Presentations

Abstract

Introduction:

Neurotensin (NTS) is an ovulation-critical paracrine signaling molecule produced in the ovulatory follicle after the luteinizing hormone surge. We have previously demonstrated a 75% reduction in ovulation after NTS neutralization in the ovulatory follicle of the cynomolgus macaque. NTS neutralization also causes dramatic red blood cell extravasation in the granulosa layer of the ovulatory follicle, indicative of disruption of the integrity of newly forming vasculature. We hypothesize that cadherin-based adherens junctions between adjacent ovarian vascular endothelial cells control permeability.

Methods:

Monkey ovarian microvascular endothelial cells (mOMECs) were isolated from follicle aspirates of adult female cynomolgus macaques (Macaca fascicularis) via CD31 magnetic bead isolation and endothelial cell selective culture conditions. In vitro cultures of >95% mOMECs (n=4) were used to assess the permeability of a ~90% confluent monolayer to the reporter-conjugated macromolecule streptavidin (60 KDa) using both a trans-well permeability assay and a vascular permeability imaging assay. Cells were treated with an NTS dose range of 0, 0.005, 0.05, 0.5, 5, and 50 mM. Thrombin (1 U/mL) and dibutyryl-cAMP (10 mM) served as positive and negative controls, respectively. To identify the cadherin profile of mOMECs, we employed RNA sequencing, qPCR, immunohistochemistry, or western blot (n=3-4). To identify the cadherins involved in the NTS-mediated effect on permeability, siRNA against N-cadherin (CDH2) and K-cadherin (CDH6) were generated. mOMECs were transfected with siRNA against either CDH2, CDH6, or control (siNC) and treated with either 0 or 5 mM NTS prior to assessment of permeability (n=4).

Results:

In the trans-well permeability assay, NTS at 0.5, 5, and 50 mM significantly decreased mOMEC permeability while 0.05 and 0.005 mM NTS had no significant effect. In the permeability imaging assay, NTS at 5 mM significantly decreased mOMEC permeability (p<0.05). As measured by both RNA sequencing and qPCR, CDH2 and CDH6 were both highly expressed in mOMECs. Surprisingly, mOMEC expression of CDH5, the primary vascular endothelial cadherin, was low to undetectable. Immunodetection confirmed that mOMECs express CDH2 and CDH6 at cell-cell junctions but lack CDH5. siRNA knockdown >65% of both CDH2 and CDH6 in mOMECs was confirmed via qPCR. In control siNC transfected mOMECs, NTS decreased permeability relative to untreated cells (p<0.05). However, in siCDH2 and siCDH6 transfected cells, the effect of NTS on permeability was abrogated.

Conclusion:

These data implicate CDH2 and CDH6 in the mechanism of NTS-mediated mOMEC permeability changes. In vitro, NTS decreases mOMEC permeability. These data indicate that neurotensin may also decrease vascular permeability in vivo to maintain vascular integrity during ovulatory angiogenesis, during which permeability is increased. mOMECs express high levels of CDH2 and CDH6 mRNA and protein, but little to no CDH5. In some highly migratory and proliferative vasculature, CDH5 expression is decreased and CDH2 expression is increased. The cadherin profile in mOMECs with high CDH2 and low CDH5 is consistent with the highly invasive phenotype of periovulatory follicular vascular endothelial cells. While CDH6 is not commonly associated with vascular endothelial cells, knockdown of CDH2 and CDH6 abrogate the NTS-induced decrease in mOMEC permeability. These data indicate that permeability-regulating adherens junctions of the ovarian follicular microvasculature may utilize CDH2 and CDH6. Supported by NIH grants HD071875 and HD097675 (DMD and TEC) and product donation from Organon, Jersey City, NJ.