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.

To study Ca(2+) signaling in the endothelium of murine feed arteries, we determined the in vitro stability of endothelial cell (EC) tubes freshly isolated from abdominal muscle feed arteries of male and female C57BL/6 mice (5-9 mo, 25-35 g). We tested the hypothesis that intracellular Ca(2+) concentration ([Ca(2+)](i)) responses to muscarinic receptor activation would increase with temperature. Intact EC tubes (length: 1-2 mm, width: 65-80 μm) were isolated using gentle enzymatic digestion with trituration to remove smooth muscle cells. A freshly isolated EC tube was secured in a chamber and superfused at 24 (room temperature), 32, or 37°C. Using fura-2 dye, [Ca(2+)](i) was monitored (ratio of fluorescence at 340- to 380-nm wavelength) at rest and in response to bolus doses of ACh (20 nmol to 200 μmol). The morphological integrity of EC tubes was preserved at 24 and 32°C. Based on the Ca(2+) K(d) values we determined for fura-2 (174 nM at 24°C and 146 nM at 32°C), resting [Ca(2+)](i) remained stable for 180 min at both 24 and 32°C (27 ± 4 and 34 ± 2 nM, respectively), with peak responses to ACh (20 μmol) increasing from ∼220 nM at 24°C to ∼500 nM at 32°C (P < 0.05). There was no difference in responses to ACh between EC tubes from male versus female mice. When EC tubes were maintained at 37°C (typical in vivo temperature), resting [Ca(2+)](i) increased by ∼30% within 15 min, and gaps formed between individual ECs as they retracted and extruded dye, precluding further study. We conclude that EC tubes enable Ca(2+) signaling to be evaluated in the freshly isolated endothelium of murine feed arteries. While Ca(2+) responses are enhanced by approximately twofold at 32 versus 24°C, the instability of EC tubes at 37°C precludes their study at typical body temperature.

Key pointsr Calcium signalling in endothelial cells of resistance arteries is integral to blood flow regulation.Oxidative stress and endothelial dysfunction can prevail during advanced age and we questioned how calcium signalling may be affected.r Intact endothelium was freshly isolated from superior epigastric arteries of Young (ß4 months) and Old (ß24 months) male C57BL/6 mice. Under resting conditions, with no difference in intracellular calcium levels, hydrogen peroxide (H 2 O 2 ) availability was ß1/3 greater in endothelium of Old mice while vascular catalase activity was reduced by nearly half. r Microvascular adaptation to advanced age may protect endothelial cells during elevated oxidative stress to preserve functional viability of the intima. Abstract Endothelial cell Ca2+ signalling is integral to blood flow control in the resistance vasculature yet little is known of how its regulation may be affected by advancing age. We tested the hypothesis that advanced age protects microvascular endothelium by attenuating aberrant Ca 2+ signalling during oxidative stress. Intact endothelial tubes (width, ß60 μm; length, ß1000 μm) were isolated from superior epigastric arteries of Young (3-4 months) and Old (24-26 months) male C57BL/6 mice and loaded with Fura-2 dye to monitor [Ca 2+ ] i . At rest there was no difference in [Ca 2+ ] i between age groups. Compared to Young, the [Ca 2+ ] i response to maximal stimulation with acetylcholine (3 μM, 2 min) was ß25% greater in Old, confirming signalling integrity with advanced age. Basal H 2 O 2 availability was ß33% greater in Old while vascular catalase activity was reduced by half. Transient exposure to elevated H 2 O 2 (200 μM, 20 min) progressively increased [Ca 2+ ] i to ß4-fold greater levels in endothelium of Young versus Old. With no difference between age groups at rest, Mn 2+ quench of Fura-2 fluorescence revealed 2-fold greater Ca 2+ influx in Young during elevated H 2 O 2 ; this effect was attenuated by ß75% using ruthenium red (5 μM) as a broad-spectrum inhibitor of transient receptor potential channels. Prolonged exposure to H 2 O 2 (200 μM, 60 min) induced ß7-fold greater cell death in endothelium of Young versus Old. Thus, microvascular endothelium can adapt to advanced age by reducing Ca 2+ influx during elevated oxidative stress. Protection from cell death during oxidative stress will sustain endothelial integrity during ageing.

Objective To test the hypothesis that Ca2+ responses to G-protein coupled receptor (GPCR) activation are coordinated between neighboring endothelial cells of resistance arteries. Methods Endothelial cell tubes were freshly isolated from superior epigastric arteries of C57BL/6 mice. Intercellular coupling was tested using microinjection of propidium iodide. Following loading with fluo-4 dye, intracellular Ca2+ responses to ACh were imaged with confocal microscopy. Results Cell-to-cell transfer of propidium iodide confirmed functional gap junction channels. 1 μM ACh evoked Ca2+ responses [9.8±0.8/min, (F/F0)=3.11±0.2] which pseudo-linescan analysis revealed to be composed of Ca2+ waves and spatially-restricted Ca2+ release events. 100 nM ACh induced Ca2+ responses of lower frequency (4.5±0.7/min) and amplitude (F/F0=1.95±0.11) composed primarily of spatially-restricted events. The interval between Ca2+ waves in Adjacent cells (0.79±0.12 s) was shorter (P<0.05) than between Nonadjacent cells (1.56±0.25 s). Spatially-restricted Ca2+ release events had similar frequencies and latencies between Adjacent and Nonadjacent cells. Inhibiting intracellular Ca2+ release with 2-APB, Xestospongin C or thapsigargin eliminated Ca2+ responses. Conclusions With moderate GPCR stimulation, localized Ca2+ release events predominate among cells. Greater GPCR stimulation evokes coordinated intercellular Ca2+ waves via the endoplasmic reticulum. Calcium signaling during GPCR activation is complex among cells, varying with stimulus intensity and proximity to actively signaling cells.