A novel mechanobiological signaling axis at endothelial cell-cell adhesions antagonizes RhoA activity and actomyosin contractility.

Introduction: Endothelial cell-cell adhesions are nexuses of mechanical and chemical signaling that regulate myriad vasculature functions including barrier function, vascular remodeling, and angiogenesis. Despite evidence linking mechanical dysregulation of cell-cell adhesions with vascular pathologies, such as atherosclerosis and pathological angiogenesis, we lack a comprehensive understanding of the molecular mechanisms facilitating the diverse signaling events converging at these interfaces. To identify novel molecular control systems regulating junctional mechanics at endothelial cell-cell adhesions, we used proximity biotinylation (BioID) to unbiasedly characterize proteins scaffolding with the intracellular domain of the endothelial cell-cell adhesion receptor, VE-cadherin (Fig. 1a). Among the most abundant VE-cadherin interacting proteins identified by mass spectrometry was Junctional Protein Associated with Coronary Artery Disease (JCAD). First described in two independent genome-wide association studies as a top genetic risk locus for coronary artery disease, JCAD is a large protein with no known cellular functions. Here, we report a novel function for JCAD in endothelial mechanics as an orchestrator of a previously unappreciated mechanobiological signaling axis antagonizing endothelial RhoA/actomyosin activities, both of which play vital roles in vascular development and homeostasis. Understanding these previously unappreciated roles for JCAD may inform how dysregulation of this axis contributes to JCAD-associated risk for vascular disease.

Materials and

Methods: Human dermal microvascular endothelial cells (hMVEC) were grown in EGM2-MV media (Lonza). For BioID analysis, hMVECs were transduced with a lentivirus encoding a VE-cadherin-BirA* transgene. These cells were treated with biotin for 12hrs., were lysed with RIPA buffer, and biotinylated proteins were isolated via streptavidin beads and identified by mass spectrometry. JCAD knockdown (JCAD KD) hMVECs were generated by lentiviral shRNA transduction. JCAD overexpression (JCAD OE) lentiviral constructs were generated by cloning the JCAD coding sequence from an hMVEC cDNA library. In vitro biomimetic vasculogenesis assays were conducted by seeding hMVECs as individual cells into a 3D extracellular matrix hydrogel and allowing them to undergo vasculogenic assembly into a perfused microvessel network. RhoA activity was assayed by affinity precipitation of RhoA-GTP using agarose beads coated with recombinant Rhotekin Rho-binding domain and immunoblotting for RhoA protein levels. For constitutive active/inactive RhoA studies, hMVECs were transduced with lentivirus encoding doxycycline-inducible RhoA(Q63L) and RhoA(T19N), respectively. JCAD and phosphorylated myosin II light chain (ppMLC) immunostaining and immunoblotting were conducted using Atlas antibody HPA017956 and Cell Signaling Technologies antibody #3674, respectively.

Results, Conclusions, and Discussions: To assess endothelial functions of JCAD, we assayed microvascular morphogenesis of JCAD KD hMVECs in a 3D-engineered biomimetic platform. Compared to control hMVECs, which form a perfused vessel network over four days, JCAD KD hMVECs fail to form a discernable vessel network (Fig. 1d). By live-cell microscopy, JCAD KD cells proliferate significantly less and exhibit defects in polarization and random migration (Fig. 2a/b). Conversely, JCAD OE results in hMVECs with spindled morphologies (Fig. 2c), resembling nonmuscle myosin-II inhibition. Together, these data suggest that JCAD plays an unappreciated role in regulating endothelial mechanics. Indeed, by assaying RhoA GTPase activity in KD and OE cells, we observed JCAD OE leads to an approximate 40% decrease in active RhoA whereas JCAD KD cells have a 70% increase in active RhoA relative to controls (Fig. 2e). Likewise, we observed an approximately 30% increase in myosin light-chain phosphorylation in JCAD KD cells (Fig. 2e).

JCAD is reported to be highly phosphorylated following treatment with thrombin (Fig. 1b), a pro-inflammatory RhoA agonist, suggesting changes in JCAD function with high RhoA/actomyosin activity. Interestingly, expression of constitutively-active RhoA results in increased levels of JCAD by immunostaining, but not by immunoblotting, suggesting putative JCAD changes in detergent-solubility with RhoA activity (Fig. 2d). Further, exogenous JCAD dynamically targets to regions of high actomyosin contractility (Fig. 2c). As JCAD lacks any known domains that regulate RhoA activity or actomyosin, we immunoprecipitated JCAD and profiled co-immunoprecipitating proteins by mass spectrometry. We identify a novel JCAD-RhoGEF interaction and ongoing work is elucidating the mechanisms and cellular contexts in which JCAD antagonizes RhoA activity through this interaction. We posit that JCAD orchestrates a feedback loop in response to high RhoA activity in the cell that, in turn, locally antagonizes RhoA and actomyosin by regulating a RhoA-GEF interaction.

Through this work, we have implicated JCAD, a major coronary artery disease risk locus, in the mechanical regulation of the vascular endothelium through antagonism of RhoA and actomyosin activity. We propose that JCAD acts as a mechanobiological rheostat to maintain cell and tissue level homeostasis and, when disrupted, may be a critical factor in JCAD-mediated disease risk.

Acknowledgements (Optional): This work was supported by the National Institutes of Health (NIH) grants R35GM150987 (to MLK) and R21AG072232 (to MLK). KAJ is supported by Tobacco-Related Disease Research Program Predoctoral Fellowship T33DT6442. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 2038436 (to KAJ). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.