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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

Characterization of potential bioactive components in HA-induced EV. Panel (a): HPMVEC were grown to confluence and switched to serum-free media and either no HA (control), 100 nM HMW-HA, 100 nM LMW-HA, or 500 ng/mL LPS for 24 hours. EVs were then isolated, run on 4–20% TBE gels, and stained with Alcian blue. Purified enlargeosomes contained HA of ~1 million Da (see arrow) consistent with HMW-HA which we have previously demonstrated to be barrier enhancing [4, 5, 7, 8]. In contrast, control, LMW-HA, or LPS-induced EV contained negligible HA. Human plasma was used as a control. Panel (b): HPMVEC were grown to confluence and switched to serum-free media and either no HA (control), 100 nM HMW-HA, or 100 nM LMW-HA for 24 hours. EVs were then isolated, run on SDS-PAGE, and immunoblotted with anti-CD44 (IM-7) (a), anti-CD44v10 (b), anti-HABP2 (c), anti-CD63 (d), or anti-AHNAK (e) antibodies. HMW-HA-induced enlargeosomes expressed the EC barrier enhancing CD44 isoform, CD44s (standard form) [4, 7]. In contrast, LMW-HA-induced exosomes expressed the EC barrier disrupting HA binding proteins, CD44 isoform CD44v10, and the extracellular serine protease, HABP2 [7, 25]. Basally secreted EV (control) had low expression of these molecules. Panel (c): isolated EVs as described in Panel (b) were subjected to RNA isolation and analysis (see Section 2). Compared to control EV, LMW-HA-induced EV had less total RNA and microRNA while HMW-HA-induced enlargeosomes had ~2-fold higher levels of total RNA and microRNA.
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fig4: Characterization of potential bioactive components in HA-induced EV. Panel (a): HPMVEC were grown to confluence and switched to serum-free media and either no HA (control), 100 nM HMW-HA, 100 nM LMW-HA, or 500 ng/mL LPS for 24 hours. EVs were then isolated, run on 4–20% TBE gels, and stained with Alcian blue. Purified enlargeosomes contained HA of ~1 million Da (see arrow) consistent with HMW-HA which we have previously demonstrated to be barrier enhancing [4, 5, 7, 8]. In contrast, control, LMW-HA, or LPS-induced EV contained negligible HA. Human plasma was used as a control. Panel (b): HPMVEC were grown to confluence and switched to serum-free media and either no HA (control), 100 nM HMW-HA, or 100 nM LMW-HA for 24 hours. EVs were then isolated, run on SDS-PAGE, and immunoblotted with anti-CD44 (IM-7) (a), anti-CD44v10 (b), anti-HABP2 (c), anti-CD63 (d), or anti-AHNAK (e) antibodies. HMW-HA-induced enlargeosomes expressed the EC barrier enhancing CD44 isoform, CD44s (standard form) [4, 7]. In contrast, LMW-HA-induced exosomes expressed the EC barrier disrupting HA binding proteins, CD44 isoform CD44v10, and the extracellular serine protease, HABP2 [7, 25]. Basally secreted EV (control) had low expression of these molecules. Panel (c): isolated EVs as described in Panel (b) were subjected to RNA isolation and analysis (see Section 2). Compared to control EV, LMW-HA-induced EV had less total RNA and microRNA while HMW-HA-induced enlargeosomes had ~2-fold higher levels of total RNA and microRNA.

Mentions: Considering the results of Figures 2 and 3, we further investigated these HA-induced EVs to determine the presence or absence of potential bioactive agents. Interestingly, Alcian blue stained TBE gels revealed that purified enlargeosomes contain HA of ~1 million Da (see arrow) consistent with HMW-HA which we have previously demonstrated to be barrier enhancing [4, 5, 7, 8]. In contrast, control, LMW-HA, or LPS-induced EV contained negligible HA (Figure 4(a)). Considering that the HA receptor, CD44, has previously been reported to be expressed on exosomes [43, 44], we next analyzed HA binding protein expressed in our purified HA-induced EV. Figure 4(b) indicates that HMW-HA-induced enlargeosomes express the EC barrier enhancing CD44 isoform, CD44s (standard form) [4, 7]. In contrast, LMW-HA-induced exosomes express the EC barrier disrupting HA binding proteins, CD44 isoform CD44v10, and the extracellular serine protease, HABP2 [7, 25]. Basally secreted EVs (control) have low expression of these molecules. In addition, EVs are believed to serve as carriers of RNAs [27, 28]. Figure 4(c) indicates that, compared to control EV, LMW-HA-induced EVs have less total RNA and microRNA while HMW-HA-induced enlargeosomes have ~2-fold higher levels of total RNA and microRNA.


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)

Characterization of potential bioactive components in HA-induced EV. Panel (a): HPMVEC were grown to confluence and switched to serum-free media and either no HA (control), 100 nM HMW-HA, 100 nM LMW-HA, or 500 ng/mL LPS for 24 hours. EVs were then isolated, run on 4–20% TBE gels, and stained with Alcian blue. Purified enlargeosomes contained HA of ~1 million Da (see arrow) consistent with HMW-HA which we have previously demonstrated to be barrier enhancing [4, 5, 7, 8]. In contrast, control, LMW-HA, or LPS-induced EV contained negligible HA. Human plasma was used as a control. Panel (b): HPMVEC were grown to confluence and switched to serum-free media and either no HA (control), 100 nM HMW-HA, or 100 nM LMW-HA for 24 hours. EVs were then isolated, run on SDS-PAGE, and immunoblotted with anti-CD44 (IM-7) (a), anti-CD44v10 (b), anti-HABP2 (c), anti-CD63 (d), or anti-AHNAK (e) antibodies. HMW-HA-induced enlargeosomes expressed the EC barrier enhancing CD44 isoform, CD44s (standard form) [4, 7]. In contrast, LMW-HA-induced exosomes expressed the EC barrier disrupting HA binding proteins, CD44 isoform CD44v10, and the extracellular serine protease, HABP2 [7, 25]. Basally secreted EV (control) had low expression of these molecules. Panel (c): isolated EVs as described in Panel (b) were subjected to RNA isolation and analysis (see Section 2). Compared to control EV, LMW-HA-induced EV had less total RNA and microRNA while HMW-HA-induced enlargeosomes had ~2-fold higher levels of total RNA and microRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Characterization of potential bioactive components in HA-induced EV. Panel (a): HPMVEC were grown to confluence and switched to serum-free media and either no HA (control), 100 nM HMW-HA, 100 nM LMW-HA, or 500 ng/mL LPS for 24 hours. EVs were then isolated, run on 4–20% TBE gels, and stained with Alcian blue. Purified enlargeosomes contained HA of ~1 million Da (see arrow) consistent with HMW-HA which we have previously demonstrated to be barrier enhancing [4, 5, 7, 8]. In contrast, control, LMW-HA, or LPS-induced EV contained negligible HA. Human plasma was used as a control. Panel (b): HPMVEC were grown to confluence and switched to serum-free media and either no HA (control), 100 nM HMW-HA, or 100 nM LMW-HA for 24 hours. EVs were then isolated, run on SDS-PAGE, and immunoblotted with anti-CD44 (IM-7) (a), anti-CD44v10 (b), anti-HABP2 (c), anti-CD63 (d), or anti-AHNAK (e) antibodies. HMW-HA-induced enlargeosomes expressed the EC barrier enhancing CD44 isoform, CD44s (standard form) [4, 7]. In contrast, LMW-HA-induced exosomes expressed the EC barrier disrupting HA binding proteins, CD44 isoform CD44v10, and the extracellular serine protease, HABP2 [7, 25]. Basally secreted EV (control) had low expression of these molecules. Panel (c): isolated EVs as described in Panel (b) were subjected to RNA isolation and analysis (see Section 2). Compared to control EV, LMW-HA-induced EV had less total RNA and microRNA while HMW-HA-induced enlargeosomes had ~2-fold higher levels of total RNA and microRNA.
Mentions: Considering the results of Figures 2 and 3, we further investigated these HA-induced EVs to determine the presence or absence of potential bioactive agents. Interestingly, Alcian blue stained TBE gels revealed that purified enlargeosomes contain HA of ~1 million Da (see arrow) consistent with HMW-HA which we have previously demonstrated to be barrier enhancing [4, 5, 7, 8]. In contrast, control, LMW-HA, or LPS-induced EV contained negligible HA (Figure 4(a)). Considering that the HA receptor, CD44, has previously been reported to be expressed on exosomes [43, 44], we next analyzed HA binding protein expressed in our purified HA-induced EV. Figure 4(b) indicates that HMW-HA-induced enlargeosomes express the EC barrier enhancing CD44 isoform, CD44s (standard form) [4, 7]. In contrast, LMW-HA-induced exosomes express the EC barrier disrupting HA binding proteins, CD44 isoform CD44v10, and the extracellular serine protease, HABP2 [7, 25]. Basally secreted EVs (control) have low expression of these molecules. In addition, EVs are believed to serve as carriers of RNAs [27, 28]. Figure 4(c) indicates that, compared to control EV, LMW-HA-induced EVs have less total RNA and microRNA while HMW-HA-induced enlargeosomes have ~2-fold higher levels of total RNA and microRNA.

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