Bladder interstitial cells:an updated review of current knowledge

The field of bladder research has been energized by the study of novel interstitial cells (IC) over the last decade. Several subgroups of IC are located within the bladder wall and make structural interactions with nerves and smooth muscle, indicating integration with intercellular communication and key physiological functions. Significant progress has been made in the study of bladder ICs' cellular markers, ion channels and receptor expression, electrical and calcium signalling, yet their specific functions in normal bladder filling and emptying remain elusive. There is increasing evidence that the distribution of IC is altered in bladder pathophysiologies suggesting that changes in IC may be linked with the development of bladder dysfunction. This article summarizes the current state of the art of our knowledge of IC in normal bladder and reviews the literature on IC in dysfunctional bladder.

Serotonin Transporter Gene Polymorphism and Myocardial Infarction - Etude Cas-te'moins de L'Infarctus du Myocarde (ECTIM)

Background— Depression is a risk factor for myocardial infarction (MI). Selective serotonin reuptake inhibitors reduce this risk. The site of action is the serotonin transporter (SLC6A4), which is expressed in brain and blood cells. A functional polymorphism in the promoter region of the SLC6A4 gene has been described. This polymorphism may be associated with the risk of MI. Methods and Results— The SLC6A4 polymorphism has been investigated by polymerase chain reaction in 671 male patients with MI and in 688 controls from the Etude Cas-Témoins de l’Infarctus du Myocarde (ECTIM) multicentric study. Percentages for LL, LS, and SS genotypes were 35.5%, 45.4%, and 19.1%, respectively, for cases versus 28.1%, 49.1%, and 22.8%, respectively, for controls. S allele frequency was 41.8% and 47.4% for cases and controls, respectively. After adjustment for age and center by using multivariable logistic regression, the odds ratio for MI associated with the LL genotype was 1.40 (95% CI 1.11 to 1.76, P=0.0047). Conclusions— The LL genotype of the SLC6A4 polymorphism is associated with a higher risk of MI. This could be attributable to the effect of the polymorphism on serotonin-mediated platelet activation or smooth muscle cell proliferation or on other risk factors, such as depression or response to stress

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes.

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 microM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 microM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in approximately 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 microM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 microM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 microM), which sensitizes the IP3R to IP3. In cells voltage clamped at -40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.

Calcium signaling in ocular arterioles

Local control of blood flow to the photoreceptors and associated neurons in the retina is largely achieved through changes in tone within the choroidal and retinal arterioles. This is primarily achieved through changes in [Ca2+] within the smooth muscle of these vessels, which regulates cell contraction and vascular constriction. Here we review some aspects of the cell physiology involved in these Ca2+-signaling processes, with particular emphasis on the molecular mechanisms involved. Ca2+-influx across the plasma membrane can occur via a variety of Ca2+-channels, including voltage-operated, store-operated, and receptor-operated channels. Ca2+ may also be released from intracellular stores via RyR-, or IP3R-gated channels in the SR membrane. Using high-speed confocal Ca2+-imaging, we have also demonstrated that the resulting signals are far from homogeneous, with spontaneous activity in retinal arterioles being characterized by both localized Ca2+-sparks and more global Ca2+-waves and oscillations. These signals may be specifically and differentially targeted, for example, to Ca2+-sensitive ion channels (stimulus-excitation coupling), or pathways regulating contraction (stimulus-contraction coupling). Exploring the role of changes in such targeting in disease states will provide exciting opportunities for future research.

Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Protein kinase B/Akt activity is involved in renal TGF-beta 1-driven epithelial-mesenchymal transition in vitro and in vivo

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-beta (TGF-beta1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-beta1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-beta1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3beta phosphorylation. TGF-beta1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and alpha-smooth muscle actin expression, consistent with the fibrotic actions of TGF-beta1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-beta1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3beta, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-beta1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.

Pharmacology of transient receptor potential melastatin channels in the vasculature.

Mammalian transient receptor potential melastatin (TRPM) non-selective cation channels, the largest TRP subfamily, are widely expressed in excitable and non-excitable cells where they perform diverse functions ranging from detection of cold, taste, osmolarity, redox state and pH to control of Mg(2+) homeostasis and cell proliferation or death. Recently, TRPM gene expression has been identified in vascular smooth muscles with dominance of the TRPM8 channel. There has been in parallel considerable progress in decoding the functional roles of several TRPMs in the vasculature. This research on native cells is aided by the knowledge of the activation mechanisms and pharmacological properties of heterologously expressed TRPM subtypes. This paper summarizes the present state of knowledge of vascular TRPM channels and outlines several anticipated directions of future research in this area.

Peptide DV-28 amide: Prototype of a novel class of bradykinin receptor antagonist isolated from the defensive skin secretion of bombinid toads

Kinestatin, isolated from the skin of the Chinese toad, Bombina maxima, was the first bradykinin B2 receptor antagonist identified in amphibians. Molecular cloning established that it is co-encoded with the bradykinin-related peptide, maximakinin, within one of several skin kininogens. To examine other species within the genus Bombina for the presence of structural homologues of kinestatin, we subjected skin secretion of the toad, Bombina orientalis, to HPLC fractionation with subsequent bioassay of fractions for antagonism of bradykinin activity using an isolated rat tail artery smooth muscle preparation. A single fraction was located that inhibited bradykinin-induced relaxation of rat arterial smooth muscle and MALDI-TOF analysis of this fraction revealed that it contained a single peptide of molecular mass 3198.5 Da. Further primary structural analysis of this peptide showed that it was a 28-mer with an N-terminal Asp (D) residue and a C-terminal Val (V) residue that was amidated. The peptide was named DV-28 amide in accordance with these primary structural attributes. Synthetic DV-28 amide replicated the observed bradykinin antagonistic effect within the smooth muscle bioassay in a dose-dependent manner. In addition, it was observed to inhibit the proliferation of human microvessel endothelial cells (HMECs) as assessed by MTT assay. Bioinformatic analysis revealed that DV-28 amide was, like kinestatin, co-encoded with a bradykinin receptor agonist on one of two skin kininogens identified in B. orientalis. DV-28 amide thus represents a novel class of bradykinin antagonist from skin secretions of bombinid toads that appear to be a rich source of such novel peptides.

Kassorins: Novel innate immune system peptides from skin secretions of the African hyperoliid frogs, Kassina maculata and Kassina senegalensis

From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC50 = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.

Endothelial Progenitors as Tools to Study Vascular Disease

Endothelial progenitor cells (EPCs) have great clinical value because they can be used as diagnostic biomarkers and as a cellular therapy for promoting vascular repair of ischaemic tissues. However, EPCs also have an additional research value in vascular disease modelling to interrogate human disease mechanisms. The term EPC is used to describe a diverse variety of cells, and we have identified a specific EPC subtype called outgrowth endothelial cell (OEC) as the best candidate for vascular disease modelling because of its high-proliferative potential and unambiguous endothelial commitment. OECs are isolated from human blood and can be exposed to pathologic conditions (forward approach) or be isolated from patients (reverse approach) in order to study vascular human disease. The use of OECs for modelling vascular disease will contribute greatly to improving our understanding of endothelial pathogenesis, which will potentially lead to the discovery of novel therapeutic strategies for vascular diseases.

Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene

Nematode parasites of the genus Trichinella are intracellular and distinct life cycle stages invade intestinal epithelial and skeletal muscle cells. Within the genus, Trichinella spiralis and Trichinella pseudospiralis exhibit species-specific differences with respect to host-parasite complex formation and host immune modulation. Parasite excretory-secretory (ES) proteins play important roles at the host-parasite interface and are thought to underpin these differences in biology. Serine proteases are among the most abundant group of T. spiralis ES proteins and multiple isoforms of the muscle larvae-specific TspSP-1 serine protease have been identified. Recently, a similar protein (TppSP-1) in T. pseudospiralis muscle larvae was identified. Here we report the cloning and characterisation of the full-length transcript of TppSP-1 and present comparative data between TspSP-1 and TppSP-1.

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.

Application of a mechanobiological simulation technique to stents used clinically

Many cardiovascular diseases are characterised by the restriction of blood flow through arteries. Stents can be expanded within arteries to remove such restrictions; however, tissue in-growth into the stent can lead to restenosis. In order to predict the long-term efficacy of stenting, a mechanobiological model of the arterial tissue reaction to stress is required. In this study, a computational model of arterial tissue response to stenting is applied to three clinically relevant stent designs. We ask the question whether such a mechanobiological model can differentiate between stents used clinically, and we compare these predictions to a purely mechanical analysis. In doing so, we are testing the hypothesis that a mechanobiological model of arterial tissue response to injury could predict the long-term outcomes of stent design. Finite element analysis of the expansion of three different stent types was performed in an idealised, 3D artery. Injury was calculated in the arterial tissue using a remaining-life damage mechanics approach. The inflammatory response to this initial injury was modelled using equations governing variables which represented tissue-degrading species and growth factors. Three levels of inflammation response were modelled to account for inter-patient variability. A lattice-based model of smooth muscle cell behaviour was implemented, treating cells as discrete agents governed by local rules. The simulations predicted differences between stent designs similar to those found in vivo. It showed that the volume of neointima produced could be quantified, providing a quantitative comparison of stents. In contrast, the differences between stents based on stress alone were highly dependent on the choice of comparison criteria. These results show that the choice of stress criteria for stent comparisons is critical. This study shows that mechanobiological modelling may provide a valuable tool in stent design, allowing predictions of their long-term efficacy. The level of inflammation was shown to affect the sensitivity of the model to stent design. If this finding was verified in patients, this could suggest that high-inflammation patients may require alternative treatments to stenting.

PsT-1: A new Tryptophyllin peptide from the skin secretion of Waxy Monkey Leaf Frog, Phyllomedusa sauvagei

The Waxy Monkey Leaf Frog, Phyllomedusa sauvagei, has been extensively-studied for many years, and a broad spectrum of bioactive peptides has been found in its skin secretions. Here we report the discovery of a novel tryptophyllin (TPH) peptide, named PsT-1, from this frog species. Skin secretions from specimens of P. sauvagei were collected by mild electrical stimulation. Peptides were identified and characterized by transcriptome cloning, and the structure was confirmed by MALDI-TOF mass spectrometry and automated Edman degradation. This novel peptide was encoded by a single precursor of 61 amino acid residues, whose primary structure was deduced from cloned skin cDNA. Analysis of different amphibian tryptophyllins revealed that PsT-1 exhibited a high degree of primary structural similarity to its homologues, PdT-1 and PdT-2, from the Mexican giant leaf frog, Pachymedusa dacnicolor. A synthetic replicate of PsT-1 was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle. It was also found that PsT-1 had an anti-proliferative effect on three different human prostate cancer cell lines (LNCaP/PC3/DU145), by use of an MTT assay coupled with direct cell counting as measures of cell growth. These data indicate that PsT-1 is a likely bradykinin receptor antagonist and its biological effects are probably mediated through bradykinin receptors. As a BK antagonist, PST-1, with antagonistic effects on BK in artery smooth muscle, inhibition of proliferation in prostate cancer cells and lack of undesirable side effects, may have potential in cardiovascular, inflammatory and anticancer therapy.

Effect of TGF-β2 and anti-TGF-β2 antibody in a new in vivo rodent model of posterior capsule opacification

PURPOSE. This study evaluated the effect of transforming growth factor (TGF)-ß2 and anti-TGF-ß2 antibody in a rodent model of posterior capsule opacification (PCO). METHODS. An extracapsular lens extraction (ECLE) was performed in 72 Sprague-Dawley rats. At the end of the procedure, 10 µL TGF-ß2 (TGF-ß2-treated group), fetal calf serum (FCS)/phosphate- buffered saline (PBS; FCS/PBS-treated control group), a human monoclonal TGF-ß2 antibody (anti-TGF-ß2-treated group), or a null control IgG4 antibody (null antibody-treated control group) was injected into the capsule. Animals were killed 3 and 14 days postoperatively. Eyes were evaluated clinically prior to euthanatization, then enucleated and processed for light microscopy and immunohistochemistry afterward. PCO was evaluated clinically and histopathologically. Student's t-test and ? were used to assess differences between groups. RESULTS. There were no statistically significant clinical or histopathological differences in degree of PCO between the TGF-ß2- and FCS/PBS-treated groups at 3 and 14 days after ECLE. Nor were there differences between the anti-TGF-ß2- and the null antibody-treated groups, with the exception of the histopathology score for capsule wrinkling 3 days after ECLE (P = 0.02). a-Smooth-muscle actin staining was observed in the lens capsular bag only in areas where there was close contact with the iris. CONCLUSIONS. No sustained effect of TGF-ß2 or anti-TGF-ß2 antibody on PCO was found in rodents at the dose and timing administered in this study. Iris cells may play a role in the process of epithelial mesenchymal transition linked to PCO. Copyright © Association for Research in Vision and Ophthalmology.