BACKGROUND AND PURPOSEElectrical conduction along endothelium of resistance vessels has not been determined independently of the influence of smooth muscle, surrounding tissue or blood. Two interrelated hypotheses were tested: (i) Intercellular conduction of electrical signals is manifest in endothelial cell (EC) tubes; and (ii) Inhibitors of gap junction channels (GJCs) have confounding actions on EC electrical and Ca 2+ signalling. EXPERIMENTAL APPROACHIntact EC tubes were isolated from abdominal muscle feed (superior epigastric) arteries of C57BL/6 mice. Hyperpolarization was initiated with indirect (ACh) and direct (NS309) stimulation of intermediate-and small-conductance Ca 2+ -activated K + channels (IKCa/SKCa). Remote membrane potential (Vm) responses to intracellular current injection defined the length constant (l) for electrical conduction. Dye coupling was evaluated following intracellular microinjection of propidium iodide. Intracellular Ca 2+ dynamics were determined using Fura-2 photometry. Carbenoxolone (CBX) or b-glycyrrhetinic acid (bGA) was used to investigate the role of GJCs. KEY RESULTSSteady-state Vm of ECs was -25 mV. ACh and NS309 hyperpolarized ECs by -40 and -60 mV respectively. Electrical conduction decayed monoexponentially with distance (l~1.4 mm). Propidium iodide injected into one EC spread into surrounding ECs. CBX or bGA inhibited dye transfer, electrical conduction and EC hyperpolarization reversibly. Both agents elevated resting Ca 2+ while bGA inhibited responses to ACh. CONCLUSIONS AND IMPLICATIONSIndividual cells were effectively coupled to each other within EC tubes. Inhibiting GJCs with glycyrrhetinic acid derivatives blocked hyperpolarization mediated by IKCa/SKCa channels, regardless of Ca 2+ signalling, obviating use of these agents in distinguishing key determinants of electrical conduction along the endothelium. AbbreviationsAFA, abdominal muscle feed artery; bGA, b-glycyrrhetinic acid; [Ca 2+ ]i,

Objective Intercellular conduction of electrical signals underlies spreading vasodilation of resistance arteries. Small and intermediate-conductance Ca2+ activated K+ channels (SKCa/IKCa) of endothelial cells serve a dual function by initiating hyperpolarization and modulating electrical conduction. We tested the hypothesis that the regulation of electrical signaling by SKCa/IKCa is altered with advancing age. Approach and Results Intact endothelial tubes (60 μm wide; 1-3 mm long) were freshly isolated from male C57BL/6 mouse (Young: 4-6 months; Intermediate: 12-14 months; Old: 24-26 months) superior epigastric arteries. Using dual intracellular microelectrodes, current was injected (±0.1-3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance: 50-2000 μm). Across age groups, greatest differences were observed between Young and Old. Resting Vm in Old (−38±1 mV) was more negative (P<0.05) than Young (−30±1 mV). Maximal hyperpolarization to both direct (NS309) and indirect (acetylcholine) activation of SKCa/IKCa was sustained (ΔVm ~ −40 mV) with age. The length constant (λ) for electrical conduction was reduced (P<0.05) from 1630±80 µm (Young) to 1320±80 μm (Old). Inhibiting SKCa/IKCa with apamin + charybdotoxin or scavenging H2O2 with catalase improved electrical conduction (P<0.05) in Old. Exogenous H2O2 (200 μM) in Young evoked hyperpolarization and impaired electrical conduction; these effects were blocked by apamin + charybdotoxin. Conclusions Enhanced current loss through KCa activation impairs electrical conduction along the endothelium of resistance arteries with aging. Attenuating the spatial domain of electrical signaling will restrict the spread of vasodilation and thereby contribute to blood flow limitations associated with advanced age.

Malignant ascites is a major source of morbidity and mortality in ovarian cancer patients. It functions as a permissive reactive tumor-host microenvironment and provides sustenance for the floating tumor cells through a plethora of survival/metastasis-associated molecules.

Epigenetic silencing of tumor suppressor genes is a new focus of investigation in the generation and proliferation of carcinomas. Secreted protein acidic and rich in cysteine (SPARC) is reportedly detrimental to the growth of ovarian cancer cells and has been shown to be epigenetically silenced in several cancers. We hypothesized that SPARC is downregulated in ovarian cancer through aberrant promoter hypermethylation. To that end, we analyzed SPARC expression in ovarian cancer cell lines and investigated the methylation status of the Sparc promoter using methylation-specific polymerase chain reaction. Our results show that SPARC mRNA expression is decreased in three (33%) and absent in four (44%) of the nine ovarian cancer cell lines studied, which correlated with hypermethylation of the Sparc promoter. Treatment with the demethylating agent 5-aza-2'-deoxycytidine rescued SPARC mRNA and protein expression. Addition of exogenous SPARC, as well as ectopic expression by an adenoviral vector, resulted in decreased proliferation of ovarian cancer cell lines. Investigation of primary tumors revealed that the Sparc promoter is methylated in 68% of primary ovarian tumors and that the levels of SPARC protein decrease as the disease progresses from low to high grade. Lastly, de novo methylation of Sparc promoter was shown to be mediated by DNA methyltransferase 3a. These results implicate Sparc promoter methylation as an important factor in the genesis and survival of ovarian carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and/or treatment modality for this disease.

The interplay between peritoneal mesothelial cells and ovarian cancer cells is critical for the initiation and peritoneal dissemination of, and ascites formation in, ovarian cancer. The production of lysophosphatidic acid (LPA) by both peritoneal mesothelial cells and ovarian cancer cells has been shown to promote metastatic phenotype in ovarian cancer. Herein, we report that exogenous addition or ectopic overexpression of the matricellular protein SPARC (secreted protein acidic and rich in cysteine) significantly attenuated LPA-induced proliferation, chemotaxis, and invasion in both highly metastatic SKOV3 and less metastatic OVCAR3 ovarian cancer cell lines. SPARC appears to modulate these functions, at least in part, through the regulation of LPA receptor levels and the attenuation of extracellular signal-regulated kinase (ERK) 1/2 and protein kinase B/AKT signaling. Moreover, our results show that SPARC not only significantly inhibited both basal and LPA-induced interleukin (IL) 6 production in both cell lines but also attenuated IL-6-induced mitogenic, chemotactic, and proinvasive effects, in part, through significant suppression of ERK1/2 and, to a lesser extent, of signal transducers and activators of transcription 3 signaling pathways. Our results strongly suggest that SPARC exerts a dual inhibitory effect on LPA-induced mesothelial-ovarian cancer cell crosstalk through the regulation of both LPA-induced IL-6 production and function. Taken together, our findings underscore the use of SPARC as a potential therapeutic candidate in peritoneal ovarian carcinomatosis.