(L-450) Polyphenols increase activity and time of elastin soluble coacervation

The protein elastin plays an important role in maintaining the elasticity of soft tissues. For example, elastin maintains mechanical integrity and provides elastic recoil in elastic arteries. The precursor to elastin, tropoelastin, has favorable properties including lifelong half-lives and reliable stability.  Once secreted from the cells, tropoelastin self-aggregates (coacervates) and forms into clumps that will interact with additional proteins necessary for elastic fiber assembly. This self-assembly process is important to elastin-based biomaterials used in tissue engineering.

Coacervation describes the liquid to solid phase change, in which hydrophobic interactions within the protein core form intermediate globules. These globules have an initial reversible change due to temperature changes, followed by an irreversible change as the protein begins to self-assemble [1]. Polyphenols like pentagalloyl glucose (PGG) and epigallocatechin gallate (EGCG) impact the coacervation process in healthy cells and increase insoluble elastin deposition [2]. However, the mechanisms in which these polyphenols interact with the process and their influence on the end product is unknown.

This project seeks to understand the impact these polyphenols have on the aggregation of soluble elastin through light microscopy and spectrometer analyses. We use a soluble elastin to model the initial coacervation of tropoelastin which precedes, and corresponds with, elastic fiber formation.  Using this model, we investigate the effect of PGG and EGCG on elastogenesis. We present novel data including analysis of varied concentrations of each polyphenol and image analysis. The results from this study can be used to better inform medical treatments which aim to impact elastic fiber formation.

To model coacervation, elastin soluble 12 (ES12, Lot #290) was diluted with distilled water to a stock concentration of 100uM and kept frozen before use. PGG and EGCG were diluted with dimethyl sulfoxide (DMSO) and distilled water, respectively, then placed in a 4.6 pH acetic acid buffer comprised of Sodium acetate (S5636-250G), and Acetic Acid (64-19-7). Polyphenols were varied to 5 concentration groups of 0, 1.06-1.09, 4.25, 7.44-7.61, and 10.6-10.9 mM, combined with 60 µM of ES12 for a total of 10 experimental groups, 5 per polyphenol.

For the absorbance measurements, ES-12 and the polyphenol were combined in a 96 well plate with a volume of 100 µL per well. A M2 Spectramax microplate reader was used to read absorbance (440 nm) of each sample every minute for 2 hours with shaking. Data was then exported into MATLAB and Excel for analysis.

Imaging was done on a Zeiss LSM 880 Confocal Microscope equipped with an incubated stage insert set to 37°C. A 63x and 40x (DIC) objective were used to image 20 µL of solution at various time points, 1 experimental group at a time. Images were taken every 5 seconds for 35 minutes total. Additional images were taken using a 488 argon laser to fluoresce Alexa 488 tagged ES-12 (A10235). Images were processed in ImageJ (NIH).

These experiments were replicated to N=9 for the spectrometer studies and N=3 for the microscopy studies to ensure repeatability. Results from the spectrometer are presented as means with standard deviation error bars.

Absorbance vs. time was plotted from spectrometer data and showed a dependence on concentration of PGG (Fig 1A). Maximum values for absorbance increased with an increased concentration of PGG and time to peak decreased. The 63x images showed globules forming at 8 minutes and dropping to the bottom of the dish around 25 minutes (Fig. 1B). Images from 40x show rugged connected globules which smooth to spherical structures at 14 minutes (Fig. 1C).

EGCG concentration was directly related to absorbance (Fig 2A), but had a lower peak compared to PGG (0.4 vs. 0.7 for 10µL, respectively). Slower than PGG, the globules formed around 11 minutes (Fig 2B) and dropped around 19 minutes (Fig 2B). At 40x smoothing occurred overtime to form spheres with concavities (Fig 2C).

From absorbance data and imaging the process, we found that polyphenols increase the coacervate production and the rate of assembly (Fig 1 & 2) consistent with previous studies [1]. Further, we found a direct relationship between coacervate production and polyphenol concentration which has not been previously defined.

We hypothesize the increase in absorbance to be a cause of globules intersecting, and relate the decay to collapse of the formed structures (Figures 1 & 2). We believe PGG may be interacting with the hydrophobic domain, whereas EGCG would differently affect the hydrophilic domains. This could suggest that the mechanism in which PGG and EGCG interact with ES12 impacts this collapse differently resulting in a different timeframe and product formation.

PGG presents a challenge to tissue engineers because of need to be dissolved in DMSO, and EGCG could provide a safer alternative. In our study, EGCG reached a lower absorbance peak compared to PGG, but, both polyphenols impacted coacervation as evident by the increased absorbance. Our imaging provides structural context to both our data, and previous literature [1].

This study was partially funded by the American Heart Association (19TPA-34910047) and the National Institutes of Health (HL164800 and HL135400).

[1] Vidal Ceballos A, Díaz A JA, Preston JM, Vairamon C, Shen C, Koder RL, Elbaum-Garfinkle S. Liquid to solid transition of elastin condensates. Proc Natl Acad Sci U S A. 2022 Sep 13;119(37):e2202240119. doi: 10.1073/pnas.2202240119. Epub 2022 Sep 6. PMID: 36067308; PMCID: PMC9477396.

[2] Sinha, Aditi et al. “Elasto-regenerative properties of polyphenols.” Biochemical and biophysical research communications vol. 444,2 (2014): 205-11. doi:10.1016/j.bbrc.2014.01.027