Gremlin-1 Is Overexpressed in Endothelial Cells of Patients with Loeys-Dietz Syndrome Due to Dysregulation of TGF-β Signalling,
Authors: Wellbrock Jasmin , Sheikhzadeh Sara , Bonk Veronika , Oliveira-Ferrer Leticia , Klaetschke Kristin , Streichert Thomas , Bokemeyer Carsten , von Kodolitsch Yskert , Fiedler Walter 
Published: October 2019

Abstract
Abstract 3269
The Loeys-Dietz syndrome (LDS) is an inherited connective tissue disorder with symptoms similar to those of Marfan syndrome and the vascular type of Ehlers-Danlos syndrome. Most patients with LDS develop severe aortic aneurysms resulting in early need of surgical intervention. Patients with LDS harbour a mutation in the transforming growth factor β (TGF-β) receptors TGFBR1 (also named ALK-5) or TGFBR2. Since the TGF-β pathway plays a crucial role in many cellular processes including angiogenesis, we focussed our analyses on endothelial cell dysfunction in patients with Loeys-Dietz syndrome.
We isolated circulating outgrowth endothelial cells (OEC) from the peripheral blood of two LDS patients (one female, 54 years; one male, 26 years old) both harbouring a mutation in the TGFBR2 gene. Gene expression profiles of OEC clones were performed using Affymetrix Human Genome U133 Plus 2.0 Arrays and confirmed by quantitative PCR analysis for genes of interest. OEC clones isolated from age- and sex-matched healthy controls served as reference subjects.
We demonstrate that several genes belonging to the TGF-β pathway had altered expression in OECs isolated from LDS patients compared to those from healthy controls. For example, mRNA levels of bone morphogenic proteins (BMP) 2 and 4 were decreased in both LDS OEC clones (mean decrease 4 and 6 fold, respectively) whereas gene expression of inhibitory downstream molecule SMAD-6 was increased 2-fold.
In both analysed OEC clones from LDS patients, gene expression of BMP antagonist Gremlin-1 (also known as Drm) showed the most prominent dysregulation with a 1136-fold and 164-fold higher expression in LDS OECs compared to healthy controls, respectively.
Interestingly, in OECs isolated from healthy donors, Gremlin-1 expression was significantly down-regulated after incubation with SB431542 (5 μM), a small molecule inhibitor of the TGF-β receptor complex (mean decrease 4 fold; t-test: p = 0.002; n = 6). In contrast, the stimulation of OEC clones with TGF-β1 (1 ng/ml) resulted in significant up-regulation of Gremlin-1 mRNA levels (mean increase 7 fold; t-test: p = 0.014; n = 6). Apparently, the up-regulation of Gremlin-1 in LDS OECs seems to mirror an activated TGF-β signalling cascade in outgrowth endothelial cells. These findings are in line with other studies published on LDS where hyperactivity of the TGF-β downstream signalling was demonstrated by higher phosphorylation levels of SMAD-2 in the aortic media of LDS patients (Loeys et al., Nat Genet. 2005 Mar;37(3):275–81). Gremlin-1 might represent a second gene supporting the concept of increased TGF-β signalling in Loeys-Dietz syndrome.
Gremlin-1 itself displays opposing effects on angiogenesis. First, it is known as a pro-angiogenic factor and was recently shown to stimulate angiogenesis via direct binding to the VEGF receptor 2 (Mitola et al., Blood. 2010 Nov 4;116(18):3677–80). On the other hand, as antagonist of bone morphogenic proteins, Gremlin-1 possesses anti-angiogenic properties by suppressing pro-angiogenic effects of BMP-2 and BMP-4.
In summary, we believe that due to its drastic up-regulation in OECs of LDS patients, Gremlin-1 represents a crucial effector of dysregulated TGF-β signalling in endothelial cells inducing vascular pathology in Loeys-Dietz syndrome.

Disclosures:
Fiedler: Pfizer: Research Funding.