Myoendothelial junction heterogeneity enables phosphatidylserine regulation of Kir2.1-mediated vasodilation.

In resistance arteries, matrix-devoid holes in the internal elastic lamina (HIEL) are presumably always the site of endothelial and smooth muscle contact (myoendothelial junction; MEJs). MEJs are signaling microdomains and we have previously demonstrated that specific protein localization regulates heterocellular communication. However, it is time consuming to localize proteins at the MEJ as it requires electron microscopy (EM) verification-it also lacks quantitative analysis for spatial localization both within endothelial cells and within the tissue. Thus, we wanted to determine if every HIEL was an MEJ without relying on any single protein marker. We first developed an in-house MATLAB analysis program to obtain quantitative data from en face images of third order mesenteric arteries, including an evaluation of spatial distribution using Monte Carlo simulations, which revealed HIEL exhibit a random spatial distribution. We then converted these metrics from en face XY views to XZ views of TEM sections, where the presence of MEJs can unequivocally be detected. We predicted an incidence of 5 - 17.5 HIEL per 1000um IEL length in TEM cross-sections (corresponding to a 190.75 HIEL/um density in en face images), and the measured incidence was 11.97, where every detected HIEL contained cellular contents. We concluded every HIEL was an MEJ. Next, we wanted to investigate the spatial distribution and function of a novel lipid marker of the MEJ, phosphatidylserine, which we previously demonstrated was enriched within in vitro MEJs by mass spectrometry, but had not been demonstrated on intact tissue. It is well described that lipid species, like PIP2, have key roles in regulating the function of channels, and thus we hypothesized that PS may function in a regulatory capacity within these signaling microdomains. We first determined PS-MEJ incidence and spatial distribution. Immunohistochemistry on en face third order mesenteric arteries revealed that PS comprised on average a 13.8% subset of total MEJs, indicating a heterogeneous nature to these microdomains. Within individual endothelial cells, an average of 9.89 MEJs were identified, and an average of 1.13 of these MEJs were PS positive. Monte Carlo analysis did not find unique PS-MEJs spatial segregation to endoplasmic reticulum, nucleus, or interendothelial junctions. It wasn't clear the biological rationale for the PS segregation in MEJs. However, recent in silico studies predicted that PS, in addition to PIP2, interacts with the inward rectifying potassium channel 2.1 (Kir2.1). Staining for Kir2.1, we found that it only localized to 1.5% of total MEJs, and yet remarkably, 83.3% of Kir2.1-MEJs localized to PS-MEJs. This contrasted with Cx40-MEJs, where only 14.6% localized to PS-MEJs. This observation led us to test the hypothesis that PS may regulate Kir2.1. Using pressurized third order mesenteric arteries, we performed dose response curves with NS309, a drug that can activate Kir2.1 channels. Application of either 1 uM or 10 uM exogenous PS demonstrated a significant reduction in dilation to the early range of NS309 doses comparable to arteries treated with the 3.6 uM ML-133 and 30 uM Ba . In addition, direct application of 10 uM PS induced a significant constriction. Thus, we hypothesize PS may provide a mechanism for counteracting the dilatory effect of PIP2. Based on this data, we conclude MEJ heterogeneity may regulate vascular function.