Abstract

Introduction

Despite significant advances in surgical and intensive care management over the past three decades, morbidity and mortality for neonates with congenital diaphragmatic hernia (CDH)-associated pulmonary hypoplasia (PH) remains high. Patients with CDH and severe PH may require cardiopulmonary support with extracorporeal membrane oxygenation (ECMO). Mortality for patients with CDH requiring ECMO approaches 50%^1,2. Animal models and human patients with CDH-associated PH may be deficient in vascular endothelial growth factor (VEGF), especially in the alveolar stage of lung development^3,4. During lung development, mesenchymal and alveolar epithelial cells secrete VEGF, which drives endothelial cell proliferation and results in coordinated bronchovascular growth^5–7. As such, increasing pulmonary VEGF during lung development, particularly during a critical period of alveolarization, much of which occurs postnatally in humans, has become an attractive therapeutic target for developmental lung diseases including CDH-associated PH, bronchopulmonary dysplasia, and respiratory distress syndrome^3,8–10.

Murine left pneumonectomy-induced compensatory lung growth (CLG) shares molecular patterning similar to developmental alveolarization and serves as a model to study pulmonary hypoplastic diseases^11,12. CLG is particularly reflective of the post-natal alveolar stage of human lung development, as it is characterized by expansion of the remaining lung to the full volume of both lungs via septal surface expansion and re-alveolarization¹². Our laboratory has previously demonstrated that CLG in mice completes at post-operative day (POD) eight to ten¹³. In accordance with the prior observation of decreased VEGF mRNA expression in patients with CDH in the alveolar stage of development, our group administered exogenous VEGF164, the most abundant murine isoform of the protein, in a murine CLG model, and demonstrated accelerated CLG to completion by POD4^13,14. However, VEGF164 contains a heparin-binding domain (HBD), which interacts with heparin. Prior studies have revealed that exogenous heparin impairs VEGF-mediated accelerated CLG¹⁵. As heparin is the most commonly used anticoagulant, especially in patients requiring ECMO, the potentially deleterious interaction between VEGF and heparin is of particular concern.

In working towards the goal of translating systemic VEGF therapy into clinical practice for PH, we hypothesized that a VEGF protein lacking heparin-binding ability may potentially be effective in accelerating CLG without interacting with exogenous heparin given clinically. The VEGF-A pre-mRNA is alternatively spliced to yield three major protein isoforms in mice and humans, VEGF120/164/188 and VEGF121/165/189, respectively. In particular, VEGF120/121, the smallest of the major isoforms, lacks exons 6 and 7, which encode the HBD¹⁶. The complete absence of heparin-binding VEGF isoforms 164/188 in a transgenic mouse model resulted in non-viable pups with completed pulmonary development but pruned lung vasculature, indicating that VEGF120 alone is not sufficient to achieve mature pulmonary function¹⁷. However, delivery of exogenous VEGF120 in the presence of normal, endogenous VEGF produces a similar pro-angiogenic response compared to provision of HBD-containing exogenous VEGF¹⁸. To our knowledge, no prior studies have assessed the efficacy of VEGF120 in the murine CLG model, or evaluated the potential for VEGF to affect functional outcomes such as exercise tolerance. The purpose of our current study was to assess the efficacy of exogenous VEGF120 in accelerating CLG in a model of murine left pneumonectomy. We also aimed to further delineate the role of the HBD in VEGF-mediated pulmonary growth, development, and function.

Results

VEGF treatment increases compensatory lung growth in a dose-dependent manner

Mice treated with VEGF164 demonstrated a dose-dependent increase in lung volume normalized to body weight compared to saline-treated control mice. While the lower dose of 0.25 mg/kg VEGF164 mildly increased lung volume compared to control, only the higher, previously validated dose of 0.5 mg/kg produced a statistically significant increase (45.7±1.7 vs. 40.1±1.3 μL/g, P=0.02) (Fig. 1A). Similarly, VEGF120 demonstrated a milder dose-dependent increase in lung volume, although neither dose reached statistical significance compared to saline control. Both the higher doses (0.0256 mmol/kg), 0.5 mg/kg VEGF164 (34.6±0.94 vs. 29.1±1.1 μL/g, P=0.03) and equimolar 0.363 mg/kg VEGF120 (33.4±1.4 vs. 29.1±1.1 μL/g, P=0.04), significantly increased total lung capacity (TLC) compared to control (Fig. 1B). However, neither of the lower doses impacted TLC. There were no significant differences in compliance among treatment groups (Fig. 1C). The increase in lung volume by water displacement and TLC was unique to active VEGF treatment. Inactive VEGF120 or VEGF164 did not produce a significant increase in lung volume or TLC at POD4 after pneumonectomy (Supplemental Fig. ^S1A,B). In addition, VEGF treatment only affected the regenerating lung, but not organs that were not regenerating such as the liver, kidney, or spleen. Non-regenerative organs had comparable weights on POD4 of VEGF120 or VEGF164 treatment compared to control treatment (Supplemental Fig. ^S2).

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Figure 1

Lung volume and pulmonary function testing. Both VEGF164 and VEGF120 increased lung volume, as measured by the water displacement method (A), and total lung capacity on pulmonary function testing (B). However, only the higher dose of VEGF164 induced a significant increase in lung volume, while both the higher dose of VEGF164 and the the equimolar dose of VEGF120 significantly increased total lung capacity. Compliance was not significantly different between VEGF-treated mice and controls (C). Statistical analysis of lung volume, total lung capacity, and compliance was performed by ANOVA with Holm-Sidak correction for multiple comparisons. Results are expressed as mean±SE. *P<0.05. Mouse cohorts of pneumonectomies were performed in three independent repeats. On any given operative day, a control group of mice was included.

VEGF164 improves alveolarization on morphometric analysis

Compared to controls (representative histology in Fig. 2E), VEGF164 significantly increased parenchymal volume (46.2±1.3 vs. 37.0±2.8 μL/g, P=0.007), alveolar volume (18.9±1.1 vs. 14.0±1.2 μL/g, P=0.01), and septal surface area (28.7±1.4 vs. 20.7±1.8 cm²/g, P=0.003) normalized for body weight (Fig. 2A–C,G). Meanwhile, VEGF120 did not affect alveolarization (Fig. 2A–C,F). Decreased mean septal thickness, which may facilitate ease of gas exchange, was observed in both VEGF120- and VEGF164-treated lungs, although this did not reach statistical significance (Fig. 2D).

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Figure 2

Morphometric analysis of lung tissue. VEGF164-treated mice exhibited significant increases in parenchymal volume (A), alveolar volume (B), and septal surface area (C), while VEGF120 did not. VEGF treatment also decreased mean septal thickness, although this did not reach statistical significance (D). Representative micrographs at×200 magnification of hematoxylin and eosin-stained, control (E), VEGF120 (F), and VEGF164 (G) lung sections demonstrate increased alveolarization with VEGF164 treatment (G). Statistical analysis of morphometrics was performed by ANOVA with Holm-Sidak correction for multiple comparisons. Results are expressed as mean±SE. *P<0.05. Results are expressed as mean±SE. *P<0.05, **P<0.01.

VEGF164 improves exercise tolerance after left pneumonectomy

Treadmill exercise tolerance testing (TETT) was used to assess functional outcomes with VEGF treatment after murine left pneumonectomy, as it may be representative of functional pulmonary outcomes in human patients. Mice were acclimated to the treadmill and underwent TETT two days prior to surgery to obtain a baseline, and again on POD4 to assess treatment effect. Distance run and time spent running on POD4 were normalized to differences in baseline values on each mouse to account for any intrinsic physiologic or behavioral differences between mice. When comparing distance run and time spent running on the treadmill post-surgery and treatment to pre-operative, VEGF164-treated mice exhibited improved exercise tolerance after left pneumonectomy compared to control mice [median 5.6 (IQR 0.9, 13.6) vs. − 0.4 (− 7.6, 2.4) minutes and 77 (11, 204) vs. − 5 (− 106, 18) meters, respectively)]. These differences were maintained after adjusting for pre-operative baseline and multiple pairwise comparisons (P=0.03 by rank-based analysis of covariance; Fig. 3). Although VEGF120-treated mice had increased exercise distance and time (median 58 (− 27, 275) meters and 4.2 (− 2.0, 18.3) minutes, respectively), the change was not statistically different from either control- (P=0.11 for distance; P=0.09 for time) or VEGF164-treated mice (P=0.64 for distance; P=0.66 for time). Additionally, VEGF120 or VEGF164 treatment did not induce a significant increase in hematocrit (Supplemental Fig. ^S1C) or endothelial cell proliferation of non-regenerative tissues (particularly skeletal muscle) (Supplemental Fig. ^S3), to alternatively account for improvement in exercise tolerance.

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Figure 3

Treadmill exercise tolerance testing. Rank-based analysis of covariance, adjusted for baseline outcome and multiple pairwise comparisons, showed greater mean change in VEGF164 vs. control (P=0.03 for distance and for time), with no differences between VEGF120 and control (P=0.11 for distance and P=0.09 for time) and no differences between VEGF120 and VEGF164 (P=0.64 and P=0.66 for distance and time, respectively) (A, B). Statistical analysis of exercise tolerance was performed by nonparametric ANCOVA of the change in distance or time (POD4 minus baseline) and adjusted for multiple pairwise corrections using the Holm-Sidak correction. Results are expressed as median and interquartile range (IQR), with mean values. *P<0.05. Mouse cohorts of pneumonectomies were performed in three independent repeats. On any given operative day, a control group of mice was included.

Exogenous VEGF stimulates downstream VEGF and paracrine signaling

Protein analysis of lung tissue was performed to characterize the effects of exogenous VEGF on pulmonary cell signaling. Both VEGF120 and VEGF164 increased pulmonary levels of VEGF ligand, although this did not reach statistical significance, in part due to differences among biological replicates (Fig. 4A,B). Similarly, there was a nonsignificant increase in activation of VEGF receptor 2 (p-Y1175t VEGFR2) with provision of exogenous VEGF compared to control. Downstream effectors of VEGF signaling, including the MAPK/ERK and AKT pathways, were also increased, as demonstrated by increased phosphorylated to total p44/42 (ERK) and phosphorylated to total AKT-T308. However, activated AKT-S473 (p/t AKT-S473) was significantly downregulated in the VEGF164-treated group compared to control (0.50±0.10 vs. 1.0±0.14-fold, P=0.03) (Fig. 4A,B).

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Figure 4

Lung tissue protein analysis. VEGF treatment with either VEGF120 or VEGF164 increased lung tissue expression of VEGF ligand, activated VEGFR2 (phosphorylated/total receptor), and downstream proliferation markers activated ERK (phosphorylated/total ERK), and activated AKT-T308 (A, B) [Full-length uncropped blots are provided in Supplementary Information File ¹.] However, activated AKT-S473 was significantly downregulated. VEGF treatment also significantly downregulated co-receptor neuropilin-2 (NRP2), while co-receptor neuropilin-1 (NRP1) expression was not significantly different between groups (C, D). [Full-length uncropped blots are provided in Supplementary Information File ².] While heparin-binding epithelial growth factor (Hb-EGF) expression was unchanged with treatment, activated epithelial growth factor receptor (phosphorylated/total EGF receptor) was upregulated, indicating potential paracrine signaling by VEGF treatment (E, F). [Full-length uncropped blots are provided in Supplementary Information File ³.] Antibodies probed on the same blot were incubated on cut membranes to facilitate specificity of binding and ability to probe multiple antibodies at one time. The β-actin loading control for each membrane is displayed below the antibodies probed on the same membrane. For membranes probed with phosphorylated and total protein, total protein antibody was used stripping the membrane as appropriate. Full length uncropped membranes are provided in the Supplementary Information Files. Statistical analysis of protein expression was performed by ANOVA with Holm-Sidak correction for multiple comparisons. Results are expressed as mean±SE. *P<0.05. Each lane represents lung protein lysate from a different mouse. Western blots were performed one to three times for quality control, and a representative image is shown.

Neuropilins (NRPs) are neuronal receptors for class 3 semaphorins, but also act as VEGF co-receptors to facilitate angiogenesis¹⁹. Provision of either VEGF120 or VEGF164 only slightly increased NRP1 expression. However, VEGF164 treatment significantly reduced NRP2 expression compared to control (0.46±0.10 vs. 1.0±0.15-fold, P=0.02) (Fig. 4C,D).

Heparin-binding epidermal growth factor-like factor (Hb-EGF) levels were mostly unchanged between VEGF-treated lung tissue and controls; however, EGFR activation, as measured by phosphorylated to total EGFR, was increased in VEGF-treated groups, although not reaching statistical significance (Fig. 4E,F).

VEGF treatment induces greater increases in total lung cell proliferation than lung endothelial cell proliferation

Immunohistochemistry (IHC) was performed to assess endothelial and epithelial cell proliferation in VEGF-treated lung tissue (Fig. 5A). Compared to controls, both VEGF120 and VEGF164 increased endothelial cell proliferation, as demonstrated by co-staining of Ki-67, a proliferative marker, and ERG, a nuclear endothelial cell marker²⁰, although not reaching statistical significance (Fig. 5B). However, when quantitating the percentage of total proliferating pulmonary cells (including both endothelial and epithelial cells), VEGF164 induced a significant increase in global pulmonary cell proliferation (endothelial and epithelial cells) compared to controls (49.2±8.2 vs. 20.0±4.8 percent, P=0.004), while VEGF120 did not (Fig. 5C).

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Figure 5

Immunohistochemistry. Representative micrographs at×200 magnification of immunofluorescence-stained lung tissue with Ki-67 (green) and ERG (red) with nuclear DAPI (blue) demonstrated increased endothelial cell proliferation and significantly increased global pulmonary proliferation with VEGF164 treatment, which is more pronounced than VEGF120 treatment, compared to control (A). Quantification of co-staining indicated more substantial increases of total cell proliferation in the lung (Ki-67/DAPI) (C) compared to endothelial cell proliferation (Ki-67/ERG/DAPI) (B), which did not reach statistical significance. Statistical analysis of immunofluorescence co-staining quantification was performed by ANOVA with Holm-Sidak correction for multiple comparisons. Results are expressed as mean±SE. *P<0.05. Results are expressed as mean±SE. **P<0.01.

The VEGF heparin-binding domain increases VEGFR2 activation

In vitro activation and induction studies were performed using human microvascular lung endothelial cells (HMVEC-L) to determine the role of the HBD in downstream VEGF and potential subsequent paracrine signaling. Increasing doses of VEGF121, the human isoform of VEGF lacking the HBD, induced a dose-dependent activation of VEGFR2, as measured by the ratio of phosphorylated (p-Y1175) to total VEGFR2 after five minutes (Fig. 6A,B, Supplemental Fig. ^S4A,B). However, VEGF165, which contains the HBD, stimulated VEGFR2 activation to a much higher degree than VEGF121 at a lower dose. The efficacy of both mouse and human isoforms of VEGF to activate VEGFR2 in HMVEC-L was verified, and again demonstrated significantly more robust receptor activation with VEGF164/5 treatment at a lower dose compared to VEGF120/1 (Supplemental Fig. ^S5).

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Figure 6

In vitro VEGFR2 activation. In human microvascular lung endothelial cells (HMVEC-L), VEGF121 activated VEGFR2 in a dose dependent manner, although with marked decreased potency compared to VEGF165 in representative immunoblots (A, B). On an induction study, increased incubation time did not improve VEGF121 receptor binding (C, D). A representative immunoblot from three independent runs for quality control is pictured (C). [Full-length uncropped blots are provided in Supplementary Information File ⁴. Three-minute exposure, as used in this main figure, is displayed in Supplementary Information File ⁴A (corresponding to A), 4C (corresponding to C), while additional 30 s exposures merged with correlating molecular weight markers, are displayed in Supplementary Information File ⁴B (corresponding to A), 4D (corresponding to B)]. The β-actin loading control from each membrane is displayed under the image of the primary antibody of interest.

To assess whether the smaller and more diffusible VEGF121 isoform may require more time to associate with its receptor, an induction study was performed in which HMVEC-L were incubated with VEGF121 for increasing durations of time. Additional time for receptor association did not significantly increase VEGFR2 activation compared to VEGF165 activation of VEGFR2, which was substantially more robust than any time point for VEGF121 (Fig. 6C,D). However, on an extended induction study, VEGF121 did demonstrate strongest receptor activation at 10 min, sustained up to 30 min, with subsequent decreased VEGFR2 activation at time points longer than 30 min (Supplemental Fig. ^S4C,D). Together, these data suggest that the VEGF HBD is required for potent and timely receptor activation in pulmonary microvascular endothelial cells, which potentially correlate to accelerated CLG with VEGF164 treatment in vivo.

Discussion

Prior work from our group demonstrated that exogenous, systemic VEGF164 therapy accelerates CLG in a murine model^13,14. However, heparin, a commonly used anticoagulant in the clinical setting, impairs VEGF-mediated pulmonary growth and development¹⁵. VEGF164/165 and VEGF188/189 are well-known to interact with exogenous heparins and endogenous heparans via their HBD. Prior clinical studies of systemic heparin infusion have demonstrated consistent downregulation of circulating plasma VEGF^21,22. As such, in this current study, we sought to circumvent the VEGF-heparin interaction by assessing the efficacy of a non-heparin-binding VEGF isoform on CLG.

Our results indicate that while both VEGF120, which lacks the HBD, and VEGF164, which contains the HBD, increased lung volume and total lung capacity in a dose-dependent manner in mice, only VEGF164 produced functionally relevant improvements. VEGF164 significantly increased treadmill exercise tolerance after pneumonectomy, while VEGF120 did not. Similarly, on the histological level, VEGF164 increased parenchymal volume, alveolar volume, and septal surface area, which represent increased functional area available for gas exchange. VEGF120 produced minimal improvements in exercise tolerance or morphometrics compared to controls. On tissue analysis, VEGF164 consistently induced more pronounced effects on VEGF signaling than VEGF120, with consequent increases in activated VEGFR2, AKT-T308, and MAPK/ERK (p44/42).

In the lungs, VEGF originates from the mesenchymal and alveolar epithelial cells, and paracrine signaling drives endothelial cell proliferation and branching of the pulmonary vasculature^5–7. However, endothelial cell growth is thought to be the primary driver of pulmonary development through microvascular endothelial cell-mediated “angiocrine” signaling²³. Downstream endothelial VEGF signaling through AKT, MAPK/ERK, and other various cascades leads to cell survival, proliferation, and paracrine signaling to alveolar epithelial cells^24–26. Our group has previously shown that provision of exogenous VEGF to HMVEC-L drives human bronchial epithelial cell proliferation in co-culture, and that addition of a neutralizing antibody to Hb-EGF abrogates this effect²⁷. Our current study demonstrates that EGFR activation following VEGF treatment in a CLG model is more pronounced than VEGFR2 activation (Fig. 4B,F). IHC demonstrating that VEGF affects total pulmonary cell proliferation more than specific endothelial cell proliferation (Fig. 5B,C) provides further evidence that VEGF-mediated paracrine signaling may be the driver of accelerated pulmonary growth. Similar to effects observed on pulmonary function and TETT, VEGF-mediated signaling and subsequent pulmonary cell proliferative effects were more robust with VEGF164 treatment than VEGF120 treatment. Increased activation of VEGFR2 in cultured HMVEC-L in response to VEGF164/5 relative to VEGF120/1 also suggests that the VEGF HBD is required for potent VEGFR2 signaling (Fig. 6, Supplemental Fig. ^S4).

Our group has previously demonstrated that the process of CLG after left pneumonectomy approaches completion by POD eight to ten¹³. Exogenous VEGF therapy consistently accelerates CLG to completion by POD4^13,14,27. However, while we have already characterized that exogenous VEGF modulates VEGFR2 activation and paracrine signaling through EGFR, P-Y1175-VEGFR2-mediated downstream signaling at POD4 in VEGF-treated mice has not previously been investigated. While a limitation of this study is that there is a significant degree of variability between biological replicates, it is important to note that, on average, VEGF164 treatment produced more significant effects in signaling downstream of VEGFR2, as well as in activating EGFR (Fig. 4B,F). Significant downregulation of pAKT-S473 and NRP2 is unique to VEGF164-treated lung tissue and is not present in control or VEGF120-treated lung tissue (Fig. 4B,D), which presumably have not completed CLG by POD4. As such, modulation of pAKT-S473 and NRP2 may represent early molecular signals of pulmonary growth completion.

The role of neuropilins (NRPs) in lung development has not been thoroughly evaluated. However, neuropilin-1 (NRP1) is known to be essential for VEGF-stimulated heterodimerization of NRP1 with VEGFR2 and downstream endothelial cell migration signaling²⁸. Previously, VEGF120 was believed to be unable to bind NRP due to the lack of the HBD (exons 6 and 7), which also contains the NRP-binding site. New evidence, including the resolved crystal structures of NRP and its bound state with VEGF, revealed that NRPs interact with VEGF exons 5, 7, and 8, the former and latter of which are still present on VEGF120/1^29,30. However, the binding affinity of VEGF120 to NRP is much less than that of VEGF164²⁹, and is inconsistent across studies³⁰. The pattern of decreased affinity for VEGF120 relative to VEGF164 is similar to the decreased activation of VEGFR2 observed in our in vitro assays with VEGF121 (Fig. 6), despite exogenous VEGF treatment having little effect on pulmonary expression of NRP1 (Fig. 4C,D). In addition, interactions with cell surface and extracellular matrix (ECM) heparans further complicate the system. Interestingly, in cell-free kinetics studies, NRP1 enhanced VEGF165 interaction with VEGFR2, but not when endogenous heparan sulfate proteoglycans (HSPGs) were missing³¹. This suggests that NRPs and endogenous HSPGs may complex with VEGF and VEGR2 to enhance receptor-ligand affinity and promote downstream signaling. In this study, we demonstrated that VEGF120/121 has reduced potency for activating VEGFR2 in vitro and in vivo compared to VEGF164/165. We therefore postulate that effective interactions with NRPs, and perhaps with endogenous HSPGs on the cell surface or ECM, may be enhanced by the presence of the HBD. Therefore, the VEGF HBD may be essential to effective exogenous VEGF/VEGFR2 binding on endothelial cells, downstream signaling, and subsequent CLG. Future research endeavors may further focus on the crosstalk between the VEGF HBD, HRPs and HSPGs by elimination or inactivation of NRPs, HSPGs, or both sequentially in both in vitro and in vivo systems.

This study should be considered in light of its limitations, chief among which are the lack of equivalency between a CLG model and CDH-associated PH, lack of additional post-operative days evaluating VEGF120 treatment, and lack of statistical significance in much of the lung tissue protein analysis. In addition, commercially available recombinant murine VEGF may have batch-to-batch variation, which may have affected the efficacy of the proteins used therapeutically. While CLG and pulmonary development are driven by similar mechanical stress responses and molecular patterning, the two processes are not identical^11,12. CDH is further driven by other genetic and diaphragmatic developmental deficiencies that may crosstalk and interfere with normal pulmonary development³². VEGF120 treatment failed to produce significant effects on exercise tolerance or molecular signaling outcomes in our model, but significantly increased TLC, and trended towards increased lung volume, activated ERK, activated EGFR, and pulmonary total cell and endothelial cell proliferation compared to controls. These data suggest that VEGF120 may accelerate CLG, but to a lesser degree than VEGF164. Expeditious pulmonary growth is essential for infant survival following surgical repair of CDH, as prolonged dependence on invasive supportive care such as ECMO or even mechanical ventilation are associated with worse outcomes and lower survival². Therefore, any therapeutic that accelerates lung growth may be worthwhile to pursue. Nevertheless, it is difficult to draw further conclusions on the efficacy of VEGF120 to accelerate lung growth without further data, and difficult to predict if such marginal effects may have a clinical impact. From a technical perspective, this manuscript is limited by VEGF120/1 and VEGF164/5 being commercially available proteins made in different organisms and purified using different methods. Although the resulting total impurities of the preparation methods were comparable (<3% versus<4% for VEGF164 and VEGF120, respectively), the observed differences in this manuscript may at least partially be accounted for by the differences in preparation, impurities, and potential immune or other extramural effects caused by exogenous protein administration.

In summary, this study provides additional evidence for the therapeutic potential of VEGF164/165. In addition to consistently demonstrating increased alveolarization on lung morphometric analysis, we now provide data to indicate improved functional outcomes with VEGF164 treatment after left pneumonectomy, as evidenced by improved pulmonary function and exercise tolerance. Further, the importance of the heparin-binding domain in potentiating downstream VEGF-mediated signaling and pulmonary proliferative effects is established in this study. However, the essentiality of the VEGF HBD raises concern for the translatability of VEGF164/165 as a therapy for children with pulmonary hypoplasia who require anticoagulation with heparin. While VEGF120/121 may avoid deleterious interactions with heparin that impair pulmonary proliferation, it lacks the potency to drive clinically relevant accelerated lung growth and development. Alternative therapeutics for pulmonary hypoplasia that either do not interact with heparin, or alternative anticoagulants which do not interact with angiogenic factors such as VEGF, deserve further expedient investigation to treat this often-fatal disease.

Methods

Supplementary Information

Acknowledgements

Abbreviations

Author contributions

Conceptualization: L.J.Y., D.T.D., M.P. Data curation: L.J.Y., V.H.K., J.D.S., A.P., B.S.C. Formal analysis: L.J.Y., P.D.M. Funding acquisition: L.J.Y., M.P. Investigation: L.J.Y., V.H.K., D.T.D., A.P. Methodology: L.J.Y., V.H.K., D.T.D., D.R.B. Project administration: A.P. Supervision: M.P. Writing—original draft: L.J.Y. Writing—review & editing: V.H.K., D.T.D., J.D.S., P.D.M., H.K., D.R.B., M.P.

Competing interests

The authors declare no competing interests.

Footnotes

References