CTL O G M
Mechanical Activation of Valve Interstitial Cells in Osteogenic Environment is Time-dependent Emily J. Farrar, M.S., Varsha Pramil, Jennifer M. Richards, Ph.D., Jonathan T. Butcher, Ph.D., Cornell University, Ithaca, NY, USA.
spring
P
δmax
500 1000 Deflection (δmax, µm)
P = 0.0122*δmax + 0.616 R2 = 0.996
0 1
13 14
3
5
7
9
11
14
Days post-treatment
f-actin, αSMA
3 mm 20µm
B
* 40 20 0 1
3
5
7
9
11
0.3
*
*
OGM
*
0.1 0 1
3
5
7
9
11
Collagen fibers
14
Days post-treatment
Figure 4. Pro-osteogenic environment disrupts native mechanical activity of VIC. A. Density of collagen fibers in the hydrogels, calculated using ImageJ. B. Alignment of collagen fibers in the hydrogels. Alignment ratio of 1 indicates perfect alignment with direction of tension in hydrogel, alignment of 0 indicates fibers perpendicular to direction of tension. C. Collagen reflectance imaging of day 14 CTL and OGM gels. Scale bar is 20µm. N ≥ 3 for all experiments, * indicates p < 0.05 versus control at the same time point. Error bars indicate SEM.
*
*
1
CTL OGM
*
* *
30 20 10 0
0 3
5
7
9
11
13 14
C
1
3
5
7
9
11 14
Days post-treatment
Day 14
Collagen
*
2
B 40
Bright field
CTL OGM
Figure 5. Nodule formation and osteogenic signaling in dynamic, mechanically active VIC hydrogels. A. Thickness of the hydrogels. B. Number of nodules formed in CTL and OGM hydrogels over 14 days. C. Brightfield and fluorescence images of OGM hydrogel nodule formation at day 14. BF scale bar is 1mm, fluorescence scale bar is 50µm. N ≥ 3 for all experiments, * indicates p < 0.05 versus control at the same time point. Error bars indicate SEM.
Fold mRNA vs. day 1 CTL
0
A
B
Stress 25
Fiber alignment 0.4
20 15 10 0
Days post-treatment
A
0.2
0.2
5
0.2
14
*
C
2
*
*
2
1
COL2A1
8
*
6 4
1
2 0
0
0
Transition phase (Day 5) kPa
*
CTL
0.4
0.4
TGF-β1
RhoA
CTL OGM CTL OGM CTL OGM CTL OGM CTL OGM Figure 6. Osteogenic VIC are more mechanically active in acute phase. A. Internal stress in hydrogels. OGM drives acute increase in VIC contractility. B. Fiber alignment. OGM hydrogels show more alignment than CTL. C. q-rtPCR profiling of most active transcripts in acute phase. RhoA and TGF-β1 drive OGM contractility, type II collagen increase may explain increased fiber alignment in OGM. N ≥ 3. * indicates p < 0.05 versus CTL. Error bars indicate SEM.
tension
OGM
Fiber alignment ratio
*
C CTL
*
ratio
kPa
100
B
C
0
MMP-9
Sox9
COL2A1
*
3
1
10
1
0.5
5
0
0
2
*
*
0
CTL OGM CTL OGM CTL OGM CTL OGM CTL OGM Figure 7. CTL and OGM VIC show similar mechanics in transition phase. A. Internal stress in hydrogels. OGM and CTL are equal, both higher than day 1. B. Fiber alignment. OGM and CTL are not statistically different. C. q-rtPCR profiling of most active transcripts in transition phase. MMP-9 is highly upregulated in OGM as cells aberrantly remodel the matrix. Sox9 and Collagen II decrease may contribute to calcification. N ≥ 3. * indicates p < 0.05 versus CTL at same time point. Error bars indicate SEM.
A
Mature pathology phase (Day 14)
B
Stress 40 30 20 10
*
0
Fiber alignment 0.4
* 0.2
0
C
Fold mRNA vs. day 1 CTL
Days post-treatment
CTL
200
12 10 8 6 4 2 0
Fiber alignment
Fold mRNA vs. day 1 CTL
11
*
A
Stress
ratio
9
*
300
tension
OGM
Cells per field 7
Days post-treatment
0 0
5
CTL OGM
1
f-actin, αSMA, DNA
C
400
0 3
C
CTL OGM
F-actin, αSMA
5
10
B
OGM
Point load (mN)
δmax
Figure 2. Calibration of micro-cantileber system to measure VIC contractility. A. Setup for empirical calibration of spring in manufactured system. B. Load versus deflection curve for 3 systems. Error bars are SEM. C. Hydrogel and VIC seeded in system.
10
*
60
Thickness (mm)
Figure 1. Design and manufacturing of micro-cantilever system to measure VIC contractility. A. Custom molds machined from polycarbonate. B. Inserts and springs were inserted into device base to form complete system (left). Temporary plug prevented downward flow of collagen hydrogel (pink) as VIC gels were applied to upper region of device (center). Over 14 days, hydrogel+VIC contracted, deflecting cantilevered spring (right) by a measurable amount. Scale bar is 1 cm.
15
20
1
Fiber density (IntDen/µm2)
Plug
B
30
*
Nodules per gel
" Base
A
*
A
Inserts
B
CTL OGM
Day 14
Figure 3. Characterization of system to measure VIC contractility. A. Internal stress in hydrogels. Stress was calculated from force exerted by VIC on the spring divided by the cross-sectional area of the gel at the thinnest point. B. Cell density in CTL and OGM conditions over 14 days of culture in the system. N ≥ 3. * indicates p < 0.05 versus CTL at same time point. Error bars indicate SEM. C. Cell morphology at day 14. OGM VIC show myofibroblastic activation and disorganization. . Scale bar is 20µm Day 14
Design & calibration of micro-cantilever system A
A Internal Stress (kPa)
Valve interstitial cells are dynamic and aggressive players in aortic valve calcification [1], but their role in modulating the changing mechanical environment of the calcifying aortic valve is not well understood. The goal of this study was to characterize the contractility of valve interstitial cells in long-term (14 day) pro-osteogenic conditions and to understand how valve interstitial cell contractility contributes to calcific degeneration of the aortic valve. Methods & Results A novel bioreactor system for quantifying aortic valve interstitial cell contractility in 3-D hydrogels was designed and characterized in control and osteogenic conditions over 14 days. Interstitial cells demonstrated a powerful ability to exert contractile force on their environment and to align collagen fibers with the direction of tension. Osteogenic environment disrupted interstitial cell contractility and led to disorganization of the collagen matrix, concurrent with increased RhoA, αSMA, TGF-β, Runx2 and calcific nodule formation. We characterized the timedependent mechanics and gene-profiling into three distinct phases describing valve interstitial cell (VIC) response: acute (day 1), transition (day 5), and mature pathology (day 14).
Three distinct stages of interstitial cell activation Acute phase (Day 1)
ratio
Interstitial cell contractility during osteogenesis
kPa
Introduction
ACTA2 30
*
Runx2 2
0
*
150 100
20 10
ALP
1 0
*
50 0
CTL OGM CTL OGM CTL OGM CTL OGM Figure 8. CTL and OGM VIC switch phenotype in mature pathology phase. A. Internal stress in hydrogels. CTL VIC exert more stress than OGM. B. Fiber alignment. OGM fibers are more disorganized. C. q-rtPCR profiling of most active transcripts in mature phase. Myofibroblastic (ACTA2) and calcific (Runx2, ALP) genes are increased in OGM. N ≥ 3. * indicates p < 0.05 versus CTL at same time point. Error bars indicate SEM. CTL OGM
Conclusions Interstitial cell contractility mediates internal stress state and organization of the aortic valve extracellular matrix. Osteogenesis disrupts interstitial cell mechanical phenotype and drives disorganization, nodule formation, and pro-calcific signaling in three distinct phases of response. Understanding these responses is key to designing effective intervention therapies. Acknowledgements: National Science Foundation (CBET-0955172, NRSEC DMR-1120296 to JTB and GRFP to EJF), National Institutes of Health (HL110328 and HL118672 to JTB). References: 1. Rajamannan NM, Evans FJ, Aikawa E, Grande-Allen KJ, Demer LL, et al. (2011) Calcific Aortic Valve Disease: Not Simply a Degenerative Process. Circulation 124: 1783–1791.
spring
P
δmax
500 1000 Deflection (δmax, µm)
P = 0.0122*δmax + 0.616 R2 = 0.996
0 1
13 14
3
5
7
9
11
14
Days post-treatment
f-actin, αSMA
3 mm 20µm
B
* 40 20 0 1
3
5
7
9
11
0.3
*
*
OGM
*
0.1 0 1
3
5
7
9
11
Collagen fibers
14
Days post-treatment
Figure 4. Pro-osteogenic environment disrupts native mechanical activity of VIC. A. Density of collagen fibers in the hydrogels, calculated using ImageJ. B. Alignment of collagen fibers in the hydrogels. Alignment ratio of 1 indicates perfect alignment with direction of tension in hydrogel, alignment of 0 indicates fibers perpendicular to direction of tension. C. Collagen reflectance imaging of day 14 CTL and OGM gels. Scale bar is 20µm. N ≥ 3 for all experiments, * indicates p < 0.05 versus control at the same time point. Error bars indicate SEM.
*
*
1
CTL OGM
*
* *
30 20 10 0
0 3
5
7
9
11
13 14
C
1
3
5
7
9
11 14
Days post-treatment
Day 14
Collagen
*
2
B 40
Bright field
CTL OGM
Figure 5. Nodule formation and osteogenic signaling in dynamic, mechanically active VIC hydrogels. A. Thickness of the hydrogels. B. Number of nodules formed in CTL and OGM hydrogels over 14 days. C. Brightfield and fluorescence images of OGM hydrogel nodule formation at day 14. BF scale bar is 1mm, fluorescence scale bar is 50µm. N ≥ 3 for all experiments, * indicates p < 0.05 versus control at the same time point. Error bars indicate SEM.
Fold mRNA vs. day 1 CTL
0
A
B
Stress 25
Fiber alignment 0.4
20 15 10 0
Days post-treatment
A
0.2
0.2
5
0.2
14
*
C
2
*
*
2
1
COL2A1
8
*
6 4
1
2 0
0
0
Transition phase (Day 5) kPa
*
CTL
0.4
0.4
TGF-β1
RhoA
CTL OGM CTL OGM CTL OGM CTL OGM CTL OGM Figure 6. Osteogenic VIC are more mechanically active in acute phase. A. Internal stress in hydrogels. OGM drives acute increase in VIC contractility. B. Fiber alignment. OGM hydrogels show more alignment than CTL. C. q-rtPCR profiling of most active transcripts in acute phase. RhoA and TGF-β1 drive OGM contractility, type II collagen increase may explain increased fiber alignment in OGM. N ≥ 3. * indicates p < 0.05 versus CTL. Error bars indicate SEM.
tension
OGM
Fiber alignment ratio
*
C CTL
*
ratio
kPa
100
B
C
0
MMP-9
Sox9
COL2A1
*
3
1
10
1
0.5
5
0
0
2
*
*
0
CTL OGM CTL OGM CTL OGM CTL OGM CTL OGM Figure 7. CTL and OGM VIC show similar mechanics in transition phase. A. Internal stress in hydrogels. OGM and CTL are equal, both higher than day 1. B. Fiber alignment. OGM and CTL are not statistically different. C. q-rtPCR profiling of most active transcripts in transition phase. MMP-9 is highly upregulated in OGM as cells aberrantly remodel the matrix. Sox9 and Collagen II decrease may contribute to calcification. N ≥ 3. * indicates p < 0.05 versus CTL at same time point. Error bars indicate SEM.
A
Mature pathology phase (Day 14)
B
Stress 40 30 20 10
*
0
Fiber alignment 0.4
* 0.2
0
C
Fold mRNA vs. day 1 CTL
Days post-treatment
CTL
200
12 10 8 6 4 2 0
Fiber alignment
Fold mRNA vs. day 1 CTL
11
*
A
Stress
ratio
9
*
300
tension
OGM
Cells per field 7
Days post-treatment
0 0
5
CTL OGM
1
f-actin, αSMA, DNA
C
400
0 3
C
CTL OGM
F-actin, αSMA
5
10
B
OGM
Point load (mN)
δmax
Figure 2. Calibration of micro-cantileber system to measure VIC contractility. A. Setup for empirical calibration of spring in manufactured system. B. Load versus deflection curve for 3 systems. Error bars are SEM. C. Hydrogel and VIC seeded in system.
10
*
60
Thickness (mm)
Figure 1. Design and manufacturing of micro-cantilever system to measure VIC contractility. A. Custom molds machined from polycarbonate. B. Inserts and springs were inserted into device base to form complete system (left). Temporary plug prevented downward flow of collagen hydrogel (pink) as VIC gels were applied to upper region of device (center). Over 14 days, hydrogel+VIC contracted, deflecting cantilevered spring (right) by a measurable amount. Scale bar is 1 cm.
15
20
1
Fiber density (IntDen/µm2)
Plug
B
30
*
Nodules per gel
" Base
A
*
A
Inserts
B
CTL OGM
Day 14
Figure 3. Characterization of system to measure VIC contractility. A. Internal stress in hydrogels. Stress was calculated from force exerted by VIC on the spring divided by the cross-sectional area of the gel at the thinnest point. B. Cell density in CTL and OGM conditions over 14 days of culture in the system. N ≥ 3. * indicates p < 0.05 versus CTL at same time point. Error bars indicate SEM. C. Cell morphology at day 14. OGM VIC show myofibroblastic activation and disorganization. . Scale bar is 20µm Day 14
Design & calibration of micro-cantilever system A
A Internal Stress (kPa)
Valve interstitial cells are dynamic and aggressive players in aortic valve calcification [1], but their role in modulating the changing mechanical environment of the calcifying aortic valve is not well understood. The goal of this study was to characterize the contractility of valve interstitial cells in long-term (14 day) pro-osteogenic conditions and to understand how valve interstitial cell contractility contributes to calcific degeneration of the aortic valve. Methods & Results A novel bioreactor system for quantifying aortic valve interstitial cell contractility in 3-D hydrogels was designed and characterized in control and osteogenic conditions over 14 days. Interstitial cells demonstrated a powerful ability to exert contractile force on their environment and to align collagen fibers with the direction of tension. Osteogenic environment disrupted interstitial cell contractility and led to disorganization of the collagen matrix, concurrent with increased RhoA, αSMA, TGF-β, Runx2 and calcific nodule formation. We characterized the timedependent mechanics and gene-profiling into three distinct phases describing valve interstitial cell (VIC) response: acute (day 1), transition (day 5), and mature pathology (day 14).
Three distinct stages of interstitial cell activation Acute phase (Day 1)
ratio
Interstitial cell contractility during osteogenesis
kPa
Introduction
ACTA2 30
*
Runx2 2
0
*
150 100
20 10
ALP
1 0
*
50 0
CTL OGM CTL OGM CTL OGM CTL OGM Figure 8. CTL and OGM VIC switch phenotype in mature pathology phase. A. Internal stress in hydrogels. CTL VIC exert more stress than OGM. B. Fiber alignment. OGM fibers are more disorganized. C. q-rtPCR profiling of most active transcripts in mature phase. Myofibroblastic (ACTA2) and calcific (Runx2, ALP) genes are increased in OGM. N ≥ 3. * indicates p < 0.05 versus CTL at same time point. Error bars indicate SEM. CTL OGM
Conclusions Interstitial cell contractility mediates internal stress state and organization of the aortic valve extracellular matrix. Osteogenesis disrupts interstitial cell mechanical phenotype and drives disorganization, nodule formation, and pro-calcific signaling in three distinct phases of response. Understanding these responses is key to designing effective intervention therapies. Acknowledgements: National Science Foundation (CBET-0955172, NRSEC DMR-1120296 to JTB and GRFP to EJF), National Institutes of Health (HL110328 and HL118672 to JTB). References: 1. Rajamannan NM, Evans FJ, Aikawa E, Grande-Allen KJ, Demer LL, et al. (2011) Calcific Aortic Valve Disease: Not Simply a Degenerative Process. Circulation 124: 1783–1791.
