Characterization of ANGPT2 mutations associated with primary lymphedema

Abstract

Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain <30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor–like domains 1 and 2) receptor complex and α5β1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)–ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin α5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.

Ang2 inhibitors and Tie2 activators: potential therapeutics in perioperative treatment of early stage cancer

“…Importantly, both Ang1 and Ang2 in dimeric form were unable to induce Tie2 phosphorylation, as was previously shown (Kim et al, 2005). The reported limitation in ligand-to-receptor stoichiometry is dependent on the coiled-coil domains, as CMP-Ang1 dimers (Ang1 with the native coiled-coil domain replaced with modified dimeric coiled-coil domain from cartilage matrix protein) can bind to Tie2 in a 2:2 complex formation (Lepp€ anen et al, 2020). The authors propose that if these asymmetrical dimers were to further cluster into higher-order forms by oligomerization of the SCD (as is the case predominantly for Ang1), it is likely that two of the four FLDs in tetrameric Ang would bind diagonally neighboring Tie2 monomers within parallel arrays of dimeric Tie2 receptors (as opposed to the two monomeric arms within a single Tie2 dimer).…”

“…1C), and a shorter N-terminal region or superclustering domain (SCD) which enables further superclustering of Ang dimers into heterogenous multimeric structures (Fig. 1D) (Davis et al, 1996;Maisonpierre et al, 1997;Davis et al, 2003;Kim et al, 2005;Lepp€ anen et al, 2020). While dimeric Ang ligands can bind Tie2, this conformation does not induce Tie2 phosphorylation (Fig.…”

Caveolae-mediated Tie2 signaling contributes to CCM pathogenesis in a brain endothelial cell-specific Pdcd10-deficient mouse model

“…However, it is now recognized that Angpt2 is able to induce Tie2 phosphorylation in a contextdependent manner 26,[48][49][50][51]65,66 . Angpt2 can act as a Tie2 agonist in lymphatics 47,65,[67][68][69][70] , and the Angpt2 agonist activity is attributed to a low level of VE-PTP expression 71 . Interestingly, it has been reported that endothelial Tie1 is essential for the agonist activity of autocrine Angpt2 by directly interacting with Tie2 to promote Angpt2-induced p-Tie2 [48][49][50][51] , promoting Angpt2-induced the enlargement of post-capillary venules and capillaries in the trachea 48,49 .…”

Crosstalk Between microRNAs and the Pathological Features of Secondary Lymphedema

“…Hereditary or primary lymphedema is associated with mutations in genes that encode for lymphatic endothelial markers; vascular endothelial growth factor-C (VEGFC) and its receptor (VEGFR), such as Fms-related receptor tyrosine kinase-4 ( FLT4) , SRY-box transcription factor 18 (SOX18) , forkhead box C2 ( FOXC2 ), and angiopoietin ( ANGPT2) ( Miaskowski et al, 2013 ; Brouillard et al, 2014 ; Leppanen et al, 2020 ). Meanwhile, studies on genetic predisposition of secondary lymphedema have demonstrated a significant association of VEGFR , RAR-related orphan receptor C ( RORC ), FOXC2 , and interleukin-6 ( IL6 ) genes with secondary lymphedema ( Newman et al, 2012 ; Miaskowski et al, 2013 ; Leung et al, 2014 ).…”

“…Meanwhile, studies on genetic predisposition of secondary lymphedema have demonstrated a significant association of VEGFR , RAR-related orphan receptor C ( RORC ), FOXC2 , and interleukin-6 ( IL6 ) genes with secondary lymphedema ( Newman et al, 2012 ; Miaskowski et al, 2013 ; Leung et al, 2014 ). However, the reported genes explain <30% of this debilitating condition ( Leppanen et al, 2020 ), hence more studies are needed to further clarify the pathways involved in lymphedema. Epigenetics can alter gene expression in the absence of genomic mutations and may contribute to the pathophysiology of lymphedema.…”