Pathology of Calcific Aortic Valve Disease: The Role of … · 2013. 12. 18. · Calcific aortic valve disease (CAVD) occurs through multiple mutually non-exclusive mechanisms that
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Pathology of Calcific Aortic Valve Disease: The Role of Mechanical and Biochemical Stimuli in Modulating the
Phenotype of and Calcification by Valvular Interstitial Cells
by
Cindy Ying Yin Yip
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of the Institute of Biomaterials and Biomedical Engineering and Cardiovascular Collaborative Sciences Program
smooth muscle actin (-SMA)57, 62-64, and are associated with increased collagen synthesis and
cellular contractility65. Pathological myofibroblast activation may lead to an unbalanced matrix
remodeling that subsequently alters the biochemical and mechanical properties of the
microenvironment within the valves. Additionally, osteoblasts-like cells are often found in
calcified aortic valves49 and likely originate from osteogenic differentiation of resident
progenitor VICs58 or bone marrow-derived hematopoietic stem cells66. VICs comprise a
phenotypically dynamic cell population (Table 2.1) and their differentiation into various
phenotypes is closely associated with defined sets of cellular functions, which presumably
modulate the progression of CAVD.
Table 2.1. VIC phenotypes and functions in normal and diseased aortic valves
Phenotype Normal aortic valves
Sclerotic/ calcified AVs
Function(s)
Quiescent VICs Abundant Less abundant than in normal valves.
Maintain valve homeostasis.
Progenitor VICs (Likely a subpopulation of quiescent VICs)
~ 50%58 Data not available
Multipotent differentiation potential. Can differentiate into multiple lineages including chondrocytes, adiopocytes and osteoblasts. If differentiated into osteoblasts, these cells can secret alkaline phosphatase and osteocalcin, and deposit calcium58.
Activated VICs (Myofibroblasts)
~1-5%67 ~ 30%61 -SMA positive contractile cells that participate in active cellular repair processes such as matrix remodeling55.
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2.3.3.3. Calcification by Valvular Interstitial Cells
The cellular mechanisms by which VICs contribute to calcification are not well defined, largely
because in vivo and ex vivo studies are limited to evaluating the end-stage of the disease.
Although large animal models such as porcine and ovine are excellent disease models with
similar lipoprotein serum levels and hemodynamic profiles to humans, it is difficult to track the
changes of individual molecular signaling pathways over the course of disease progression partly
due to the lack of imaging modalities that can monitor valve pathological development in a time-
dependent manner. Alternatively, ex vivo studies with human aortic valves are often limited to
those at the end-stage rather than at the onset of the disease, and therefore these studies are not
capable of identifing molecular mechanisms responsible for disease initiation. Hence in vitro cell
culture systems are often used to study disease-related molecular and cellular events in hopes of
deciphering the underlying mechanisms of valve calcification, as these cell culture models often
display in vivo characteristics of the disease (Table 2.2).
Table 2.2. A comparison of the cellular characteristics between calcification by VICs in vivo
and in vitro
Characteristics Calcified aortic valves In vitro calcification by VICs
Response Matrix stiffness Cell type Reference Osteogenic differentiation increases with substrate stiffness involving activation of MAPK pathway downstream of RhoA and ROCK.
Soft substrate ~ 7 kPa Stiff substrate ~ 160 kPa
MC3T3-E1 Khatiwala et al. 2009164
Greater cell spreading with higher expression of actin stress fiber and focal adhesion on stiff substrate.
Soft substrate ~ 1 kPa Stiff substrate ~ 8 kPa
Smooth muscle cells
Engler et al. 2004136,
155
Cell shape and spreading unaffected by substrate stiffness.
Soft substrate ~ 2 kPa Stiff substrate ~ 1012 Pa
Neutrophils Yeung et al. 2005129
Cells differentiated to neuronal, myogenic and osteogenic lineages when cultured on soft, immediate and stiff substrate, respectively.
Formed striations only when cultured on intermediate (~ 11 kPa) stiffness.
Substrates at 1, 8, 11 and 17 kPa
Myoblasts Engler et al. 2004136
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Table 2.6. Advantages and disadvantages of various culture systems for studying the effect
of substrate stiffness
Substrate Advantages Disadvantages Collagen - Easy to handle
- Physiologic relevant - Readily adhesive to cells - Can support 3D culture - Can decouple substrate chemistry from mechanics if stiffness is adjusted by changing the geometry in 2D culture
- Cannot support a wide range of stiffness, unless concentration of collagen is adjusted
MatrigelTM - Can tune the stiffness by cross-linking the gels using glutaraldehyde
- Heterogeneous composition and batch-to-batch variability - Cannot decouple substrate chemistry from mechanics as stiffness is adjusted by altering the concentration of MatrigelTM
Fibrin - Can support 3D culture - Cannot decouple substrate
chemistry from mechanics
Agarose - Easy to handle - Not readily adhesive to cells, requires surface modification - Cannot decouple substrate chemistry from mechanics
Polyacrylamide - Can adjust to a wide range of stiffness
- Limited to 2D culture - Cross-linker is toxic to cells - Requires surface functionalization
Poly(ethylene glycol) - Can adjust to a wide range of stiffness - Can generate 3D culture - Has physical characteristics similar to those of soft tissues, e.g., permeable to oxygen, nutrients and water-soluble metabolites
- Requires surface functionalization - When used in 3D culture, substrate chemistry and mechanics cannot be decoupled
Polydimethylsiloxane - Can adjust to wide range of stiffness - Surface chemistry and substrate stiffness are decoupled
- Surface functionalization is difficult - Can only support short culture periods of a few days
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Substrate stiffness modulates proliferation by interfering with cell cycle progression (i.e., the
cycle of G1 phase, S phase, G2 phase and M phase). Focal adhesion kinase (FAK) and actin
polymerization were found to regulate the expression of the cell cycle regulatory proteins and
cylcin-dependent kinases (cdk) through extracellular signal-regulated kinase (ERK), Ras and
Rho signaling pathways depending on the level of compliance. For example, cells cultured on
free-floating collagen gels exhibited reduced cell proliferation due to G1 phase arrest, which was
accompanied by reduced FAK autophosphorylation, ERK activity, and absence of cyclin
expression and upregulation of cdk inhibitor expression. Stiffer substrates favour the formation
of mature focal adhesions and FAK activation, which is associated with ERK activation and
ERK-dependent induction of cyclin expression, hence permitting the cells to enter the S phase.
Of note, ERK- and FAK-independent induction of cyclin has also been reported and further work
is required to identify the interplay among the different mechanisms by which substrate stiffness
alters cyclin and cdk expression171.
Without doubt, cell-ECM contacts are an important aspect of stiffness sensing. It is also known
that the lack of a firm adhesive substrate contributes to anoikis (a type of apoptosis which is
induced by the detachment of cells from their adherent surfaces). Presumably, the higher number
of mature focal adhesions in cells cultured on stiffer substrates provides a firm adhesive substrate
and protects the cells from apoptosis. This notion is supported by the reduction in apoptosis of
pre-osteoblasts when cultured in stiffer substrates174. It is known that apoptosis is associated
with integrin signaling and cytoskeleton content175, but the direct relation between stiffness-
mediated cytoskeleton reorganization and apoptosis remains unknown.
2.4.4.3. Cell Phenotype and Differentiation
While the potential of mesenchymal stem cells to differentiate into a diverse range of lineages is
well-known, other cell types in the body such as VICs can also undergo phenotypic changes
during pathologic differentiation47, 49. Recent studies demonstrate that cellular differentiation is
not only regulated by soluble factors and matrix composition, but also depends on substrate
stiffness. Notably, cells appear to express their differentiated phenotype in vitro on substrates
that are similar to the stiffness of their native ECM130, 136. It has therefore been postulated that
changes in tissue mechanics during pathologic development may partly contribute to cellular
pathological differentiation16, 176. Further, matrix stiffness-induced phenotypic alterations may
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influence the interpretation of in vitro studies, which are often conducted with glass or TCPS that
are orders of magnitude stiffer than any tissue. Lastly, the fundamental understanding of matrix
mechanics in regulating differentiation has substantial influence on the development of
biomaterials for tissue engineering as material stiffness may influence cell differentiation and
functions.
The ability to differentiate stem cells into a desired lineage is important in tissue engineering and
regenerative medicine. Interestingly, Engler’s study showed that mesenchymal stem cells
differentiate into different lineages as a function of substrate stiffness130. Mesenchymal stem
cells grown on substrates with brain-like compliance (E ~ 0.1-1 kPa) underwent neuronal
differentiation. Myogenic differenation was observed in cells cultured on substrates of
intermediate stiffness (E ~ 8-17 kPa) and osteogenic differentiation was found in cells grown on
relatively stiff substrates (E ~ 25-40 kPa). These data suggest that cells are able to recognize
physiologically relevant substrate stiffnesses and differentiate accordingly. Engler et al further
demonstrated that soluble factors and matrix stiffness synergistically guide MSC commitment to
particular lineages130.
The effect of substrate stiffness on differentiation is not limited to progenitor cells, but also
impacts on various cell lines and committed mammalian cells (e.g., pre-osteoblastic cells,
hepatocytes, myofibroblasts, VICs). One of the most frequently studied cell lines is the pre-
osteoblastic MC3T3-E1 cell line, which displays substrate stiffness-dependent osteogenic
differentiation and calcium deposition. When MC3T3-E1 cells were cultured on substrates
coated with RGD rather than full-length type I collagen, osteogenic differentiation was optimal
on more compliant substrates (E = 20 kPa)177. However, the same cells cultured on type I
collagen-coated substrates deposited more bone mineral on stiff substrates (E > 20 kPa)132.
These differences may result from the unique engagement of integrins to particular ECM
proteins (i.e., typically αvβ3 and α5β1 integrins interact with RGD and α2β1 interacts with type I
collagen), which together with matrix stiffness differentially regulate cellular differentiation.
This emphasizes the importance of decoupling surface chemistry from substrate mechanics.
Similar matrix stiffness effects on cell-mediated calcification were also found in VICs. An
increase in calcium deposition by VICs was identified when cultured on fibrin-modified TCPS
(i.e., a stiff substrate) in comparison to those cultured on fibrin-modified soft polyethylene
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glycol178. These results demonstrate that matrix stiffness regulates cell-mediated calcification.
Calcification by cultured cells is associated with integrin binding and FAK activation149.
Presumably, substrate stiffness may regulate calcification by influencing integrin binding, focal
adhesion signaling, and possibly cytoskeleton reorganization, although further studies are
necessary to decipher the precise molecular mechanisms.
Matrix stiffness also regulates and maintains the differentiation of committed cell types. This has
implications in disease development, where ECM composition and tissue compliance are often
altered and phenotypic changes of cells are observed. For example, hepatocytes cultured on soft
substrates (e.g., collagen gels and MatrigelTM of G′~ 34 Pa) remain differentiated179, whereas
those cultured on stiff substrates (e.g., collagen film and cross-linked MatrigelTM of G′~118 Pa)
adopt a dedifferentiated phenotype179, 180. Another example is the differentiation of fibroblasts to
contractile myofibroblasts in wound repair, in which matrix stiffness, cell tension, and TGF-β
release are all required to promote and maintain myofibroblast differentiation that is responsible
for wound closure (reviewed in137, 181). In vitro studies of myofibroblasts cultured on stiff
constrained collagen gels (E ~ 20 kPa) responded to TGF-β and expressed a high level of
-SMA and abundant stress fibers. In contrast, cells cultured on free floating (more compliant)
collagen gels (E ~ 8 kPa) had reduced -SMA expression and stress fibers regardless of the
presence or absence of TGF-β, suggesting that matrix stiffness regulates cellular sensitivity to
soluble factors related to myofibroblast differentiation12.
2.4.5. Matrix Stiffness and Pathologies
Regulation of cellular responses by substrate stiffness is not just an in vitro phenomenon, but
also has relevant clinical implications. Studies have linked matrix stiffness to various pathologies
such as cancer, osteoporosis and atherosclerosis. Significant stiffening of tumour tissue has been
correlated with an increase in tumour cell migration and proteolysis182, which has been suggested
to partially explain the failure of protease inhibitors as cancer therapies183, 184. This result
emphasizes the value of understanding the effect of matrix stiffness on the response of cells to
biochemical factors including therapeutics.
Fibrosis and tissue stiffening are often found as liver disease progresses. The differentiation of
portal fibroblasts and hepatic stellate cells into hepatic myofibroblasts is the key pathological
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mediator. Interestingly, studies with rat models found that liver stiffening occurs prior to
fibrosis7, 185, suggesting that matrix mechanics may induce myofibroblast differentiation in early
nm). Samples were subsequently mounted on microscope slides with PermaFluor mounting
medium and examined by fluorescence microscopy immediately (Olympus Model IX71,
Olympus, Center Valley, PA). The cell contours were identified based on the images of
phalloidin-stained cells. Cell spreading was estimated by tracing the cell contours and measuring
the cell area with ImageJ (NIH, Bethesda, MD).
5.1.4. Staining of Viable, Dead and Apoptotic Cells
VICs cultured on compliant or stiff matrices were quickly rinsed with sterile PBS. Viable cells
were determined by fluorescent labeling with 4 M Calcein AM (excitation/emission
wavelengths: 494 nm/517 nm) and dead cells were labeled with 2 M Ethidium Homodimer-1
(excitation/emission wavelengths: 528 nm/617 nm; LIVE/DEAD® Viability/Cytotoxicity Kit for
mammalian cells, Invitrogen, Burlington, ON). Cells were incubated with fluorescent dye for one
hour at 37 oC and then the nuclei were counterstained with Hoechst 33242 dye
(excitation/emission wavelengths: 350 nm/461 nm) for five minutes. Samples were subsequently
mounted on microscope slides with PermaFluor mounting medium and examined by
fluorescence microscopy immediately. As a negative control, cells were killed by formalin
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fixation prior to Calcein AM staining and nuclear counterstaining to confirm that the Calcein
AM staining was specific to viable cells and not simply binding to calcium.
Apoptotic cells were identified by cellular uptake of APOPercentageTM dye (Biocolor Ltd,
Carrickfergus, UK) as a result of apoptosis-induced membrane phosphatidylserine and
phosphatidylcholine translocation. In brief, samples were quickly rinsed with sterile PBS with
calcium and magnesium prior to incubation with APOPercentageTM dye diluted 1:20 in
supplemented DMEM at 37 °C for 30 minutes. Samples were then mounted on microscope slides
with PermaMount mounting medium and images were captured under the microscope. Positive
controls were achieved by chemically-induced apoptosis of cells using 5 mM hydrogen peroxide
for three hours at 37 °C prior to staining. Intense staining, typically bright pink or purple
depending on the culture substrate and cell density, was observed in the positive controls,
ensuring the validity of the apoptosis detection method in VICs. Negative controls were achieved
by incubating samples without the APOPercentageTM dye.
5.1.5. Polymerase Chain Reaction for Expression of Osteogenic Markers
VICs were released from collagen matrices via collagenase digestion. Cell pellets were obtained
by centrifugation, followed by aspiration of the supernatant. Total RNA was isolated from cell
pellets following standard protocols of the Micro RNeasy System (Qiagen, Mississauga, ON).
Subsequently, total RNA was reverse transcribed with oligo-(dT)12-18 primers (Invitrogen,
Burlington, ON) and Superscript III reverse transcriptase (Invitrogen). cDNA was quantified
with a NanoDrop Spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE), and
then used as the template for real-time PCR using SYBR Green, an annealing temperature of 60 oC, and 35 cycles. Two osteoblast-related transcripts, osteonectin (Accession number:
AW436132, forward primers: 5’-tccggatctttcctttgctttcta-3’ and reverse primer 5’-
ccttcacatcgtggcaagagtttg-3’) and osteocalcin (Accession number: AW346755, forward primers:
5’-tcaaccccgactgcgacgag-3’ and reverse primer 5’-ttggagcagctgggatgatgg-3’) were tested222.
VICs were cultured in calcifying media to promote osteogenic differentiation. Transcript levels
of Cbfa-1/Runx2, osteonectin and osteocalcin after three, eight and sixteen days of culture in
calcifying media with or without CNP were measured using qRT-PCR. ALP and osteocalcin
staining was performed after 21 days of culture in calcifying media. Calcium deposition was
determined by ARS staining after 14 days of culture. In addition, a colony forming unit-ALP
(CFU-ALP) assay was used to determine the frequency of VIC osteoprogenitor differentiation
under the influence of CNP58. Briefly, viable primary VICs were seeded at 0.2 cells/well into
96-well plates. The cells were cultured for three days in complete media to permit cell adhesion,
at which point the media were replaced with calcifying media with or without CNP and changed
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every two days for three weeks. The cells were subsequently fixed with 10% NBF and stained
for ALP. For each plate, the number of wells without ALP-positive aggregates was recorded.
The ratio of wells without ALP-positive aggregates to the total number of wells was calculated.
Based on Poisson’s distribution, the negative natural logarithm of the ratio of wells without
aggregates to the total number of wells is the CFU-ALP frequency or equivalently, the expected
number of osteoprogenitors per plate under the specific culture conditions.
6.1.7. Evaluation of Myofibroblast Differentiation
After seven days of culture in complete DMEM with or without CNP, cells were fixed with 10%
NBF, permeablized and immunostained for α-SMA, followed by nuclear counterstaining.
Expression of α-SMA was quantified by densitometry of the immunoblots. In addition, CNP
expression in cultured VICs was evaluated by immunoflurescence staining after plating and after
five days of treatment with 1 ng/mL TGF-1 to induce myofibroblast differentiation.
To investigate changes in myofibroblast function, collagen deposition and cell contractility were
analyzed. Collagen content was measured with Sirius Red dye release method as described
previously241. Briefly, cultured cells were fixed with 10% NBF, followed by one hour incubation
at room temperature with 0.1% Sirius Red F3BA reconstituted in saturated picric acid. Stained
samples were washed five times with 10 mM HCl and then rinsed with distilled water. For
quantification, Sirius Red dye was released by 0.1 M NaOH for five minutes. The absorbance of
the supernatant containing the released dye was measured at 540 nm. Absorbance was
normalized by total cell number determined by DNA content, which was measured using
PicoGreen (Molecular Probes, Eugene, OR). Contractility was measured using standard stress-
relaxation collagen gel model224. In brief, VICs were cultured on constrained, compliant collagen
gels for four days in complete media with (100 nM) or without CNP at a cell density of 10,000
cell/cm2. Myofibroblast differentiation of VICs was induced by treating the cells with 1 ng/mL
of TGF-1 for 48 hours, after which the gels were released to allow for contraction. Images of
the collagen gels were taken every 30 minutes and the gel areas were determined using ImageJ.
6.1.8. Statistical Analysis
Results are presented as mean standard error. Samples sizes were at least three in all cases,
and experiments were repeated at least three times. Unpaired Student’s t-test was used for
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comparisons between two groups. ANOVA and Fisher’s least significant difference test were
used to evaluate statistically significant differences in multiple group comparisons.
6.2. Results
6.2.1. Expression of Pathological Markers and CNP in Normal and Sclerotic Aortic Valves
Immunofluorescent staining of normal and sclerotic aortic valve leaflets revealed that the
expression of CNP is spatially mutually exclusive with that of -SMA and Runx2/Cbfa-1
(Figure 6.1). In normal valves, CNP expression was abundant throughout the interstitium, with
slightly higher expression in the ventricularis (Figure 6.1: A). There were few -SMA positive
cells in normal leaflets (Figure 6.1: D). Numerous cells in sclerotic valves stained strongly
positive for -SMA, particularly near lesions (Figures 6.1: E and F), indicative of VIC
myofibroblast differentiation. In contrast, weak or absent CNP staining was observed in these
regions in sclerotic valves (Figures 6.1: B and C). Cbfa-1/ Runx2, an osteochondral transcription
factor, was not detected in normal leaflets (Figure 6.1: G), but was abundant in sclerotic valves
(Figure 6.1: H and I) accompanied by low expression of CNP.
6.2.2. Molecular Components of CNP Signaling
Natriuretic receptors (NPR-A, NPR-B, NPR-C) are expressed in cardiac atria and ventricles, but
their expression in VICs is unknown. Of the three receptors, CNP has the highest binding affinity
with NPR-B. NPR-B is linked to a guanylyl cyclase domain, and CNP binding with NPR-B
receptor induces cGMP synthesis, which mediates downstream cellular responses (reviewed in 242). To determine if VICs are equipped with molecular components to detect and to process CNP
signaling, we identified the expression of NPR-B mRNA by VICs as well as the induction of
cGMP synthesis by CNP. Primary VICs indeed express transcripts of NPR-B (Figure 6.2: A) and
cGMP synthesis by VICs was observed within 10 minutes of treatment with 100 nM of CNP
(Figure 6.2: B).
6.2.3. Dose Response of CNP
Induction of cGMP synthesis by CNP showed a threshold effect. VICs appeared to be highly
responsive to 100 nM of CNP (Figure 6.2: B). We further investigated if there was a dose-
dependent response by measuring the expression level of -SMA with Western blotting. By
79
treating the cells with CNP concentrations of 0, 0.1, 1, 10 and 100 nM, it was found that
suppression of -SMA was most prominent with 100 nM of CNP (Figure 6.3: A and B).
Together with the cGMP synthesis data, we decided that in this in vitro culture model, 100 nM of
CNP was the most effective in mediating detectable cellular responses and all subsequent
experiments were conducted with 100 nM of CNP.
Figure 6.1. Expression of CNP, -SMA and Runx2/Cbfa-1 in normal and sclerotic porcine
aortic valves
(A-C) Immunofluorescent staining of CNP (red) and nucleus (blue). (D-F) Immunostaining of -
SMA (red) and nucleus (blue). (G-I) Immunostaining of Runx2/Cbfa-1 (red) and nucleus (blue).
“AO” denotes the aortic side and “V” denotes the ventricular side of the normal leaflets. Only
the aortic side of the sclerotic leaflets was shown.
Normal Sclerotic
CN
P
-SM
AR
un
x2
/Cb
fa1
Boxed Area
Boxed Area
G
Boxed Area
A B C
D E F
H I
100 m
AO
V
AO
V
V
AO
Normal Sclerotic
CN
P
-SM
AR
un
x2
/Cb
fa1
Boxed Area
Boxed Area
G
Boxed Area
A B C
D E F
H I
100 m
AO
V
AO
V
V
AO
CN
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-SM
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/Cb
fa1
Boxed Area
Boxed Area
G
Boxed Area
A B C
D E F
H I
100 m
AO
V
AO
V
V
AO
80
NPR-B (142 \bp)
0 1 100
CNP concentration (nM)
cGM
P c
onc
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tion
(fm
ol/w
ell)
No
t de
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No
t de
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0
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12*
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NPR-B (142 \bp)
0 1 100
CNP concentration (nM)
cGM
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onc
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(fm
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ell)
No
t de
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No
t de
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0
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12*
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A.
NPR-B (142 \bp)
0 1 100
CNP concentration (nM)
cGM
P c
onc
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(fm
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ell)
No
t de
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No
t de
tec
ted
0
3
6
9
12*
B.
0
3
6
9
12*
B.
A.
Figure 6.2. Expression of NPR-B and activation of cGMP by CNP
(A) Primary VICs from three different isolations expressed transcript for NPR-B (lane 1,2,3).
(B) Treatment of VICs with 100 nM of CNP significantly induced cGMP synthesis in
comparison to those treated with 1 nM of CNP and the untreated culture, * P < 0.05
6.2.4. Cellular Proliferation and Morphological Changes
CNP had little effect on proliferation whether cells were cultured in complete media
(Figure 6.4: A) or calcifying media (Figure 6.4: B). In complete media, VICs displayed typical
fibroblastic morphology with elongated processes, and no substantial morphological differences
were observed between CNP-treated and untreated cells (data not shown). However, when cells
were cultured in calcifying media for more than ten days, formation of multicellular aggregates
was prominent only in the absence of CNP (Figures 6.4: C and D). Since CNP had no effect on
cellular proliferation in all culture conditions (P ≥ 0.4 between untreated and CNP treated
samples at each time point), cell density was similar in all cases and was unlikely to contribute to
the observed differences in aggregation or the phenotypic differences described below.
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Figure 6.3. Dose-dependent -SMA expression by VICs
Immunoblots of -SMA and GAPDH and the corresponding densitometric quantification for
culture after seven days with CNP concentrations of 0, 0.1, 1, 10 and 100 nM. ** P < 0.001 and
* P < 0.05 in comparison to no CNP treatment.
6.2.5. CNP Inhibits Calcification and Osteogenic Differentiation of VICs
Consistent with previous studies58, VICs formed multicellular aggregates when cultured in
calcifying media (Figure 6.4: D). In untreated cultures, the aggregates contained calcium as
shown with the intense positive staining of ARS (Figure 6.5: A and B). CNP-treated VICs
stained diffusely for calcium with minimal aggregate formation (Figure 6.5: C). The few
aggregates that did form in the CNP-treated culture stained weakly for ARS (Figure 6.5: D).
Quantification of the number of ARS-positive aggregates confirmed that CNP treatment
inhibited calcification (Figure 6.5: E).
SMA (42 kDa)
GAPDH (36 kDa)
0 0.1 1 10 100
Concentration of CNP (nM)
0
20
40
60
80
100
120
0 0.1 1 10 100
Concentration of CNP (nM)
Pe
rce
nta
ge
of S
MA
e
xpre
ssio
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ela
tive
t o n
o tr
ea
tme
nt *
** **
**
SMA (42 kDa)
GAPDH (36 kDa)
0 0.1 1 10 100
Concentration of CNP (nM)
0
20
40
60
80
100
120
0 0.1 1 10 100
Concentration of CNP (nM)
Pe
rce
nta
ge
of S
MA
e
xpre
ssio
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ela
tive
t o n
o tr
ea
tme
nt *
** **
**
82
Figure 6.4. Proliferation and morphology of cells with or without CNP treatment
(A) Proliferation of VICs from four hours to 15 days in complete media and (B) calcifying
media. (C) In calcifying media, few aggregates formed in CNP-treated cultures, (D) whereas
abundant aggregate formation was observed in untreated cultures.
Formation of calcified aggregates occurs in vitro through at least two possible processes, one
associated with myofibroblast apoptosis and the other associated with osteoblast
differentiation79. Both types of aggregates were observed in the untreated cultures, indicating
calcification in the untreated VICs was due in part to osteoblast differentiation. Notably,
transcript expression of Cbfa-1/Runx2, osteonectin and osteocalcin in CNP-treated cells was
lower than that of untreated samples over the culture duration, with a significant reduction in
osteonectin expression after as little as eight days of culture (Figures 6.6: A, B and C).
Consistent with the transcriptional profile, expression of bone-related proteins was also reduced
with CNP treatment. ALP activity (Figure 6.7: A and B) and osteocalcin expression were low
Ce
ll nu
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er(
1x1
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)
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4 hrs 5 days 10 days 15 daysCulture duration
Complete media + 100nM CNPComplete media
A. B.
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Culture duration
OS media + 100nM CNPOS media
C. Calcifying media + 100 nM CNP D. Calcifying media
Culture duration
Calcifying media + 100 nM CNPCalcifying media
Complete media + 100 nM CNPComplete media
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Culture duration
Calcifying media + 100 nM CNPCalcifying media
Complete media + 100 nM CNPComplete media
83
within CNP-treated cultures (Figure 6.7: C), but high within multicellular aggregates in the
untreated cultures (Figure 6.7: D).
To further understand the cellular target of the anti-osteogenic effect of CNP, we determined the
CFU-ALP frequency as a measure of the differentiation of single osteoprogenitor cells58. Under
CNP treatment, the CFU-ALP frequency was significantly reduced in comparison to the
untreated culture (Figure 6.8). These data suggest that the anti-osteogenic effect of CNP was
mediated by suppressing the osteogenic differentiation of progenitor cells in the VIC population.
6.2.6. Inhibition of Myofibroblast Differentiation by CNP
Myofibroblast differentiation can be induced by biochemical (e.g., cytokines such as TGF-165)
and mechanical stimuli (e.g., a rigid culture surface55, 79). To evaluate the myofibroblast
differentiation of VICs, we cultured freshly isolated VICs on stiff TCPS with complete medium.
As in Chapter Five, we aimed to achieve a cell population that reflected that of native valves, and
therefore the effect of CNP was only tested on primary VICs, as subculturing induces
myofibroblast differentiation55. Freshly isolated VICs did not express -SMA, indicative of an
undifferentiated cell population (Figure 6.9: A). After seven days of culture, cells treated with
CNP (Figure 6.9: B) expressed little -SMA compared to untreated cells, which had prominent
-SMA stress fibers (Figure 6.9: C). Western blotting of CNP-treated and untreated cultures
further confirmed significantly lower -SMA expression with CNP treatment (Figures 6.10: A
and B).
84
Figure 6.5. CNP modulates calcification by VICs
(A and B) Untreated cultures stained intensely with ARS, indicating high and localized
concentration of calcium deposition. (C) CNP-treated cultures displayed diffuse ARS staining
and (D) only weak ARS staining even in the few aggregates that formed. (E) CNP inhibited
formation of calcified aggregates after fourteen days in osteogenic medium, * P < 0.05.
A. B.
0
20
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60
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120
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Nu
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A. B.
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Nu
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E.
C. D.
85
Figure 6.6. Expression of bone-related transcripts
(A) Transcript expression of Runx2/Cbfa-1, (B) osteonectin and (C) osteocalcin in CNP-treated
cultures relative to that of untreated samples. * P < 0.05. (A relative gene expression level of less
than one indicates lower expression with CNP treatment relative to that without CNP treatment).
A.
B.
Re
lati
ve R
un
x2/C
bfa
-1
exp
ress
ion
(AU
) *
0.00.20.40.60.81.01.21.41.6
Day 3 Day 8 Day 16
*
0.00.20.40.60.81.01.2
Day 3 Day 8 Day 16
**
Re
lati
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ste
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ctin
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ress
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(AU
)
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)
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A.
B.
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-1
exp
ress
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(AU
) *
0.00.20.40.60.81.01.21.41.6
Day 3 Day 8 Day 16
*
0.00.20.40.60.81.01.2
Day 3 Day 8 Day 16
**
Re
lati
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ste
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ctin
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ress
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(AU
)
C.
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(AU
)
0.0
1.0
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Day 8 Day 16
*
86
Figure 6.7. Expression of bone-related proteins
(A) At the protein level, CNP-treated cells had low levels of ALP activity and (C) weak diffuse
staining for osteocalcin. (B) Untreated cultures displayed high levels of localized ALP activity as
well as (D) osteocalcin within the aggregates (inset represents no primary antibody control; black
arrow indicates aggregate).
To investigate if CNP regulates biochemically-induced myofibroblast differentiation of VICs, we
compared the difference in CNP expression by VICs before and after five days of culture with
TGF-1. VICs expressed CNP but not -SMA after one day in culture (Figure 6.11: A). After
five days of induction with TGF-β1, the majority of VICs differentiated into -SMA positive
myofibroblasts as expected (Figure 6.11: B). Similar to the mutually exclusive expression of
-SMA and CNP observed in the histological analysis, -SMA positive myofibroblasts did not
express CNP. A few cells that did not differentiate into myofibroblasts with TGF-1 induction
stained positive for CNP (Figure 6.11: B, arrow). Co-expression of -SMA and CNP was
therefore rarely observed in vivo or in vitro.
A. B.
C. D.
A. B.
C. D.
87
Figure 6.8. Effect of CNP on osteoprogenitor subpopulation
The CFU-ALP frequency was reduced in CNP-treated cultures, suggesting that CNP inhibits
osteogenic differentiation of the valve progenitor subpopulation (* P < 0.05).
Myofibroblasts typically have increased collagen synthesis and cellular contractility55, 59, 65. CNP
treatment suppressed collagen synthesis compared with untreated cells (Figure 6.12). To
evaluate the effect of CNP on myofibroblast-induced contractility, we cultured TGF-1-treated
VICs on constrained collagen gels and then measured gel contraction by VICs upon gel release.
Gels treated with CNP contracted significantly less than untreated gels, suggesting that CNP
suppressed TGF-1-induced myofibroblast differentiation (Figure 6.13: A and B).
6.3. Discussion
CNP is expressed in disease-protected regions of normal porcine valves6 and in normal human
valves19, but its expression is down-regulated in calcified aortic valves19 and in advanced
atherosclerotic lesions243. In the current study, we identified the expression of NPR-B receptor
and cGMP activity in VICs, which are components of CNP signaling. We found mutually
exclusive spatial expression of CNP and disease-related VIC phenotypes in vivo, and confirmed
in vitro that CNP inhibits differentiation of VICs to myofibroblasts and osteoblasts, phenotypes
associated with CAVD. Our current findings provide a cellular basis responsible for the
protective of CNP against valve calcification.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
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CF
U-A
LP
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ncy
*
0
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100 nM CNP No CNP
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U-A
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*
88
Figure 6.9. CNP inhibits expression of myofibroblast marker
(A) Immunostaining of freshly isolated VICs, (B) CNP-treated VICs and (C) untreated VICs
for -SMA (red) and nucleus (blue).
The influence of CNP on osteogenic differentiation and calcification appear to be tissue and
cell-type specific. Several animal models with either targeted disruption of NPPC244, the gene
for natriuretic peptide precursor C, or loss-of-function mutation in NPR-B receptor107, 245
display skeletal defects due to disturbed chondrogenesis during endochondral ossification.
CNP-dependent skeletal growth was also demonstrated in cell culture studies with pre-
osteoblastic cells188, 246, 247 and calvaria cells248. Treatment of pre-osteoblastic cells with CNP
has been reported to increase calcium deposition and the expression of ALP and osteocalcin
via NPR-B/cGMP signaling, indicative of CNP-induced osteoblast differentiation188, 246, 247.
Although CNP promotes ossification in bone cells, the reciprocal effect was found in
vascular cells, suggesting its response is cell-type specific. Vascular smooth muscle cells
treated with CNP displayed reduced calcium deposition and ALP expression187. Here, by
manipulating the culture conditions to promote osteogenic differentiation, CNP inhibited the
differentiation of VICs into osteoblasts, as demonstrated by reduced calcium deposition and
A.
B. C.
A.
B. C.
89
No
rma
lize
d
-SM
A e
xpre
ssio
n-SMA (42 kDa)
GAPDH (36 kDa)
100nM CNP no CNP
0.0
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*
No
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GAPDH (36 kDa)
100nM CNP no CNP
0.0
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CNP + CNP -100nM CNP no CNP
*
-SMA (42 kDa)
GAPDH (36 kDa)
100nM CNP no CNP
0.0
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1.0
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CNP + CNP -100nM CNP no CNP
*
lowered expression of bone-related transcripts and proteins in whole cell populations,
providing additional evidence to support the paradoxical effect of CNP in the vascular
system and the skeletal system.
Figure 6.10. Quantification of -SMA expression
Western blot of -SMA and GAPDH and the corresponding densitometric quantification for
culture after seven days with or without CNP treatment. * P < 0.05.
We have previously identified a large subpopulation of progenitor cells in VICs with multipotent
differentiation potential58, and therefore investigated their responsiveness to CNP using single-
cell clonal assays. By treating cells at the start of the experiments, we tested the response of
uncommitted, undifferentiated valve progenitors to CNP. We found that CNP attenuated
osteogenic differentiation of the valve progenitor subpopulation significantly. Although the
effect of CNP on valve progenitors at different stages of committment will require further
investigation, our initial findings suggest the ability of CNP to prevent osteogenic differentiation
of at least the undifferentiated valve progenitor cells. Commitment of cells to specific lineages
has been shown to influence the responsiveness of cells to CNP treatment. For example, ROB-
C26 cells induced by BMP-2 to undergo osteoblast differentiation displayed high levels of CNP-
mediated cGMP activity, whereas the same cells committed to the adipogenic lineage with
dexamathesone treatment exhibited marked reduction of CNP-mediated cGMP activity249.
90
Presumably VICs, including the subpopulation of osteoprogenitors, undergo pathological
differentiation during CAVD pathogenesis, leading to their commitment to myofibroblast or
osteoblast cell lineages, which may ultimately alter their response to CNP. Hence, the
effectiveness of CNP against CAVD in vivo may depend on the stage of the disease. Therefore,
future work on the therapeutic application of CNP for CAVD should explore the stage-related
effect of CNP treatment as a function of the disease progression.
Figure 6.11. Mutually exclusive expression of CNP and -SMA in cultured VICs.
(A) After one day in culture, VICs expressed CNP (red). (B) After five days of growth in media
containing TGF-β1, the majority of VICs differentiated into myofibroblasts that expressed -
SMA (green); however a few cells that did not express -SMA stained positive for CNP (white
arrow).
VICs can undergo myofibroblast differentiation, which is closely associated with apoptosis-
dependent calcification in vitro as described in Chapter Five. CNP is widely recognized to
regulate fibrosis in other tissues. For example, administration of CNP in animal models reduced
fibrosis associated with vascular intimal thickening110, pulmonary fibrosis111, and myocardial
infarction112. We observed that CNP attenuated myofibroblast differentiation of quiescent VICs
as reflected by the down-regulation of -SMA and loss of myofibroblast-related functions. Co-
expression of -SMA and CNP was rarely observed in vivo or in vitro. Although the influence
of CNP on apoptosis varies with cell type250-252, an anti-apoptotic effect of CNP has been
reported in some cell types such as pulmonary endothelial cells252. In our culture, notable cell
death was not observed as majority of the cells were well-spread on TCPS and plasma membrane
A. B.A. B.
91
blebbing resulting from cleavage of cytoskeleton proteins by caspases during apoptosis253, 254
was not evident. These observations clearly demonstrate that CNP is not pro-apoptotic in VICs.
Figure 6.12. CNP affects function associated with activated myofibroblasts
Collagen production of CNP-treated cells was significantly less than the
untreated culture. * P < 0.05.
It has been well documented that the actions of CNP are modulated through membrane-bound
receptors, mainly NPR-B and NPR-C, of which only NPR-B is linked to the cGMP-dependent
signaling cascade. We found that VICs express the transcript of NPR-B and synthesize cGMP in
response to CNP treatment. Additional studies are required to determine if the observed CNP-
mediated effects on VIC differentiation involve NPR-B/cGMP pathway. Because of the lack of
an NPR-B antagonist, siRNA-based knockdown of NPR-B would be one approach to test its
role.
Co
llag
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duc
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(A
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*
92
Figure 6.13. Contractility of VICs with or without CNP treatment.
Cells were seeded on the surface of constrained collagen gels. Contractility was recorded every
half an hour after gel release. Untreated VICs were more contractile than those treated with CNP
(* P < 0.05, ** P < 0.06).
In summary, the results of this study demonstrate that VICs express components of CNP
signaling. CNP inhibits myofibroblast and osteoblast differentiation of VICs, which may prevent
calcification. These findings provide a cellular mechanism by which CNP maintains valve
homeostasis and protects against aortic valve calcification in vivo. This fundamental knowledge
regarding CNP enables future studies aimed at the identification of the molecular mechanisms of
its putative protective actions, both in vitro and in vivo.
0 hr 0.5 hr 1 hr 1.5 hrs 2.0 hrs
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93
Chapter 7
7. The Combined Effects of Mechanical and Biochemical
Cues on the Transcriptional Regulation of VICs
It is well accepted that matrix stiffness regulates phenotypic drift and functions of a wide range
of cell types55, 221. Recent studies further suggest that substrate stiffness may modulate the release
of and the response to biochemical factors by cells12. The correlative link between cellular
response, mechanical cues and biochemical cues from the microenvironment has yet to be
studied thoroughly. Little is known regarding the impact of the combined effects of mechanical
and biochemical cues on VIC biology, despite increasing evidence of close relationships between
changes in tissue mechanics, soluble factors and disease progression7-9, 176.
Matrix stiffness regulates the responses of cells to biochemical factors to ultimately define cell
behaviour. The first demonstration of this was the differential effects of TGF-β on
myofibroblasts by matrix stiffness11. Myofibroblasts cultured on more compliant substrates were
insensitive to TGF-β, whereas those cultured on stiffer substrates were highly responsive to
TGF-β. Similar differential effects of TGF-β were also observed in VICs cultured on compliant
and stiff matrices as described in Chapter Five, suggesting that matrix stiffness may also
modulate the response of VICs to soluble factors. Others have also reported growth factors
mediate cellular response in a matrix stiffness-dependent manner169, 170. In addition, soluble
factors and matrix stiffness have been shown to synergistically guide stem cell commitment to
particular lineages130.
We have demonstrated the ability of matrix stiffness or CNP alone to modulate the pathological
differentiation of VICs into myofibroblasts and osteoblasts. In this chapter, we investigated the
combined effect of matrix stiffness and CNP on the transcriptional regulation of VICs. We
identified the impact of matrix stiffness on CNP-dependent transcript expression. The evaluation
of cell response to biochemical cues in the context of the cellular mechanical environment will
provide a more complete understanding of valve cell biology.
94
7.1. Materials and Methods
7.1.1. Cell Culture
Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (Oakville, ON,
Canada). Detailed protocols are described in Appendix A. Assays that followed the protocols
from the manufacturers without any modification are not listed in Appendix A; these protocols
can be found on the websites of the respective suppliers.
7.1.2. Sample Preparation
Primary VICs were isolated from porcine aortic valves by collagenase digestion as described in
Chapter Five. Compliant and stiff collagen matrices were constructed following procedures
described in Chapter Four. VICs were seeded on collagen matrices at 10,000 cells/cm2 in
calcifying media with (100 nM) or without CNP. A total of four different experimental
conditions were tested:
I. VICs cultured on compliant collagen matrices with calcifying media
II. VICs cultured on stiff collagen matrices with calcifying media
III. VICs cultured on compliant collagen matrices with calcifying media and 100 nM
CNP
IV. VICs cultured on stiff collagen matrices with calcifying media and100 nM CNP
After nine days in culture, VICs were released from collagen matrices by collagenase digestion.
Cell pellets were obtained by centrifugation, followed by aspiration of the supernatant. Total
RNA was isolated from cell pellets following standard protocols of the Micro RNeasy System
(Qiagen, Mississauga, ON). A total of 16 RNA samples from the four culture conditions using
cells from four separate VIC isolations were collected (N = 4). Universal reference RNA was
obtained by extracting RNA directly from freshly isolated VICs. RNA samples were quantified
with a NanoDrop Spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE).
Sample integrity based on the 28S:18S ribosomal RNA ratio was determined using Agilent 2100
Bioanalyzer (Agilent Technologies Canada, Mississauga, ON). All microarray samples had RNA
integrity number (RIN) of at least nine.
95
7.1.3. Microarray Experiments
RNA samples from the four culture conditions were labeled with Cy5 and the universal reference
RNA labeled with Cy3. Labeled RNA samples were competitively hybridized onto 44k 60-mer
(BMP2) and mitogen-activated protein kinase phosphatase-1 (MKP-1). NFKBIA encodes for a
protein that negatively regulates Notch signaling pathway. Notch signaling has been shown to
regulate osteogenesis, including its inhibitory effect on the differentiation of mesenchymal
progenitor cells to osteoblast lineage 256. Notch1, part of the Notch signaling system, represses
the activation of Cbfa-1/Runx2257, which is a transcription factor associated with osteogenic
differentiation. Expression of Cbfa-1/Runx2 was found to be upregulated in mouse and rabbit
models of valvular calcification258, 259 and in cells cultured on compliant collagen matrices
(described in Chapter Five). Negative regulation of Notch signaling by NFKBIA might possibly
promote Runx2 activation. Similarly, BMP2260 and MKP-1261 signaling for osteogenesis require
Cbfa-1/Runx2 activity (see Discussion).
Coupled with the relative high expression of osteoinductive transcripts, the expression of
transforming growth factor beta 3 (TGFB3) was downregulated in cells grown on compliant
matrices. TGFB3 has been shown to inhibit osteogenic differentiation of mesenchymal stem
cells262. The upregulation of osteoinducive transcripts and downregulation of osteorepressive
transcript on cells cultured on compliant matrices presumably contributes to the pro-osteogenic
nature of these matrices. In addition, the expression of transcripts related to cell adhesion and
actin-myosin cytoskeleton system were downregulated in VICs cultured on the more compliant
matrices.
100
Table 7.1. A subset of transcripts with higher expression in VICs cultured on compliant
matrices relative to those cultured on stiff matrices
Gene name* C/S fold change‡
P-value Biological process
CSF2 59.48 5.39 x 10-4 Immune Response IL8 41.88 2.19 x 10-6 Inflammatory response CCL20 27.58 1.21 x 10-3 Immune Response BTG2 15.24 1.91 x 10-5 Negative regulation of apoptosis SELE 9.67 2.2 x 10-3 Cell adhesion SPY2 7.27 2.27 x 10-4 Negative regulation of MAP kinase
activity NFKBIA 4.32 1.14 x 10-3 Negative regulation of Notch signaling
pathway MKP-1 3.45 1.09 x 10-3 Protein amino acid dephosphorylation BMP2 3.25 3.00 x 10-3 Growth C-JUN 2.95 1.05 x 10-4 Regulation of transcription SLN 2.79 4.22 x 10-3 Regulation of calcium ion transport PIAP 2.58 3.76 x 10-3 Regulation of apoptosis
nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha; MKP-1,
mitogen-activated protein kinase phosphatase-1; BMP2, bone morphogenetic proteins; C-JUN,
C-JUN protein; SLN, sarcolipin; PIAP, inhibitor of apoptosis-like. Sequence mapped to genome of other species (e.g. Homo sapiens, Bos taurus, Mus musculus) ‡ Fold changes of genes expressed by cells cultured on compliant matrices relative to those
cultured on stiff matrices
7.2.3. Differential Gene Expression by CNP
The effect of CNP on altering transcript expression in VICs cultured on compliant and stiff
collagen matrices was evaluated. For VICs cultured on compliant matrices, CNP treatment
influenced the expression of 181 sequences, with higher expression of 73 sequences and lower
expression of 108 sequences. When VICs were cultured on stiff collagen matrices, CNP
treatment altered the expression of 237 transcripts, with higher expression of 139 genes and
lower expression of 98 genes in CNP-treated samples relative to untreated cells. The majority of
the transcripts displayed expression fold differences of less than five (Figures 7.4 and 7.5).
101
CNP treatment affects a wide variety of genes that are associated with various biological
processes. When cells were cultured on compliant matrices, CNP upregulated transcripts related
to metabolic processes such as gluconeogenesis, lipid catabolic process, collagen catabolic
process and ATP biosynthetic process (Table 7.3). CNP downregulated the expression of
transcripts related to ion binding and transport (Table7.4). Similarly when cells were cultured on
stiff matrices, CNP treatment also upregulated several transcripts related to metabolic processes
including glycogen metabolic process and lipid catabolic process (Table 7.5). CNP
downregulated transcripts associated with the actin-myosin cytoskeleton system when cells were
cultured on stiff matrices (Table 7.6).
Table 7.2. A subset of transcripts with lower expression in VICs cultured on compliant
matrices relative to those cultured on stiff matrices
Gene name* C/S fold change‡ P-value Biological classification GP38K 11.75 3.31 x 10-5 Carbohydrate metabolic process TNFSF10 6.58 5.85 x 10-3 Immune response ITIH4 5.10 1.67 x 10-3 Acute phase response CDH5 4.77 2.39 x 10-3 Cell adhesion WNT2B 3.46 2.68 x 10-4 Wnt receptor signaling pathway,
calcium modulating pathway CNN1 3.27 9.89 x 10-5 Actomyosin structure organization TIMP1 2.76 3.12 x 10-3 Erythrocyte maturation TNNC2 2.59 3.06 x 10-3 Calcium ion binding COL5A1 2.52 9.43 x 10-4 Cell adhesion TGFB3 2.37 5.41 x 10-5 Growth, positive regulation of cell
division DDC 2.37 6.07 x 10-3 Metabolic process, catecholamine
biosynthetic process TPM1 2.08 8.61 x 10-4 Actin binding
superfamily member 10; ITIH4, inter-alpha (globulin) inhibitor H4 (plasma kallikrein-sensitive
glycoprotein); CHD5, cadherin 5; WNT2B, wingless-type MMTV integration site family,
member 2B;CNN1, calponin 1 basic smooth muscle; TIMP1, TIMP metallopeptidase inhibitor 1;
TNNC2, troponin C type 2; COL5A1, collage type V alpha 1; TGFB3, transforming growth
factor beta 3; DDC, dopa decarboxylase; TPM1, tropomyosin 1. Sequence mapped to genome of other species (e.g. Homo sapiens, Bos taurus, Mus musculus) ‡ Fold changes of genes expressed by cells cultured on compliant matrices relative to those
cultured on stiff matrices
102
0
10
20
30
40
50
60
70
2.5 5 10 20 30 40+Fold difference
Nu
mb
er
of e
ntr
ies
UpregulatedDownregulated
Figure 7.4. The distribution of sequences differentially expressed with CNP treatment in
cultures on compliant matrices
Figure 7.5. The distribution of sequences differentially expressed with CNP treatment in
cultures on stiff matrices
0102030405060708090
2.5 5 10 20 30 40+
Fold difference
Nu
mb
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of e
ntr
ies Upregulated
Downregulated
103
Table 7.3. A subset of transcripts with higher expression in CNP-treated VICs relative to
untreated cells when cultured on compliant matrices.
Gene name T/U fold change‡ P value Biological classification PAH 11.47 2.06 x 10-3 Aromatic amino acid family metabolic
complement component 1;VDR, vitamin D (1,25-dihydroxyvitamine D3) receptor; PTGFR,
prostaglandin F receptor; LDL, lipoprotein lipase; ATP9A, ATPase class II type 9A; PC,
pyruvate carboxylase. Sequence mapped to genome of other species (e.g. Homo sapiens, Bos taurus, Mus musculus) ‡Fold changes of genes expressed by CNP-treated cells relative to untreated cells
7.2.4. The Combined Effect of Matrix Stiffness and CNP on Transcriptional Regulation
Venn diagrams were constructed to identify the genes commonly and exclusively modulated by
matrix stiffness and by CNP. We first compared the list of differentially expressed sequences by
104
matrix stiffness alone and by CNP on cells cultured on compliant matrices. Matrix stiffness alone
affected five times more transcripts than those regulated by CNP treatment. Of the 998
transcripts modulated by matrix stiffness, the expression of only 18 transcripts was also regulated
by CNP when cells were cultured on compliant matrices (Figure 7.6), suggesting the majority of
the transcripts were exclusively regulated by either matrix stiffnss or by CNP.
A similar trend was observed when comparing the number of sequences affected by matrix
stiffness and by CNP treatment on VICs cultured on stiff matrices. The expression of only a
small portion (i.e., 31 entries) of the sequences was influenced by matrix stiffness as well as by
CNP, further confirming that the majority of the transcripts were exclusively regulated by either
mechanical and biochemical cues (Figure 7.7).
Table 7.4. A subset of transcripts with lower expression in CNP-treated VICs relative to
untreated cells when cultured on compliant matrices.
Gene name T/U fold change‡ P value Biological classification PBD-1 7.39 9.65 x 10-4 Defense response PALMD 2.57 2.16 x 10-3 Regulation of cell shape LIM 2.41 1.35 x 10-2 Metal ion binding, zinc ion
binding SRPK3 2.17 3.78 x 10-3 Protein amino acid
phosphorylation KCNN4 2.10 7.40 x 10-5 Potassium ion transport ALDH1A3 2.05 2.56 x 10-2 Positive regulation of apoptosis,
oxidation reduction, metabolic process
GPR183 2.05 1.36 x 10-2 G-protein coupled receptor protein signaling pathway, immune response
UPP1 2.04 1.52 x 10-3 Nucleotide catabolic process *PBD-1, prepro-beta-defensin 1; PALMD, palmdelphin ; LIM, alpha-actinin-2-associated LIM
protein; SRPK3, SFRS protein kinase 3; KCNN4, potassium intermediate/small conductance
calcium-activated channel subfamily N member 4; ALDH1A, aldehyde dehydrogenase family 1
subfamily A3;GPR183, G-protein coupled receptor 183 ; UPP1, uridine phosphorylase 1. Sequence mapped to genome of other species (e.g. Homo sapiens, Bos taurus, Mus musculus) ‡Fold changes of genes expressed by CNP-treated cells relative to untreated cells
105
Table 7.5. A subset of transcripts with higher expression in CNP-treated VICs relative to
untreated cells when cultured on stiff matrices
Gene name T/U fold
change‡
P value Biological classification
HP 5.05 7.72 x 10-3 Proteolysis PKHA1 3.34 2.61 x 10-2 Glycogen metabolic process CD36 2.83 9.67 x 10-3 Cell adhesion ANGPTL4 2.25 2.75 x 10-3 Cell differentiation, angiogenesis, signal
transduction CXCL12 2.22 1.55 x 10-2 Immune response ALB 2.17 3.62 x 10-2 Cellular response to starvation,
maintenance of mitochondrion location, negative regulation of apoptosis, transport
PLA2G7 2.16 2.38 x 10-2 Lipid catabolic process FABP4 2.10 9.28 x 10-2 Transport (lipid binding) IGF1 2.06 6.79 x 10-4 Positive regulation of DNA replication ANGPT1 2.04 2.24 x 10-2 Signal transduction, angiogenesis, cell
differentiation SERPINA6 2.03 3.01 x 10-2 Transport NEO 2.02 2.56 x 10-2 Cell adhesion, regulation of
transcription, myoblast fusion RAMP1 2.00 1.79 x 10-2 Regulation of G-protein coupled
receptor protein signaling pathway, intracellular protein transport, transport
protein-coupled) activity modifying protein 1. Sequence mapped to genome of other species (e.g. Homo sapiens, Bos taurus, Mus musculus) ‡Fold changes of genes expressed by CNP-treated cells relative to untreated cells
106
Table 7.6. A subset of transcripts with lower expression in CNP-treated VICs relative to
untreated VICs cells when cultured on stiff matrices
Gene name T/U fold change‡ P value Biological classification ACTA1 6.69 9.05 x 10-3 Skeletal muscle fiber development,
muscle thin filament assembly CNN1 2.83 5.89 x 10-3 Actomyosin structure organization NPY1R 2.44 3.61 x 10-2 Signal transduction, G-protein coupled
receptor protein signaling pathway GNAO1 2.30 2.00 x 10-3 Locomotory behaviour, regulation of
binding family B member 1 interacting protein. Sequence mapped to genome of other species (e.g. Homo sapiens, Bos taurus, Mus musculus) ‡Fold changes of genes expressed by CNP-treated cells relative to untreated cells
107
Figure 7.6. Transcript expression modulated by matrix stiffness and/or by CNP in cultures
on compliant matrices
Blue region represents transcript expression altered by matrix stiffness. Yellow region represents
transcripts that were changed in VICs cultured on compliant matrices with CNP treatment
relative to the untreated cultures. The union region represents genes that were non-exclusively
regulated by matrix stiffness.
When cells were cultured on compliant or stiff matrices, CNP treatment influenced
approximately 150-200 genes (Figure 7.8). The expression of only 30 CNP-regulated genes was
not affected by matrix stiffness (Figure 7.8, union region), indicating matrix mechanics
significantly modulated the response of VICs to CNP treatment. CNP-modulated, but non-
mechanically regulated genes were related to diverse biological processes including cell division,
replication of DNA, cellular amino acid biosynthetic process (Table 7.7).
980 genesregulated by matrix stiffness
163 genes regulated by CNP (cells cultured on compliant matrices)
18 genes
980 genesregulated by matrix stiffness
163 genes regulated by CNP (cells cultured on compliant matrices)
18 genes
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Figure 7.7. Transcript expression modulated by matrix stiffness and/or by CNP in cultures
on stiff matrices
Blue region represents sequences regulated by matrix stiffness. Yellow region represents
transcripts that were changed in VICs cultured on stiff matrices with CNP treatment relative to
the untreated cultures. The union region represents genes that were non-exclusively regulated by
matrix stiffness.
967 genesregulated by matrix stiffness
208 genes regulated by CNP (cells cultured on stiff matrices)
31 genes967 genesregulated by matrix stiffness
208 genes regulated by CNP (cells cultured on stiff matrices)
31 genes
109
Table 7.7. A partial list of CNP-regulated, mechanically-insensitive genes
HP Haptoglobin Proteolysis SAA1 Serum amyloid A1, transcript
variant 1 Positive regulation of interleukin-1 secretion, regulation of protein secretion, positive regulation of cell adhesion, acute-phase response, platelet activation, negative regulation of inflammatory response, chemotaxis, elevation of cytosolic calcium ion concentration
Sequence mapped to annotation of other species (e.g. Homo sapiens, Bos taurus, Mus musculus)
† Incomplete GO annotation lacking defined biological process(es). Molecular functions: FST, TGF-
signaling pathway; SULT1A1, transferase activity.
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A three-way Venn diagram was constructed to further identify any genes that were regulated by
matrix stiffness and/or CNP. The regulation of only one transcript, follistatin, was altered by
matrix stiffness and CNP (Figure 7.8). Interestingly, in the absence of CNP, the expression of
follistatin transcript was 2.4-fold higher on compliant matrices relative to cells cultured on stiff
matrices, indicative of mechanically-regulated follistatin transcript expression in certain
biochemical environments. On the compliant matrices that favored osteogenic differentiation of
VICs, expression of follistatin was 2.0-fold higher in CNP-treated cells relative to the untreated
cells. On the stiff matrices that preferentially promoted myofibroblast differentiation, CNP-
treatment on VICs led to a 3.3-fold upregulation in the expression of follistatin. These data
suggest that the regulation of follistatin transcript expression by VICs in the presence of CNP
was independent of matrix stiffness, while the majority of CNP-regulated transcripts were
sensitive to modulation by matrix mechanics.
7.3. Discussion
Although matrix stiffness is recognized to play critical roles in regulating cell functions and
differentiation, its contribution in regulating cellular response to biochemical factors is poorly
understood. Further, the effect of matrix stiffness on CNP-mediated cellular response has never
been studied. By evaluating the transcriptional profile of VICs cultured on compliant and stiff
collagen matrices in the presence or absence of CNP, we identified a subset of mechanically- and
biochemically-regulated transcripts. The differential gene expression profile suggests that the
majority of CNP-regulated transcripts are sensitive to matrix stiffness. These data demonstrate
the significance of matrix stiffness in modulating the response of VICs to CNP, and the
combined effect of mechanical and biochemical cues in regulating VICs at the transcriptional
level, which ultimately may be important in understanding valve pathology and in determining
VIC response to potential therapeutics, as valve tissues mechanics changes during disease
progression.
111
Figure 7.8. A three-way Venn diagram showing the commonly and exclusively modulated
genes by matrix stiffness and by CNP
Blue region represents transcriptional regulation by matrix stiffness alone. Yellow region
represents CNP-regulated genes in VICs cultured on stiff matrices. Green region represents
CNP-regulated genes in VICs cultured on compliant matrices. The center area in which all three
regions overlap indicates a transcript (follistatin) that was regulated by matrix stiffness and CNP.
In the absence of CNP, higher expression of osteoinductive transcripts (e.g., BMP-2, NFKB1A,
MKP-1) was observed on compliant matrices, substrates that favored osteogenic differentiation
of VICs. The expression of BMP-2 has been observed in VICs differentiated to osteoblast-like
cells in vitro68 and in calcified valvular tissue48, 84. Addition of BMP-2 to VICs in culture
increased their expression of osteoblast-related markers and their rate of calcific aggregate
formation54, 73. BMP-2-induced osteoblastic differentiation has been shown to be mediated in a
MKP-1-dependent manner263. Similarly, we also found an upregulation of MKP-1 in the pro-
osteogenic, compliant matrices. Further, Notch signaling is involved in early stage of valve
formation as well as inhibition of the mediators of osteogenic-dependent valvular calcification257.
950 genesregulated by matrix stiffness
208 genes regulated by CNP (cells cultured on stiff matrices)
30
134 genes regulated by CNP (cells cultured on compliant matrices)
17
1
29
950 genesregulated by matrix stiffness
208 genes regulated by CNP (cells cultured on stiff matrices)
30
134 genes regulated by CNP (cells cultured on compliant matrices)
17
1
29
950 genesregulated by matrix stiffness
208 genes regulated by CNP (cells cultured on stiff matrices)
30
134 genes regulated by CNP (cells cultured on compliant matrices)
17
1
29
112
Presumably, the negative regulator of Notch signaling pathway, NFKB1A, may regulate
osteogenic differentiation indirectly by mitigating the osteorepressive effect of Notch signaling;
however the direct contribution of NFKB1A in CAVD will require further investigation.
When cells were cultured on the stiffer matrices that promoted myofibroblast differentiation,
higher expression of transcripts associated with actin-myosin cytoskeleton system including
calponin 1 (CNN1) and tropomyosin 1 (TPM-1) was observed. These findings are consistent
with a previous study by Chambers et al. in which expression profiling identified upregulation of
genes associated with contractile phenotype and cytoskeletal organization in myofibroblasts264
when compared to quiescent fibroblasts. The transition of fibroblasts to myofibroblasts is
closely related to maturation of focal adhesions265, which depends on the force applied to the
ECM-integrin-cytoskeleton connections either externally (e.g., ECM motion, substrate rigidity)
or internally (e.g., actin polymerization). A stiffer culture surface would presumably permit the
generation of greater traction forces, which facilitates focal adhesion maturation and enables the
transition of fibroblasts to myofibroblasts. The dependency of the transition of VICs into
myofibroblasts on matrix stiffness has previously been reported in the study by Pho et al.55 and
likely plays a role in the phenotypic drift of VICs in stiffened sclerotic valves.
Matrix stiffness alone affected the expression of ~ 1000 transcripts, whereas the expression of
only ~ 200 transcripts was affected by CNP. Notably, CNP upregulated various transcripts
associated with metabolic processes, including lipoprotein lipase (LPL) and phospholipase A2
(PLA2G7). LPL is the rate-limiting enzyme of triglyceride removal from plasma and has been
implicated in atherosclerosis. The expression of LPL transcript was downregulated in
atherosclerotic patients266, whereas statin treatment significantly increased LPL activity in
patients267. Our microarray data suggest a link between lipoprotein catabolic processes and CNP
signaling. Intriguingly, initial work found an upregulation of CNP transcript expression in statin-
treated VICs (Appendix B1); however whether there exists molecular relationships among
lipoprotein, statins and CNP awaits to be determined.
A striking observation from the two-way Venn diagram was the relatively small number of CNP-
dependent transcripts that were insensitive to matrix stiffness. The expression of 97% of all the
differentially expressed CNP-dependent transcripts was regulated by matrix stiffness. These data
emphasize the contribution of matrix mechanics in modulating cellular response to biochemical
113
factors. Also notable in the three-way Venn diagram analysis was the expression of only one
transcript, follistatin, which was non-exclusively regulated by matrix stiffness or CNP. One
possible explanation is that follistatin may regulate both osteoblast and myofibroblast
differentiation. Follistatin is a 34-kDa soluble protein that binds activin with high affinity to
inhibit the activation of TGF- signaling268, 269. In the absence of follistatin, activins bind to the
activin type IIA and type IIB receptors, leading to the recruitment and phosphorylation of type I
receptor, and subsequently the phosphorylation of Smad2/3270. Activation of Smad2/3 signaling
has been shown to increase -SMA expression and myofibroblast differentiation271. In addition
to activin signalling, follistatin has also been demonstrated to form a trimeric complex with BMP
and receptors of BMP to inhibit osteogenic effect mediated by BMP activity272. VICs
preferentially differentiated to pathological osteoblasts when cultured on compliant matrices and
CNP treatment would presumably prevent the osteogenic differentiation of these VICs. The
upregulation of follistatin in CNP-treated cells cultured on compliant matrices may facilitate the
inhibition of osteogenic differentiation by mitigating BMP activity272. CNP repressed
myofibroblast differentiation of VICs on stiff matrices. The higher expression of follistatin on
CNP-treated cells cultured on stiff matrices would likely mitigate TGF- signaling and Smad2/3
phosphorylation, which are required for the activation of fibroblasts to myofibroblasts.
Clearly, matrix stiffness has been shown to modulate various cell behaviours in vitro. Here, we
demonstrated the significance of matrix stiffness not only in modulating VIC phenotype, but also
their response to CNP at the transcriptional level. We identified a subset of mechanically- and
biochemically-regulated transcripts. The differential gene expression profile suggests that the
majority of CNP-regulated transcripts are sensitive to matrix stiffness. These data demonstrate
that matrix stiffness significantly affects the response of VICs to biochemical cues, and the
combined effect of mechanical and biochemical cues may ultimately govern the functions and
phenotypes of VICs. These findings may impact the response of cells to therapeutics in diseases
with substantial tissue matrix remodeling, where changes in tissue mechanics may define cellular
response to soluble factors. For example, significant stiffening of tumour tissue has been
correlated to an increase in proteolysis182, which has been suggested to partially explain the
failure of protease inhibitors as cancer therapies. The aortic valve undergoes significant
pathological matrix remodeling, which may alter local matrix mechanics. It is possible that local
matrix mechanics regulates valve homeostasis and functions of VICs in vivo, which may affect
114
their response to biochemical factors and the effectiveness of therapeutics against CAVD in a
stage-dependent manner. While our findings are suggestive of a correlative link between matrix
mechanics, VIC phenotypes and transcriptional regulation, further investigations are required to
demonstrate causality of this link.
115
Chapter 8 8. Conclusions and Recommendations
8.1. Conclusions
Pathological differentiation of VICs alters cell functions and is closely associated with valve
calcification. It is well accepted that biochemical factors such as TGF-1 induce the pathological
differentiation of VICs69, but little is known regarding factors that can inhibit the differentiation
of VICs into undesirable phenotypes. Further, the role of mechanical stimuli in regulating VIC
phenotype and functions has been overlooked and has yet to be investigated, despite the
observed influence of matrix stiffness on the differentiation of and mineralization by other non-
valve cell types132, 185. Alterations in tissue stiffness have been reported to occur prior to
substantial cellular and histological changes in diseases such as liver fibrosis7 and
atherosclerosis16, suggesting correlative links between tissue mechanics, disease progression and
the regulation of cell response. The aortic valve undergoes significant pathological matrix
remodeling and stiffens when calcified; it is likely that matrix stiffness may modulate VIC
behaviour and response to biochemical factors. Hence, the overall goal of this thesis was to
investigate the effect of matrix stiffness on modulating the response of VICs to pro- and anti-
calcific biochemical factors, which would provide further insights in valve pathology.
The first objective was to implement and characterize a culture system with tunable stiffness.
The morphological and molecular changes in VICs cultured on compliant and stiff matrices were
evaluated. In addition, the ability of CNP, a putative anti-sclerotic and anti-calcific agent, to
suppress pathological differentiation of VICs was tested in vitro. Lastly, the combined effect of
matrix stiffness and CNP on the transcriptional regulation of VICs was investigated.
VICs were found to be highly responsive to matrix stiffness. In conjunction with pro-calcific
biochemical factors, VICs preferentially underwent osteogenic differentiation and calcified when
cultured on the more compliant matrices. In contrast, the stiffer matrix favored myofibroblast
differentiation of VICs, contributing to contraction-mediated calcification that downregulated
Akt activity and was associated with apoptosis. Similarly, microarray study of cells cultured on
compliant and stiff matrices with pro-calcific biochemical factors identified upregulation of
116
osteoinductive transcripts and downregulation of osteorepressive transcripts on the more
compliant matrices relative to those cultured on the stiffer matrices. The ability to distinguish
two calcification processes by simply changing the matrix stiffness provides a useful research
tool to dissect the fundamental mechanisms of cell-mediated calcification.
The protective effect of CNP on VICs was also evident in the cell culture study. CNP inhibited
myofibroblast and osteoblast differentiation of VICs and suppressed in vitro calcification by
VICs. A striking finding was the small number of transcripts that were commonly regulated by
CNP and by matrix stiffness. The microarray results clearly demonstrate that the combined
effects of mechanical and biochemical cues govern transcriptional regulation of VIC, which
further emphasizes the necessity to consider both biochemical and mechanical factors in valve
studies in order to improve our fundamental understanding of VIC biology and valve pathology.
This thesis work contributes to the field of mechanobiology and valve biology. It provides an
improved understanding of VIC-matrix interactions, which is required to aid in interpretation of
VIC calcification studies in vitro; to guide the selection of biomaterials with appropriate
mechanical properties for valve tissue engineering; and to assess if alterations in extracellular
matrix mechanics that occur with disease modulate pathologic changes in VIC phenotypes and
calcification processes. In addition, the current study identifies for the first time the ability of
VICs to respond to CNP and provides a cellular explanation responsible for the protective effect
of CNP against calcification. These fundamental findings are essential for future mechanistic
studies of CNP at the molecular level and may perhaps eventually lead to the development of a
new treatment option.
8.2. Future Work
Given the current results, there are a number of suggested directions for future investigations, as
detailed in the following sections.
8.2.1. Determination of Changes in Valve Matrix Mechanics in vivo
The significance of matrix mechanics in modulating phenotype and transcriptional regulation of
VICs is evident in this thesis. To bridge the gap between our in vitro findings and the in vivo
relevancy, it will be important to identify the association between changes in valve matrix
117
mechanics and the progression of CAVD. Changes in mechanical properties of valve tissue
during CAVD can be determined using a micropipette aspiration technique similar to that
described by Matsumoto et al.16. AVs at different stages of disease development can be obtained
from porcine animal model fed with an atherogenic diet for various durations. The local elastic
moduli of normal AVs (i.e., animal fed a normal diet) and those of early-, intermediate- and late-
disease stage AVs can be measured by the micropipette aspiration method.
Immunohistochemical staining can be performed to characterize the pathological differentiation
of VICs temporally with respect to disease progression. Changes in the mechanical properties of
normal and diseased valve tissues can then be correlated with the extent of pathological
differentiation of VICs.
8.2.2. Improvement of the Cell Culture System
Although the collagen-based cell culture system was functional for all tests conducted, there
exist a number of limitations. Some of the limitations are: 1) the duration of cell culture was
limited to prevent substantial collagen degradation; 2) there exists a difference in the total
amount of collagen available on the two matrices, which may affect the ability of cells to spread
at later time points; and 3) fine tuning of stiffness is not possible with the existing system.
Presumably, changes in valve matrix mechanics involve a wide range of stiffness. To study the
effect of a range of physiologically relevant stiffness identified from Section 8.2.1, a culture
system that can be fine-tuned to provide a wide range of stiffness while maintaining surface
chemistry and to provide substrates with the same total collagen available over the given culture
conditions is needed. Polyacrylamide (PA) substrates are promising candidate materials as they
can provide a wide range of stiffnesses, while maintaining similar surface chemistry (reviewed in 10). However, initial efforts in our lab with standard surface modification methods failed to
provide appropriate surface adhesiveness to VICs. Recently, we successfully modified the
surface modification procedure and improved the adhesiveness to primary VICs. Our
preliminary study with primary VICs cultured on collagen-coated PA substrates with stiffnesses
of 11 kPa, 22 kPa, 50 kPa and 144 kPa showed that calcification by VICs was more prominent
on substrates with stiffnesses of 22 kPa and 50 kPa in comparison to those cultured on substrate
with stiffnesses of 11 kPa and 144 kPa (Figure B.3. in Appendix B). This preliminary result
suggests the possibility of culturing VICs on PA substrates, which can be tuned to the stiffness of
valve tissues measured at various disease stages. Such an in vitro study will provide a means to
118
identify the molecular determinants for mechanically regulated phenotypic drift of and
calcification by VICs on materials that closely resemble the mechanical properties of native
normal and diseased valves.
8.2.3. Effect of CNP treatment at Different Stages of Disease Progression
This thesis identified a cellular basis responsible for the protective effects of CNP against
CAVD. The next logical step is to identify the molecular mechanism responsible for the
inhibitory effects of CNP in the pathological differentiation of VICs. We hypothesized that CNP
mediated its cellular response via the NPR-B/cGMP signaling pathway. To address this
hypothesis, we have begun siRNA transfection experiments to manipulate the expression of
NPR-B receptor in VICs. Once those are completed, it will be important to evaluate the effect of
CNP: 1) in vitro by culturing cells on matrices with stiffnesses that represent various disease
stages; and 2) in vivo to evaluate the effectiveness of CNP treatment given at different stages of
CAVD. Because of the heterogeneity of VICs and the ability of these cells to differentiate into
various phenotypes over the disease progression, their response to CNP may vary depending on
the time at which treatment is administered. Studies have reported that the responsiveness of
cells to CNP depends on their commitment to certain lineages249. Presumably, the ability of VICs
to differentiate into various phenotypes is regulated in part by matrix stiffness and is altered as
the disease progresses. To test this, CNP can be applied to VICs cultured on matrices with
stiffnesses that represent early-, immediate- and late-stages of the disease. Such a culture system
can provide information regarding the ability of CNP to suppress pathological changes of VICs
when subjected to matrix stiffnesses that are physiologically relevant. Subsequently, these data
may help determine the preferred CNP treatment time point over the course of disease
development. Such data can serve as an initial guideline for the selection of treatment regimen
for in vivo tests. Mice models may be suitable for in vivo CNP studies, because genetic mutation
with mice can be done with ease. Initial work has verified the feasibility of dissecting mouse
aortic valves and isolating VICs from the valve leaflets (Figure B.4 and B.5 in Appendix B).
8.2.4. Identification of Transcriptional Pathways that Regulate Pathological
Differentiation of VICs
The current microarray study identified differential regulation of transcripts by matrix stiffness
and CNP. The study further confirmed the pro-osteogenic nature of our compliant matrices.
119
However, to fully benefit from the hypothesis generating power of microarray experiments and
to understand the transcriptional regulatory pathways that are involved in mechanical and
biochemical modulations, gene expression network analysis can be done to reveal important
phenomenological link between the expression of different genes. To do so, re-annotation of all
entries of the porcine microarray chips based on BLAST and cross-referencing of porcine
sequences to the human genome is necessary. Based on the re-annotation, canonical pathway
analysis can be performed to identify transcript networks with putative significance in CAVD.
120
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Appendix A A. Protocols
A.1. Fabrication of Type I Collagen Matrices
(Modified from Bellows CG, Melcher AH, Aubin JE. Contraction and organization of collagen
gels by cells cultured from periodontal ligament, gingiva and bone suggest functional differences
between cell types. J Cell Sci. 1981;50:299-314.)
Purpose: To synthesize collagen matrices with different mechanical properties, but similar
biochemical properties.
Reagents:
10x concentrated sterile DMEM (Sigma D7777) made with distilled water
0.25 M sterile NaHCO3 (Sigma 223530) buffer made with distilled water
0.01 M sterile NaOH (Sigma S2770) made with distilled water
A.11. Western Blot Purpose: To quantify the expression of a protein of interest
Reagents:
Cold Sterile PBS with calcium chloride and magnesium chloride (Sigma P5655)
10x lysis buffer
100 mM Phenylmethanesulfonyl fluoride (Sigma P7626, reconstituted in 100% ethanol)
Micro BCA Protein Assay Reagent (Pierce 23235)
30% acrylamide and bis-crylamide(acry/bis)
Resolving gel buffer (1.5 M Tris-base)
158
- 18.15 g Tris-base (Sigma 77-86-1)
- 60 mL Deionized H2O
- Adjust pH with 6 N HCl to pH 8.8
- Bring volume to 100mL and store at 4oC
Stacking gel buffer
- 6 g Tris-base (Sigma 77-86-1)
- 60 mL Deionized H2O
- Adjust pH with 6 N HCl to pH 6.8
- Bring volume to 100mL and store at 4oC
0.5% (wt/vol) and 10% (wt/vol) sodium dodecyl sulfate (SDS, Sigma L3771, reconstituted in
deionized H2O)
10% APS
TEMED (Sigma
10x running buffer
- 30.3 g Tris-base (Sigma 77-86-1)
- 144 g Glycine (Sigma G8898)
- Bring to 1 litre with deionized H2O, store at 4oC
1M Tris-base
- 12 g Tris-base (Sigma 77-86-1)
- Adjust pH with 6 N HCl to pH 6.8
- Bring volume to 100 mL with deionized H2O, store at 4oC
5x Laemmli loading dye
- 1 M Tris-base
- 10 g SDS (Sigma L3771)
- 50 mL Glycerol
- 250 mg Bromophenol blue
- 20 mL -mercaptoethanol (Sigma M3148)
- Store in glass bottle at room temperature
Protein ladder (Fermentas SM0671)
Methanol (Sigma 179957)
Protein transfer buffer (** must be made one day in advance)
- 11.64 g Tris-base
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- 5.86 g Glycine
- 20% Methanol
- 7.5 mL 10% SDS
- Bring the volume to 1 litre with deionized H2O and store at 4 oC
10x TBS
- Tris-base
- NaCl
- Bring the volume to 1 litre with deionized H2O and store at 4 oC
1% Bovine serum albumin (BSA Sigma A9647, reconstituted in 1x TBST buffer)
Amersham ECL Plus™ chemilumiscece western blotting detection reagent
Equipment:
Pre-chilled cell scrapers
Pre-chilled eppendorf tubes
96 well plate
Filter papers
Polyvinylidene fluoride (PVDF) transfer membranes (BioRad 1620177)
SNAP i.d.TM protein detection system
Western blotting film
Procedures:
I. Protein extraction
1. Prepare fresh 1x lysis buffer for each protein extraction. For 1 mL of 1x lysis buffer,
combine:
a. 100 L 10x lysis buffer
b. 10 L 100 mM PMSF
c. 890 L deionized H2O
2. Place culture on ice
3. Remove media
4. Rinse (2-3 times) with cold PBS
5. Add 1x lysis buffer
a. For 24 well plate, add 30 L per well
b. For 12 well plate, add 100 L per well
c. For 6 well plate, add 200 L per well
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6. Remove cells from culture by using a pre-chilled cell scrape. Rinse cell scrape with cold
PBS between samples.
7. Transfer lysate to a pre-chilled eppendorf tubes
8. Incubate on ice and place on a shaker set at maximum speed for 30 minutes
9. Vortex the samples every 5 minutes
II. Quantification of protein concentration
10. Prepare diluted BSA albumin standards
Vial Volume of diluent Volume and source of BSA Final BSA concentration
A 450 L 50 L of stock (2 mg/mL) 200 g/mL
B 400 L 100 L of vial A dilution 40 g/mL
C 250 L 250 L of vial B dilution 20 g/mL
D 250 L 250 L of vial C dilution 10 g/mL
E 250 L 250 L of vial D dilution 5 g/mL
F 250 L 250 L of vial E dilution 2.5 g/mL
G 300 L 200 L of vial F dilution 1 g/mL
H 250 L 250 L of vial G dilution 0.5 g/mL
I 500 L -- 0 g/mL
11. Prepare BCA working reagent by mixing 25 parts of micro BCA reagent MA, 24 parts
Reagent MB with 1 part of Reagent MC (25:24:1, Reagent MA:MB:MC)
12. Pipette 50 L of each standard or unknown sample into a microplate well
13. Add 50 L of working reagent to each well and mix plate thoroughly on a plate shaker
for 30 seconds
14. Incubate at 37oC for 2 hours
15. Cool plate to room temperature
16. Measure the absorbance at or near 562 nm on a plate reader
17. Subtract the average 562 nm absorbance reading of the Blank standard replicates from
the 562 nm reading of all other individual standard and unknown sample replicates
18. Prepare a standard curve by plotting the average Blank-corrected 562 nm reading for
each BSA standard versus its concentration in g/mL. Use the standard curve to
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determine the protein concentration of each unknown sample. The linear working range
is 2-40 g/mL.
III. Protein electrophoresis
19. Cast 10% resolving gel by combining the reagents in the following order:
a. 7.9 mL deionized H2O
b. 6.7 mL 30% acryl/bis
c. 5 mL resolving gel buffer
d. 200 L 10% SDS
e. 200 L of 10% APS
f. 8 L TEMED
g. Mix gently on ice and immediately pour into the gel cassette. Fill the gel cassette
up to 1 cm under the comb.
h. Overlay the gel with 0.5% SDS
i. Allow the gel to polymerize for 45-60 minutes
j. Rinse the gel completely with deionized H2O to remove SDS
k. Remove excess water with filter paper
20. Cast 5% stacking gel by combining:
a. 76.8 mL deionized H2O
b. 1.7 mL 30% acryl/bis
c. 1.25 mL stacking gel buffer
d. 100 L 10% SDS
e. 100 L of 10% APS
f. 10 L TEMED
g. Mix gently on ice and immediately pour on top of the polymerized resolving gel
h. Place gel comb into the cassette
i. Overlay the gel with 0.5% SDS
l. Allow the gel to polymerize for 45-60 minutes
m. Rinse the gel completely with deionized H2O to remove SDS
n. Wrap the gel with plastic wrap and then store in humidifying chamber at 4oC or
proceed to protein electrophoresis
21. Prepare 1x running buffer by combining:
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a. 100 mL 10x running buffer
b. 10 mL 10% SDS
c. 890 mL Deionized H2O
d. Warm buffer to room temperature
22. Boil a beaker of water to 95%
23. Dilute samples to the desire concentration with 1x lysis buffer and 5x loading dye. Keep
samples on ice at all time. (e.g. For 60 g/ 40 L in a lane, combine X L of sample, 8
L of 5x loading dye and 40 L - 8 L - X L of 1x lysis buffer)
24. Pipette up and down to mix
25. Boil diluted samples at 95 oC for 5 minutes
26. Cool to room temperature and spin down the samples with a bench top centrifuge
27. Load the samples to the gel (i.e. 40 l per lane for a 1.5 mm gel)
28. Load 10 l of ladder to the gel
29. Run gel with running buffer for 1.5 hour at 170 V and 0.04 A
IV. Protein transfer to membrane
30. Prepare filter pads, filter papers and membrane for protein transfer
a. Filter pads:
i. Rinse with warm tap H2O
ii. Soak in distilled H2O for 5 minutes
iii. Soak in deionized H2O for 5 minutes
iv. Soak in cold transfer buffer for at least 30 minutes
b. Filter papers
i. Soak in distilled H2O for 5 minutes
ii. Soak in deionized H2O for 5 minutes
iii. Soak in cold transfer buffer for at least 30 minutes
c. PDVF membranes (*do not touch the membrane with gloves, use tweezers)
i. Soak in distilled H2O for 5 minutes
ii. Soak in deionized H2O for 5 minutes
iii. Soak in cold transfer buffer for at least 30 minutes
31. Assemble the transfer tank on ice with stir bar according the following diagram
32. Run transfer tank at 100 V, 0.35 A for 1hour and 5 minutes
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33. In the mean while, prepare 1x TBST by combing:
a. 1 mL Tween 20
b. 100 mL 10x TBS buffer
c. Bring the volume to 1 litre with deionized H2O, store at 4oC
34. Upon completion of protein transfer, rinse membrane twice with 1x TBST
35. Membranes can be wrapped with plastic wrap and store at –20oC or proceed to
immunoblot
Figure A.1. Assembly of the protein transfer tank
V. Immunoblot
36. Rinse membrane twice with 1x TBST
37. Prepare the SNAP i.d.TM protein detection system accordingly to user’s manual
38. Prepare 1% (wt/vol) BSA with 1x TBST (i.e. the blocking agent, ~ 50 mL per membrane)
39. Dilute primary antibody with 1% BSA and store on ice (see dilution chart for details, ~3
mL per membrane)
40. Dilute secondary antibody with 1% BSA and store on ice (see dilution chart for details,
~3mL per membrane)
41. Place membranes onto SNAP i.d.TM protein detection system
42. Block membranes with 1% BSA for 20-30s
43. Add 3 mL of diluted primary antibody to each membrane and incubate for 10 minutes at
room temperature. At this point, prepare the developing machine (i.e. ensure there is
sufficient developer, fixer, water and turn on the machine) and warm the
chemiluminescence reagents to room temperature.
164
44. Wash membranes (3-5 times) with approximately 30 mL of 1x TBST. Turn on the
vacuum pump for 20-30 seconds in-between each wash to remove the reagents.
45. Add 3 mL of diluted secondary antibody to each membrane and incubate for 10 minutes
at room temperature
46. Wash membranes (3-5 times) with approximately 30 mL of 1x TBST. Turn on the
vacuum pump for 20-30 seconds in-between each wash to remove the reagents.
47. Remove the membranes to plastic tray and keep membranes in 1x TBST
VI. Chemiluminescence detection (* must be done in the dark)
48. For each membrane, mix 4 mL of Amersham ECL Plus™ Reagent A with 100 L of
Reagent B in the dark. Store the detection mixture in a tube wrapped with tin foil.
49. In the dark room, prepare three pieces of clean plastic wrap
50. Place membrane onto a clean plastic wrap
51. Pour 4 mL of detection mixture onto each membrane
52. Incubate for 1-5 minutes
53. Remove membrane with tweezers and remove excess detection reagents by tapping the
edge of the membrane on a paper towel
54. Place the membranes onto another clean plastic wrap
55. Wrap the membrane with the plastic wrap and fold the edges to prevent the membrane
from drying up
56. Place the wrapped membrane onto a cassette
57. Carefully place a piece of film on top. Expose the film what an appropriate length of
time.
58. Develop the film immediate
59. Repeat the procedure for optimal exposure time
60. Rinse (2-3 times) membranes with 1x TBST, wrap with plastic wrap and store at –80oC
(* membranes can be used again in the future with proper handling and storage)
VII. Image analysis
61. Scan the developed film
62. Load the image into Image J
63. Select “analyze” function
165
64. Select “gel” function
65. Set the location of each lane
66. Select “plot lanes”
67. Measure area of each lane by using the “wand” tool
68. Do the same with the housekeeping protein
69. Normalize protein expression with the housekeeping protein expression level
VIII. Antibody dilution chart
Primary antibody Secondary antibody
Name and supplier Dilution factor
Expected product size
(kDa)
Name and supplier
Dilution factor
GAPDH (Stressgen CSA-335E)
1:3000
36 HRP anti-mouse 1:3000
Akt (Cell signal #9272)
1:1000 60 HRP anti-rabbit 1:3000
Phosphorylated Akt (Cell signal #9271)
1:1000 60 HRP anti-rabbit 1:3000
P38 (Cell signal #9212)
1:1000 43 HRP anti-rabbit 1:3000
Phosphorylated P38 (Cell signal #9211)
1:1000 43 HRP anti-rabbit 1:3000
-SMA (Sigma A2547)
1:10000 42 HRP anti-mouse 1:3000
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A.12. Primer Sequences for PCR and qRT-PCR
Gene name and accession
number
Primer sequence Annealing temperature
( oC)
Product size (bp)
NPR-B DQ487044.1
Left primer: 5’-agcattaccgtaccctggtg-3’ Right primer: 5’-tagtgaggccggtcatcatgt-3’
60 142
CNP, M64758.1
Left primer: 5’-accgactccagca-3’ Right primer: 5’-ataaagtggccag-3’
60 103
Osteonectin, AW436132
Left primer: 5’-tccggatctttcctttgctttcta-3’ Right primer: 5’-ccttcacatcgtggcaagagtttg-3’
60 187
Osteocalcin, AW346755
Left primer: 5’-tcaaccccgactgcgacgag-3’ Right primer 5’-ttggagcagctgggatgatgg-3’
60 106
GAPDH, AF017079
Left primer: 5’-tgtaccaccaactgcttggc-3’ Right primer 5’-ggcatggactgtggtcatgag-3’
60 86
TGF-1 receptor I, AB182260.1
Left primer: 5’-gacggcattccagtgtttct-3’ Right primer: 5’-tgcacatacaaatggcctgt-3’
60 169
TGF-1 receptor II, EF396957.1
Left primer: 5’-cagggaagaacgttcatggt-3’ Right primer 5’-ccaaccaaagctgagtccat-3’
60 128
167
Appendix B B. Preliminary Data
B.1. The Effect of Statins on the Expression of CNP by VICs Objective: Statins are lipoprotein-lowering agents and are potential therapeutics for CAVD.
Intriguingly, statins display similar effects as CNP on the differentiation of VICs, inhibiting
myofibroblast120, 233 and osteoblast differentiation of VICs in vitro92. This prompts the question
of whether there exists a possible molecular association between CNP and statins, leading to
similar biological effects on VICs. We therefore evaluated the expression of CNP in VICs
treated with or without simvastatin as a means to provide a mechanistic explanation of statin-
mediated protective effects on VICs.
Methods: The effect of statin treatment on CNP expression by VICs was evaluated. Simvastatin
was activated prior to use by alkaline hydrolysis with NaOH and ethanol273. Cells were cultured
in complete media or calcifying media with (1 M) or without activated simvastatin for up to 14
days. Morphological changes were evaluated with bright field microscopy. RNA was extracted
after three days of treatment and qRT-PCR was performed with primers for CNP (Accession
number: M64768, forward primer: 5’-accgactccagca-3’ and reverse primer: 5’-ataaagtggccag-3’).
Transcriptional expression was quantified by the comparative Ct method as previously described.
Results: A three-day treatment with simvastatin significantly upregulated CNP transcript
expression in cells cultured in complete media (Figure B.1). Statin treatment had no detectable
effect on CNP transcript expression in cells cultured on calcifying media (Figure B.1), despite its
ability to inhibit aggregate formation after 14 days in culture (Figure B.2: A, B and D).
168
Figure B.1. Expression of CNP transcript after three days of simvastatin treatment
A relative gene expression level of less than one indicates lower exprssion with simvastatin
treatment relative to that without treatment. * P < 0.05.
Discussion: Similar to CNP, statins have recently been shown to suppress aggregate formation
that is associated with myofibroblast or osteoblast differentiation of VICs in vitro. We found that
expression of CNP by VICs cultured in calcifying media was not augmented by simvastatin,
despite the observed inhibition of calcific aggregate formation with simvastatin treatment. These
data suggest that statins might regulate osteogenic differentiation of VICs via a signaling
pathway independent of CNP. Statins could interfere with other signaling pathways that regulate
osteogenic differentiation of VICs. For instance, statins promote the breakdown of extracellular
ATP to adenosine and trigger signaling via the P1 purinergic receptor, which leads to the
inhibition of osteogenic differentiation of VICs in vitro73. In contrast, we identified an
upregulation of CNP expression by simvastatin, when VICs were cultured in conditions that
favoured myofibroblast differentiation. Although this result is intriguing, additional investigation
is necessary to further identify if CNP signaling mediates, in part, the anti-myofibrogenic effect
of statins. Whether CNP signaling pathway interacts with the HMG-CoA reductase pathway has
yet to be investigated, but statins have been shown to decrease aggregate formation associated
with myofibroblasts independent of the HMG-CoA reductase pathway120. While little is known
regarding the regulation of CNP production by VICs, CNP expression by endothelial cells has
been studied extensively274. It has been shown that oxidized LDL and its extracted lipids reduce
secretion of CNP by cultured vascular endothelial cells. Various in vitro studies suggest that
CNP could act to suppress the atherogenic activity of both oxidized LDL and the bioactive
0.0
0.5
1.0
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Complete media Osteogenic media
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lativ
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/un
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lati
ve C
NP
exp
ress
ion
(A
U)
*
Complete media Calcifying media
0.0
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1.0
1.5
2.0
2.5
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Complete media Osteogenic media
Re
lativ
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xpre
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n(T
rea
ted
/un
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ate
d)
Re
lati
ve C
NP
exp
ress
ion
(A
U)
*
Complete media Calcifying media
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NP
exp
ress
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(A
U)
*
Complete media Calcifying media
169
lysophospholipids275, 276. Lipophilic signaling molecules known to be associated with high-
density lipoprotein (HDL) such as sphingosine-1-phosphate have also been showed to suppress
CNP/NPR-B signaling277. Data from these studies suggest a close relation between lipoproteins
and CNP signaling, which should be investigated in the future in order to improve our
fundamental understanding of CNP and its involvement in valve cell biology.
B.2. Culturing Primary VICs on Polyacrylamide Substrates
Objective: An alternative cell culture system that can be fine-tuned to a wider range of stiffness
was implemented for culturing of VICs. Previously, we found poor adhesion of primary VICs to
polyacrylamide (PA) substrates coated with monomeric collagen. It is evident that VICs adhere
well on fibrillar type I collagen matrices, and therefore we tested if coating PA substrates with
thin fibrillar type I collagen matrices could improve the adhesivness of primary VICs to the PA
substrates.
Method: The process to fabricate the PA gels was described in the study by
Khatiwala et al132. Briefly, stock solutions of 40% acrylamide and 2% bis-acrylamide (Bio-Rad
Laboratories) were used. Different volumes of acrylamide and bis-acrylamide were mixed with
sterile de-ionized water and 10 M HEPES (pH 7.5). The ratio of acrylamide and bis-acrylamide
determines the stiffness of PA gels. Next, photoinitiator (10% ammonium persulfate; Bio-Rad
Laboratories) and radical stabilizer TEMED (Bio-Rad Laboratories) were added at 1/200 volume
and 1/2000 volume respectively. Immediately, the solution was syringe filtered using a 0.22 μm
filter. The mixture was pipetted onto an adhesive film and then covered with a surfasil-treated
top coverslip. The gels were polymerized in sterile biosafety cabinet for 10-15 minuties. Once
polymerized, the top cover slips were removed and the gels were surface functionalized with N-