Lymphedema (LE) afflicts hundreds of millions people worldwide. Manual lymph drainage, a specialized form of massage therapy, is an integral component - along with compression bandaging (CB) - of combined physical therapy (CPT), an internationally recommended optimal treatment. Experimental data from a rat model of obstructive LE suggests that massage alone can reduce LE volume as effectively as CB and CPT, at least early in the pathologic process. In a preliminary retrospective clinical study, it was also found that massage alone in a cohort of patients with mild stable upper-limb LE was as effective as CPT in volume reduction (massage 80.6 /spl plusmn/ 0.2% decrease compared to CPT 72.6 /spl plusmn/ 0.2%, mean /spl plusmn/ standard deviation, not significant). These initial studies lay the groundwork and evidence-based rationale for the design and implementation of the expanded, prospective randomized clinical trial of massage alone in various types and stages of LE in children and adults.

Connexin proteins form gap junctions controlling exchange of ions and small molecules between cells and play an important role in movement of lymph within lymphatic vessels. Connexin47 (CX47) is highly expressed in lymphatic endothelial cells and CX47 missense mutations, i.e., R260C, cosegregate with primary lymphedema in humans. However, studies utilizing CX47 knockout mice have failed to demonstrate any lymphatic anomalies. To unravel the lymphatic consequences of expressing a mutant CX47 protein, we used CRISPR technology to create a mouse carrying a Cx47 missense mutation (Cx47R259C) equivalent to the human CX47R260C missense mutation associated with human primary lymphedema. Intradermal Evans Blue dye injection identified a 2-fold increase in regional lymph nodes in homozygous Cx47R259C mice compared to wildtype, particularly in the jugular region (4.8 ± 0.4 and 2.0 ± 0.0, respectively, p<0.01). Associated lymphatic channels were increased in Cx47R259C mice and mesenteric lymph reflux occurred in homozygous Cx47R259C mice but not in wildtype. Contractility of superficial cervical lymphatics, assessed by pressure myography, was reduced in homozygous Cx47R259C mice compared to wildtype. In conclusion, our data are the first to demonstrate a role for the Cx47 protein in lymphatic anatomy and function. This phenotype is similar to that found with other valve deficient mouse mutants, e.g., in Foxc2. Of significance, this study is the first to use CRISPR technology to develop a pre-clinical model of primary lymphedema and demonstrates the importance of distinguishing between lack of and presence of mutant protein when developing clinically relevant animal models for translation of pre-clinical findings.