane. Moreover, the N-terminal regions of ERM interact with Rho GDI, an inhibitory regulator of all the Rho family members, and reduce its inhibitory action, finally initiating the activation of the Rho family members. We show here that the N-terminal region of radixin furthermore interacts with Dbl, a stimulatory GDP/GTP exchange protein of the Rho family members. This interaction does not affect the Dbl activity to stimulate the GDP/GTP exchange reaction of RhoA, a member of the Rho subfamily. Dbl does not interact with radixin which is precomplexed with Rho GDI, and Rho GDI displaces Dbl from radixin. Thus, radixin plays an important role in activation of the Rho family members by recruiting their positive and negative regulators.

Interactions of drebrin and gephyrin with profilin.

Profilin is an actin monomer-binding protein which stimulates actin polymerization. Recent studies have revealed that profilin interacts with VASP, Mena, Bnilp, Bnrlp, and mDia, all of which have the proline-rich domain. Here, we isolated three profilin-binding proteins from rat brain cytosol by glutathione S-transferase-profilin affinity column chromatography and identified them as Mena, drebrin, and gephyrin. These proteins had a proline-rich domain and directly interacted with profilin.

Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein.

The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. We have reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Here we report that Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system.

Involvement of a sperm protein sensitive to sulfhydryl-depleting reagents in mouse sperm-egg fusion.

Effects of various sulfhydryl (SH)-depleting reagents on sperm-egg fusion were demonstrated. When sperm were treated with three plasma membrane-permeable SH-depleting reagents, N-ethyl-maleimide, sodium tetrathionate and 5,5'-dithiobis (2-nitro-benzoic acid), the rates of cleavage of eggs were significantly lower than those with control sperm. Neither the motility, penetration of the zona pellucida, acrosomal status of the sperm, nor sperm-egg binding was affected by the SH-depleting reagents. Fusion of sperm and zona-free egg was estimated by the sperm nuclear incorporation into the eggs, intracellular calcium mobilization, and cortical granule exocytosis in the eggs. Sperm-egg fusion was blocked dose-dependently when sperm were exposed to membrane-permeable SH-depleting reagents, but was not blocked by a membrane-impermeable SH-depleting reagent, eosin-5-maleimide. Blockage of fusion by sodium tetrathionate was completely reversed by an SH-reductant, dithiothreitol. These results suggest that a protein which is sensitive to SH-depleting reagents may play an important role in mouse sperm-egg fusion and that the functional SH region of the protein may be located at an intracellular site.

Involvement of Rabphilin-3A in cortical granule exocytosis in mouse eggs.

Rabphilin-3A is a putative target protein for Rab3A, a member of the small GTP-binding protein superfamily that has been suggested to play a role in regulated exocytosis in presynapses. In this study we determined the expression and the function of Rabphilin-3A in mouse eggs at fertilization. Rabphilin-3A mRNA and protein were detected by reverse transcriptase-PCR and immunoblot analysis, respectively, in metaphase II mouse eggs. Immunofluorescence analysis showed that Rabphilin-3A protein was distributed in the cortical region in eggs. Sperm induces cortical granule (CG) exocytosis via an increase in cytosolic Ca2+ at fertilization. We microinjected the NH2- or COOH-terminal fragment of recombinant Rabphilin-3A into metaphase II eggs. Neither treatments altered the sperm-induced cytosolic Ca2+ increase, but both inhibited CG exocytosis in a dose-dependent manner. The NH2-terminal fragment was more effective than the COOH-terminal fragment. Full-length Rabphilin-3A did not affect CG exocytosis, but it attenuated the inhibition of CG exocytosis by the NH2-terminal fragment. These results show that Rabphilin-3A is involved in Ca(2+)-dependent CG exocytosis at fertilization in mouse eggs.

Reactive oxygen species block sperm-egg fusion via oxidation of sperm sulfhydryl proteins in mice.

The effects of reactive oxygen species (ROS) on mouse sperm-egg fusion were determined. Sperm were treated with superoxide and hydrogen peroxide generated by addition of xanthine oxidase (XO: 10-200 mlU/ml) to hypoxanthine (HX: 1 mM). While XO at concentrations higher than 100 mlU/ml decreased the motility and lipid peroxidation of sperm, XO at less than 50 mlU/ml had no such effect. However, 20-50 mlU/ml XO significantly suppressed sperm fusion with zona-free eggs. Two ROS scavengers, superoxide dismutase and catalase, attenuated the inhibition of sperm-egg fusion by HX-XO. The sulfhydryl (SH) reductant, dithiothreitol, also reversed the inhibition. The sperm SH-rich fusion-related proteins were highly sensitive to ROS. These results suggest that ROS at low concentrations may inhibit sperm-egg fusion via oxidation of the SH-proteins in the sperm membrane, without causing loss of motility.

Dynamics of cortical granule exocytosis at fertilization in living mouse eggs.

Sperm-egg fusion induces an intracellular free calcium concentration ([Ca2+]i) increase and exocytosis of cortical granules (CGs). Recently we used an impermeable fluorescent membrane probe, 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), to develop a method to evaluate the kinetics of exocytosis in single living cells. In this study we used digital imaging and confocal laser scanning microscopy to evaluate CG exocytosis in living mouse eggs with TMA-DPH. Time-related changes of CG exocytosis were estimated as the percent increase of TMA-DPH fluorescence. The increase of fluorescence in the egg started after sperm attachment, continued at an almost uniform rate, and ceased at 45-60 min. Whereas the [Ca2+]i increase at fertilization was transient or oscillatory, exocytosis was not always induced concomitantly with each [Ca2+]i peak. Next we used this method to determine some intracellular mediators of exocytosis in the egg. An intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and a microfilament inhibitor, cytochalasin B, blocked sperm-induced exocytosis. A guanosine 5'-triphosphate-binding protein activator, AlF4-, induced exocytosis. These results suggest that [Ca2+]i, microfilament, and guanosine 5'-triphosphate-binding proteins may be involved in CG exocytosis. In conclusion, this method has significant advantages for studying exocytosis in living eggs.

Inhibition of mouse placental lactogen-II release from placental cells by interleukin-1 after mid-pregnancy.

The effects of interleukin (IL)-1 and granulocyte-macrophage colony stimulating factor (GM-CSF), which are present in the mouse placenta, on the secretion of mouse placental lactogen (mPL)-1 and mPL-II by placental cells were tested in vitro. IL-1 alpha and IL-1 beta, 2.5 nmol/l each, significantly inhibited mPL-II secretion by cells from days 9 and 12 of pregnancy, but did not affect mPL-II secretion by cells from day 7 of pregnancy or mPL-I secretion by cells from days 7, 9 or 12 of pregnancy. GM-CSF had no effect on mPL-I and mPL-II secretion by cells from days 7, 9 or 12 of pregnancy. The inhibitory effects of IL-1 alpha and IL-1 beta on mPL-II secretion were completely eliminated by the addition of antibodies to IL-1 alpha and IL-1 beta respectively. Western blot analysis for mPL-II indicated that IL-1 alpha significantly reduced the intensity of the mPL-II band. Steady-state levels of mPL-II mRNA, assessed by Northern blot analysis, were reduced by incubation of placental cells from day 12 of pregnancy with 2.5 nmol/l IL-1 alpha for 5 days. Co-incubation of 0.25 pmol/l IL-1 alpha, 25 pmol/l IL-6, and 25 pmol/l tumor necrosis factor-alpha, each of which did not significantly inhibit mPL-II secretion by itself, together inhibited mPL-II secretion. These results suggest that IL-1, but not GM-CSF, is a potent inhibitor of mPL-II secretion after mid-pregnancy, and that the combined action of cytokines can inhibit mPL-II secretion.

Regulation of mouse soluble interleukin-6 receptor secretion by decidua.

Regulation of msIL-6R secretion by mIL-6 and 8-bromo cAMP was examined using primarily cell culture of mouse decidua. Mouse decidua on day 11 of pregnancy was digested by collagenase and decidual cells were cultured for up to 3 days. Addition of mIL-6 and 8-bromo cAMP resulted in significant inhibition of msIL-6R secretion from cultured mouse decidual cells by the 2nd day in culture. The effects were dose-dependent and the lowest concentrations of mIL-6 and 8-bromo cAMP which cause significant inhibition of msIL-6R secretion were 250 pM and 50 microM, respectively. Northern blot analysis indicated that treatment of decidual cells with 8-bromo cAMP significantly decreased the steady-state level of mIL-6R mRNA. However, treatment of decidual cells with mIL-6 did not affect the steady-state level of mIL-6R mRNA. These results suggest that mIL-6 decreases msIL-6R secretion without changing of the steady-state level of mIL-6R mRNA, and that cAMP is one of the second messengers in mouse decidual cells involved in this down regulation of msIL-6R secretion.

Scanning electron microscope assessment of exocytotic changes in purified gonadotropes.

These studies were undertaken to characterize the exocytotic changes in purified gonadotropes by three-dimensional imaging using scanning electron microscopy. Rat gonadotropes were purified using a fluorescence-activated cell sorter and an argon laser treatment system. The purified gonadotropes were stimulated with GnRH under various conditions and fixed for scanning electron microscopy. After the GnRH stimulation, many 'hole' structures (diameter 0.1-0.5 micron) were observed on the cell surface, and notably the population of cells with 10 or more holes was clearly increased. The pattern of the time-course of the changes in this population was perfectly consistent with the LH secretory profile of pituitary cells, and their formation of the cells with 10 or more holes was completely inhibited by pretreatment with a GnRH antagonist. Our data suggest that the hole structure represents an exocytotic opening site and that regulated exocytosis in purified gonadotropes can be evaluated by scanning electron microscopy. This method may be widely applicable to other endocrine cells.