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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

Identification of LMW-HA and HMW-HA-induced EV from human EC. Panel (a): biomarker analysis of HA-induced EV. 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-CD9 (a), anti-CD63 (b), anti-CD81 (c), anti-AHNAK (d), antiannexin II (e), or anti-caveolin-1 (f) antibodies. LMW-HA-induced EV expressed exosome markers while HMW-HA-induced EV expressed enlargeosome markers. EV also expressed caveolin-1, a crucial component of caveolin-enriched microdomains (CEM). Panel (b): topographical images of HA-induced EV using atomic force microscopy (AFM). Isolated HA-induced EVs as described in Panel (a) were plated on mica and subjected to AFM analysis (see Section 2). The vesicles were never dried and are shown as imaged under buffer. Control EV and exosomes were round in shape and had a relatively smooth surface. In contrast, enlargeosomes were relatively round in shape but had a rough uneven surface topology. Control EV and exosomes had a diameter of ~50 nm which is consistent with the accepted exosome size range of 30–100 nm [20]. HMW-HA-induced enlargeosomes appeared slightly larger in comparison to exosomes.
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fig2: Identification of LMW-HA and HMW-HA-induced EV from human EC. Panel (a): biomarker analysis of HA-induced EV. 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-CD9 (a), anti-CD63 (b), anti-CD81 (c), anti-AHNAK (d), antiannexin II (e), or anti-caveolin-1 (f) antibodies. LMW-HA-induced EV expressed exosome markers while HMW-HA-induced EV expressed enlargeosome markers. EV also expressed caveolin-1, a crucial component of caveolin-enriched microdomains (CEM). Panel (b): topographical images of HA-induced EV using atomic force microscopy (AFM). Isolated HA-induced EVs as described in Panel (a) were plated on mica and subjected to AFM analysis (see Section 2). The vesicles were never dried and are shown as imaged under buffer. Control EV and exosomes were round in shape and had a relatively smooth surface. In contrast, enlargeosomes were relatively round in shape but had a rough uneven surface topology. Control EV and exosomes had a diameter of ~50 nm which is consistent with the accepted exosome size range of 30–100 nm [20]. HMW-HA-induced enlargeosomes appeared slightly larger in comparison to exosomes.

Mentions: We next characterized HA-induced EV using biomarker analysis and atomic force microscopy (AFM). The results of Figure 2(a) indicate that LMW-HA induces EV with biomarkers consistent with exosomes (CD9, CD63, and CD81). In contrast, HMW-HA induces EV with biomarkers consistent with a novel vesicle called an enlargeosome. Enlargeosomes are specialized vesicles enriched in AHNAK and annexin II that have been observed intracellularly, fusing to the plasma membrane and shedding from the plasma membrane [21–23]. Intrigued with these results, we further analyzed the HA-induced EV with AFM. Figure 2(b) indicates control EV and exosomes are round in shape and have a relatively smooth surface. In contrast, enlargeosomes are relatively round in shape but have a rough uneven surface topology. Control EV and exosomes have a diameter of ~50 nm which is consistent with the accepted exosome size range of 30–100 nm [20]. HMW-HA-induced enlargeosomes appear slightly larger in comparison to exosomes.


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)

Identification of LMW-HA and HMW-HA-induced EV from human EC. Panel (a): biomarker analysis of HA-induced EV. 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-CD9 (a), anti-CD63 (b), anti-CD81 (c), anti-AHNAK (d), antiannexin II (e), or anti-caveolin-1 (f) antibodies. LMW-HA-induced EV expressed exosome markers while HMW-HA-induced EV expressed enlargeosome markers. EV also expressed caveolin-1, a crucial component of caveolin-enriched microdomains (CEM). Panel (b): topographical images of HA-induced EV using atomic force microscopy (AFM). Isolated HA-induced EVs as described in Panel (a) were plated on mica and subjected to AFM analysis (see Section 2). The vesicles were never dried and are shown as imaged under buffer. Control EV and exosomes were round in shape and had a relatively smooth surface. In contrast, enlargeosomes were relatively round in shape but had a rough uneven surface topology. Control EV and exosomes had a diameter of ~50 nm which is consistent with the accepted exosome size range of 30–100 nm [20]. HMW-HA-induced enlargeosomes appeared slightly larger in comparison to exosomes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Identification of LMW-HA and HMW-HA-induced EV from human EC. Panel (a): biomarker analysis of HA-induced EV. 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-CD9 (a), anti-CD63 (b), anti-CD81 (c), anti-AHNAK (d), antiannexin II (e), or anti-caveolin-1 (f) antibodies. LMW-HA-induced EV expressed exosome markers while HMW-HA-induced EV expressed enlargeosome markers. EV also expressed caveolin-1, a crucial component of caveolin-enriched microdomains (CEM). Panel (b): topographical images of HA-induced EV using atomic force microscopy (AFM). Isolated HA-induced EVs as described in Panel (a) were plated on mica and subjected to AFM analysis (see Section 2). The vesicles were never dried and are shown as imaged under buffer. Control EV and exosomes were round in shape and had a relatively smooth surface. In contrast, enlargeosomes were relatively round in shape but had a rough uneven surface topology. Control EV and exosomes had a diameter of ~50 nm which is consistent with the accepted exosome size range of 30–100 nm [20]. HMW-HA-induced enlargeosomes appeared slightly larger in comparison to exosomes.
Mentions: We next characterized HA-induced EV using biomarker analysis and atomic force microscopy (AFM). The results of Figure 2(a) indicate that LMW-HA induces EV with biomarkers consistent with exosomes (CD9, CD63, and CD81). In contrast, HMW-HA induces EV with biomarkers consistent with a novel vesicle called an enlargeosome. Enlargeosomes are specialized vesicles enriched in AHNAK and annexin II that have been observed intracellularly, fusing to the plasma membrane and shedding from the plasma membrane [21–23]. Intrigued with these results, we further analyzed the HA-induced EV with AFM. Figure 2(b) indicates control EV and exosomes are round in shape and have a relatively smooth surface. In contrast, enlargeosomes are relatively round in shape but have a rough uneven surface topology. Control EV and exosomes have a diameter of ~50 nm which is consistent with the accepted exosome size range of 30–100 nm [20]. HMW-HA-induced enlargeosomes appear slightly larger in comparison to exosomes.

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