Reduced blood pressure after smooth muscle EFNB2 deletion and the potential association of EFNB2 mutation with human hypertension risk

Abstract

Ephrin B2 (EFNB2) is a ligand for erythropoietin-producing hepatocellular kinases (EPH), the largest family of receptor tyrosine kinases. It has critical functions in many biological systems, but is not known to regulate blood pressure. We generated mice with a smooth muscle cell (SMC)-specific deletion of EFNB2 and investigated its roles in blood pressure regulation and vascular SMC (VSMC) contractility. Male Efnb2 knockout (KO) mice presented reduced blood pressure, whereas female KO mice had no such reduction. Both forward signaling from EFNB2 to EPHs and reverse signaling from EPHs to EFNB2 were involved in regulating VSMC contractility, with EPHB4 serving as a critical molecule for forward signaling, based on crosslinking studies. We also found that a region from aa 313 to aa 331 in the intracellular tail of EFNB2 was essential for reverse signaling regulating VSMC contractility, based on deletion mutation studies. In a human genetic study, we identified five SNPs in the 3′ region of the EFNB2 gene, which were in linkage disequilibrium and were significantly associated with hypertension for male but not female subjects, consistent with our findings in mice. The coding (minor) alleles of these five SNPs were protective in males. We have thus discovered a previously unknown blood pressure-lowering mechanism mediated by EFNB2 and identified EFNB2 as a gene associated with hypertension risk in humans. European Journal of Human Genetics (2016) 24, 1817-1825; doi:10.1038/ejhg.2016.105; published online 17 August 2016 INTRODUCTION Erythropoietin-producing hepatocellular kinases (EPH) are the largest family of receptor tyrosine kinases. In the basis of sequence homology, they are divided into A and B subfamilies. 1 Their ligands, called ephrins (EFNs), are also cell surface molecules. 2 EFNs are also divided into A and B subfamilies, based on the way they anchor on the cell surface: the A subfamily anchors on the cell surface through glycophosphatidylinositol, and the B subfamily, through a transmembrane domain. 2 The interactions between EPH kinases and EFNs are promiscuous, but EPHA kinases preferably interact with EFNA ligands, and EPHB kinases with EFNB ligands, which have three members, EFNB1, EFNB2 and EFNB3. 2 Although EPH members and EFN members share homology with their respective members, each member has its distinct function in different cellular processes. [3][4][5][6][7] In general, the EPH kinases interact with their EFN ligands on neighboring cells, because EPHs and EFNs are all cell surface molecules. 2 These molecules could be cleaved from the cell surface by enzymes such as ADAM10, 8,9 an unspecified matrix metalloproteinase, 10 or γ-secretase; 11 therefore, it is possible that the shed soluble fragments of EPH and EFN might be able to influence cells and tissues at a distance by blocking the interaction of EPHs and EFNs there.

Evidence from single nucleotide polymorphism analyses of ADVANCE study demonstrates EFNB3 as a hypertension risk gene

“…These genes are expressed in vascular smooth muscle cells (VSMCs), providing a molecular framework for their function in these cells. Through smooth muscle-specific gene deletion and in vitro mechanistic dissection, we have proved that VSMCs are the major targets through which EPHs/EFNs exert their effect on BP modulation262728293031. The BP phenotype in the knock out (KO) mice is often sex- and sex hormone-level dependent.…”

“…The BP phenotype in the knock out (KO) mice is often sex- and sex hormone-level dependent. For example, hypertension in EPHB6 KO is only observed in castrated mice26; hypertension after Efnb3 KO is present in females but not males; the BP phenotype in Efnb3 KO mice is reversed after gonadectomy: castrated KO males become hypertensive, while ovariectomized KO females, normotensive29; on the other hand, male but not female Efnb2 and Ephb4 KO mice are hypotensive2730.…”

Observational analyses from ADVANCE and ADVANCE‐ON

“…Several ADVANCE analyses have addressed genotypic associations with raised blood pressure [67][68][69][70] or kidney disease. 71,72 We have also identified the PROX1 gene CC genotype to be related to early-onset of diabetes.…”

Guidance Molecules in Vascular Smooth Muscle

“…Proliferative influences by Ephs and ephrins may be unique to the EphA subfamily, as deletion of ephrinB or EphB family members show no alterations in vascular smooth muscle proliferation ( Wang et al, 2016a ). In contrast, EphB-ephrinB signaling predominantly regulates cell adhesion to modulate pericyte recruitment and vascular wall assembly ( Figure 3 ).…”