Extracellular Vesicles from Caveolin-Enriched Microdomains Regulate Hyaluronan-Mediated Sustained Vascular Integrity

Defects in vascular integrity are an initiating factor in several disease processes. We have previously reported that high molecular weight hyaluronan (HMW-HA), a major glycosaminoglycan in the body, promotes rapid signal transduction in human pulmonary microvascular endothelial cells (HPMVEC) leading to barrier enhancement. In contrast, low molecular weight hyaluronan (LMW-HA), produced in disease states by hyaluronidases and reactive oxygen species (ROS), induces HPMVEC barrier disruption. However, the mechanism(s) of sustained barrier regulation by HA are poorly defined. Our results indicate that long-term (6–24 hours) exposure of HMW-HA induced release of a novel type of extracellular vesicle from HLMVEC called enlargeosomes (characterized by AHNAK expression) while LMW-HA long-term exposure promoted release of exosomes (characterized by CD9, CD63, and CD81 expression). These effects were blocked by inhibiting caveolin-enriched microdomain (CEM) formation. Further, inhibiting enlargeosome release by annexin II siRNA attenuated the sustained barrier enhancing effects of HMW-HA. Finally, exposure of isolated enlargeosomes to HPMVEC monolayers generated barrier enhancement while exosomes led to barrier disruption. Taken together, these results suggest that differential release of extracellular vesicles from CEM modulate the sustained HPMVEC barrier regulation by HMW-HA and LMW-HA. HMW-HA-induced specialized enlargeosomes can be a potential therapeutic strategy for diseases involving impaired vascular integrity

The mu opioid receptor (MOR) is recruited to the EGF receptor with EGF stimulation but does not regulate EGF receptor phosphorylation.

<p><b>Panel A</b>: Human H358 non-small cell lung cancer (NSCLC) cells were treated with no (control) or 100 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoprecipitated with anti-EGFR antibody. Immunoblots were performed on total cell lysates (left) and immunoprecipitated material (right) using anti-MOR (a) and anti-EGFR (b) antibodies. The mu opioid receptor is recruited to the EGFR with EGF stimulation. <b>Panel B</b>: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone, 10 ng/ml EGF for 5, 15 or 30 minutes, or 100 nM MNTX and 10 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoblotted using anti-pY⁸⁴⁵ EGFR (a), anti-pY⁹⁹² EGFR (b), anti-pY¹⁰⁴⁵ EGFR (c), anti-pY¹⁰⁶⁸ EGFR (d), anti-EGFR (e) and anti-actin (e) antibodies. MNTX does not inhibit EGF-induced EGFR tyrosine phosphorylation.</p

Schematic diagram illustrating mu opioid receptor (MOR) regulation of growth factor receptor signaling and human lung cancer proliferation, migration and epithelial mesenchymal transition (EMT).

<p>Activation of growth factor receptors promotes complex formation with the mu opioid receptor (MOR) and Grb-2 as well as Src activation (1). These events induce recruitment of the scaffolding protein, Gab1, to the plasma membrane (2) and consequent recruitment/activation of PI3 kinase, Akt and STAT3 signaling (3) which are required for human lung cancer proliferation, migration and epithelial mesenchymal transition (EMT) (4). Inhibition of MOR (siRNA, shRNA and/or the peripheral MOR antagonist, methylnaltrexone (MNTX)) blocks morphine and DAMGO binding to the mu opioid receptor (5). In addition, inhibiting MOR attenuates growth factor-induced phosphorylation/activation of Src, Gab-1, PI3 kinase, Akt and STAT3 with consequent inhibition of human lung cancer proliferation, migration and EMT (6).</p

The peripheral mu opioid receptor antagonist, methylnaltrexone (MNTX), inhibits EGF-induced phosphorylation of Src, PI3 kinase and STAT3 in human lung cancer cells.

<p><b>Panel A</b>: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone, 10 ng/ml EGF for 5, 15 or 30 minutes, or 100 nM MNTX and 10 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoblotted using anti-phospho-Src (pY⁴¹⁶) (a), anti-phospho-p85/p55 PI3 kinase (pY⁴⁵⁸/pY¹⁹⁹) (b,c), anti-phospho-STAT3 (pY⁷⁰⁵) (d) and anti-actin (e) antibodies. <b>Panel B</b>: Graphical quantitation of immunoreactivity of experiments performed described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091577#pone-0091577-g003" target="_blank">Figure 3-B</a> and Panel A with normalization to total specific protein and n = 3 independent experiments per condition. An asterisk (*) indicates a statistically significant difference (p<0.05) from control. The error bars = standard deviation.</p

The peripheral mu opioid receptor antagonist, methylnaltrexone (MNTX), inhibits EGF-induced recruitment/activation of the linker protein, Grb-2, and the scaffolding protein, Gab-1, in human lung cancer cells.

<p><b>Panel A</b>: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX (1 hour pre-incubation), 10 ng/ml EGF for 15 minutes, or 100 nM MNTX and 10 ng/ml EGF. Cell lysates were obtained and immunoprecipitated with anti-EGFR antibody. Immunoblots were performed on total cell lysates and immunoprecipitated material using anti-Grb-2 (a,c) and anti-EGFR (b,d) antibody. MNTX inhibits EGF-induced recruitment of Grb-2 to the EGFR. <b>Panel B</b>: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone, 10 ng/ml EGF for 5, 15 or 30 minutes, or 100 nM MNTX and 10 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoblotted using anti-pY⁶²⁷ Gab-1 (a), anti-pY³⁰⁷ Gab-1 (b) and anti-actin (c) antibodies. MNTX attenuates EGF-induced Gab-1 tyrosine phosphorylation.</p

Inhibiting the mu opioid receptor (MOR), Src, Gab-1, PI3 kinase or STAT3 attenuates opioid and growth factor-induced epithelial mesenchymal transition (EMT) in human lung cancer cells.

<p><b>Panel A</b>: Graphical representation of the % control vimentin expression. H358 human NSCLC cells were either untreated, treated with 100 ng/ml epidermal growth factor (EGF), 100 nM DAMGO, morphine or fentanyl or 100 ng/ml insulin growth factor (IGF) for 96 hours with or without pretreatment of cells with the peripheral MOR antagonist, methylnaltrexone (MNTX, 100 nM), MOR siRNA, Gab-1 siRNA, Src siRNA, the PI3 kinase inhibitor LY294002 (10 uM), Akt Inhibitor X (5 uM), the Src family kinase inhibitor PP2 (100 nM) or the STAT3 inhibitor Stattic (10 uM). Cell lysates were then obtained and immunoblotted with the EMT marker anti-vimentin antibody. Experiments were repeated in triplicate and immunoreactive bands were analyzed using computer-assisted densitometry. There is a statistically significant difference (p<0.05 indicated by asterisks (*)) between control and treatment groups with error bars = standard deviation. An increase in vimentin expression is suggestive of an epithelial mesenchymal transition. <b>Panel B</b>: Graphical representation of the % control claudin-1 expression. H358 human NSCLC cells were either untreated, treated with 100 ng/ml epidermal growth factor (EGF), 100 nM DAMGO, morphine or fentanyl or 100 ng/ml insulin growth factor (IGF) for 96 hours with or without pretreatment of cells with the peripheral MOR antagonist, methylnaltrexone (MNTX, 100 nM), MOR siRNA, Gab-1 siRNA, Src siRNA, the PI3 kinase inhibitor LY294002 (10 uM), Akt Inhibitor X (5 uM), the Src family kinase inhibitor PP2 (100 nM) or the STAT3 inhibitor Stattic (10 uM). Cell lysates were then obtained and immunoblotted with the EMT marker anti-claudin-1 antibody. Three independent experiments per condition were performed and immunoreactive bands were analyzed using computer-assisted densitometry. There is a statistically significant difference (p<0.05 indicated by asterisks (*)) between control and treatment groups with error bars = standard deviation. A decrease in claudin-1 expression is suggestive of an epithelial mesenchymal transition.</p