Shehata Marker: A Promising Tool for Assessing Graft Endothelial Dysfunction and Hypoxic Injury

Abstract

Background: The integrity of the graft vasculature is essential for maintaining homeostasis in transplantation,
regulating perfusion, selective permeability, preventing thrombosis, and facilitating immune surveillance.
However, despite extensive studies on graft rejection and ischemia-reperfusion injury, the molecular responses
of vascular effectors to hypoxic stress during transplantation remain largely uncharacterized.
Objective: This study addresses a critical knowledge gap by evaluating the collective response of established
vascular effector molecules to hypoxic injury, a condition commonly encountered in transplantation. The objective is to assess the potential of these molecules, when analyzed as a panel, to serve as early, non-invasive
biomarkers for graft function and viability.
Methods: In this study, hypoxia-inducible factor-1 alpha (HIF-1α) was chemically induced using cobalt chloride
(CoCl₂), a well-established hypoxia mimetic. Unlike conventional methods such as ELISA, which are typically
used for detecting subtle changes at the picogram level, immunoblotting was employed in this hypothesis-generating, proof-of-concept pilot investigation to capture more prominent alterations in vascular effectors at the
preclinical stage. The study included three independent biological replicates, in which varying concentrations
of CoCl₂ were compared to untreated controls under normoxic conditions.
Results: Hypoxic stimulation led to significant upregulation of certain key vascular effectors, including eNOS,
VEGF-A, sVEGFR1, and endothelin-1. Most notably, a novel and unexpected finding regarding nostrin, a regulator of nitric oxide signaling, is reported: its expression and secretion were differentially modulated by hypoxia
in a cell-type-specific manner. While nostrin expression decreased in HuH7 and HEK293T cells, it accumulated
in HUVECs—a divergence not documented in prior literature. Furthermore, nostrin secretion exhibited an inverse pattern: a trend toward increased levels in tissue cells, but a significant decrease in endothelial cells
under hypoxic conditions—a phenomenon not previously reported.
Conclusion: This study provides the first evidence of differential nostrin regulation across cell types in response
to hypoxia, unveiling a novel layer of complexity in vascular biology within the transplant setting. By integrating a novel methodological approach with the discovery of uncharted hypoxia-induced vascular signatures, the
findings open new avenues for developing non-invasive biomarkers for graft monitoring, particularly in the
early post-transplant phase and during ex vivo perfusion.