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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

Analysis of HMW-HA-induced AHNAK redistribution in human EC. HPMVEC were grown to confluence on glass coverslips and treated with 100 nM HMW-HA for 6 hours. 50 μM ATP (six hours) and 3 μM ionomycin (10 minutes) were used as controls due to their previously reported ability to stimulate enlargeosome exocytosis [21–24]. The samples were then fixed in 4% paraformaldehyde and subjected to immunocytochemical analysis using AHNAK antibodies (green). Plasma membranes were labeled with Alexa Fluor 594 wheat germ agglutinin (red) and nuclei were stained with Hoechst 33342 (blue). Control HPMVEC exhibited diffuse cytoplasmic AHNAK staining. When stimulated with HMW-HA, intracellular AHNAK redistributed to a “vesicular” pattern which is similar to treatment with ATP and ionomycin.
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fig3: Analysis of HMW-HA-induced AHNAK redistribution in human EC. HPMVEC were grown to confluence on glass coverslips and treated with 100 nM HMW-HA for 6 hours. 50 μM ATP (six hours) and 3 μM ionomycin (10 minutes) were used as controls due to their previously reported ability to stimulate enlargeosome exocytosis [21–24]. The samples were then fixed in 4% paraformaldehyde and subjected to immunocytochemical analysis using AHNAK antibodies (green). Plasma membranes were labeled with Alexa Fluor 594 wheat germ agglutinin (red) and nuclei were stained with Hoechst 33342 (blue). Control HPMVEC exhibited diffuse cytoplasmic AHNAK staining. When stimulated with HMW-HA, intracellular AHNAK redistributed to a “vesicular” pattern which is similar to treatment with ATP and ionomycin.

Mentions: To further investigate HMW-HA-induced enlargeosome dynamics in HLMVEC, confluent monolayers were treated with HMW-HA for 6 hours. ATP and ionomycin were used as controls due to their previously reported ability to stimulate enlargeosome exocytosis [21–24]. The samples were then fixed and subjected to immunocytochemical analysis using AHNAK antibodies (green). Plasma membranes were labeled with Alexa Fluor 594 wheat germ agglutinin (red) and nuclei were stained with Hoechst 33342 (blue). The results of Figure 3 indicate that control HPMVEC exhibit diffuse cytoplasmic AHNAK staining. When stimulated with HMW-HA, intracellular AHNAK redistributes to a “vesicular” pattern which is similar to treatment with ATP and ionomycin.


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)

Analysis of HMW-HA-induced AHNAK redistribution in human EC. HPMVEC were grown to confluence on glass coverslips and treated with 100 nM HMW-HA for 6 hours. 50 μM ATP (six hours) and 3 μM ionomycin (10 minutes) were used as controls due to their previously reported ability to stimulate enlargeosome exocytosis [21–24]. The samples were then fixed in 4% paraformaldehyde and subjected to immunocytochemical analysis using AHNAK antibodies (green). Plasma membranes were labeled with Alexa Fluor 594 wheat germ agglutinin (red) and nuclei were stained with Hoechst 33342 (blue). Control HPMVEC exhibited diffuse cytoplasmic AHNAK staining. When stimulated with HMW-HA, intracellular AHNAK redistributed to a “vesicular” pattern which is similar to treatment with ATP and ionomycin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Analysis of HMW-HA-induced AHNAK redistribution in human EC. HPMVEC were grown to confluence on glass coverslips and treated with 100 nM HMW-HA for 6 hours. 50 μM ATP (six hours) and 3 μM ionomycin (10 minutes) were used as controls due to their previously reported ability to stimulate enlargeosome exocytosis [21–24]. The samples were then fixed in 4% paraformaldehyde and subjected to immunocytochemical analysis using AHNAK antibodies (green). Plasma membranes were labeled with Alexa Fluor 594 wheat germ agglutinin (red) and nuclei were stained with Hoechst 33342 (blue). Control HPMVEC exhibited diffuse cytoplasmic AHNAK staining. When stimulated with HMW-HA, intracellular AHNAK redistributed to a “vesicular” pattern which is similar to treatment with ATP and ionomycin.
Mentions: To further investigate HMW-HA-induced enlargeosome dynamics in HLMVEC, confluent monolayers were treated with HMW-HA for 6 hours. ATP and ionomycin were used as controls due to their previously reported ability to stimulate enlargeosome exocytosis [21–24]. The samples were then fixed and subjected to immunocytochemical analysis using AHNAK antibodies (green). Plasma membranes were labeled with Alexa Fluor 594 wheat germ agglutinin (red) and nuclei were stained with Hoechst 33342 (blue). The results of Figure 3 indicate that control HPMVEC exhibit diffuse cytoplasmic AHNAK staining. When stimulated with HMW-HA, intracellular AHNAK redistributes to a “vesicular” pattern which is similar to treatment with ATP and ionomycin.

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