EXPRESS: Gremlin1 blocks vascular endothelial growth factor signalling in the pulmonary microvascular endothelium

Abstract

The bone morphogenetic protein (BMP) antagonist gremlin 1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension (HPH). Recently, non-canonical functions of gremlin 1 have been identified, including specific binding to the vascular endothelial growth factor receptor-2 (VEGFR2). We tested the hypothesis that gremlin 1 modulates VEGFR2 signaling in the pulmonary microvascular endothelium.

We examined the effect of gremlin 1 haploinsufficiency on the expression of VEGF responsive genes and proteins in the hypoxic (10% O₂) murine lung in vivo. Using human microvascular endothelial cells in vitro we examined the effect of gremlin 1 on VEGF signaling.

Gremlin 1 haploinsufficiency (Grem1^+/–) attenuated the hypoxia-induced increase in gremlin 1 observed in the wild-type mouse lung. Reduced gremlin 1 expression in hypoxic Grem1^+/– mice restored VEGFR2 expression and endothelial nitric oxide synthase (eNOS) expression and activity to normoxic values. Recombinant monomeric gremlin 1 inhibited VEGFA-induced VEGFR2 activation, downstream signaling, and VEGF-induced increases in Bcl-2, cell number, and the anti-apoptotic effect of VEGFA in vitro.

These results show that the monomeric form of gremlin 1 acts as an antagonist of VEGFR2 activation in the pulmonary microvascular endothelium. Given the previous demonstration that inhibition of VEGFR2 causes marked worsening of HPH, our results suggest that increased gremlin 1 in the hypoxic lung, in addition to blocking BMP receptor type-2 (BMPR2) signaling, contributes importantly to the development of PH by a non-canonical VEGFR2 blocking activity.

Fig. 1.

Gremlin 1 haploinsufficiency reduces gremlin 1 expression and restores VEGFR2 expression and eNOS expression and activity in the hypoxic lung in vivo. (a) Representative western blot and densitometric analysis of gremlin 1 expression in normoxic and hypoxic wild-type (^+/+) and Grem1^+/– lung lysate. (b) Representative western blot and densitometric analysis of VEGFR2 expression in normoxic and hypoxic wild-type ^(+/+) and Grem1^+/– lung lysate. (c) Immunohistochemical localization of eNOS in the lungs of normoxic and hypoxic wild-type and Grem1^+/– mice. (d) Representative western blot and densitometric analysis of eNOS expression in normoxic and hypoxic wild-type and Grem1^+/– lung lysate. (e) Representative western blot and densitometric analysis of VASP phosphorylation in normoxic or hypoxic wild-type and Grem1^+/– lung lysate. Densitometry values are normalized to a pooled standard. See Supplemental material for details. Bars represent median ± IQR.

Fig. 2.

Monomeric gremlin 1 is not a VEGF agonist but blocks VEGFA-induced VEGFR2 phosphorylation and downstream signaling in vitro. (a) Representative western blot and densitometric analysis of VEGFR2 phosphorylation in human microvascular endothelial cells from the lung (HMVEC-L) exposed to VEGFA (16 ng/mL) for 2 min, gremlin 1 (2 µg/mL) for 60 min, or VEGFA (16 ng/mL) for 2 min after pre-incubation with gremlin 1 (2 µg/mL) for 60 min. (b) Representative western blot and densitometric analysis of eNOS serine1177 phosphorylation in HMVEC-L exposed to VEGFA (16 ng/mL) or BMP2 (100 ng/mL) stimulation for increasing periods from 2 to 60 min. VEGFA response was maximal after 2 min while the maximum BMP2 response was not observed until 60 min of stimulation. The image is representative of three independent experiments. (c) Representative western blot and densitometric analysis of eNOS phosphorylation in HMVEC-L exposed to VEGFA (16 ng/mL) for 2 min, gremlin 1 (2 µg/mL) for 60 min, or VEGFA (16 ng/mL) for 2 min after gremlin 1 (2 µg/mL) preincubation for 60 min. (d) Representative western blot and densitometric analysis of p44/42 MAPK (Erk 1/2) phosphorylation in HMVEC-L exposed to VEGFA (16 ng/mL) for 2 min, gremlin 1 (2 µg/mL) for 60 min, or VEGFA (16 ng/mL) for 2 min after gremlin 1 (2 µg/mL) preincubation for 60 min. Bars represent median ± IQR.

Fig. 3.

Gremlin 1 blocks the expression of the VEGFA responsive gene Bcl-2 in a BMP independent manner. (a) Relative Bcl-2 gene expression in human microvascular endothelial cells from the lung (HMVEC-L) after stimulation with increasing concentrations of VEGFA (1–16 ng/mL) for 24 h. (b) Relative Bcl-2 gene expression in HMVEC-L stimulated with increasing concentrations of VEGFA (1–16 ng/mL) and gremlin 1 (2 µg/mL) together for 24 h. (c) Relative Bcl-2 gene expression in HMVEC-L stimulated for 24 h with VEGFA (16 ng/mL) alone, gremlin 1 (2 µg/mL) alone, VEGFA (16 ng/mL) and gremlin 1 (2 µg/mL) together, BMP2 (100 ng/mL) alone, or BMP4 (80 ng/mL) alone. (d) Relative caspase activity in human microvascular endothelial cells from the lung (HMVEC-L) after stimulation with VEGF (16 ng/mL), gremlin 1 (2 µg/mL) alone, or VEGF and gremlin 1 together for 24 h. Caspase 3/7 activity is expressed as fold change in luminescence relative to vehicle-treated cells. Bars represent median ± IQR.

Fig. 4.

Gremlin 1 attenuates VEGFA induced increases in human lung microvascular endothelial cell number. (a) Numbers of human microvascular endothelial cells from the lung (HMVEC-L) after exposure for 24 h to vehicle alone, vehicle and gremlin 1 (2 µg/mL), VEGFA (1 ng/mL) alone, VEGFA (1 ng/mL) together with gremlin 1 (2 µg/mL), VEGFA (8 ng/mL) alone, or VEGFA (8 ng/mL) together with gremlin 1 (2 µg/mL). Cell counts are expressed relative to control number (vehicle) in each experiment. (b) Numbers of HMVEC-L after exposure to vehicle alone, vehicle and gremlin 1 (2 µg/mL), BMP2 (50 ng/mL) alone, BMP2 (50 ng/mL) together with gremlin 1 (2 µg/mL), BMP2 (100 ng/mL) alone, or BMP2 (100 ng/mL) together with gremlin 1 (2 µg/mL) for 24 h. Cell counts are expressed relative to control number (vehicle) in each experiment. Bars represent mean ± SD.

Fig. 5.

Proposed blocking action of gremlin 1 on both BMP and VEGF signaling during the development of hypoxic pulmonary hypertension (HPH). As confirmed in the present study, the canonical action of increased gremlin 1 during the development of HPH is to prevent BMP2 and BMP4 binding to BMPRs thus blocking BMP dependent signaling. Increased gremlin 1 simultaneously acts to block VEGF signaling by binding to VEGFR2.⁵³ Reduced BMPR signaling^, and reduced VEGFR2 signaling^, both contribute importantly to the development of HPH. Thus, gremlin 1 mediated blockade of both pathways causes vasoconstriction and vascular remodeling. The signaling pathways illustrated in the schematic are only those used in the present study to demonstrate the separate blocking actions of gremlin 1 on BMPR signaling and on VEGFR2 signaling. Both agonists also exert multiple other actions that are not shown here.