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Biophysical induction of vascular smooth muscle cell podosomes.

Kim NY, Kohn JC, Huynh J, Carey SP, Mason BN, Vouyouka AG, Reinhart-King CA - PLoS ONE (2015)

Bottom Line: Both microtopographical cues and imposed pressure mimicking stage II hypertension induce podosome formation in A7R5 rat aortic smooth muscle cells.Notably the effect of each of these biophysical stimuli on podosome stimulation can be inhibited using a Src inhibitor.Together, these data indicate that physical cues can induce podosome formation in VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Vascular smooth muscle cell (VSMC) migration and matrix degradation occurs with intimal hyperplasia associated with atherosclerosis, vascular injury, and restenosis. One proposed mechanism by which VSMCs degrade matrix is through the use of podosomes, transient actin-based structures that are thought to play a role in extracellular matrix degradation by creating localized sites of matrix metalloproteinase (MMP) secretion. To date, podosomes in VSMCs have largely been studied by stimulating cells with phorbol esters, such as phorbol 12,13-dibutyrate (PDBu), however little is known about the physiological cues that drive podosome formation. We present the first evidence that physiological, physical stimuli mimicking cues present within the microenvironment of diseased arteries can induce podosome formation in VSMCs. Both microtopographical cues and imposed pressure mimicking stage II hypertension induce podosome formation in A7R5 rat aortic smooth muscle cells. Moreover, wounding using a scratch assay induces podosomes at the leading edge of VSMCs. Notably the effect of each of these biophysical stimuli on podosome stimulation can be inhibited using a Src inhibitor. Together, these data indicate that physical cues can induce podosome formation in VSMCs.

No MeSH data available.


Related in: MedlinePlus

Physical barriers induce formation of podosomes.(A) Representative confocal image of A7R5 aortic smooth muscle cells seeded on a micromolded 30 kPa collagen-coated polyacrylamide (PA) gel containing a vertical wall. A schematic of the top and side views of the molded PA gel are illustrated in the inset in the upper right corner. Podosomes were identified based on the colocalization of actin (green) and cortactin (red). Nuclei (blue) were stained with DAPI. The cell denoted by the asterisk (*) is not in contact with a wall whereas the cell denoted by the crosshatch (#) is adhered to a wall (dotted white line). The white arrowhead indicates punctate podosome formation. Scale bar, 30 μm. (B and C) Intensity profiles of actin and cortactin staining of the yellow dotted line (20 μm) (depicted in A) drawn in the cell without wall contact (B) and with wall contact (C). (D) Percentage of cells exhibiting podosomes when in contact or not in contact with a wall. Data are mean ± S.D. * P < 0.05 (Student’s t-test, n = 4).
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pone.0119008.g001: Physical barriers induce formation of podosomes.(A) Representative confocal image of A7R5 aortic smooth muscle cells seeded on a micromolded 30 kPa collagen-coated polyacrylamide (PA) gel containing a vertical wall. A schematic of the top and side views of the molded PA gel are illustrated in the inset in the upper right corner. Podosomes were identified based on the colocalization of actin (green) and cortactin (red). Nuclei (blue) were stained with DAPI. The cell denoted by the asterisk (*) is not in contact with a wall whereas the cell denoted by the crosshatch (#) is adhered to a wall (dotted white line). The white arrowhead indicates punctate podosome formation. Scale bar, 30 μm. (B and C) Intensity profiles of actin and cortactin staining of the yellow dotted line (20 μm) (depicted in A) drawn in the cell without wall contact (B) and with wall contact (C). (D) Percentage of cells exhibiting podosomes when in contact or not in contact with a wall. Data are mean ± S.D. * P < 0.05 (Student’s t-test, n = 4).

Mentions: Several mechanical and structural cues exist in diseased arteries due to matrix rearrangements that occur during disease progression and in treated arteries due to stent geometry [21]. As both cell adhesion and migration have been shown to be affected by topographical cues [22,23], we hypothesized that cell contact with a physical barrier, analogous to VSMCs contacting a stent in vivo, may induce podosome formation. To create three-dimensional substrate features, cells were seeded on 30 kPa polyacrylamide (PA) gels patterned with 10 μm tall vertical walls. Cells were considered “in contact” with a wall when any portion of the cell body was in contact with a wall, regardless of contact area. When cells were not in contact with micropost walls, cortactin, a widely used podosome marker [8], was diffuse throughout the cells (Fig. 1A, left), exhibiting no colocalization with actin. However, when cells contacted micropost walls (Fig. 1A, right), cortactin co-localized with actin in punctate spots, indicating the presence of podosomes. To more rigorously identify podosomes, a line segment was drawn across the cell edge (Fig. 1A, dotted line) and the intensity of red and green fluorescence was quantified (Figs. 1B and C). Overlapping curves (Fig. 1C) indicate the presence of a podosome. Podosomes were found primarily at the cell edge when contacting the wall. It is interesting to note that no podosomes were observed along the top horizontal surface of the wall. The average number of cells that exhibit podosomes was quantified and compared between the conditions in which cells were and were not in contact with micropost walls (Fig. 1D). We found an approximate 9-fold increase in the number of cells with podosomes that were in contact with a vertical wall as compared to those not in contact with a wall.


Biophysical induction of vascular smooth muscle cell podosomes.

Kim NY, Kohn JC, Huynh J, Carey SP, Mason BN, Vouyouka AG, Reinhart-King CA - PLoS ONE (2015)

Physical barriers induce formation of podosomes.(A) Representative confocal image of A7R5 aortic smooth muscle cells seeded on a micromolded 30 kPa collagen-coated polyacrylamide (PA) gel containing a vertical wall. A schematic of the top and side views of the molded PA gel are illustrated in the inset in the upper right corner. Podosomes were identified based on the colocalization of actin (green) and cortactin (red). Nuclei (blue) were stained with DAPI. The cell denoted by the asterisk (*) is not in contact with a wall whereas the cell denoted by the crosshatch (#) is adhered to a wall (dotted white line). The white arrowhead indicates punctate podosome formation. Scale bar, 30 μm. (B and C) Intensity profiles of actin and cortactin staining of the yellow dotted line (20 μm) (depicted in A) drawn in the cell without wall contact (B) and with wall contact (C). (D) Percentage of cells exhibiting podosomes when in contact or not in contact with a wall. Data are mean ± S.D. * P < 0.05 (Student’s t-test, n = 4).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4364673&req=5

pone.0119008.g001: Physical barriers induce formation of podosomes.(A) Representative confocal image of A7R5 aortic smooth muscle cells seeded on a micromolded 30 kPa collagen-coated polyacrylamide (PA) gel containing a vertical wall. A schematic of the top and side views of the molded PA gel are illustrated in the inset in the upper right corner. Podosomes were identified based on the colocalization of actin (green) and cortactin (red). Nuclei (blue) were stained with DAPI. The cell denoted by the asterisk (*) is not in contact with a wall whereas the cell denoted by the crosshatch (#) is adhered to a wall (dotted white line). The white arrowhead indicates punctate podosome formation. Scale bar, 30 μm. (B and C) Intensity profiles of actin and cortactin staining of the yellow dotted line (20 μm) (depicted in A) drawn in the cell without wall contact (B) and with wall contact (C). (D) Percentage of cells exhibiting podosomes when in contact or not in contact with a wall. Data are mean ± S.D. * P < 0.05 (Student’s t-test, n = 4).
Mentions: Several mechanical and structural cues exist in diseased arteries due to matrix rearrangements that occur during disease progression and in treated arteries due to stent geometry [21]. As both cell adhesion and migration have been shown to be affected by topographical cues [22,23], we hypothesized that cell contact with a physical barrier, analogous to VSMCs contacting a stent in vivo, may induce podosome formation. To create three-dimensional substrate features, cells were seeded on 30 kPa polyacrylamide (PA) gels patterned with 10 μm tall vertical walls. Cells were considered “in contact” with a wall when any portion of the cell body was in contact with a wall, regardless of contact area. When cells were not in contact with micropost walls, cortactin, a widely used podosome marker [8], was diffuse throughout the cells (Fig. 1A, left), exhibiting no colocalization with actin. However, when cells contacted micropost walls (Fig. 1A, right), cortactin co-localized with actin in punctate spots, indicating the presence of podosomes. To more rigorously identify podosomes, a line segment was drawn across the cell edge (Fig. 1A, dotted line) and the intensity of red and green fluorescence was quantified (Figs. 1B and C). Overlapping curves (Fig. 1C) indicate the presence of a podosome. Podosomes were found primarily at the cell edge when contacting the wall. It is interesting to note that no podosomes were observed along the top horizontal surface of the wall. The average number of cells that exhibit podosomes was quantified and compared between the conditions in which cells were and were not in contact with micropost walls (Fig. 1D). We found an approximate 9-fold increase in the number of cells with podosomes that were in contact with a vertical wall as compared to those not in contact with a wall.

Bottom Line: Both microtopographical cues and imposed pressure mimicking stage II hypertension induce podosome formation in A7R5 rat aortic smooth muscle cells.Notably the effect of each of these biophysical stimuli on podosome stimulation can be inhibited using a Src inhibitor.Together, these data indicate that physical cues can induce podosome formation in VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Vascular smooth muscle cell (VSMC) migration and matrix degradation occurs with intimal hyperplasia associated with atherosclerosis, vascular injury, and restenosis. One proposed mechanism by which VSMCs degrade matrix is through the use of podosomes, transient actin-based structures that are thought to play a role in extracellular matrix degradation by creating localized sites of matrix metalloproteinase (MMP) secretion. To date, podosomes in VSMCs have largely been studied by stimulating cells with phorbol esters, such as phorbol 12,13-dibutyrate (PDBu), however little is known about the physiological cues that drive podosome formation. We present the first evidence that physiological, physical stimuli mimicking cues present within the microenvironment of diseased arteries can induce podosome formation in VSMCs. Both microtopographical cues and imposed pressure mimicking stage II hypertension induce podosome formation in A7R5 rat aortic smooth muscle cells. Moreover, wounding using a scratch assay induces podosomes at the leading edge of VSMCs. Notably the effect of each of these biophysical stimuli on podosome stimulation can be inhibited using a Src inhibitor. Together, these data indicate that physical cues can induce podosome formation in VSMCs.

No MeSH data available.


Related in: MedlinePlus