Nadja is a rising senior at the University of Puerto Rico at Mayaguez, majoring in Mechanical Engineering. She is currently working in Rebecca Well’s Lab on the study of lipid droplets and their effect on the development of liver diseases. After finishing her Bachelor’s Degree, Nadja is determined in pursuing a Ph.D. in Biomedical Engineering and continuing a career in academic or industry research. In her free time, she enjoys singing, dancing, and spending time with friends and family.
Quantifying repair factors in lipid loaded hepatocytes through image analysis
Nadja M. Maldonado Luna, Mariah A. Turner
Introduction: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, often occurs in people with chronic liver diseases, and the presence of cirrhosis is seen as the most significant risk factor . Cirrhosis is characterized by alterations in the extracellular matrix (ECM), including increased deposition of collagen and alignment of the ECM architecture, which drastically increases tissue stiffness . Although HCC is often associated with cirrhosis, it can also arise in non-cirrhotic livers in the context of non-alcoholic fatty liver disease (NAFLD) . Lipid accumulation in the liver cells, characteristic in NAFLD, fills the cell cytoplasm and compresses the nucleus. Based on this morphology, the Wells Lab has hypothesized that the lipid droplets act as a mechanical stress on the nucleus, functioning similarly to tissue stiffness . Nuclear deformation from external sources of mechanical stress, such as migration through constricted environments or culture on stiff substrates, has been shown to increase the frequency of nuclear rupture, leading to depletion of nuclear repair factors and the accumulation of DNA damage . We suggest that deformation due to lipid droplets may similarly lead to the depletion of important repair factors and increased accumulation of double-stranded DNA breaks, which may increase the risk of HCC development in NAFLD livers. The objective of this study was to quantify the impact of lipid accumulation in liver cells (hepatocytes) on the amount of DNA damage repair factor in the cells.
Materials and Methods: Cell culture: Primary human hepatocytes (PHH) were seeded onto collagen-coated polyacrylamide (PAA) gels with storage modulus values of 500 and 10 kPa that are representative of the stiffness of normal and cirrhotic livers, respectively. Cells were also cultured on glass, which is non-physiologically stiff. Fatty acid treatment: After the seeding period, PHH cells were incubated for 48 h in the presence of 400µM oleic acid and 0.5% bovine serum albumin (BSA) solution in DMEM. Oleate is the second most common fatty acid in the human diet and is easily packaged into lipid droplets. Cell staining, microscopy, and image analysis: To identify the lipids and assess gross nuclei morphology, cells were stained with BODIPY and DAPI, respectively. To look at the amount of repair factor, BSA control and oleate-treated cells were stained for Ku80. Cells were imaged using a confocal microscope. Nuclei morphology (area, circularity, and solidity) was analyzed using semi-automated image segmentation and detection of individual nuclei in ImageJ. Ku80 mean intensity and integrated density of the nucleus was measured. One-way ANOVAs were used to test the statistical significance of lipid accumulation on nuclear deformation and on Ku80 staining intensity.
Results and Discussion: Nuclear area, circularity, and solidity for oleate-loaded cells tended to decrease compared to controls, consistent with previous work . Also, Ku80 repair factor means intensity and integrated density decreased with lipid loading of cells seeded on the stiffest substrates. Ku80 mean intensity was associated with the nuclear area, however no strong association was seen for any of the measured shape parameters.
Conclusions: The results of these experiments were consistent with previous work from the lab, showing increased nuclear deformation and compression, and decreased repair factor intensity in oleate-treated cells. Ku80 mean intensity was also correlated with nuclear area, suggesting that nuclear compression may be a contributor to Ku80 decrease in oleate-treated cells. Furthermore, differences between the groups were highest on the stiffest substrates, indicating that some level of tissue stiffening may be required for lipid droplets to act as a mechanical stress. Future work aims to determine if lipid- droplet accumulation increases DNA damage accumulation, through a number of different potential mechanisms.
References:  Masuzaki+2009 J Hep.  Asselah+2009 Gut.  Kanwal+2018 Gastroenterology.  Chin+2020 AJP-Gastrointest Liver Physiol.  Ivanovska+2019 Biophysical Journal.