1 We studied the effect of a new angiotensin II type 1 (AT 1 ) receptor antagonist, olmesartan medoxomil (olmesartan), on the fibrogenic responses in rat hepatic stellate cells (HSCs) and liver fibrogenesis. 2 Olmesartan (1 mg kg À1 per day) was orally administered to fibrotic rats, induced by bile duct ligation. Liver hydroxyproline content, the mRNA expression of collagen a1(I) and a-smooth muscle actin (a-SMA), and plasma levels of transforming growth factor-b1 (TGF-b1) were significantly reduced by olmesartan treatment, suggesting that olmesartan improved liver fibrosis. Interestingly, AT 1 receptors were found to be expressed in a-SMA-positive cells in the fibrotic area of livers in bile duct-ligated rats by immunohistochemical analysis. Olmesartan treatment reduced the number of these cells. 3 In vitro experiments showed that angiotensin II (Ang II) treatment induced proliferation and collagen synthesis, and upregulated the profibrogenic cytokines, TGF-b1 and connective tissue growth factor (CTGF), in rat primary HSCs. Olmesartan blocked all these effects of Ang II. 4 Based on these results, since activated HSCs were found to express AT 1 receptors and Ang II is thought to play an important role in the pathogenesis of liver fibrosis by binding to these receptors, olmesartan may act as a potent antifibrotic drug to suppress the proliferation, collagen synthesis and the expression of profibrogenic cytokines in activated HSCs by blocking these receptors.

We report the construction of a single-component optogenetic Rac1 (opto-Rac1) to control actin polymerization by dynamic membrane recruitment.

Using dephosphorylated neurofilament (NF) proteins as substrates, the kinase with a higher activity for the dephosphorylated NF-H than the phosphorylated form of NF-H was searched for in the porcine brain extract. Most NF-H kinase activity in the brain extract pelleted with microtubules. The NF-H kinase purified from a high salt extract of the microtubule pellets was composed of cdk5 and a 26 kDa protein, a fragment of the 35 kDa regulatory subunit of cdk5. In contrast to the association of the active kinase with microtubules, each of uncomplexed cdk5 and the 35 kDa regulatory subunit was differently distributed in the supernatant fraction and the pellet, respectively, by ultracentrifugation of the brain extract. Dephosphorylated forms of NF-H and NF-M became reactive to antibodies recognizing in vivo phosphorylation sites (SMI31, 34, and 36, JJ31 and 51) by phosphorylation with cdk5/p26. cdk5/p26 showed similar enzymatic properties to p34cdc2/cyclin B kinase; the substrate specificity and inhibition by a p34cdc2 kinase specific inhibitor, butyrolactone I. However, p34cdc2/cyclin B kinase was distinguished from cdk5/p26 by its binding to p13suc1 protein and by its reactivity to anti-p34cdc2 antibodies. In spite of similar enzymatic properties of cdk5/p26 and p34cdc2/cyclin B kinase, cdk5/p26 did not display M-phase promoting activity when assayed with a cell-free system of Xenopus egg extract.

Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/Dimethylarginine dimethylaminohydrolase (DDAH)/Asymmetric Dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by iNOS) could play a role in idiopathic pulmonary fibrosis (IPF), sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high throughput screen for DDAH modulators.

The preparation of hepatocyte spheroids by adding a water-soluble synthetic polymer as an artificial matrix was performed in a cell suspension system. Cell-aggregation was promoted without cytotoxicity by adding Eudragit (a copolymer of methacrylic acid and methylmethacrylate) to the culture medium. Spheroid-like cell aggregates, whose liver functions were enhanced, were effectively formed in the presence of 0.1% Eudragit, independent of the cultural substratum. Moreover, the mass preparation of spheroids could be achieved with a high production yield by means of a suspension culture in a spinner flask. In this case, the polymer protected the cells from damage due to agitation. The spheroids induced with Eudragit expressed high liver functions, such as albumin secretion, ammonia removal, and urea synthesis. On histological observation, the spheroids showed a well-developed cell adhesion apparatus and bile canaliculi. In addition, a higher calcium ion concentration in the cells of spheroids was observed compared with in monolayer cells.

We studied spheroid (multicellular aggregate) formation by hepatocytes and the expression of liver-specific functions such as albumin secretion when hepatocytes were cultured with various extracellular matrices. Hepatocytes cultured on Primaria(R) and poly-D-lysine coated dishes, and in the presence of a polymer, Eudragit, formed spheroids, and they also exhibited higher liver-specific functions and poor growth compared to monolayer cultures. The results indicated that the cell morphological change and cell-cell interaction caused by the spheroid formation were key factors promoting the expression of the liver-specific functions. To elucidate the mechanism underlying the poor growth in spheroids, we examined the HGF signaling pathway. Phosphorylation and down-regulation of the HGF receptor (c-Met proto-oncogene product) were observed for the cells from both monolayer and spheroid cultures, but Ras activation was partly blocked in spheroids. Furthermore, we found that CDK inhibitors, p21 and p27, were highly expressed in spheroids. These results suggested that the reduced Ras signaling and high expression of the CDK inhibitors might cause the lower growth in spheroids. We then examined the relationship between liver-enriched transcription factors (C/EBPalpha and beta) and liver-specific functions. The results revealed that the high expression of C/EBPalpha was maintained during cultures when hepatocytes formed spheroids. Antisense oligonucleotides of C/EBPalpha repressed albumin secretion and the expression of p21, suggesting that the transcription factor, C/EBPalpha, may play a crucial role in the growth and differentiation of hepatocytes in spheroids.

to the extracellular matrix (ECM) via focal adhesions. [3] Thus, new tools for inducible control over RhoA activity may greatly enhance understanding of cytoskeletal dynamics and mechanotransduction. [4,5] Optogenetics is highly attractive for this purpose owing to its high spatiotemporal precision versus pharmacological and genetic techniques that can be encumbered by slow uptake/washout kinetics and pleotropic effects. Because small GTPases and their activating guanine nucleotide exchange factors (GEFs) signal at the plasma membrane, optogenetic membrane localization techniques are effective for inducible control over their function, where cytosol-sequestered proteins are dynamically recruited to the cytosol-facing inner leaflet of the plasma membrane to upregulate their signaling. [6] Based on earlier reported chemically inducible dimerization (CID)-based approaches for RhoA membrane recruitment, [7,8] optogenetic heterodimerization and photoactivatable chemically induced dimerization between a photo-responsive protein and a protein-binding partner (one of which is membrane localized) have been widely used to control upstream RhoA-activating GEFs [9][10][11][12][13][14] and phosphatases. [15] The heterodimerization strategy is sensitive to the stoichiometry of the two components, and thus may require expression leveltuning by clonal cell line selection and/or multiple fluorescent tags (i.e., separate for each component) at the expense of optical bandwidth otherwise useful for visualizing other fluorescent probes. [16][17][18] Previously, we reported the direct optogenetic control over RhoA GTPase itself by another mechanism of inducible clustering of RhoA fused to an oligomerizing form of plant cryptochrome, which presumably increases the binding avidity of the GTPase to membrane GEFs. However, this system has limited spatial resolution due to extensive cytosolic diffusion beyond the optical stimulation field prior to stable membrane localization post-oligomerization. [19] Recently, we reported that BcLOV4, a light-oxygen-voltage (LOV) flavoprotein from Botrytis cinerea, rapidly translocates to the plasma membrane in mammalian cells via a blue lightregulated electrostatic protein-lipid interaction (PLI) with the inner leaflet. [20,21] This direct interaction with the membrane itself is powerful for creating "single-component" tools for dynamic membrane recruitment of fused peripheral membrane proteins, without the obligate heterodimerization-or Optogenetic tools are created to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activator ARHGEF11, is fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these proteins induces potent contractile signaling sufficient to separate adherens junctions with as little as one pulse of blue light. Induced cytoskeletal morphology changes are dependent o...

AimsGenetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor, asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element, we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin sensitivity.Methods and ResultsIn this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls.ConclusionOur study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.