PMC

SMCs were grown in either growth media (A, B; DMEM+10% serum) or growth-arrest media (C, D; DMEM+0.4% serum), with (B, D) or without (A,C) heparin, for 7 days, then fixed and stained for elastin as in previous experiments. Elastin is indicated in red, nuclei in blue (DAPI).

hAoSMC were plated onto silk films. Samples were allowed to grow for 9 days on silk films (A) or silk+heparin films (B) and were then fixed and stained by immunofluorescence for elastin (in red with phase contrast overlay). Longer timepoint samples, grown for 18 days on silk films (D) and silk+heparin films (F) were stained by van Gieson stain. Nonspecific response to van Gieson stain was ruled out by silk (C), or silk+heparin (E), films stained without cells.

hAoSMC cultured for 7 days with 300 µg/ml DTAF-heparin. Confocal sections were taken, and different wavelength images of the same z-sections were reported. (A) shows the distribution of labeled heparin. As shown in (B), the DTAF-heparin retains biological activity, as it promotes elastic fiber formation. As seen in (C), the heparin and nascent elastin fibers colocalize extensively.

Adult human aortic smooth muscle (hAoSMC, A) or endothelial (HAEC, B) cells were seeded onto silk films with or without heparin incorporated. At indicated timepoints, cells were trypsinized and counted. Data are the average of three experiments, with standard error of the mean (SEM) indicated by error bars. Stars indicate significance of P< 0.01.

In two types of silk film processing, heparin is incorporated by admixing silk and heparin solution. Caste films have higher total amount of silk (A,C), while layer-by-layer films contain less silk in thinner layers(B,D). In each situation, the heparin is fairly evenly distributed throughout the film, as shown by the dark blue staining in ‘with heparin’ samples (C, D). Some pale blue background staining arises from nonspecific affinity of silk for alcian blue in ‘silk only’ samples (A, B).

15,000 hAoSMC/cm² were plated and grown in medium containing 10% serum with the indicated concentrations of heparin. After 5 days, the cells were fixed and processed for immunofluorescence using an anti-elastin antibody as described in Materials and Methods. More extensive elastin fibers are seen with increasing heparin treatment (arrows). Some potential sheets of extracellular elastin are visible in the highest heparin concentration samples (arrowhead).

Adult human aortic SMCs were grown in the absence (A,B) or presence (C,D) of 300 µg/ml heparin for 7 days, then fixed and processed for immunofluorescence microscopy. Non-permeabilized samples were labeled with polyclonal anti-elastin antibody (red), CellTracker Green for cell membrane, and DAPI (blue) for nuclear stain (A,C); or with van Gieson stain for elastin (B,D). Significant extracellular elastin fibers are shown in the heparin treated samples.

15,000 hAoSMC/cm² were plated and grown in medium containing 10% serum alone (A), or medium containing 10% serum plus 300 µg/ml chondroitin sulfate (B), 300 µg/ml heparin (C), or 300 µg/ml LMW-heparin (D). After 5 days, cells were fixed and processed for immunofluorescence microscopy as described in Materials and Methods. Few elastic fibers are seen with chondroitin treatment, similar to the untreated control, whereas numerous fibers are seen in heparin treated samples.

SMCs were plated at 5,000, 15,000 and 30,000 cells/cm² on day 0. Media was changed on day 1, with or without 300 µg/ml heparin. Cells were grown for 7 days, fixed and processed by immunofluorescence using an anti-elastin primary antibody and Cy3-labeled secondary antibody. Elastin fibers (white arrows) are more prevalent in the 15,000 cells/cm² condition, while minimal in cultures plated more and less densely, despite complete confluence of all cultures at the time of fixation.

15,000 hAoSMCs/cm² were treated with or without heparin, and samples were harvested at 6 hrs, 24 hrs, 2 days, 4 days, and 6 days. RNA was extracted, reverse-transcribed, and qPCR was used to measure relative amounts of elastin mRNA expression. Each sample was normalized to GAPDH expression, then to elastin expression in the untreated sample at the corresponding time point. Results are given as delta-delta Ct values, and are the average of 3 independent experiments. Standard deviation indicated by error bars. Elastin mRNA levels rise, beginning at 24 hrs post-heparin treatment and remain elevated for the duration of the experiment.