Online Appendix for the following JACC article
TITLE: Cardiovascular and Systemic Microvascular Effects of Anti-VEGF Therapy for Cancer
AUTHORS: J. Todd Belcik, BS, RDCS, Yue Qi, MD, Beat A. Kaufmann, MD, Aris Xie, BS,
Sherry Bullens, BA, Terry K. Morgan, MD, PhD, Susan P. Bagby, MD, Ganesh Kolumam, PhD,
Joe Kowalski, BS, Jon A. Oyer, PhD, Stuart Bunting, PhD, Jonathan R. Lindner, MD
APPENDIX
Methods - Echocardiography
Echocardiography (Vevo-770, Visualsonics Inc.) was performed in the parasternal longaxis and mid-ventricular short-axis planes at 40 MHz. Images were obtained by ECG gating of
sequential M-mode sweeps for a 2-D frame rate of 1,000-1,100 Hz. Diastolic dimension of the
interventricular septum and posterior wall, and LV internal diameter at end-diastole (LVIDd) and
end-systole (LVIDs) in the minor axis were measured. Left ventricular ejection fraction (LVEF)
and LV mass were calculated from cavity and wall dimensions as previously described (Gardin,
et al.). Velocity of circumferential fiber shortening (VcF) was measured by dividing fractional
shortening by the ejection time. Peak systolic (S′) and early diastolic (E′) radial velocities in the
short-axis view were measured by tissue Doppler imaging with a sample volume placed in the
posterior endocardium. Stroke volume (SV) was measured by the product of the LV outflow
tract area and angle-corrected time-velocity integral measured by pulsed-wave Doppler (<20).
Dimension of the mid-ascending aorta was measured with M-mode imaging at a 90 angle of
incidence.
Measurements from three consecutive cardiac cycles were averaged for all
echocardiographic parameters and data were generated from 14-18 animals from each treatment
group.
2
Reference: Gardin JM, Siri FM, Kitsis RN, Edwards JG, Leinwand LA. Echocardiographic
assessment of left ventricular mass and systolic function in mice. Circ Res 1995;76:907-14.
Methods - PCR for -myosin Heavy Chain, ANP, and BNP mRNA
Total RNA was isolated from ventricle tissue (~100 mg) from 5 mice from each treatment group
by homogenization in Trizol reagent (Invitrogen) followed by chloroform extraction and ethanol
precipitation/wash according to the manufacturer’s instructions. For each sample, 1 g of
purified RNA was used as template for first strand cDNA synthesis with oligo-dT primers and
SuperScript III reverse transcriptase (Invitrogen) following the manufacturer’s standard protocol.
cDNA quantities were evaluated by quantitative real-time PCR measuring SybrGreen
fluorescence on an ABI 7900HT. Following activation of the hot start polymerase at 95° C for 10
minutes, reactions were cycled 40 times at 95° C for 15 seconds and 60° C for 1 minute.
Experimental cDNA samples were run in triplicate with quantities calculated from standard
curves generated from serial dilutions of cDNA from a control sample. Primer sequences used to
amplify transcripts were: GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) (forward: 5’TTG CCA TCA ACG ACC CCT TC -3’ and reverse: 5’- TTG TCA TGG ATG ACC TTG GC 3’) ; β-MHC (forward: 5’- CTA CAG GCC TGG GCT TAC CT -3’ and reverse: 5’- TCT CCT
TCT CAG ACT TCC GC -3’) (Nishi, et al.); ANP: (forward: 5’- CAA GAA CCT GCT AGA
CCA CC-3’ and reverse: 5’-AGC TGT TGC AGC CTA GTC C-3’) and BNP: (forward: 5’CCA GAG ACA GCT CTT GAA GG -3’ and reverse: 5’- TCC GAT CCG GTC TAT CTT G 3’) (Armstrong). Expression of cDNA was normalized to GAPDH levels to control for variation
in reaction inputs.
3
References:
Nishi H, Ono K, Horie T, et al. MicroRNA-27a regulates beta cardiac myosin heavy chain gene
expression by targeting thyroid hormone receptor beta1 in neonatal rat ventricular myocytes.
Mol Cell Biol 2011;31:744-755.
Armstrong DWJ, et al. Altered expression of the natriuretic peptide system in genetically
modified heme oxygenase-1 mice treated with high dietary salt. Mol Cell Biochem 2011;346:57.
Methods - eNOS Phosphorylation
Cardiac muscle tissue samples were weighted and homogenized in lysis buffer containing
1mM phenylmethylsulfonyl fluoride. The samples were then centrifuged and the supernatant
was evaluated for phosphorylated eNOS using an enzyme-linked immunosorbent assay
(PathScan ELISA, Cell Signaling Tech.) according to manufacturer’s instructions.
Spectrophotometric absorbance at 450 nm was determined 30 min after adding the STOP
solution.
4
Online Figure A
-MHC
Control
G6-31
1.0
0.8
0.6
0.4
0.2
1.2
mRNA Realative to GAPDH
1.2
*
1.0
0.8
0.6
0.4
0.2
0
1
2
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.0
0.0
Control
G6-31
1.4
1.4
mRNA Realative to GAPDH
mRNA Realative to GAPDH
Control
G6-31
*
1.4
BNP
ANP
0
1
2
0
1
2
Mean (±SEM) data on rtPCR for b-MHC, ANP, and BNP from the left ventricle of control and
G6-31-treated mice (n=5 for each group with each sample performed in triplicate). *p<0.05 vs
control by Mann-Whitney test. Differences in BNP were of borderline significance (p=0.09).
Online Figure B
Examples of picrosirius staining (yellow) of the myocardium using polarized light. Regions of
interstitial and perivascular collagen are illustrated on low magnification (right) whereas
interstitial collagen (perimysium) is shown on high magnification (left)
5
Online Figure C
Examples of smooth muscle -actin staining of non-capillary intramyocardial microvessels.
using polarized light. Examples are illustrative of data from a control (left) and G6-31-treated
mouse. These examples also illustrate myocyte cross sectional area differences.
Online Figure D
CD31
Fibrin
Overlay
3-D
confocal
Control
G6-31
Dual fluorescent immunohistochemistry illustrating glomerular capillary endothelium by CD31
staining (red) and fibrin deposition (green) in the glomeruli in G6-31-treated mice. Overlay by
fluorescent microscopy and 3-D confocal microscopy illustrate glomerular colocalization in the
G6-31 mice.