Heparin Impairs Compensatory Lung Growth by Sequestering VEGF into Extracellular Matrix and Reducing Mitogenic Signaling of the Vascular Endothelial Growth Factor Receptor 2
Duy T. Dao, Lorenzo Anez-Bustillos, Jared Ourieff, Amy Pan, Hiroko Kishikawa, Paul D. Mitchell, Gillian L. Fell, Meredith A. Baxter
Boston Children's Hospital, Boston, MA
Objective: Children with hypoplastic lung diseases such as congenital diaphragmatic hernia often require extracorporeal membrane oxygenation and systemic heparinization. Mixed data suggest that heparin can impair angiogenesis. This study aims to investigate the interactions of heparin with VEGF and its effects on organ growth in a model of compensatory lung growth (CLG).
Design: Human lung microvascular endothelial cells (HMVEC-L) were plated on Matrigel®-coated plates in basal EBM-2 medium + 0.5% fetal bovine serum and starved for 24 hours. Cells were then treated with VEGF-165 in different heparin concentrations ranging from 0 to 1 unit/mL. Activation of VEGFR2 was determined with Western blot after 5 minutes of incubation. Cell density was measured with a tetrazolium salt assay after 3 days of incubation. Eight-week old C57Bl6 male mice underwent left pneumonectomy and were randomized into two groups. The control group received 100 microliters of intraperitoneal (IP) normal saline daily. The heparin group received 500 units/kg of IP heparin every 12 hours for the first 48 hours followed by every 24 hours. Mice were euthanized on post-operative day 4 for lung volume measurement. Morphometric analyses were performed with a point-counting technique. Frozen lung tissue was used for Western blot and polymerase chain reaction (PCR) array of the VEGF receptor (VEGFR2) signaling pathway.
Outcome: Lung volume measurements Morphometric analyses Protein and mRNA expression studies Activation and mitogenic activity assays
Results: Compared to the absence of heparin, heparin at 0.5 and 1 unit/mL decreased VEGF’s activation of VEGFR2 (P < 0.05 and P < 0.01, respectively) and mitogenic activity on HMVEC-L (P < 0.01 and P < 0.001, respectively). Compared to the control group, mice in the heparin group displayed lower lung volume/body weight (0.054 ± 0.001 vs. 0.049 ± 0.001 mL/g, P < 0.05), septal surface area (17.2 ± 0.9 vs. 14.1 ± 0.7 cm2/g, P < 0.05) and alveolar density (3.1 ± 0.2 vs. 2.6 ± 0.1 x 107 per cm3, P < 0.05). Lungs of heparinized mice displayed increased VEGF tissue levels (P < 0.001), reduced P-Y1175-VEGFR2 (P < 0.05) (pro-mitogen pathway), and increased P-Y1214-VEGFR2 (P < 0.05) (pro-migration pathway) compared to the control group. PCR array of VEGFR2 signaling cascade showed more than 2-fold reduction in mRNA level of the mitogen-activated protein kinase (MAPK) genes in the heparin group compared to the control group.
Conclusions: Systemic heparin sequestered VEGF into lung tissue and caused a shift in VEGFR2 signaling from a pro-mitogen to a pro-migration pathway. As a result, heparin impaired CLG as evident by decreased lung volume, septal surface area, and alveolar density. These findings raise concern for the clinical use of heparin in the setting of organ growth or regeneration.
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