Off-target comparison modulation of nuclease Zinc-finger protein 36 (ZFP36) by cytosolic (ATB-346) and mitochondrial (AP-39) hydrogen sulfide donors on the pro-thrombotic and pro-inflammatory phenotype transition of endothelial cells

• Grant number: 24/12229-7
• Support Opportunities: Scholarships in Brazil - Support Program for Fixating Young Doctors
• Start date: August 01, 2024
• End date: June 30, 2026
• Field of knowledge: Biological Sciences - Pharmacology - Cardiorenal Pharmacology
• Agreement: CNPq
• Principal Investigator: Stephen Fernandes de Paula Rodrigues
• Grantee: Paula Rodrigues de Barros
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 24/02250-9 - Off-target comparison modulation of nuclease Zinc-finger protein 36 (ZFP36) by cytosolic (ATB-346) and mitochondrial (AP-39) hydrogen sulfide donors on the pro-thrombotic and pro-inflammatory phenotype transition of endothelial cells, AP.R

Abstract

Endothelial cells have a physiological role in maintaining hemostasis with antiplatelet, fibrinolytic, and antithrombotic effects. However, in pathological situations the endothelial phenotype can convert into a pro-thrombotic state with local and systemic consequences. The use of hydrogen sulfide (H2S), an important endogenous gaseous mediator, demonstrated antithrombotic and regulatory potential by inhibiting platelet activation. Newly synthesized drugs act as rapid donors of H2S via the mitochondrial (AP-39) or cytosolic (ATB-346) pathway. These drugs have anti-inflammatory effects with no gastrointestinal adverse reactions. But their mechanisms and optimal dosage in certain cell types remain unexplored in literature. A potential target of H2S-mediated actions are the Zinc-finger proteins, an DNA linkage protein rich in cysteine residues, sensitive to the redox system and important regulator of gene transcription. ZFP36 has an important anti-inflammatory function, suppressing mRNA and expression of NF-ºB and the cytokine IL-6, pro-inflammatory mediators. In this context, we intend to evaluate if the anti-thrombotic/antiplatelet and anti-inflammatory effects of ATB-346 and AP -39 are dependent on the expression and activity of ZFP36 protein. To test our hypothesis, three specific aims are stablished: 1) Determine whether treatment with H2S donors can modulate the transcription and expression of ZFP36 in endothelial cells. For this, endothelial cells treated with increasing doses of the drugs will be extracted for mRNA quantification and DNA sequencing. 2) Evaluate whether the ZFP36 protein is necessary for ATB-346 and AP-39 anti-inflammatory effect. Endothelial cells treated with interfering RNAs (siRNAs) for ZFP36 in the presence or absence of the drugs will be evaluated in in vitro functional assays for their pro-thrombotic endothelial phenotype through the activity of the tissue factor pathway inhibitor (TFPI), endothelial cell migration and permeability, pro-inflammatory cytokine production by ELISA and evaluation of the expression of proteins related to the pro-inflammatory and coagulation process. 3) And to evaluate the ex vivo anti-thrombotic/anti-platelet potential exerted by the H2S donors ATB346 and AP39, we will perform the platelet adhesion assay on endothelial cells treated with interfering RNAs (siRNAs) for the ZPF36 protein. With these results we intend to elucidate whether the H2S/persulfidation signaling axis mediates the inflammatory and antithrombotic response via a signaling pathway dependent on the ZPF36 protein with further maintenance of the anti-inflammatory and anti-platelet aggregation phenotype of the endothelial cell (AU)