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Expression of Angiogenic and Vasculogenic Factors in Human Lymphedema Tissue
Rafael A. Couto, BA, Ann M. Kulungowski, MD, Aditya S. Chawla, BA, Steven J. Fishman, MD, John B. Mulliken, MD, Arin K. Greene, MD, MMSc.
Children's Hospital Boston/ Harvard Medical School, Boston, MA, USA.

BACKGROUND: Lymphedema is the progressive distention of tissue due to lymphatic dysfunction. The affected area enlarges over time due to fibroadipose deposition, causing morbidity. Because increasing tissue mass requires neovascularization, we hypothesized that angiogenesis or vasculogenesis might be upregulated in lymphedema.
METHODS: Lymphedematous tissue was collected prospectively from nine patients after resection: upper extremity (n=1), lower extremity (n=3), penis/scrotum (n=5). Neovascularization was compared to normal tissue. Specimens were analyzed using immunohistochemistry for α-smooth muscle actin (pericyte marker), CD31 (microvascular density), CD31/Ki67 (proliferating endothelial cells), and CD34/CD133 (endothelial progenitor cells). Quantitative real-time PCR (qRT-PCR) was used to determine mRNA expression of progenitor cells (CD133) and factors that recruit them: vascular endothelial growth factor-A (VEGF-A), hypoxia-inducible factor 1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), and stromal-cell derived factor 1α (SDF-1α). Angiopoetin-1,-2 (ANG-1,-2), matrix metalloproteinase-2 (MMP-2), and VEGF receptors (VEGFR1,2) were quantified using qRT-PCR.
RESULTS: There was no difference in microvascular density, pericytic density, or endothelial proliferation between lymphedematous and normal tissue (p=0.1). Endothelial progenitor cells (EPCs) were not present in lymphedema or normal specimens (p<0.01). VEGF-A (1.3 fold), HIF-1α (0.8 fold), SDF-1α (2.1 fold), VEGFR2 (0.09 fold), and CD133 (0.02 fold) expression were not elevated compared to normal tissue (p=0.2). ANG-1 (5.6 fold), ANG-2 (2.5 fold), MMP-2 (3.9 fold), MMP-9 (33.4 fold), and VEGFR1 (12.8 fold) mRNA were increased in lymphedematous tissue compared to control (p<0.05).
CONCLUSION: Lymphedematous tissue does not exhibit upregulation of angiogenesis or vasculogenesis; the vasculature is stable. Neovascularization is unlikely to be involved in the pathogenesis of this condition.


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