title: Wnt5a Regulates the Assembly of Human Adipose Derived Stromal Vascular Fraction-Derived Microvasculatures.
abstract: Human adipose-derived stromal vascular fraction (hSVF) cells are an easily accessible, heterogeneous cell system that can spontaneously self-assemble into functional microvasculatures in vivo. However, the mechanisms underlying vascular self-assembly and maturation are poorly understood, therefore we utilized an in vitro model to identify potential in vivo regulatory mechanisms. We utilized passage one (P1) hSVF because of the rapid UEA1+ endothelium (EC) loss at even P2 culture. We exposed hSVF cells to a battery of angiogenesis inhibitors and found that the pan-Wnt inhibitor IWP2 produced the most significant hSVF-EC networking decrease (~25%). To determine which Wnt isoform(s) and receptor(s) may be involved, hSVF was screened by PCR for isoforms associated with angiogenesis, with only WNT5A and its receptor, FZD4, being expressed for all time points observed. Immunocytochemistry confirmed Wnt5a protein expression by hSVF. To see if Wnt5a alone could restore IWP2-induced EC network inhibition, recombinant human Wnt5a (0-150 ng/ml) was added to IWP2-treated cultures. The addition of rhWnt5a significantly increased EC network area and significantly decreased the ratio of total EC network length to EC network area compared to untreated controls. To determine if Wnt5a mediates in vivo microvascular self-assembly, 3D hSVF constructs containing an IgG isotype control, anti-Wnt5a neutralizing antibody or rhWnt5a were implanted subcutaneously for 2w in immune compromised mice. Compared to IgG controls, anti-Wnt5a treatment significantly reduced vessel length density by ~41%, while rhWnt5a significantly increased vessel length density by ~62%. However, anti-Wnt5a or rhWnt5a did not significantly affect the density of segments and nodes, both of which measure vascular complexity. Taken together, this data demonstrates that endogenous Wnt5a produced by hSVF plays a regulatory role in microvascular self-assembly in vivo. These findings also suggest that manipulating Wnt signaling could enhance control of hSVF vascularization in tissue engineering applications.
authors.affiliation: Division of Cardiovascular Therapeutics, Cardiovascular Innovation Institute, University of Louisville, Louisville, Kentucky, United States of America.|Department of Physiology, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: Venkat M
authors.initials: VM
authors.lastname: Ramakrishnan
authors.affiliation: Division of Cardiovascular Therapeutics, Cardiovascular Innovation Institute, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: Kevin T
authors.initials: KT
authors.lastname: Tien
authors.affiliation: Division of Cardiovascular Therapeutics, Cardiovascular Innovation Institute, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: McKinley
authors.affiliation: Division of Cardiovascular Therapeutics, Cardiovascular Innovation Institute, University of Louisville, Louisville, Kentucky, United States of America.|Georgetown College, Georgetown, Kentucky, United States of America.
authors.forename: Braden R
authors.initials: BR
authors.lastname: Bocard
authors.affiliation: Division of Plastic Surgery, Department of Surgery, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: Terry M
authors.initials: TM
authors.lastname: McCurry
authors.affiliation: Division of Cardiovascular Therapeutics, Cardiovascular Innovation Institute, University of Louisville, Louisville, Kentucky, United States of America.|Department of Physiology, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: Stuart K
authors.initials: SK
authors.lastname: Williams
authors.affiliation: Division of Cardiovascular Therapeutics, Cardiovascular Innovation Institute, University of Louisville, Louisville, Kentucky, United States of America.|Department of Physiology, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: James B
authors.initials: JB
authors.lastname: Hoying
authors.affiliation: Division of Cardiovascular Therapeutics, Cardiovascular Innovation Institute, University of Louisville, Louisville, Kentucky, United States of America.|Department of Physiology, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: Nolan L
authors.initials: NL
authors.lastname: Boyd
mesh_terms: D017667:Adipocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D055806:Microvessels / Q000187:drug effects* / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000494:pharmacology*; D051153:Wnt Proteins / Q000494:pharmacology*; D060449:Wnt Signaling Pathway / Q000187:drug effects* / Q000502:physiology; D000071818:Wnt-5a Protein
chemical_list: D011518:Proto-Oncogene Proteins; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein