Rapamycin reversal of VEGF-C–driven lymphatic anomalies in the respiratory tract
Peter Baluk, … , Young-Kwon Hong, Donald M. McDonald
Peter Baluk, … , Young-Kwon Hong, Donald M. McDonald
Published August 17, 2017
Citation Information: JCI Insight. 2017;2(16):e90103. https://doi.org/10.1172/jci.insight.90103.
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Research Article Vascular biology

Rapamycin reversal of VEGF-C–driven lymphatic anomalies in the respiratory tract

Abstract

Lymphatic malformations are serious but poorly understood conditions that present therapeutic challenges. The goal of this study was to compare strategies for inducing regression of abnormal lymphatics and explore underlying mechanisms. CCSP-rtTA/tetO-VEGF-C mice, in which doxycycline regulates VEGF-C expression in the airway epithelium, were used as a model of pulmonary lymphangiectasia. After doxycycline was stopped, VEGF-C expression returned to normal, but lymphangiectasia persisted for at least 9 months. Inhibition of VEGFR-2/VEGFR-3 signaling, Notch, β-adrenergic receptors, or autophagy and antiinflammatory steroids had no noticeable effect on the amount or severity of lymphangiectasia. However, rapamycin inhibition of mTOR reduced lymphangiectasia by 76% within 7 days without affecting normal lymphatics. Efficacy of rapamycin was not increased by coadministration with the other agents. In prevention trials, rapamycin suppressed VEGF-C–driven mTOR phosphorylation and lymphatic endothelial cell sprouting and proliferation. However, in reversal trials, no lymphatic endothelial cell proliferation was present to block in established lymphangiectasia, and rapamycin did not increase caspase-dependent apoptosis. However, rapamycin potently suppressed Prox1 and VEGFR-3. These experiments revealed that lymphangiectasia is remarkably resistant to regression but is responsive to rapamycin, which rapidly reduces and normalizes the abnormal lymphatics without affecting normal lymphatics.

Authors

Peter Baluk, Li-Chin Yao, Julio C. Flores, Dongwon Choi, Young-Kwon Hong, Donald M. McDonald

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Figure 1

Lymphatics in tracheas of CCSP/VEGF-C mice before and after doxycycline.

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Lymphatics in tracheas of CCSP/VEGF-C mice before and after doxycycline....
(A) VEGF-C mRNA expression (relative to β-actin) in trachea increased on doxycycline and returned almost to baseline after 14-day doxycycline washout period. (B) Number of Prox1-positive nuclei before and during doxycycline administration. (C–E) Overview of tracheal whole mounts stained for LYVE-1 (green) and Prox1 (red) from mice given 0.01 mg/ml doxycycline for 7 days (P21–P28) and perfused at P28 (C and D) or after 14-day washout period (P42) (E). (C) Trachea from a control (single-transgenic) mouse, with normal lymphatics restricted to intercartilage spaces and over the trachealis muscle at the center. (D) Trachea from a double-transgenic CCSP/VEGF-C mouse after doxycycline for 7 days, where lymphatics cover a much larger area and increase in abundance toward the caudal end. (E) Trachea from a P42 double-transgenic mouse 14 days after doxycycline withdrawal; lymphatics are less numerous than at P28, especially in central muscular region, but most of the lymphatic abnormality persists. Boxed regions are shown at higher magnification below. The middle row shows the boxed region stained for LYVE-1 showing normal lymphatics (C), lymphangiectasia at P28 (D), and spontaneous regression between P28 and P42 (E). Some lymphatics are narrowed regions (arrows). The lower row shows Prox1-positive nuclei at the beginning (C) and end of doxycycline treatment (D) and 14 days after withdrawal (E). Scale bar: 1 millimeter (top row); 50 μm (middle and bottom rows). (F) Extent of LYVE-1 lymphatics in trachea before doxycycline (blue box), after doxycycline (red box), and after doxycycline withdrawal (gray boxes). Values show the lymphatic expansion during doxycycline and some regression during the 2-week withdrawal period (P28–P42) but none thereafter throughout the 9-month study period (gray boxes were not significantly different from each other). n = 3 to 10 mice/group. *P < 0.05 vs. baseline group, †P < 0.05 vs. P28, ANOVA. Box and whisker plots show the median, first and third quartiles, and maximum and minimum.