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

Mirzapoiazova T, Lennon FE, Mambetsariev B, Allen M, Riehm J, Poroyko VA, Singleton PA - Int J Cell Biol (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Pulmonary and Critical Care, Pritzker School of Medicine, The University of Chicago, Chicago, IL, USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Inhibiting enlargeosome release attenuates HMW-HA-mediated sustained human EC barrier enhancement. Panel (a): inhibition of annexin II expression using siRNA in HPMVEC. Human EC were plated at ~50% confluence and treated with either no siRNA (control), siControl, or siAnnexin II with or without 100 nM LMW-HA or 100 nM HMW-HA for 48 hours. EC lysates were then obtained, run on SDS-PAGE, and immunoblotted with anti-annexin II (a) or anti-actin (b) antibodies. Panel (b): graphical representation of the role of annexin II inhibition in HA-mediated EV production from human EC. HPMVEC were treated as described in Panel (a) and EVs were analyzed utilizing nanosight nanoparticle tracking analysis (NTA). Silencing of annexin II, which has previously been reported to be crucial for enlargeosome exocytosis [23], significantly reduces HMW-HA-, but not LMW-HA-, mediated EV secretion from human EC with n = 3 per group and error bars = standard deviation. Panel (c): HPMVEC previously treated with either no siRNA (control), siControl, or annexin II siRNA were plated on transendothelial electrical resistance (TER) electrodes, grown to confluence, and switched to serum-free media and 100 nM LMW-HA was then added. Silencing of annexin II did not affect the sustained human EC barrier disruptive effects of LMW-HA with n = 3 per group and error bars = standard deviation. Panel (d): HPMVEC previously treated with either no siRNA (control), siControl, or annexin II siRNA were plated on transendothelial electrical resistance (TER) electrodes, grown to confluence, and switched to serum-free media and 100 nM HMW-HA was then added. In contrast to LMW-HA, silencing annexin II almost completely inhibited the sustained barrier enhancing effects of HMW-HA with n = 3 per group and error bars = standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4581561&req=5

fig6: Inhibiting enlargeosome release attenuates HMW-HA-mediated sustained human EC barrier enhancement. Panel (a): inhibition of annexin II expression using siRNA in HPMVEC. Human EC were plated at ~50% confluence and treated with either no siRNA (control), siControl, or siAnnexin II with or without 100 nM LMW-HA or 100 nM HMW-HA for 48 hours. EC lysates were then obtained, run on SDS-PAGE, and immunoblotted with anti-annexin II (a) or anti-actin (b) antibodies. Panel (b): graphical representation of the role of annexin II inhibition in HA-mediated EV production from human EC. HPMVEC were treated as described in Panel (a) and EVs were analyzed utilizing nanosight nanoparticle tracking analysis (NTA). Silencing of annexin II, which has previously been reported to be crucial for enlargeosome exocytosis [23], significantly reduces HMW-HA-, but not LMW-HA-, mediated EV secretion from human EC with n = 3 per group and error bars = standard deviation. Panel (c): HPMVEC previously treated with either no siRNA (control), siControl, or annexin II siRNA were plated on transendothelial electrical resistance (TER) electrodes, grown to confluence, and switched to serum-free media and 100 nM LMW-HA was then added. Silencing of annexin II did not affect the sustained human EC barrier disruptive effects of LMW-HA with n = 3 per group and error bars = standard deviation. Panel (d): HPMVEC previously treated with either no siRNA (control), siControl, or annexin II siRNA were plated on transendothelial electrical resistance (TER) electrodes, grown to confluence, and switched to serum-free media and 100 nM HMW-HA was then added. In contrast to LMW-HA, silencing annexin II almost completely inhibited the sustained barrier enhancing effects of HMW-HA with n = 3 per group and error bars = standard deviation.

Mentions: To determine whether HMW-HA-induced enlargeosomes affect sustained human EC barrier function, we silenced the expression of annexin II which has previously been reported to be crucial for enlargeosome exocytosis [23]. Figure 6(a) indicates that we can successfully inhibit annexin II expression with siRNA in human EC. Silencing of annexin II significantly reduces HMW-HA-, but not LMW-HA-, mediated EV secretion from human EC (Figure 6(b)). Importantly, silencing of annexin II did not affect the sustained human EC barrier disruptive effects of LMW-HA (Figure 6(c)) but almost completely inhibited the sustained HMW-HA barrier enhancing effects (Figure 6(d)).


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

Mirzapoiazova T, Lennon FE, Mambetsariev B, Allen M, Riehm J, Poroyko VA, Singleton PA - Int J Cell Biol (2015)

Inhibiting enlargeosome release attenuates HMW-HA-mediated sustained human EC barrier enhancement. Panel (a): inhibition of annexin II expression using siRNA in HPMVEC. Human EC were plated at ~50% confluence and treated with either no siRNA (control), siControl, or siAnnexin II with or without 100 nM LMW-HA or 100 nM HMW-HA for 48 hours. EC lysates were then obtained, run on SDS-PAGE, and immunoblotted with anti-annexin II (a) or anti-actin (b) antibodies. Panel (b): graphical representation of the role of annexin II inhibition in HA-mediated EV production from human EC. HPMVEC were treated as described in Panel (a) and EVs were analyzed utilizing nanosight nanoparticle tracking analysis (NTA). Silencing of annexin II, which has previously been reported to be crucial for enlargeosome exocytosis [23], significantly reduces HMW-HA-, but not LMW-HA-, mediated EV secretion from human EC with n = 3 per group and error bars = standard deviation. Panel (c): HPMVEC previously treated with either no siRNA (control), siControl, or annexin II siRNA were plated on transendothelial electrical resistance (TER) electrodes, grown to confluence, and switched to serum-free media and 100 nM LMW-HA was then added. Silencing of annexin II did not affect the sustained human EC barrier disruptive effects of LMW-HA with n = 3 per group and error bars = standard deviation. Panel (d): HPMVEC previously treated with either no siRNA (control), siControl, or annexin II siRNA were plated on transendothelial electrical resistance (TER) electrodes, grown to confluence, and switched to serum-free media and 100 nM HMW-HA was then added. In contrast to LMW-HA, silencing annexin II almost completely inhibited the sustained barrier enhancing effects of HMW-HA with n = 3 per group and error bars = standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4581561&req=5

fig6: Inhibiting enlargeosome release attenuates HMW-HA-mediated sustained human EC barrier enhancement. Panel (a): inhibition of annexin II expression using siRNA in HPMVEC. Human EC were plated at ~50% confluence and treated with either no siRNA (control), siControl, or siAnnexin II with or without 100 nM LMW-HA or 100 nM HMW-HA for 48 hours. EC lysates were then obtained, run on SDS-PAGE, and immunoblotted with anti-annexin II (a) or anti-actin (b) antibodies. Panel (b): graphical representation of the role of annexin II inhibition in HA-mediated EV production from human EC. HPMVEC were treated as described in Panel (a) and EVs were analyzed utilizing nanosight nanoparticle tracking analysis (NTA). Silencing of annexin II, which has previously been reported to be crucial for enlargeosome exocytosis [23], significantly reduces HMW-HA-, but not LMW-HA-, mediated EV secretion from human EC with n = 3 per group and error bars = standard deviation. Panel (c): HPMVEC previously treated with either no siRNA (control), siControl, or annexin II siRNA were plated on transendothelial electrical resistance (TER) electrodes, grown to confluence, and switched to serum-free media and 100 nM LMW-HA was then added. Silencing of annexin II did not affect the sustained human EC barrier disruptive effects of LMW-HA with n = 3 per group and error bars = standard deviation. Panel (d): HPMVEC previously treated with either no siRNA (control), siControl, or annexin II siRNA were plated on transendothelial electrical resistance (TER) electrodes, grown to confluence, and switched to serum-free media and 100 nM HMW-HA was then added. In contrast to LMW-HA, silencing annexin II almost completely inhibited the sustained barrier enhancing effects of HMW-HA with n = 3 per group and error bars = standard deviation.
Mentions: To determine whether HMW-HA-induced enlargeosomes affect sustained human EC barrier function, we silenced the expression of annexin II which has previously been reported to be crucial for enlargeosome exocytosis [23]. Figure 6(a) indicates that we can successfully inhibit annexin II expression with siRNA in human EC. Silencing of annexin II significantly reduces HMW-HA-, but not LMW-HA-, mediated EV secretion from human EC (Figure 6(b)). Importantly, silencing of annexin II did not affect the sustained human EC barrier disruptive effects of LMW-HA (Figure 6(c)) but almost completely inhibited the sustained HMW-HA barrier enhancing effects (Figure 6(d)).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Pulmonary and Critical Care, Pritzker School of Medicine, The University of Chicago, Chicago, IL, USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus