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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

Determination of extracellular vesicle (EV) release kinetics and caveolin-enriched microdomain (CEM) dependence in human endothelial cells (EC). Panel (a): graphical representations of the kinetics of HA-induced EV release from human pulmonary microvascular endothelial cells (HPMVEC). Human EC monolayers were placed in serum-free media and the resulting media containing EV were collected at 0, 1, 2, 3, 6, 12, and 24 hours. The concentrations of EV were determined utilizing nanosight nanoparticle tracking analysis (NTA). HPMVEC exhibit basal secretion of EV (control). However, addition of 100 nM HMW-HA or 100 nM LMW-HA dramatically increased the production of EV starting at ~6 hours posttreatment. N = 3 per group and error bars = standard deviation. Panel (b): graphical representation of the role of CEM in HA-mediated EV production from human EC. HPMVEC monolayers were either untreated or treated with 5 mM methyl-β-cyclodextrin (MβCD, a CEM disrupting agent) with or without addition of 100 nM LMW-HA or 100 nM HMW-HA (24 hours). EVs were analyzed as described in Panel (a). Inhibiting CEM formation significantly inhibited both LMW-HA and HMW-HA-mediated EV secretion with n = 3 per group and error bars = standard deviation.
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fig1: Determination of extracellular vesicle (EV) release kinetics and caveolin-enriched microdomain (CEM) dependence in human endothelial cells (EC). Panel (a): graphical representations of the kinetics of HA-induced EV release from human pulmonary microvascular endothelial cells (HPMVEC). Human EC monolayers were placed in serum-free media and the resulting media containing EV were collected at 0, 1, 2, 3, 6, 12, and 24 hours. The concentrations of EV were determined utilizing nanosight nanoparticle tracking analysis (NTA). HPMVEC exhibit basal secretion of EV (control). However, addition of 100 nM HMW-HA or 100 nM LMW-HA dramatically increased the production of EV starting at ~6 hours posttreatment. N = 3 per group and error bars = standard deviation. Panel (b): graphical representation of the role of CEM in HA-mediated EV production from human EC. HPMVEC monolayers were either untreated or treated with 5 mM methyl-β-cyclodextrin (MβCD, a CEM disrupting agent) with or without addition of 100 nM LMW-HA or 100 nM HMW-HA (24 hours). EVs were analyzed as described in Panel (a). Inhibiting CEM formation significantly inhibited both LMW-HA and HMW-HA-mediated EV secretion with n = 3 per group and error bars = standard deviation.

Mentions: In order to identify potential mechanism(s) of hyaluronan- (HA-) mediated sustained human endothelial cell (EC) barrier function, we first determined the kinetics of HA-induced extracellular vesicle (EV) release. Human pulmonary microvascular EC (HPMVEC) were grown to confluence and placed in serum-free media, and no HA (control), 100 nm HMW-HA, or 100 nm LMW-HA were added for 0, 1, 2, 3, 6, 12, or 24 hours. Treated media were then collected and analyzed using nanosight nanoparticle tracking analysis (NTA) to determine EV concentrations (see Section 2). The results of Figure 1(a) indicate that HPMVEC secrete basal levels of EV. However, there is a dramatic increase in EV release from HPMVEC with either LMW-HA or HMW-HA addition starting at ~6 hours.


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)

Determination of extracellular vesicle (EV) release kinetics and caveolin-enriched microdomain (CEM) dependence in human endothelial cells (EC). Panel (a): graphical representations of the kinetics of HA-induced EV release from human pulmonary microvascular endothelial cells (HPMVEC). Human EC monolayers were placed in serum-free media and the resulting media containing EV were collected at 0, 1, 2, 3, 6, 12, and 24 hours. The concentrations of EV were determined utilizing nanosight nanoparticle tracking analysis (NTA). HPMVEC exhibit basal secretion of EV (control). However, addition of 100 nM HMW-HA or 100 nM LMW-HA dramatically increased the production of EV starting at ~6 hours posttreatment. N = 3 per group and error bars = standard deviation. Panel (b): graphical representation of the role of CEM in HA-mediated EV production from human EC. HPMVEC monolayers were either untreated or treated with 5 mM methyl-β-cyclodextrin (MβCD, a CEM disrupting agent) with or without addition of 100 nM LMW-HA or 100 nM HMW-HA (24 hours). EVs were analyzed as described in Panel (a). Inhibiting CEM formation significantly inhibited both LMW-HA and HMW-HA-mediated EV secretion with n = 3 per group and error bars = standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581561&req=5

fig1: Determination of extracellular vesicle (EV) release kinetics and caveolin-enriched microdomain (CEM) dependence in human endothelial cells (EC). Panel (a): graphical representations of the kinetics of HA-induced EV release from human pulmonary microvascular endothelial cells (HPMVEC). Human EC monolayers were placed in serum-free media and the resulting media containing EV were collected at 0, 1, 2, 3, 6, 12, and 24 hours. The concentrations of EV were determined utilizing nanosight nanoparticle tracking analysis (NTA). HPMVEC exhibit basal secretion of EV (control). However, addition of 100 nM HMW-HA or 100 nM LMW-HA dramatically increased the production of EV starting at ~6 hours posttreatment. N = 3 per group and error bars = standard deviation. Panel (b): graphical representation of the role of CEM in HA-mediated EV production from human EC. HPMVEC monolayers were either untreated or treated with 5 mM methyl-β-cyclodextrin (MβCD, a CEM disrupting agent) with or without addition of 100 nM LMW-HA or 100 nM HMW-HA (24 hours). EVs were analyzed as described in Panel (a). Inhibiting CEM formation significantly inhibited both LMW-HA and HMW-HA-mediated EV secretion with n = 3 per group and error bars = standard deviation.
Mentions: In order to identify potential mechanism(s) of hyaluronan- (HA-) mediated sustained human endothelial cell (EC) barrier function, we first determined the kinetics of HA-induced extracellular vesicle (EV) release. Human pulmonary microvascular EC (HPMVEC) were grown to confluence and placed in serum-free media, and no HA (control), 100 nm HMW-HA, or 100 nm LMW-HA were added for 0, 1, 2, 3, 6, 12, or 24 hours. Treated media were then collected and analyzed using nanosight nanoparticle tracking analysis (NTA) to determine EV concentrations (see Section 2). The results of Figure 1(a) indicate that HPMVEC secrete basal levels of EV. However, there is a dramatic increase in EV release from HPMVEC with either LMW-HA or HMW-HA addition starting at ~6 hours.

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