Shankar Chakkera

Helios Scholar

Non-alcoholic fatty liver disease(NAFLD), a disease typically associated with obesity and Type 2 diabetes, affects up to 25% of adults worldwide. The conditions of NAFLD range from excess liver fat and inflammation to liver cirrhosis(scarring) and failure. Although studies have analyzed potential biomarkers to detect NAFLD, little to no research has studied extracellular vesicle(EV)-associated markers of this disease and its potential implications. As organ-specific biomarkers, EVs can be more accurate and informative compared to protein/apoptosis biomarkers and significantly less invasive compared to a liver biopsy.

Our experimental study aims to focus on analyzing extracellular vesicles to determine unique EV-associated proteins associated with non-alcoholic steatohepatitis(NASH), a condition of NAFLD. These proteins were selected based on our previous proteomics study that indicated a change in the concentration of these proteins at a statistically significant p-value. Our experimental procedure consisted of three major steps: a mounted capture immunoassay, EV fixation and permeabilization, and immunofluorescence staining of target proteins. Through immunofluorescence, we identified the particle count of three proteins(SHBG, ApoA1, and Afamin) at each capture site(CD63, CD81, and CD9), which are the most common EV associated markers.

The analysis revealed that SHBG is positively associated with NAFLD and Apo-A1 is negatively associated with NAFLD. In addition, an increase in SHBG at CD9, decrease of Apo-A1 at CD63, and increase in Afamin at CD81, was significantly associated with NAFLD, revealing a high degree of binding site specificity and functionality. These results align with the proteins’ physiological functions and should be further explored to influence future diagnostics and potential therapies.