DEFECTS IN LYSOSOMAL DEGRADATION CONTRIBUTE TO IMPAIRED FIBRONECTIN MATRIX ASSEMBLY IN PREECLAMPSIA

Objectives: The glycoprotein,fibronectin (FN), is a fundamentalcomponent of the extracellular matrix (ECM), driving trophoblast inva-sion and angiogenesis in the developing placenta. These events arecompromised in preeclampsia (PE), a pathology typified by impairedangiogenesis. FN undergoes extensive intracellular processing, from itsdimerization and secretion, to signalling, endocytosis and lysosomaldegradation. To date, the mechanisms controlling FN trafficking anddeposition in PE remain unknown, prompting us to investigate FN ma-trix assembly in the human placenta in physiological and pathologicalconditions.Methods: Placentae were collected fromfirst trimester, preeclamptic(n¼17), pre-term (n¼14) and term (n¼15) pregnancies. Placental mesen-chymal cells (pMCs) were isolated and characterized by FACS. Organelleswere isolated using sucrose density gradients and ultracentrifugation.pMCs were exposed to either cycloheximide (protein synthesis inhibitor),Brefeldin A (ER-Golgi transport inhibitor), or the lysosomal inhibitors,NH4Cl and Bafilomycin. Concanavalin A lectin-binding was used to assessFN glycosylation.Results: FN monomers and dimers accumulated in the Golgi and lyso-somal compartments in PE placentae and in PE pMCs relative to age-matched controls, whileFN1mRNA was unchanged. FN intracellularglycosylation was markedly reduced in PE pMCs, accompanied by aberrantER-Golgi transit and secretion. Assessment of protein half-life by cyclo-heximide revealed distinct impairments in FN matrix turnover in PE, whilein controls, FN was sequentially processed from cell-associated solubledimers to matrix-associated insolublefibrils. Confirming impairments inits clearance, lysosomal inhibition by NH4Cl or Bafilomycin in control pMCsstabilized intracellular FN and attenuated integrin-mediated signalling,while in PE, FN failed to be stabilized and continued to aberrantly signaldownstream.Conclusion: This study highlights profound disruptions in key stages offibronectin matrix assembly in preeclampsia, particularly its depositionand lysosomal degradation. Given the importance of pMC-derived ECM intriggering villous angiogenesis, this work sheds new light on the mecha-nisms contributing to abnormal vascular development in PE