Regulation and consequences of differential gene expression in diabetic kidney disease

DIN (diabetic nephropathy) is the leading cause of end-stage renal disease worldwide and develops in 25-40% of patients with Type 1 or Type 2 diabetes mellitus. Elevated blood glucose over long periods together with glomerular hypertension leads to progressive glomerulosclerosis and tubulointerstitial fibrosis in susceptible individuals. Central to the pathology of DIN are cytokines and growth factors such as TGF-beta (transforming growth factor beta) superfamily members, including BMPs (bone morphogenetic protein) and TGF-beta 1, which play key roles in fibrogenic responses of the kidney, including podocyte loss, mesangial cell hypertrophy, matrix accumulation and tubulointerstitial fibrosis. Many of these responses can be mimicked in in vitro models of cells cultured in high glucose. We have applied differential gene expression technologies to identify novel genes expressed in in vitro and in vivo models of DN and, importantly, in human renal tissue. By mining these datasets and probing the regulation of expression and actions of specific molecules, we have identified novel roles for molecules such as Gremlin, IHG-1 (induced in high glucose-1) and CTGF (connective tissue growth factor) in DIN and potential regulators of their bioactions.

Rhodopsin and the Others: A Historical Perspective on Structural Studies of G Protein-Coupled Receptors

The role of rhodopsin as a structural prototype for the study of the whole superfamily of G protein-coupled receptors (GPCRs) is reviewed in an historical perspective. Discovered at the end of the nineteenth century, fully sequenced since the early 1980s, and with direct three-dimensional information available since the 1990s, rhodopsin has served as a platform to gather indirect information on the structure of the other superfamily members. Recent breakthroughs have elicited the solution of the structures of additional receptors, namely the beta 1- and beta 2-adrenergic receptors and the A(2A) adenosine receptor, now providing an opportunity to gauge the accuracy of homology modeling and molecular docking techniques and to perfect the computational protocol. Notably, in coordination with the solution of the structure of the A(2A) adenosine receptor, the first "critical assessment of GPCR structural modeling and docking" has been organized, the results of which highlighted that the construction of accurate models, although challenging, is certainly achievable. The docking of the ligands and the scoring of the poses clearly emerged as the most difficult components. A further goal in the field is certainly to derive the structure of receptors in their signaling state, possibly in complex with agonists. These advances, coupled with the introduction of more sophisticated modeling algorithms and the increase in computer power, raise the expectation for a substantial boost of the robustness and accuracy of computer-aided drug discovery techniques in the coming years.

Integrin expression at the bone/biomaterial interface

The aim of this study was to visualize integrin expression by cells in interface tissue in relation to their ligands. Tissue samples were obtained from 25 patients undergoing revision of aseptically loose total joint replacements. Serial sections were immunolabeled for the integrins alpha (2)beta (1) alpha (v)beta (3), alpha (4)beta (1) alpha (L)beta (2) (CD11a), alpha (M)beta (2) (CD11b), and alpha (x)beta (2) (CD11c), and the ligands fibronectin, laminin, vitronectin, intercellular adhesion molecule-1, and vascular adhesion molecule-1. Most cells were found to express alpha (2)beta (1) most macrophages and giant cells expressed CD11b, and the majority of CD11a was found on perivascular T lymphocytes. From the small amount of alpha (4)beta (1) and vascular adhesion molecule-1 expression in the interface tissue and the combination of CD11a, CD11b, and intercellular adhesion molecule-1 expression, it would seem that macrophages use beta (2) integrins to transmigrate. (C) 2001 John Wiley & Sons, Inc.

Role of Delta Np63 gamma in Epithelial to Mesenchymal Transition

Although members of the p63 family of transcription factors are known for their role in the development and differentiation of epithelial surfaces, their function in cancer is less clear. Here, we show that depletion of the Delta Np63 alpha and beta isoforms, leaving only Delta Np63 gamma, results in epithelial to mesenchymal transition (EMT) in the normal breast cell line MCF10A. EMT can be rescued by the expression of the Delta Np63 alpha isoform. We also show that Delta Np63 gamma expressed in a background where all the other Delta Np63 are knocked down causes EMT with an increase in TGF beta-1, -2, and -3 and downstream effectors Smads2/3/4. In addition, a p63 binding site in intron 1 of TGF beta was identified. Inhibition of the TGF beta response with a specific inhibitor results in reversion of EMT in Delta Np63 alpha- and beta-depleted cells. In summary, we show that p63 is involved in inhibiting EMT and reduction of certain p63 isoforms may be important in the development of epithelial cancers.

Differential expression of steroid 5 alpha-reductase isozymes and association with disease severity and angiogenic genes predict their biological role in prostate cancer

The biological role of steroid 5 alpha-reductase isozymes (encoded by the SRD5A1 and SRD5A2 genes) and angiogenic factors that play important roles in the pathogenesis and vascularization of prostate cancer (PC) is poorly understood. The sub-cellular expression of these isozymes and vascular endothelial growth factor (VEGF) in PC tissue microarrays (n=62) was examined using immunohistochemistry. The effect of SRD5A inhibition on the angiogenesis pathway genes in PC was also examined in prostate cell lines, LNCaP, PC3, and RWPE-1, by treating them with the SRD5A inhibitors finasteride and dutasteride, followed by western blot, quantitative PCR, and ELISA chip array techniques. In PC tissues, nuclear SRD5A1 expression was strongly associated with higher cancer Gleason scores (P=0.02), higher cancer stage (P=0.01), and higher serum prostate specific antigen (PSA) levels (P=0.01), whereas nuclear SRD5A2 expression was correlated with VEGF expression (P=0.01). Prostate tumor cell viability was significantly reduced in dutasteride-treated PC3 and RWPE-1 cells compared with finasteride-treated groups. Expression of the angiogenesis pathway genes transforming growth factor beta 1 (TGFB1), endothelin (EDN1), TGF alpha (TGFA), and VEGFR1 was upregulated in LNCaP cells, and at least 7 out of 21 genes were upregulated in PC3 cells treated with finasteride (25 mu M). Our findings suggest that SRD5A1 expression predominates in advanced PC, and that inhibition of SRD5A1 and SRD5A2 together was more effective in reducing cell numbers than inhibition of SRD5A2 alone. However, these inhibitors did not show any significant difference in prostate cell angiogenic response. Interestingly, some angiogenic genes remained activated after treatment, possibly due to the duration of treatment and tumor resistance to inhibitors. Endocrine-Related Cancer (2010) 17 757-770

Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions

Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. we have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha 2 beta 1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where 0 is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

GPVI levels in platelets: relationship to platelet function at high shear

We have investigated the density of the collagen receptors glycoprotein VI (GPVI) and alpha(2)beta(1) on human platelets and their relationship to polymorphisms within the GPVI gene. GPVI levels varied 1.5-fold and showed a weak correlation (r = 0.35) with the levels of alpha(2)beta(1), which varied 3-fold. GPVI genotype had a significant effect on receptor levels with carriers of the proline 219 allele (approximately 22% of the population) having 10% lower GPVI levels than the more common serine homozygotes. GPVI and alpha(2)beta(1) levels were found to be significantly decreased on platelets from patients with myeloproliferative disorders (MPDs). In both the MPD and the control group, GPVI levels were found not to affect platelet function under high shear in whole blood. Similarly murine platelets that express up to 5-fold lower levels of GPVI showed no significant difference than controls in thrombus formation on a high-density collagen-coated surface. However platelets lacking the GPVI/Fc receptor gamma-chain (FcR gamma-chain) complex or a functional FcR gamma-chain (immunoreceptor tyrosine-based activation motif [ITAM] point mutant) exhibited severely abrogated thrombus formation at 800 s(-1) and 1500 s(-1). These results demonstrate that GPVI levels are tightly controlled and play a critical role in thrombus formation on collagen; nevertheless, a range of receptor densities can support platelet function under high shear. (C) 2003 by The American Society of Hematology.

Dependence of the kinetics of liquid-phase photocatalyzed reactions on oxygen concentration and light intensity

The initial kinetics of the oxidation of 4-chlorophenol, 4-CP, photocatalyzed by titania films and aqueous dispersions were studied as a function of oxygen partial pressure, P-O2, and incident light intensity, I. The reaction conditions were such that the kinetics were independent of [4-CP] but strongly dependent on PO2-a situation that allowed investigation of the less-often studied kinetics of oxygen reduction. The observed kinetics fit a pseudo-steady-state model in which the oxygen is Langmuir-adsorbed on the titania photocatalyst particles before being reduced by photogenerated electrons. The maximum rate of photocatalysis depends directly on I-beta, where, beta = 1 for films and 0.7 for dispersions of titania, indicating that the kinetics are dominated by the surface reactions of the photogenerated electrons and holes for the films and by direct recombination for the powder dispersions. Using the pseudo-first-order model, for both titania films and dispersions, the apparent Langmuir adsorption constant, K-LH, derived from a Langmuir-Hinshelwood analysis of the kinetics, appears to be largely independent of incident light intensity, unlike KLH for 4-CP Consequently, similar values are obtained for the Langmuir adsorption constant, K-ads, extracted from a pseudosteady-state analysis of the kinetics for oxygen on TiO2 dispersions and films in aqueous solution (i.e., ca. 0.0265 +/- 0.005 kPa(-1)), and for both films and dispersions, oxygen appears to be weakly adsorbed on TiO2 compared with 4-CP, at a rate that would take many minutes to reach equilibrium. The value of Kads for oxygen on titania particles dispersed in solution is ca. 4.7 times lower than that reported for the dark Langmuir adsorption isotherm; possible causes for this difference are discussed. (c) 2006 Elsevier Inc. All rights reserved.

Kinetics of liquid phase semiconductor photoassisted reactions: Supporting observations for a pseudo-steady-state model

The kinetics of liquid phase semiconductor photocatalytic and photoassisted reactions are an area of some debate, reignited recently by an article by Ollis(1) in which he proposed a simple pseudo- steady- state model to interpret the Langmuir- Hinshelwood type kinetics, commonly observed in such systems. In the current article, support for this model, over other models, is provided by a reinterpretation of the results of a study, reported initially in 1999,2 of the photoassisted mineralization of 4- chlorophenol, 4-CP, by titania films and dispersions as a function of incident light intensity, I. On the basis of this model, these results indicate that 4- CP is adsorbed more strongly on P25 TiO2 when it is in a dispersed, rather than a film form, due to a higher rate constant for adsorption, k(1). In addition, the kinetics of 4- CP removal appear to depend on I-beta where, beta = 1 or 0.6 for when the TiO2 is in a film or a dispersed form, respectively. These findings are discussed both in terms of the pseudo- steady- state model and other popular kinetic models.

Cytokine phenotype, genotype, and renal outcomes at cardiac surgery

Cardiac surgery modulates pro- and anti-inflammatory cytokine balance involving plasma tumour necrosis factor alpha (TNFa) and interleukin-10 (IL-10) together with urinary transforming growth factor beta-1 (TGFß1), interleukin-1 receptor antagonist (IL1ra) and tumour necrosis factor soluble receptor-2 (TNFsr2). Effects on post-operative renal function are unclear. We investigated if following cardiac surgery there is a relationship between cytokine (a) phenotype and renal outcome; (b) genotype and phenotype and (c) genotype and renal outcome. Since angiotensin-2 (AG2), modulates TGFß1 production, we determined whether angiotensin converting enzyme insertion/deletion (ACE I/D) genotype affects urinary TGFß1 phenotype as well as renal outcome.

Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2

NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-beta(1) activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.

Functional characterization of alpha-adrenoceptors mediating pupillary dilation in rats

Previously, we reported that the alpha(1A)-adrenoceptor, but not the alpha(1D)-adrenoceptor, mediates pupillary dilation elicited by sympathetic nerve stimulation in rats. This study was undertaken to further characterize the alpha-adrenoceptor subtypes mediating pupillary dilation in response to both neural and agonist activation. Pupillary dilator response curves were generated by intravenous injection of norepinephrine in pentobarbital-anesthetized rats. Involvement of alpha(1)-adrenoceptors was established as mydriatic responses were inhibited by systemic administration of nonselective alpha-adrenoceptor antagonists, phentolamine (0.3-3 mg/kg) and phenoxybenzamine (0.03-0.3 mg/kg), as well as by the selective alpha(1)-adrenoceptor antagonist, prazosin (0.3 mg/kg). The alpha(2)-adrenoceptor antagonist, rauwolscine (0.5 mg/kg), was without antagonistic effects. alpha(1A)-Adrenoceptor selective antagonists, 2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane (WB-4101; 0.1-1 mg/kg) and 5-methylurapidil (0.1-1 mg/kg), the alpha(1B)-adrenoceptor selective antagonist, 4-amino-2-[4-[1-(benzyloxycarbonyl)-2(S)- [[(1,1-dimethylethyl)amino]carbonyl]-piperazinyl]-6,7-dimethoxyquinazoline (L-765314; 0.3-1 mg/kg), as well as the alpha(1D)-adrenoceptor selective antagonist, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY-7378; 1 mg/kg), were used to delineate the adrenoceptor subtypes involved. Mydriatic responses to norepinephrine were significantly antagonized by intravenous administration of both WB-4101 and 5-methylurapidil, but neither by L-765314 nor by BMY-7378. L-765314 (0.3-3 mg/kg, i.v.) was also ineffective in inhibiting the mydriasis evoked by cervical sympathetic nerve stimulation. These results suggest that alpha(1B)-adrenoceptors do not mediate sympathetic mydriasis in rats, and that the alpha(1A)-adrenoceptor is the exclusive subtype mediating mydriatic responses in this species.

Contractile properties of human renal cell carcinoma recruited arteries and their response to nicotinamide

Background and purpose: The manipulation of tumour blood supply and thus oxygenation is a potentially important strategy for improving the treatment of solid tumours by radiation. Increased knowledge about the characteristics that distinguish the tumour vasculature from its normal counterparts may enable tumour blood flow to be more selectively modified, Nicotinamide (NA) causes relaxation of preconstricted normal and tumour-supply arteries in rats. It has also been shown to affect microregional blood flow in human tumours. Direct effects of NA on human tumour supply arteries have not previously been reported. This paper describes our evaluation of the effects of NA on two parameters: 'spontaneous', oscillatory contractile activity and agonist (phenylephrine)-induced constriction in the arteries supplying human renal cell carcinomas.

Materials and methods: Isolated renal cell carcinoma feeder vessels were perfused in an organ bath with the alpha(1)-adrenoceptor agonist phenylephrine (PE). When the arteries had reached a plateau of constriction, nicotinamide (8.2 mM) was added to the perfusate and changes in perfusion pressure were measured.

Results: PE (10 mu M) induced a sustained constriction in the majority of the renal cell carcinoma feeder vessels examined, demonstrating that they retain contractile characteristics, at least in response to this alpha(1)-adrenoceptor agonist. In combination with NA (8.2 mM) the constriction was significantly attenuated in half of the preparations. In addition, seven arteries exhibited spontaneous contractile activity which was significantly attenuated by NA in six of them.

Conclusions: NA can significantly attenuate both 'spontaneous' and agonist-induced constrictions in tumour-recruited human arteries, though not all arteries are sensitive. Published by Elsevier Science Ireland Ltd.

Osteoprotegerin Expression in the Dental Pulp

Objectives: The inflammatory response to pulpal injury or infection has major clinical significance. Osteoprotegerin (OPG) is a soluble decoy receptor for Receptor Activator of NF kappa B Ligand (RANKL), preventing ligand binding to its receptor (RANK), thus inhibiting clastic cell formation. The aim of the study is to investigate the expression of OPG in human dental pulp and the effects of inflammatory mediators. This study will specifically investigate the effects of Transforming Growth Factor Beta-1 (TGF-β1) and Interleukin 1-Beta (IL-1β) on the expression of OPG on pulp fibroblasts in vitro. Method: Five primary pulp fibroblast populations were obtained by explant culture of healthy pulp tissue. Triplicate cultures were grown to confluence in 12-well plates and stimulated for 48 hours with IL-1β (10ng/ml) or TGF-β1 (10ng/ml). The conditioned media was collected and OPG levels detected by ELISA (R+D Systems, UK). Results: All fibroblast populations produced quantifiable levels of OPG in a time-dependant fashion. IL-1β significantly increased the expression of OPG (p<0.05) in all cultures. In contrast, TGF-β1 had no significant effect on OPG expression levels. In addition, previous work in our laboratory demonstrated both TGF-β1 and IL-1β stimulated OPG expression by periodontal ligament fibroblasts. Conclusion: These data indicate that IL-1β-regulated expression of OPG by pulpal fibroblasts may mediate hard tissue turnover in the inflamed dental pulp.

Neuropeptide Receptors in the Dental Pulp

Objectives: The inflammatory response to pulpal injury or infection has major clinical significance. The aim of the study is to investigate the presence and regulation of expression of neuropeptide receptors on human pulp fibroblasts and whole pulp tissue. This study will investigate the expression of Substance P (NK-1) and Neuropeptide Y (NPY-Y1) receptors on pulp fibroblasts, determine the effects of Transforming Growth Factor Beta-1 (TGF-b1) and Interleukin 1-Beta (IL-1b) on the expression of NK-1 and NPY-Y1 receptors on pulp fibroblasts and examine the levels of receptor expression in whole pulp samples. Methods: Primary pulp fibroblast cell lines were obtained from patients undergoing extractions for orthodontic reasons. The cells were grown to confluence and stimulated for 5 days with IL-1b or TGF-b1. Pulp tissue fragments were obtained from freshly extracted sound and carious teeth, snap frozen in liquid nitrogen and cracked open using a vice. The monolayer was removed with cell scrapers and pelleted. The cell membranes of the cultured cells and the whole tissue were isolated using a Mem-PER® Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce, UK). The membrane proteins were separated by SDS-PAGE and Western blotting was used to detect the presence of NK-1 and NPY-Y1. Results: Initial results demonstrated the presence of NK-1 and NPY-Y1 in cultured pulp fibroblasts. Following the 5 day incubation with TGF-b1, the cells appeared not to express NK-1. IL-1b had a slight stimulatory effect on NK-1 expression. The NPY-Y1 expression was not affected by either TGF-b1 or IL-1b. In whole pulp samples, levels of NK-1 were increased in carious teeth compared to caries-free teeth. The NPY-Y1 levels were similar in carious and non-carious teeth. Conclusion: These findings give an insight into how pulp cells react to inflammatory stimuli with regards to neuropeptide receptor expression and their roles in health and disease