1. health and fitness
2. disease

MATERIALS AND METHODS
All reagents were purchased from Sigma-Aldrich unless otherwise specified.
Animals – C57BL/6J mice (wild-type, WT) or matrix Gla protein (MGP)-deficient (Mgp-/-, KO)
mice up to 4.5 weeks of age were used in this study. Mgp-/- mice were courteously provided by
Gerard Karsenty, Columbia University, New York, New York 1. Mice were genotyped using
standard PCR. All procedures were approved by the institutional animal care and use
committee at the University of Maryland Medical School and conducted in compliance with
National Institutes of Health guidelines for the care and use of laboratory animals.
Cell culture and luciferase assay – Primary mouse vascular smooth muscle cells (VSMCs) from
C57BL/6J (WT) or Mgp-/- (KO) mice were obtained by a modification of the explant method
originally described by Ross 2. Smooth muscle cell identity was confirmed by positive
immunostaining for α-smooth muscle actin according to the above procedure (data not shown).
For all experiments, primary cells were used between passages 2 and 4. For Wnt16 knockdown
studies, WT VSMCs were treated with lentiviral particles containing either shRNA to Wnt16 or
control scrambled shRNA (Santa Cruz Biotechnology). For luciferase studies in primary VSMCs,
replicates of WT or KO primary cells were infected with lentivirus expressing either a Notchdependent luciferase reporter under the control of RBP-J promoter or GFP under the control of
a constitutive (CMV) promoter (as a control for infection efficiency).
For all other luciferase analyses, the A10 clonal embryonic rat aortic smooth muscle cell line
(ATCC; Manassas VA) was used. VSMCs were maintained in complete growth medium [DMEM
supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin
(Invitrogen)]. Stable pathway-specific luciferase reporter cell lines were established by
transducing A10 cells with transcription response element (TRE)-responsive Cignal Lentiviral
luciferase reporter constructs (SA Biosciences) , according to manufacturer’s protocol, followed
by a 2-week selection with puromycin (10 g/mL). The BMP/TGF-dependent luciferase
reporter cell line was established by transducing A10 cells with the SMAD-responsive luciferase
reporter, Notch-dependent luciferase reporter cell line with the RBP-J-responsive reporter, and
Wnt-dependent luciferase reporter cell line with the TCF/LEF-responsive reporter. Luciferase
activity in whole cell lysates was measured in a 96-well plate luminometer (Harta Instruments,
Bethesda MD) using the Promega Luciferase Assay Kit and was normalized to the total lactate
dehydrogenase (LDH) present in whole cell lysates, measured using a commercial LDH activity
kit (BioVision, San Francisco CA). LDH activity in the culture medium was also measured to
determine cell viability.
VSMC chondrogenesis in micromass cultures – To induce chondrogenic transformation, primary
mouse VSMCs or rat A10 reporter VSMCs were seeded as high density micromasses 3 (2.5x105
cells in 10 L volume) in DMEM or DMEM-S [DMEM supplemented with 10−7 mmol/L
dexamethasone, 0.1 mmol/L ascorbic acid (Wako Chemicals, VA), 1% insulin, transferrin,
selenium (ITS) premix (BD Biosciences, NJ)] containing 1% fetal bovine serum (Hyclone) and
1% Pencillin–Streptomycin (Invitrogen). Recombinant mouse TGF-1 (10 ng/mL), TGF-2 (10
ng/mL), or TGF-3 (10 ng/mL) (ProSpec, NJ), recombinant mouse Noggin (10 ng/mL) (R&D
Systems), LDN193189 (5 nmol/L) (Cayman Chemical), SB431542 (100 nmol/L) (Cayman
Chemical), LY2157299 (60 nmol/L) (Cayman Chemical), or N-[N-(3,5-Difluorophenacetyl-Lalanyl)]-S-phenylglycine t-Butyl Ester (DAPT, 250 nmol/L) (EMD Biosciences) were added as
described in the text.
Medium was changed twice a week for up to 14 days. Sulfated glycosaminoglycan (GAG)
synthesized by VSMCs in micromass cultures was detected by fixing micromasses in 4% PFA
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and staining with 1% Alcian blue (8GX) dissolved in 0.1 mol/L HCl, according to standard
protocols 4. For quantitative analysis, Alcian blue was extracted with 4 mol/L guanidine
hydrochloride and absorbance at 590 nm was measured using an Optima spectrophotometer
(PolarStar). GAG was then normalized to relative cell number. Cell number was estimated using
either crystal violet to stain cell nuclei [(0.25% crystal violet dissolved in 3% acetic acid)
extracted with Sorenson’s solution (30 mmol/L sodium nitrate, 0.02 mol/L HCl, 50% ethanol)
and measured at 540 nm] or WST [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt] substrate to measure total intracellular LDH
activity (Dojindo). Cytotoxicity in micromass cultures was monitored by measuring LDH release
into culture medium using a commercial LDH activity kit (Biovision).
Wnt16 expression vector – Untagged human Wnt16 expressed from pCDNA3.1/v5-His-TOPO
with a stop codon introduced between the open reading frame of Wnt16 and the His sequence
was a kind gift from Professor Dell’Acci (Queen Mary University of London). For all studies on
the effects of Wnt16 on chondrogenesis, Cos-7 cells were transfected with Wnt16 plasmid
(pWnt16) or mock transfected without plasmid and then DMEM-S without additional growth
factors was pre-conditioned on either the mock- or pWnt16-transfected Cos-7 cells. Secretion of
Wnt16 into the conditioned medium was determined by Western blot and activity of Wnt16 in the
conditioned DMEM-S was verified by luciferase activity assay with a Wnt-responsive reporter.
Real-time PCR – mRNA was isolated from VSMCs or whole mouse aortae. Tissue from both
genders was analyzed as a combined sample. Quantitative real-time PCR was performed
according to the standard protocol with EVA green chemistry in a CFX96 thermocycler (BioRad) using the primers in Tables 1 and 2. Relative changes in gene expression were calculated
by the Ct method using Microsoft Excel. By this method, gene expression within individual
samples was first normalized to housekeeping genes in the same sample and then expressed
as fold change compared to a control sample set as =1.0. RNeasy kit (Qiagen) was used ofr
mRNA isolation and first strand synthesis was performed using the Maxima RT kit (ThermoFisher) according to manufacturer’s instructions in a DYAD thermocycler (MJ Research).
Table 1: Mouse genes analyzed by real-time PCR
Gene Target
Accession #
Wnt16
NM_053116
Chondrogenic markers
Sox9
NM_011448
Collage type II NM_031163
(Col II)
Aggrecan
NM_007424
(Agg)
Transglutamin NM_009373
ase 2 (TG2)
Smooth muscle markers
Calponin
NM_009922
Myosin heavy NM_001161775
chain (MHC)
Sm22-alpha
NM_011526
(sm22a)
Smooth
NM_007392
muscle actin
(smAct)
Forward primer
cggcatgtggttcagcagaaagtt
Reverse primer
tcatggctagcaggactctgcttt
gttgtaacaccagcagcgtcaag
caccgaaagtttaagcacaccca
tgacatactccactttggccacct
aaataaccctgcccacactcttg
atgccacaagtcacagaaaccacg
aaggcagtcacagcattgttgagc
aggtgtccctgaagaacccacttt
ttccacagacttctgctccttggt
ctgcctatagggttacggtttg
cggcaactggtatccaatct
gggacacccagttctatgttg
gtctctctcatccgcatacttg
ctaatggctttgggcagtttg
ctgtctgtgaagtccctcttatg
tctttcattgggatggagtcag
gacaggacgttgttagcataga
2
Osteogenic markers
Bone
NM_008318
sialoprotein
(BSP)
Osteopontin
NM_001204201
(OPN)
Notch ligands/receptors
Notch1
NM_008714
Notch2
NM_010928
Delta-like 1
NM_007865
(Dll1)
Jagged 1
NM_013822
(Jag1)
Jagged 2
NM_010588
(Jag2)
Notch targets
Hey1
NM_010423
Hey2
NM_013904
Hes1
NM_008235
NM_001080927
RBP-J
Housekeeping genes
Ribosomal
NM_009078
protein L19
beta-actin
NM_007393
tggtgctggtgccgttgac
aatggcctgtgctttctcgatga
tggacgacgatgatgacgatgat
ggctgccctttccgttgttg
tgatggcacaactccactgatcct
aacttgctcaagaagcaatcgccc
acggagaaggttgctctgtgttct
gagcacaacagcagcatccacatt
aaatggcaagggcaaatggagagg
tcatcacaccctggccagacagatt
caaatgagtgcgaggccaaacctt
agccaggaaggcaatcacagtagt
ctggaagggcatcaactgccaaat
acacacactggtacccattgacca
gcgcggacgagaatggaaa
aaggctactttgatgcccatgctc
ccagccagtgtcaacacga
ttctggctctctgggcttctgaaa
tcaggtgatccacagtcatctg
acctagccacttctgtcaagcact
aatgccgggagctatctttct
gagtgaaagcagcaacgctggaaa
aagaggaagggtactgccaatgct
tgaccttcaggtacaggctgtgat
taatttctgaatggcccaggtct
ctggctgcctcaacacctcaa
Table 2: Rat genes analyzed by real-time PCR
Gene Target
Wnt16
Ribosomal
protein L19
Accession #
NM_001109223
NM_031103
Forward primer
gatgtccagtacggcatgtggt
agcacatccacaaactgaaggca
Reverse primer
catggctagcaggactctgctt
cgctttcgtgcttccttggtc
Immunostaining & histology – For immunohistochemistry analyses, frozen 10 m sections of
freshly-dissected aortas, non-perfusion fixed in 4% paraformaldehyde, were labeled using a
fluorescein isothiocyanate (FITC)-conjugated anti--smooth muscle actin mouse monoclonal
antibody (1:200; Abcam), and a rabbit anti-Wnt16 antibody (1:50; Santa Cruz), a rabbit
polyclonal antibody against phospho-Smad 1/5 (1:100; Cell Signaling Technologies), or a rabbit
polyclonal antibody against phosphor-Smad2 (Ser465/476) (1:100; One World Laboratory, San
Diego, CA) overnight at 4 degrees. Rabbit primary antibodies were visualized using a goat antirabbit secondary antibody conjugated to Dylight-555 (1:400; Jackson Immunoresearch). Nuclei
were counterstained with DAPI. Images were collected using a Leica DMIL inverted microscope
equipped with a SPOT RT3 real-time CCD camera (Diagnostic Instruments). As a positive
control for phospho-Smad 1/5 labeling, sections of wild-type mouse developing limb bud and
vertebrae from day 0 (newborn) pups were also stained. For histologic analysis of aortae,
sections were stained for proteoglycan deposition using Alcian blue stain and for calcified matrix
using von Kossa silver nitrate method, according to standard protocols 4.
Western blot – Aortic tissue was cut into small pieces on dry ice and lysed by freeze-thaw
cycles in RIPA buffer containing EDTA-free Protease and Phosphatase Inhibitors (Thermo
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Fisher). For VSMCs, cells were lysed in RIPA buffer containing Protease/Phosphatase inhibitors
at 4 degrees. Denatured 40 g protein samples were separated by SDS-PAGE, transferred to
PVDF membranes (Bio-Rad) and Western blot was performed using the standard protocol.
Primary antibody was rabbit anti-Wnt16 (1:1000, Santa Cruz Biotechnology). Proteins were
detected using HRP-conjugated secondary goat anti-rabbit antibody (1:3000, Millipore). As a
loading control, membranes were incubated in HRP-conjugated mouse anti-GAPDH (1:35,000).
Signal was visualized with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher).
RNA deep sequencing – Rat A10 VSMCs were treated with lentiviral particles containing either
shRNA to MGP or control scrambled shRNA (Santa Cruz Biotechnology). RNA was prepared
using Qiagen RNeasy kit. The samples were sequenced using the Illumina HiSeq platform
rendering 101 base pair paired end reads populated into separate FASTQ format files. The
reads obtained from the sequencing platforms were aligned to the Rattus norvegicus genomic
reference sequence for each of the sequencing datasets using the TopHat v1.4 read alignment
tool 5. In the alignment phase, up to two mismatches per 25 bp segment were allowed and
reads that aligned to more than 20 genomic locations were removed. TopHat alignments were
used to generate read counts for each gene in the reference genome annotation using HTSeq,
and the counts generated by HTSeq were used to generate the Differential expression results
using the R package DESeq 6.
Statistical analysis – The data are presented as mean ± standard error of the mean (SEM). For
experiments containing 2 groups, significance was determined by comparison using WilcoxonMann-Whitney test. For experiments containing more than 2 groups, Levene’s test was used to
determine equality of variance (homoscedasticity) followed by 1-way or 2-way analysis of
variance (ANOVA) and Tukey-Kramer post-hoc analysis for comparison between groups. A pvalue of < 0.05 was considered to be statistically significant. *, p <0.05; **, p < 0.01; ***,
p<0.001.
SUPPLEMENTARY DATA
MGP deficiency is associated with activation of the TGF signaling pathway in vitro and in vivo
To determine whether MGP deficiency in VSMCs causes autocrine activation of TGF signaling,
expression of MGP in the A10 VSMC line was down-regulated with shRNA which causes an
80% reduction in MGP protein 7. High throughput mRNA deep sequencing of samples from
VSMCs with reduced MGP levels and control cells transfected with scrambled shRNA identified
a greater than 2-fold change in expression in 55 genes including a 60-fold reduction in MGP
mRNA. Ingenuity Pathway Analysis of a mechanistic network of TGF signaling with 16 other
pathways detected significant changes in 33 genes dependent on TGF (Supplemental Fig. IV)
indicating induction of the TGF signaling pathway in VSMCs with reduced levels of MGP. In
agreement with this, activated phosphorylated Smad2 protein is detected by
immunohistochemistry in the cartilaginous metaplasia of MGP-null arteries (Supplemental Fig.
V, A). Of note, the high throughput mRNA deep sequencing analysis did not identify activation
of the BMP signaling pathway by down-regulation of MGP, and we did not detect Smad1/5
phosphorylation which would be indicative of the activation of BMP signaling (Supplemental Fig.
V, B). We conclude from these data that downregulation of MGP itself is sufficient to stimulate
the TGF signaling in VSMCs, further supporting the hypothesis that elevated endogenous
active TGF growth factors likely drive chondrogenic transformation of MGP-null VSMCs.
Wnt16 stabilizes contractile phenotype in VSMCs
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In wild-type VSMCs infected with lentivirus expressing shRNA to Wnt16, the expression of
contractile markers indicative of differentiated VSMCs was significantly reduced compared to
wild-type cells exposed to control scrambled shRNA (Supplemental Fig. VIII, A). Similarly,
expression of the contractile smooth muscle markers was restored by exogenous Wnt16 in
TGF3-treated VSMCs (Supplemental Fig. VIII, B). Since both chondrogenic and osteogenic
transformations of VSMCs can contribute to vascular calcification, we also analyzed expression
of the osteogenic markers in micromasses. Specific markers of mature osteoblasts, bone
sialoprotein and osteopontin were not affected by either TGF3 or Wnt16 (Supplemental Fig.
VIII, C), suggesting that TGF induced VSMC transformation toward a chondrogenic rather
than osteogenic lineage in the absence of Wnt16. Endpoint cell numbers remained similar in
both mock-treated and Wnt16-treated micromasses (Supplemental Fig. VIII, D-E).
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