(28ak) Investigating the Role of H2s in Liver Fibrosis

Small gaseous molecules such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) have historically been known as potent and toxic¹. However, recent developments have suggested that these molecules may have important biological functions. H2S has been discovered to be an important gastrotransmitter, with additional roles in vascular regulation, reducing oxidative stress, anti-inflammation, edema, anti-clotting, and angiogenesis. The numerous functions that implicate H2S can impact all organs and tissues in the body¹. However, the exact nature of its role in healthy and diseased liver function remains cryptic.

A variety of stimuli, including viruses and toxicants, or other health-related impacts, such as obesity and diabetes may initiate fibrosis in the liver. Fibrosis ultimately leads to an excess amount of secreted extracellular matrix (ECM) material in an uncontrolled manner. Effects of fibrosis can lead to disruption of blood flow, dedifferentiation, and disruption of hepatic architecture, and cirrhosis. Severe inflammation can also result due to fibrosis, which can disrupt signaling between the hepatic cells and communication with other systems.

H2S holds potential for being a therapeutic agent in liver fibrosis. However, further investigation is needed to accurately determine the effects and mechanisms caused by this signaling molecule. The role of H2S may have varying effects on the different cell types in the liver, including hepatocytes, hepatic stellate cells (HSCs), and even Kupffer cells (KCs). Therefore, our goal is to investigate how this small molecule impacts the hepatic environment under normal and fibrotic conditions. We will use tunable H2S releasing agents from amino acid-based C-substituted N- thiocarboxyanhydrides (NTAs) to study their effects in hepatic organoids². NTAs release H2S by nucleophilic substitution that results in the formation of carbonyl sulfide (COS). Carbonic anhydrase, an enzyme produced within eukaryotic cells, converts COS to H2S. The only byproduct of the decomposition of the NTA is the amino acid from which it was derived². The release of H2S can be caused by ROS, which are present during hepatocellular injury.

Methods

Primary rat hepatocytes were seeded on Type 1 (1.1 mg/ mL) collagen hydrogels. A second layer of collagen was added to the cultures 24 hours later to assembly a collagen sandwich model. Five days after collagen sandwich assembly (Day 6), Alanine-NTA (ala-NTA) and valine-NTA (val-NTA) were administered to the cultures at 100 uM in the cell media for two sequential days. Cultures were then ended 24 hours later for analysis (Day 8).

Results

Initial results suggest that ala-NTA does not significantly lower normalized urea secretion, potentially indicating that the H₂S does not affect the hepatocytes. However, upon val-NTA administration, significant decreases in normalized urea secretion are exhibited. There is a ~1.2-1.6-fold decrease in normalized urea secretion on Day 7 and Day 8, compared to Day 6, the day NTAs were first administered. This could potentially indicate that the release of H₂S from val-NTA may affect hepatocyte function.

Conclusions and Future Work

Initial results suggest that iHLCs do not exhibit significant decreases in function upon H₂S administration. Ongoing work includes investigation of H₂S releasing agents on hepatic cells, such as hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs). Future work will include assembling engineered 3D liver organoids with hepatocytes, LSECs, Kupffer cells, and HSCs. The mechanical environment of engineered 3D liver organoids will be manipulated by tuning the stiffness of the hydrogel to initiate fibrosis³. NTAs will be administered to the fibrotic cultures to investigate if attenuation occurs.

References

1. Kolluru GK, Shen X, Bir SC, Kevil CG. Hydrogen sulfide chemical biology: pathophysiological roles and detection. Nitric Oxide. 2013 Nov 30;35:5-20. doi: 10.1016/j.niox.2013.07.002. Epub 2013 Jul 9. PMID: 23850632; PMCID: PMC4077051.
2. Kaur K, Enders P, Zhu Y, Bratton AF, Powell CR, Kashfi K, Matson JB. Amino acid-based H2S donors: N-thiocarboxyanhydrides that release H2S with innocuous byproducts. Chem Commun (Camb). 2021 Jun 3;57(45):5522-5525. doi: 10.1039/d1cc01309b. PMID: 33956024; PMCID: PMC8178226.
3. Orbach SM, Ford AJ, Saverot SE, Rajagopalan P. Multi-cellular transitional organotypic models to investigate liver fibrosis. Acta Biomater. 2018 Dec;82:79-92. doi: 10.1016/j.actbio.2018.10.010. Epub 2018 Oct 11. PMID: 30316024.