Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.

Control of ocular blood flow occurs predominantly at the level of the retinal and choroidal arterioles. The present article provides an overview of the Ca2 + handling mechanisms and plasmalemmal ion channels involved in the regulation of retinal and choroidal arteriolar smooth muscle tone. Increases in global intracellular free Ca2 + ([Ca2 +]i) involve multiple mechanisms, including agonist-dependent release of Ca2 + from intracellular stores through activation of the inositol trisphosphate (IP3) pathway. Ca2 + enters by voltage-dependent L-type Ca2 + channels and novel dihydropyridine-sensitive store-operated nonselective cation channels. Ca2 + extrusion is mediated by plasmalemmal Ca2 +-ATPases and through Na+/Ca2+ exchange. Local Ca2 + transients (Ca2 + sparks) play an important excitatory role, acting as the building blocks for more global Ca2 + signals that can initiate vasoconstriction. K+ and Cl- channels may also affect cell function by modulating membrane potential. The precise contribution of each of these mechanisms to the regulation of retinal and choroidal perfusion in vivo warrants future investigation.

Role of calcium influx through voltage-operated calcium channels and of calcium mobilization in the physiology of Schistosoma mansoni muscle contractions

We tested the hypothesis that voltage-operated Ca2+ channels mediate an extracellular Ca2+ influx in muscle fibres from the human parasite Schistosoma mansoni and, along with Ca2+ mobilization from the sarcoplasmic reticulum, contribute to Muscle contraction. Indeed, whole-cell voltage clamp revealed voltage-gated inward currents carried by divalent ions with a peak current elicited by steps to + 20 mV (from a holding potential of -70 mV). Depolarization of the fibres by elevated extracellular K+ elicited contractions that were completely dependent on extracellular Ca2+ and inhibited by nicardipine (half inhibition at 4(.)1 mu M). However these contractions were not very sensitive to other classical blockers of voltage-gated Ca2+ channels, indicating that the schistosome Muscle channels have an atypical pharmacology when compared to their mammalian counterparts. Furthermore, the contraction induced by 5 mM caffeine was inhibited after depletion of the sarcoplasmic reticulum either with thapsigargin (10 mu M) or ryanodine (10 mu M). These data suggest that voltage-operated Ca2+ channels docontribute to S. mansoni contraction as does the mobilization of stored Ca2+, despite the small volume of sarcoplasmic reticulum in schistosome smooth muscles.

Recapitulation of embryological programmes in renal fibrosis - The importance of epithelial cell plasticity and developmental genes

Chronic fibrosis represents the final common pathway in progressive renal disease. Myofibroblasts deposit the constituents of renal scar, thus crippling renal function. It has recently emerged that an important source of these pivotal effector cells is the injured renal epithelium. This review concentrates on the process of epithelial-mesenchymal transition (EMT) and its regulation. The role of the developmental gene, gremlin, which is reactivated in adult renal disease, is the subject of particular focus. This member of the cysteine knot protein superfamily is critical to the process of nephrogenesis but quiescent in normal adult kidney. There is increasing evidence that gremlin expression reactivates in diabetic nephropathy, and in the diseased fibrotic kidney per se. Known to antagonize members of the bone morphogenic protein (BMP) family, gremlin may also act downstream of TGF-beta in induction of EMT. An increased understanding of the extracellular modulation of EMT and, in particular, of the gremlin-BMP axis may result in strategies that can halt or reverse the devastating progression of chronic renal fibrosis. Copyright (c) 2006 S. Karger AG, Basel.

Inward rectifier potassium channels in the HL-1 cardiomyocyte-derived cell line.

HL-1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K(+) channels. Our aim was to identify and characterize inward rectifier K(+) channels in HL-1 cells. External Ba(2+) (100?µM) inhibited 44?±?0.05% (mean?±?s.e.m., n?=?11) of inward current in whole-cell patch-clamp recordings. The reversal potential of the Ba(2+)-sensitive current shifted with external [K(+)] as expected for K(+)-selective channels. The slope conductance of the inward Ba(2+)-sensitive current increased with external [K(+)]. The apparent Kd for Ba(2+) was voltage dependent, ranging from 15?µM at -150 ?mV to 148?µM at -75 ?mV in 120 ?mM external K(+). This current was insensitive to 10?µM glybenclamide. A component of whole-cell current was sensitive to 150?µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), although it did not correspond to the Ba(2+)-sensitive component. The effect of external 1 mM Cs(+) was similar to that of Ba(2+). Polymerase chain reaction using HL-1 cDNA as template and primers specific for the cardiac inward rectifier K(ir)2.1 produced a fragment of the expected size that was confirmed to be K(ir)2.1 by DNA sequencing. In conclusion, HL-1 cells express a current that is characteristic of cardiac inward rectifier K(+) channels, and express K(ir)2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype.

Runx2, p53, and pRB Status as Diagnostic Parameters for Deregulation of Osteoblast Growth and Differentiation in a New Pre-Chemotherapeutic Osteosarcoma Cell Line (OS1)

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS I have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS I had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio, (P

Vascular Endothelial Cell Growth–Activated XBP1 Splicing in Endothelial Cells Is Crucial for Angiogenesis

BACKGROUND - : Vascular endothelial cell growth factor plays a pivotal role in angiogenesis via regulating endothelial cell proliferation. The X-box binding protein 1 (XBP1) is believed to be a signal transducer in the endoplasmic reticulum stress response. It is unknown whether there is crosstalk between vascular endothelial cell growth factor signaling and XBP1 pathway. 

METHODS AND RESULTS - : We found that vascular endothelial cell growth factor induced the kinase insert domain receptor internalization and interaction through C-terminal domain with the unspliced XBP1 and the inositol requiring enzyme 1 α in the endoplasmic reticulum, leading to inositol requiring enzyme 1 α phosphorylation and XBP1 mRNA splicing, which was abolished by siRNA-mediated knockdown of kinase insert domain receptor. Spliced XBP1 regulated endothelial cell proliferation in a PI3K/Akt/GSK3β/β- catenin/E2F2-dependent manner and modulated the cell size increase in a PI3K/Akt/GSK3β/β-catenin/E2F2-independent manner. Knockdown of XBP1 or inositol requiring enzyme 1 α decreased endothelial cell proliferation via suppression of Akt/GSK3β phosphorylation, β-catenin nuclear translocation, and E2F2 expression. Endothelial cell-specific knockout of XBP1 (XBP1ecko) in mice retarded the retinal vasculogenesis in the first 2 postnatal weeks and impaired the angiogenesis triggered by ischemia. Reconstitution of XBP1 by Ad-XBP1s gene transfer significantly improved angiogenesis in ischemic tissue in XBP1ecko mice. Transplantation of bone marrow from wild-type o XBP1ecko mice could also slightly improve the foot blood reperfusion in ischemic XBP1ecko mice. 

CONCLUSIONS - : These results suggest that XBP1 can function via growth factor signaling pathways to regulate endothelial proliferation and angiogenesis. 

Coexpressing shRNA with fluorescence tags for quantification of cell migration studies

Understanding migration of cells has many implications in human physiology; some examples include developmental biology, healing, immune responses and tissue remodeling. On the other hand, invasive migration by tumor cells is pathological and is a major cause of mortality amongst cancer sufferers. Cell migration assays have been widely used to quantify potentially metastatic genes. In recent years, the use of RNAi has significantly increased the tools available in cell migration research due to its specific gene targeting for knockdown. The inability to ensure 100% transfection/transduction efficiency reduces the sensitivity of cell migration assays because cells not successfully transfected/transduced with the RNAi are also included in the calculations. This study introduces a different experimental setup mathematically expressed in our named normalized relative infected cell count (N-RICC) that analyses cell migration assays by co-expressing retrovirally transduced shRNA with fluorescence tags from a single vector. Vectors transduced into cells are visible under fluorescence, thus alleviating the problems involved with transduction efficiency by individually identifying cells with targeted genes. Designed shRNAs were targeted against a list of potentially metastatic genes in a highly migratory breast cancer cell line model, MDA-MB-231. We have successfully applied N-RICC analysis to show greater sensitivity of integrin alpha5 (ITGA5) and Ras homologue A (RhoA) in cell metastasis over conventional methods in scratch-wound assays and migration chambers assays.

A morphologic and autoradiographic study of cell death and regeneration in the retinal microvasculature of normal and diabetic rats

Cell loss and regeneration were investigated and compared in the retinal microvasculature of age- and sex-matched normal and streptozotocin diabetic rats. Selective pericyte loss in the diabetic rat was characterized by changes in the pericyte to endothelial cell ratio in retinal capillaries isolated for microscopy by the trypsin digest technique. A comparison of 3- and 9-month-old normal rats showed no significant change in the pericyte to endothelial cell ratio (1:2.7). In diabetic animals the ratio was reduced to 1:4.03, which was statistically significant (P less than .001). Premitotic retinal vascular cells in normal and diabetic rats were labelled with tritiated thymidine and the labelling indices calculated from cell counts of trypsin digest preparations. Methyl H3 thymidine was infused continuously over an eight-day period using osmotic mini pumps. The labelling index of endothelial cells (0.33%) in normal rats increased to 0.91% in diabetic animals (P less than .05). The labelling index of pericyte cells in normal animals (0.16%) did not increase significantly (P greater than .05) in diabetic animals (0.19%). A special stain was used to exclude labelled polymorphonuclear leukocytes from the cell counts.

The effect of melanocortin peptides on Interleukin 1-beta induced chondrocyte cell death and inflammation

ntroduction: Osteoarthritis (OA) is a degenerative joint disease affecting more than 8.5 million people in the UK. Disruption in the catabolic and anabolic balance, with the catabolic cytokine Interleukin 1 beta (IL-1β) being involved in the initiation and progression of OA (1). Melanocortin peptides (α-MSH and D[Trp8]-γ-MSH) exert their anti-inflammatory effects via activation of melanocortin receptors (MC), with both MC1 and MC3 being identified as promising candidates as novel targets for OA (2). This study aims to assess the chondroprotective and anti-inflammatory effects of the pan melanocortin receptor agonist α-MSH and MC3 agonist D[Trp8]-γ-MSH following IL-1β chondrocyte stimulation. Methods: RT-PCR/ Western Blot: Human C-20/A4 chondrocytic cell-line were cultured in 6 well plates (1x106 cells/well) and harvested to determine MC and IL-1β expression by RT-PCR, and Western Blot. Cell-Culture: Cells were cultured in 96 well plates (1x106 cells/well) and stimulated with H2O2 (0.3%), TNF-α (60 pg/ml) or IL-1β (0-5000pg/ml) for 0-72h and cell viability determined. Drug Treatment: In separate experiments cells were pre-treated with 3 μg/ml α-MSH (Sigma-Aldrich Inc. Poole, UK), or D[Trp8]-γ-MSH (Phoenix Pharmaceuticals, Karlsrhue, Germany) (all dissolved in PBS) for 30 minutes prior to IL-1β (5000pg/ml) stimulation for 6-24h. Analysis: Cell viability was determined by using the three cell viability assays; Alamar Blue, MTT and the Neutral Red (NR) assay. Cell-free supernatants were collected and analysed for Interleukin -6 (IL-6) and IL-8 release by ELISA. Data expressed as Mean ± SD of n=4-8 determination in quadruplicate. *p≤ 0.05 vs. control. Results: Both RT-PCR, and Western Blot showed MC1 and MC3 expression on C-20/A4 cells. Cell viability analysis: IL-1β stimulation led to a maximal cell death of 35% at 6h (Alamar Blue), and 40% and 75% with MTT and Neutral Red respectively at 24h compared to control. The three cell viability assays have different cellular uptake pathways, which accounts for the variations observed in cell viability in response to the concentration of IL-1β, and time. Cytokine analysis by ELISA: IL-1β (5000pg/ml) stimulation for 6 and 24h showed maximal IL-6 production 292.3 ±3.8 and 275.5 ±5.0 respectively, and IL-8 production 353.3 ±2.6 and 598.3 ±8.6 respectively. Pre-treatment of cells with α-MSH and D[Trp8]-γ-MSH caused significant reductions in both IL-6 and IL-8 respectively following IL-1β stimulation at 6h. Conclusion: MC1/3 are expressed on C-20/A4 cells, activation by melanocortin peptides led to an inhibition of IL-1β induced cell death and pro-inflammatory cytokine release.

Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals' toxic effects observed.

T cell Maturation and Regulatory T Cell Differentiation:From the Thymus to the Periphery

Immunological tolerance, that is, the failure to mount an immune response to an otherwise immunogenic molecule, is one of the fundamental questions in immunology. The fact that lymphocytes express antigen receptors that are generated randomly and have the potential to recognize any conceivable antigen, adds another puzzle to the physiology of immunological tolerance. The other side of the coin, the general absence of immune responses to self antigens, is ensured by a tight regulation and several selection steps during T and B cell differentiation. One of these processes is the differentiation of regulatory T cells (Treg). While developing in the thymus, T cell clones bearing receptors with high affinity/avidity to antigens present at the time of differentiation may be eliminated by apoptosis or, alternatively, express Foxp3 and become Treg. Treg are key players in the regulation of immunological tolerance since humans and mice with complete loss of function variants of this gene develop fatal autoimmune conditions early in life.(...)