Biophysical Assessment of Single Cell Cytotoxicity: Diesel Exhaust Particle-Treated Human Aortic Endothelial Cells

Exposure to diesel exhaust particles (DEPs), a major source of traffic-related air pollution, has become a serious health concern due to its adverse influences on human health including cardiovascular and respiratory disorders. To elucidate the relationship between biophysical properties (cell topography, cytoskeleton organizations, and cell mechanics) and functions of endothelial cells exposed to DEPs, atomic force microscope (AFM) was applied to analyze the toxic effects of DEPs on a model cell line from human aortic endothelial cells (HAECs). Fluorescence microscopy and flow cytometry were also applied to further explore DEP-induced cytotoxicity in HAECs. Results revealed that DEPs could negatively impair cell viability and alter membrane nanostructures and cytoskeleton components in a dosage- and a time-dependent manner; and analyses suggested that DEPs-induced hyperpolarization in HAECs appeared in a time-dependent manner, implying DEP treatment would lead to vasodilation, which could be supported by down-regulation of cell biophysical properties (e.g., cell elasticity). These findings are consistent with the conclusion that DEP exposure triggers important biochemical and biophysical changes that would negatively impact the pathological development of cardiovascular diseases. For example, DEP intervention would be one cause of vasodilation, which will expand understanding of biophysical aspects associated with DEP cytotoxicity in HAECs

Effects of Cyclic Strain and Growth Factors on Vascular Smooth Muscle Cell Responses

Under physiological and pathological conditions, vascular smooth muscle cells (SMC) are exposed to different biochemical factors and biomechanical forces. Previous studies pertaining to SMC responses have not investigated the effects of both factors on SMCs. Thus, in our research we investigated the combined effects of growth factors like Bfgf (basic fibroblast growth factor), TGF-β (transforming growth factor β) and PDGF (platelet-derived growth factor) along with physiological cyclic strain on SMC responses. Physiological cyclic strain (10% strain) significantly reduced SMC proliferation compared to static controls while addition of growth factors bFGF, TGF-β or PDGF-AB had a positive influence on SMC growth compared to strain alone. Microarray analysis of SMCs exposed to these growth factors and cyclic strain showed that several bioactive genes (vascular endothelial growth factor, epidermal growth factor receptor, etc.) were altered upon exposure. Further work involving biochemical and pathological cyclic strain stimulation will help us better understand the role of cyclic strain and growth factors in vascular functions and development of vascular disorders

Maps of cell mechanics including adhesion force (<i>F</i>_ad, nN) and Young's modulus (<i>E</i>, kPa) of live HAECs, which were measured on single cells in culture medium.

<p>These maps are only to exhibit heterogeneous property in cell mechanics and do not reflect alteration tendency of <i>F</i>_ad or <i>E</i> because of individuality of cell-cell. Column 1, untreated (0 µg/ml) cells; columns 2–4, images of cells treated with different concentrations of DEPs for 4 hours. Row 1 shows AFM deflection mode images; rows 2 and 3 are their corresponding maps of adhesion force and Young's modulus. The color bars showing at the right of maps display the value scale of <i>F</i>_ad and <i>E</i>. The scanning size of AFM images is marked on each image of row 1.</p

Representative fluorescence images of cell viability evaluation.

<p>HAECs cells were exposed to DEPs with three different concentrations: 10 µg/ml (row 1), 50 µg/ml (row 2), 100 µg/ml (row 3), and four treatment durations: 4 hours (column 1), 8 hours (column 2), 24 hours (column 3), and 48 hours (column 4), respectively. Cells are stained with Invitrogen LIVE/DEAD Viability/Cytotoxicity Assay Kit. Green fluorescence presents live cells, whereas red fluorescence shows dead or membrane-damaged cells. All images were obtained with 10× lens. These fluorescence images together revealed that DEPs impaired cell viability in a dosage- and a time-dependent manner. And the corresponding bright-field picture of each fluorescent image is shown as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036885#pone.0036885.s005" target="_blank">Information S5</a>, from which DEPs can be clearly seen.</p

Representative observations of fixed HAECs using the coupled AF/FL microscope in PBS.

<p>Column 1 shows images of untreated (0 µg/ml) group; columns 2–4 are images of cells treated with different concentrations of DEPs for 4 hours. Row 1 shows bright-field images, row 2 is fluorescence images, and row 3 exhibits AFM images of the same cells. Optical images were obtained by the 60× OIL (or 100× OIL for 10 µg/ml group); scanning size of AFM images is marked on the respective images. The black dots representatively pointing by green arrows are DEPs attached on cell membrane.</p

Representative images of DEP (10 µg/ml) -treated HAECs (fixed cells) in PBS obtained by the coupled AF/FL microscope.

<p>The panel arrangement is similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036885#pone-0036885-g006" target="_blank">Figure 6</a>. Optical images were obtained using a 100× oil objective. The exposure time of DEPs is marked on respective optical images. It is seen that fluorescence intensity slightly decreased, although cytoskeletal structures can be seen after 48 hours of treatment. Interestingly, cellular mitosis was still progressing and a dividing cell nucleus can be seen (column 4), implying this low dosage did not completely inhibit cell activities, which coincided with assessment of cell viability. Additionally, DEPs attached on cell membrane surface are seen for all four experimental groups, as indicated by green arrows.</p

Representative AFM deflection mode images of HAECs treated with 10 µg/ml DEPs for different time (cells were fixed prior to observe in PBS).

<p>Column 1 shows images of cells treated with DEPs for 4 hours; column 2, 8 hours; column 3, 24 hours; column 4, 48 hours. Row 1 shows deflection mode images of single cells; rows 2 and 3 show images of membrane surface ultrastructures. Particles on cells representatively indicated by green arrows are DEP. Scanning size of row 1 is marked on respective image; and that of row 2 and row 3 (ultrastructures): 10 µm×10 µm. This group of images indicated that when treatment time increased from 4 hours to 48 hours, cell membrane damage appeared in a time-dependent manner.</p

Statistical analysis of adhesion force (<i>F</i>_ad, nN) and Young's Modulus (<i>E</i>, kPa) of live HAECs.

<p>Decreases of adhesion force implicates depression of cell membrane adhesion behavior in the presence of DEP, and down-regulation of Young's modulus suggests cells become softer in the context of cytoskeleton losing induced by DEP treatment. The data of histograms were obtained from multiple cells (N_cell = 12 for each group, and 26 datum points on each cell). Error bar: standard error (SE). (*, P<0.01).</p

Representative AFM deflection mode images of fixed HAECs obtained in PBS.

<p>Column 1, images of untreated cells (0 µg/ml group); columns 2–4, images of cells treated with different concentrations of DEPs for 4 hours. Row I, image of single cells, whose scanning size is marked on respective image; rows II and III, images of membrane surface ultrastructures, whose scanning size is 10 µm×10 µm. Particles (representatively shown by green arrows) on cells are DEPs. This group of images indicated that when DEP concentration increased from 0 µg/ml to 100 µg/ml, cytoskeletal structures became gradually degraded, suggesting that cell membrane damage appeared in a dosage-dependent manner. And poor resolution of ultra-structures of 100 µg/ml DEP treated cells is mainly resulted from influences of the large amount of DEPs on membrane surface.</p

Enhanced Endothelialization on Surface Modified Poly(l-Lactic Acid) Substrates

Improved biodegradable vascular grafts and stents are in demand, particularly for pediatric patients. Poly(l-lactic acid) (PLLA) is an FDA-approved biodegradable polymer of potential use for such applications. However, tissue culture studies have shown that endothelial cell (EC) attachment and growth occurs relatively slowly on PLLA surfaces. This slow growth has been attributed to the fact that PLLA represents a hydrophobic substrate, relatively devoid of active functional groups. As a result, the slow EC recovery on the luminal side of PLLA stents provides an increased risk of induced thrombosis. In the present study, surface modification of PLLA substrates has been examined as a potential route to enhance EC growth. For this purpose, PLLA surfaces were modified via pulsed plasma deposition of thin films of poly(vinylacetic acid). The –COOH surface groups, introduced by the plasma deposition, were employed to conjugate fibronectin (FN), followed by attachment of vascular endothelial growth factor to FN. Pig Aorta ECs (PAE) and kinase-insert domain-containing receptor (KDR)-transfected PAE showed increased cell adhesion and proliferation, as well as substantially improved cell retention under fluidic shear stress on surface-modified PLLA compared with untreated PLLA. Although KDR-transfected PAE exhibited better cell proliferation than PAE, normal EC functions, including EC morphology, nitric oxide production, and KDR expression, were observed when cells were grown on surface-modified PLLA. The results obtained clearly indicate that this combined surface modification technique using poly(vinylacetic acid) deposition, FN conjugation, and vascular endothelial growth factor surface delivery can enhance endothelialization on PLLA, particularly when employed in conjunction with the growth of KDR-transfected ECs