Targeting uPARAP Modifies Lymphatic Vessel Architecture and Attenuates Lymphedema
Abstract
BACKGROUND: Lymphedema is an incurable disease associated with lymphatic dysfunction that causes tissue swelling and fibrosis. We investigated whether lymphedema could be attenuated by interfering with uPARAP (urokinase plasminogen activator receptor-associated protein; Mrc2 gene), an endocytic receptor involved in fibrosis and lymphangiogenesis. METHODS: We generated mice with lymphatic endothelial cell (LEC)–specific uparap deficiency and compared them with constitutive knockout mice by applying a preclinical model of secondary lymphedema (SL). Computerized methods were applied for 2-dimensional and 3-dimensional image quantifications. Cellular effects of uPARAP deletion on lymphatic permeability were assessed by small interfering RNA–mediated silencing in human dermal LECs and a pharmacologic treatment targeting ROCK (Rho-associated coiled coil containing kinase), an established regulator of cell junctions. The uPARAP and vascular endothelial cadherin partnership was investigated through proximity ligation assay, coimmunoprecipitation, and immunostaining. An in silico model was generated to analyze the fluid-absorbing function of the lymphatic vasculature. To interfere with uPARAP, its downregulation was achieved in vivo through a gapmer approach. RESULTS: uparap deficiency mitigated several key pathologic features of SL, including hindlimb swelling, epidermal thickening, and the accumulation and size of adipocytes. In both global and LEC-conditional uparap -deficient mice, induction of SL led to a distinctive labyrinthine vasculature, defined herein by twisted and hyperbranched vessels with overlapping cells. This topology, mainly composed of pre–collecting vessels, correlated with reduced SL, but not with change in fibrosis, highlighting the importance of uPARAP in regulating LEC functions in a lymphedematous context. In vitro, uPARAP knockdown in LECs impaired vascular endothelial growth factor C–mediated endosomal trafficking of vascular endothelial cadherin and induced overlapping cell junctions. The pharmacologic inhibition of ROCK recapitulated cell superimposition in vitro and the labyrinthine vasculature in vivo with attenuated SL. Computational modeling of labyrinthine lymphatic vasculature supported the observation on their improved fluid-absorbing function in comparison with a normal hierarchic network. These data provide proof of concept of inducing a labyrinthine topology to treat SL. For therapeutic purposes, we validated the use of an anti-uPARAP gapmer to induce a labyrinthine vasculature and attenuate SL formation. CONCLUSIONS: Our findings provide evidence that downregulating uPARAP expression can induce a beneficial remodeling of lymphatic vasculature that attenuates lymphedema through a cell junction–based mechanism, offering a novel therapeutic pathway for lymphedema.
Article Details
Authors (20)
Fabrice Gucciardo
From the Laboratory of Tumor and Development Biology, GIGA (F.G., A.L., S.P., F.B., E.I., S.B., J.D., L.B., A.P., C.M., M.G.-I., A.N.), University of Liège, Sart-Tilman, Belgium.
Alizée Lebeau
From the Laboratory of Tumor and Development Biology, GIGA (F.G., A.L., S.P., F.B., E.I., S.B., J.D., L.B., A.P., C.M., M.G.-I., A.N.), University of Liège, Sart-Tilman, Belgium.
Sébastien Pirson
From the Laboratory of Tumor and Development Biology, GIGA (F.G., A.L., S.P., F.B., E.I., S.B., J.D., L.B., A.P., C.M., M.G.-I., A.N.), University of Liège, Sart-Tilman, Belgium.
Florence Buntinx
From the Laboratory of Tumor and Development Biology, GIGA (F.G., A.L., S.P., F.B., E.I., S.B., J.D., L.B., A.P., C.M., M.G.-I., A.N.), University of Liège, Sart-Tilman, Belgium.
Elitsa Ivanova
From the Laboratory of Tumor and Development Biology, GIGA (F.G., A.L., S.P., F.B., E.I., S.B., J.D., L.B., A.P., C.M., M.G.-I., A.N.), University of Liège, Sart-Tilman, Belgium.
Silvia Blacher
From the Laboratory of Tumor and Development Biology, GIGA (F.G., A.L., S.P., F.B., E.I., S.B., J.D., L.B., A.P., C.M., M.G.-I., A.N.), University of Liège, Sart-Tilman, Belgium.
Pascal Brouillard
Human Molecular Genetics, de Duve Institute, University of Louvain, Brussels, Belgium (P.B., M.V.).
Jonathan Deroye
From the Laboratory of Tumor and Development Biology, GIGA (F.G., A.L., S.P., F.B., E.I., S.B., J.D., L.B., A.P., C.M., M.G.-I., A.N.), University of Liège, Sart-Tilman, Belgium.
Louis Baudin
From the Laboratory of Tumor and Development Biology, GIGA (F.G., A.L., S.P., F.B., E.I., S.B., J.D., L.B., A.P., C.M., M.G.-I., A.N.), University of Liège, Sart-Tilman, Belgium.
Alexandra Pirnay
From the Laboratory of Tumor and Development Biology, GIGA (F.G., A.L., S.P., F.B., E.I., S.B., J.D., L.B., A.P., C.M., M.G.-I., A.N.), University of Liège, Sart-Tilman, Belgium.
Florent Morfoisse
U1297-Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Toulouse, France (F.M.).
Claire Villette
Christophe Nizet
Departments of Plastic and Reconstructive Surgery (C.N.), University of Liège, Sart-Tilman, Belgium.
François Lallemand
Carine Munaut
Kari Alitalo
Wihuri Research Institute and Translational Cancer Medicine Program, Biomedicum, University of Helsinki, Finland (K.A.).
Liesbet Geris
Miikka Vikkula
Human Molecular Genetics, de Duve Institute, Université Catholique de Louvain
Marine Gautier-Isola
Agnès Noel
From the Laboratory of Tumor and Development Biology, GIGA (F.G., A.L., S.P., F.B., E.I., S.B., J.D., L.B., A.P., C.M., M.G.-I., A.N.), University of Liège, Sart-Tilman, Belgium.