Estrogen-producing steroidogenic pathways in parietal cells of the rat gastric mucosa

Recently we demonstrated the presence of aromatase (P450(arom)), estrogen synthetase, and the active production of estrogen in parietal cells of the rat stomach. We therefore investigated the steroidogenic pathways of estrogen and also other steroid metabolites in the gastric mucosa of male rats, by showing the mRNA expression of steroidogenic enzymes using RT-PCR and in situ hybridization histochemistry, and by measuring the blood concentration of steroids in the artery and the portal vein. RT-PCR analysis showed the strong mRNA expression of 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), 17beta-hydroxysteroid dehydrogenase (HSD) type III and P450(arom), and the weak mRNA expression of 17beta-HSD type II, 5alpha-reductase type I and 3alpha-HSD. The other mRNAs of steroidogenic enzymes examined were not detected. In situ hybridization histochemistry demonstrated the localization of mRNAs for P450(17alpha), 17beta-HSD type III and P450(arom) in the parietal cells. Higher levels of progesterone and testosterone were found in the artery compared with the portal vein. Higher amounts of estrone and 17beta-estradiol, by contrast, were present in the portal vein compared with the artery. These results indicate that parietal cells of rat stomach convert circulating progesterone and/through androstenedione and testosterone to synthesize both estrone and 17beta-estradiol, which then enter the liver via the portal vein.     

Cloning, purification, and characterization of a non-collagenous anti-angiogenic protein domain from human alpha1 type IV collagen expressed in Sf9 cells

alpha1(IV)NC1, a cleavage fragment of the carboxy terminal non-collagenous human alpha1 chain of type IV collagen, is derived from the extracellular matrix specifically by MMP-2. Recently we determined the in vitro and in vivo anti-angiogenic activity of alpha1(IV)NC1 and presently, its role in cancer therapy is under evaluation. To characterize alpha1(IV)NC1 as a potential candidate for drug development and to test its efficacy in animal models, an effective method to produce a purified active form of alpha1(IV)NC1 is needed. In the present study, expression of alpha1(IV)NC1 in Sf9 cells using baculovirus expression system was discussed, this method was found to be effective in the production of a functionally active soluble form of the recombinant protein. The purified protein showed its characteristic activities such as inhibiting cell proliferation, migration, and tube formation in endothelial cells.     

Fibroblast nemosis induces angiogenic responses of endothelial cells

Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-κB. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.     

Rho-kinase mediates lysophosphatidic acid-induced IL-8 and MCP-1 production via p38 and JNK pathways in human endothelial cells

Lysophosphatidic acid (LPA), an inflammatory mediator that is elevated in multiple inflammatory diseases, is a potent activator of Rho kinase (ROCK) signaling and of chemokine production in endothelial cells. In this study, LPA activated ROCK, p38, JNK and NF-κB pathways and induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) mRNA and protein expression in human endothelial cells. We mapped signaling events downstream of ROCK, driving chemokine production. In summary, MCP-1 production was partly regulated by ROCK acting upstream of p38 and JNK and mediated downstream by NF-κB. IL-8 production was largely driven by ROCK through p38 and JNK activation, but with no involvement of NF-κB.     

Intracellular proteolytic activity of cathepsin B is associated with capillary-like tube formation by endothelial cells in vitro

The lysosomal cysteine protease cathepsin B is implicated in degradation of extracellular matrix (ECM), a crucial step in a variety of physiological and pathological processes, including tumor dissemination and angiogenesis. In this study, we analyzed the contribution of extracellular and intracellular cathepsin B activity on the formation of capillary-like tubular structures by human umbilical vein endothelial cells (HUVECs) grown on Matrigel matrix, using general and specific cysteine protease inhibitors. We demonstrated, by confocal assay using quenched fluorescent protein substrate DQ-collagen IV, that endothelial cells degrade ECM both intracellularly and pericellularly. Intracellular cathepsin B activity detected by degradation of Z-Arg-Arg cresyl violet substrate was co-localized with the products of DQ-collagen IV degradation in the perinuclear region and in the capillary-like tubular structures. Treatment of cells with membrane-permeable CA-074 Me effectively abolished intracellular cathepsin B activity, and resulted in reduced tube length (32.3+/-9.4% at 10 microM), total tubule area (49.6+/-12.4% at 10 microM), and the number of branch points of tubules (47.5+/-7.7% at 10 microM) in a dose-dependent manner. In contrast, CA-074 (0.1-10 microM), a membrane-impermeable cathepsin B specific inhibitor, general cysteine protease inhibitors chicken cystatin (5 microM) and E-64 (10 microM), and the metalloprotease inhibitor Minocycline (10 microM) showed no significant inhibitory effect in our angiogenesis model. These results show that, besides multiple regulatory molecules, intracellular cathepsin B also contributes to the neovascularization process and should be considered as a potential therapeutic target.     

Computational Phlebology: The Simulation of a Vein Valve

We present a three-dimensional computer simulation of the dynamics of a vein valve. In particular, we couple the solid mechanics of the vein wall and valve leaflets with the fluid dynamics of the blood flow in the valve. Our model captures the unidirectional nature of blood flow in vein valves; blood is allowed to flow proximally back to the heart, while retrograde blood flow is prohibited through the occlusion of the vein by the valve cusps. Furthermore, we investigate the dynamics of the valve opening area and the blood flow rate through the valve, gaining new insights into the physics of vein valve operation. It is anticipated that through computer simulations we can help raise our understanding of venous hemodynamics and various forms of venous dysfunction.     

Heat-shock protein 27 is a major methylglyoxal-modified protein in endothelial cells

In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d-glucose produced approximately 2-fold higher levels of methylglyoxal but not 3-deoxyglucosone and glyoxal, as compared to 5 mM d-glucose. Under hyperglycaemic conditions, the methylglyoxal-arginine adduct argpyrimidine as detected with a specific antibody, but not N(e)-(carboxymethyl)lysine and N(e)-(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARgamma ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine-modified protein. With in-gel digestion and mass spectrometric analysis, we identified this major protein as heat-shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.     

Differential leaf expansion can enable hydraulic acclimation to sun and shade

Although leaf size is one of the most responsive plant traits to environmental change, the functional benefits of large versus small leaves remain unclear. We hypothesized that modification of leaf size within species resulting from differences in irradiance can allow leaves to acclimate to different photosynthetic or evaporative conditions while maintaining an efficient balance between hydraulic supply (vein density) and evaporative demand. To test this, we compared the function and anatomy of leaf hydraulic systems in the leaves of a woody angiosperm (Toona ciliata M. Roem.) grown under high and low irradiance in controlled conditions. Our results confirm that in this species, differential leaf expansion regulates the density of veins and stomata such that leaf hydraulic conductance and stomatal conductance remain proportional. A broader sample of field-grown tree species suggested that differences in leaf venation and stomatal traits induced by sun and shade were not regulated by leaf size in all cases. Our results, however, suggest that leaf size plasticity can provide an efficient way for plants to acclimate hydraulic and stomatal conductances to the contrasting evaporative conditions of sun and shade.