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Direct isolation, culture and transplant of mouse skeletal muscle derived endothelial cells with angiogenic potential.

Ieronimakis N, Balasundaram G, Reyes M - PLoS ONE (2008)

Bottom Line: By utilizing multicolor fluorescent-activated cell sorting (FACS), we have isolated a distinct population of Sca-1(+), CD31(+), CD34(dim) and CD45(- )cells from skeletal muscles of C57BL6 mice.Characterization of this population revealed these cells are functional EC that can be expanded several times in culture without losing their phenotype or capabilities to uptake acetylated low-density lipoprotein (ac-LDL), produce nitric oxide (NO) and form vascular tubes.When transplanted subcutaneously or intramuscularly into the tibialis anterior muscle, EC formed microvessels and integrated with existing vasculature.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: Although diseases associated with microvascular endothelial dysfunction are among the most prevalent illnesses to date, currently no method exists to isolate pure endothelial cells (EC) from skeletal muscle for in vivo or in vitro study.

Methodology: By utilizing multicolor fluorescent-activated cell sorting (FACS), we have isolated a distinct population of Sca-1(+), CD31(+), CD34(dim) and CD45(- )cells from skeletal muscles of C57BL6 mice. Characterization of this population revealed these cells are functional EC that can be expanded several times in culture without losing their phenotype or capabilities to uptake acetylated low-density lipoprotein (ac-LDL), produce nitric oxide (NO) and form vascular tubes. When transplanted subcutaneously or intramuscularly into the tibialis anterior muscle, EC formed microvessels and integrated with existing vasculature.

Conclusion: This method, which is highly reproducible, can be used to study the biology and role of EC in diseases such as peripheral vascular disease. In addition this method allows us to isolate large quantities of skeletal muscle derived EC with potential for therapeutic angiogenic applications.

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Related in: MedlinePlus

EC of the skeletal muscle form microvessels in matrigel injected subcutaneously over the dorsum.A–B, Tissue sections of the matrigel plug recovered from the dorsal skin of C57BL6 mice analyzed 14 days post injection reveal microvessel formation. Staining for anti-CD31 and anti-vWF followed with a secondary Alexa 647 antibody highlight mature vessels formed within the matrigel by injected cells (PKH26+). C, Conjugated FITC anti-SMA staining indicates the recruitment of endogenous smooth muscle cells in the formation of microvessels. Scale bar = 50 µm.
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pone-0001753-g006: EC of the skeletal muscle form microvessels in matrigel injected subcutaneously over the dorsum.A–B, Tissue sections of the matrigel plug recovered from the dorsal skin of C57BL6 mice analyzed 14 days post injection reveal microvessel formation. Staining for anti-CD31 and anti-vWF followed with a secondary Alexa 647 antibody highlight mature vessels formed within the matrigel by injected cells (PKH26+). C, Conjugated FITC anti-SMA staining indicates the recruitment of endogenous smooth muscle cells in the formation of microvessels. Scale bar = 50 µm.

Mentions: We first tested the angiogenic capacity of muscle EC with the widely used dorsal matrigel plug technique [20]. To track cells in vivo, muscle EC were labeled with PKH26, a red fluorescent dye that irreversible binds the cell membrane and has successfully been used for in vivo transplantation of EC [37]. 14 days after transplant PKH26+ EC formed new vessels that stained positive for CD31 and vWF. PKH26+ EC made “bonafide” microvessels as endogenous smooth muscle cells, identified as SMA positive but PKH26 negative, were seen wrapping PKH26+ vessels (figure 6). Thus, we hypothesized that muscle EC may exhibit angiogenic potential if transplanted back into their tissue of origin, the skeletal muscle.


Direct isolation, culture and transplant of mouse skeletal muscle derived endothelial cells with angiogenic potential.

Ieronimakis N, Balasundaram G, Reyes M - PLoS ONE (2008)

EC of the skeletal muscle form microvessels in matrigel injected subcutaneously over the dorsum.A–B, Tissue sections of the matrigel plug recovered from the dorsal skin of C57BL6 mice analyzed 14 days post injection reveal microvessel formation. Staining for anti-CD31 and anti-vWF followed with a secondary Alexa 647 antibody highlight mature vessels formed within the matrigel by injected cells (PKH26+). C, Conjugated FITC anti-SMA staining indicates the recruitment of endogenous smooth muscle cells in the formation of microvessels. Scale bar = 50 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2262143&req=5

pone-0001753-g006: EC of the skeletal muscle form microvessels in matrigel injected subcutaneously over the dorsum.A–B, Tissue sections of the matrigel plug recovered from the dorsal skin of C57BL6 mice analyzed 14 days post injection reveal microvessel formation. Staining for anti-CD31 and anti-vWF followed with a secondary Alexa 647 antibody highlight mature vessels formed within the matrigel by injected cells (PKH26+). C, Conjugated FITC anti-SMA staining indicates the recruitment of endogenous smooth muscle cells in the formation of microvessels. Scale bar = 50 µm.
Mentions: We first tested the angiogenic capacity of muscle EC with the widely used dorsal matrigel plug technique [20]. To track cells in vivo, muscle EC were labeled with PKH26, a red fluorescent dye that irreversible binds the cell membrane and has successfully been used for in vivo transplantation of EC [37]. 14 days after transplant PKH26+ EC formed new vessels that stained positive for CD31 and vWF. PKH26+ EC made “bonafide” microvessels as endogenous smooth muscle cells, identified as SMA positive but PKH26 negative, were seen wrapping PKH26+ vessels (figure 6). Thus, we hypothesized that muscle EC may exhibit angiogenic potential if transplanted back into their tissue of origin, the skeletal muscle.

Bottom Line: By utilizing multicolor fluorescent-activated cell sorting (FACS), we have isolated a distinct population of Sca-1(+), CD31(+), CD34(dim) and CD45(- )cells from skeletal muscles of C57BL6 mice.Characterization of this population revealed these cells are functional EC that can be expanded several times in culture without losing their phenotype or capabilities to uptake acetylated low-density lipoprotein (ac-LDL), produce nitric oxide (NO) and form vascular tubes.When transplanted subcutaneously or intramuscularly into the tibialis anterior muscle, EC formed microvessels and integrated with existing vasculature.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: Although diseases associated with microvascular endothelial dysfunction are among the most prevalent illnesses to date, currently no method exists to isolate pure endothelial cells (EC) from skeletal muscle for in vivo or in vitro study.

Methodology: By utilizing multicolor fluorescent-activated cell sorting (FACS), we have isolated a distinct population of Sca-1(+), CD31(+), CD34(dim) and CD45(- )cells from skeletal muscles of C57BL6 mice. Characterization of this population revealed these cells are functional EC that can be expanded several times in culture without losing their phenotype or capabilities to uptake acetylated low-density lipoprotein (ac-LDL), produce nitric oxide (NO) and form vascular tubes. When transplanted subcutaneously or intramuscularly into the tibialis anterior muscle, EC formed microvessels and integrated with existing vasculature.

Conclusion: This method, which is highly reproducible, can be used to study the biology and role of EC in diseases such as peripheral vascular disease. In addition this method allows us to isolate large quantities of skeletal muscle derived EC with potential for therapeutic angiogenic applications.

Show MeSH
Related in: MedlinePlus