JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO.

7, 2020

ª 2020 THE AUTHORS. PUBLISHED BY ELSEVIER ON BEHALF OF THE AMERICAN

COLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER

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PRECLINICAL RESEARCH

Paclitaxel Drug-Coated Balloon

Angioplasty Suppresses Progression and
Inflammation of Experimental
Atherosclerosis in Rabbits
Mohammed M. Chowdhury, MB CHB, PHD,a,d,* Kanwarpal Singh, PHD,b,* Mazen S. Albaghdadi, MD, MSC,a
Haitham Khraishah, MD,a,e Adam Mauskapf, BS,a Chase W. Kessinger, PHD,a Eric A. Osborn, MD, PHD,a,e
Stephan Kellnberger, PHD,a Zhonglie Piao, PHD,b Christian L. Lino Cardenas, PHARMD, PHD,a Madeleine S. Grau, BA,a
Michael R. Jaff, DO,f Kenneth Rosenfield, MD,a Peter Libby, MD,g Elazer R. Edelman, MD, PHD,g,h
Mark E. Lindsay, MD, PHD,a Guillermo J. Tearney, MD, PHD,b,c,y Farouc A. Jaffer, MD, PHDa,b,y

VISUAL ABSTRACT

Chowdhury, M.M. et al. J Am Coll Cardiol Basic Trans Science. 2020;5(7):685–95.

From the aCardiovascular Research Center, Division of Cardiology, Massachusetts General Hospital, Harvard Medical School,
Boston, Massachusetts; bWellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston,
Massachusetts; cDepartment of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts;
d
Department of Vascular and Endovascular Surgery, Department of Surgery, Addenbrooke’s Hospital, University of Cambridge,

ISSN 2452-302X https://doi.org/10.1016/j.jacbts.2020.04.007

686 Chowdhury et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 7, 2020

Paclitaxel-DCB Angioplasty Suppresses Inflammatory Plaque Progression JULY 2020:685–95

ABBREVIATIONS HIGHLIGHTS
AND ACRONYMS
 Restenosis limits the efficacy of PTA or stent treatment of atherosclerosis in peripheral and coronary artery disease.
 Paclitaxel DCBs effectively reduce restenosis; however, recently, their overall safety profile has been called into
2D = 2-dimensional
question.
CSA = cross-sectional area
 In an in vivo molecular-structural imaging study of 25 rabbits with experimental atherosclerosis, DCB-PTA, plain
DCB = drug-coated balloon
PTA, or sham-PTA was investigated using serial intravascular NIRF-OCT and IVUS.
EEM = external elastic  DCB-PTA reduced lesion inflammation on NIRF-OCT and halted lesion progression on IVUS, compared with PTA or
membrane
sham-PTA.
IVUS = intravascular
 These findings indicated the potential for DCBs to serve effectively and safely as a regional anti-atherosclerosis therapy.
ultrasound

NIRF = near-infrared
fluorescence SUMMARY
OCT = optical coherence
tomography
Paclitaxel drug-coated balloons (DCBs) reduce restenosis, but their overall safety has recently raised concerns.
PAD = peripheral arterial
This study hypothesized that DCBs could lessen inflammation and reduce plaque progression. Using 25 rabbits
disease
with cholesterol feeding- and balloon injury-induced lesions, DCB-percutaneous transluminal angioplasty
PAV = percent atheroma
volume
(PTA), plain PTA, or sham-PTA (balloon insertion without inflation) was investigated using serial intravascular
near-infrared fluorescenceoptical coherence tomography and serial intravascular ultrasound. In these ex-
PB = plaque burden
periments, DCB-PTA reduced inflammation and plaque burden in nonobstructive lesions compared with PTA or
PTA = percutaneous
transluminal angioplasty sham-PTA. These findings indicated the potential for DCBs to serve safely as regional anti-atherosclerosis
PTX = paclitaxel
therapy. (J Am Coll Cardiol Basic Trans Science 2020;5:685–95) © 2020 The Authors. Published by Elsevier on
behalf of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND
TAV = total atheroma volume
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Cambridge, United Kingdom; eCardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,
Massachusetts; fDepartment of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts;
g
Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; and the hInstitute for
Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts. *Drs. Chowdhury and Singh
contributed equally to this work and are joint first authors. yDrs. Tearney and Jaffer contributed equally to this work and are joint
senior authors. Juan Granada, MD, served as Guest Editor for this paper. Dr. Chowdhury is supported by fellowships from the
Royal College of Surgeons of England, the British Heart Foundation (BHF; FS/16/29/31957), the University of Cambridge (Parke-
Davies Fellowship Award), and the President’s Early Career Award from the Circulation Foundation (UK Vascular Charity–
awarded to Mr P. Coughlin, Vascular Surgery Department, University of Cambridge). Dr. Jaffer is supported by the National In-
stitutes of Health (NIH) (grants R01 HL122388, R01 HL137913, R01 HL150538) and an MGH Hassenfeld Research Scholar Award. Dr.
Osborn is supported by the NIH (grant K08 HL130465). Dr. Kellnberger is supported by an American Heart Association (AHA)
postdoctoral fellowship award (17POST33670288). Drs. Cardenas and Lindsay are supported by the Toomey Fund for Aortic
Dissection and the Fredman Fellowship. Dr. Libby is supported by the National Heart, Lung, and Blood Institute (R01HL080472),
AHA (18CSA34080399), and RRM Charitable Fund. Dr. Edelman was supported in part by a grant from the NIH (R01GM49039). Dr.
Albaghdadi has taken part in sponsored research from Kowa, Pfizer, and Sanofi. Dr. Olsen has been a consultant for Abbott
Vascular; and has been a member of the scientific advisory board for Dyad Medical. Dr. Jaff has been a noncompensated advisor to
Boston Scientific; has been a compensated advisor to Medtronic Vascular, Philips/Volcano, Biotronik, Vactronix, EFemoral, and R3
Vascular; and has been an equity shareholder in Vascular Therapies, EFemoral, and R3 Vascular. Dr. Rosenfield has been a
member of the scientific advisory board and a consultant for Abbott Vascular, Access Vascular, Boston Scientific-BTG, Volcano-
Philips, Surmodics, Cruzar, Magneto, Micell, Summa Therapeutics, and Thrombolex; holds stocks/stock options in Access
Vascular, Accolade, Contego, Endospan, Embolitech, Eximo, JanaCare, PQ Bypass, Primacea, MD Insider, Shockwave, Silk Road,
Summa Therapeutics, Cruzar Systems, Capture Vascular, and Magneto; and has been a Board Member for Viva Physicians and
National PERT Consortium. Dr. Libby has received research funding in the last 2 years from Novartis; has been an unpaid
consultant to, or involved in clinical trials for, Amgen, AstraZeneca, Esperion Therapeutics, Ionis Pharmaceuticals, Kowa Phar-
maceuticals, Novartis, Pfizer, Sanofi-Regeneron, and XBiotiech, Inc; and has been a member of scientific advisory board for
Amgen, Athera Biotechnologies, Cordivia Therapeutics, DalCor Pharmaceuticals, IFM Therapeutics, Kowa Pharmaceuticals, Olatec
Therapuetics, Medimmune, Novartis, and XBiotech, Inc. Dr. Tearney has taken part in sponsored research from VivoLight, Canon,
and AstraZeneca; has received royalties and catheter components from Terumo; and has a financial/fiduciary interest and consults
for SpectraWAVE, a company developing an OCT-NIRS intracoronary imaging system and catheter. Dr. Jaffer has taken part in
sponsored research from Canon and Siemens; has been a consultant for Boston Scientific, Abbott Vascular, Siemens, Biotronik,
Philips, and Acrostak; and holds equity interest in Intravascular Imaging, Inc. Drs. Tearney and Jaffer have received royalties for
Terumo, Canon, and Spectrawave for a patent pending with Massachusetts General Hospital.
The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’ in-
stitutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit
the JACC: Basic to Translational Science author instructions page.

Manuscript received February 12, 2020; revised manuscript received April 13, 2020, accepted April 13, 2020.
JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 7, 2020 Chowdhury et al. 687
JULY 2020:685–95 Paclitaxel-DCB Angioplasty Suppresses Inflammatory Plaque Progression

C ompared with percutaneous transluminal

angioplasty (PTA) using standard balloons,
paclitaxel (PTX)eluting, drug-coated bal-
loons (DCBs) have recently shown improved efficacy
followed by imaging. Animals were sacrificed 4 weeks
later at week 10 (Supplemental Appendix).
In brief, atherosclerosis was induced in the aortas
of male and female New Zealand White rabbits (aorta
in treating peripheral arterial disease (PAD) or coro- diameter: 3 to 3.5 mm; weight: 3 to 4 kg; Charles River
nary artery diseasebased restenosis and have Laboratories; Wilmington, Massachusetts) by local
become the primary endovascular treatment for fem- balloon arterial injury and high-cholesterol diet. After
oropopliteal PAD (1–5). However, at present, scant a 2-week, lead-in atherogenic diet (1% cholesterol, 5%
data exist regarding the effects of DCB-PTA on peanut oil, Research Diets, New Brunswick, New
atherosclerosis progression in vivo or the mecha- Jersey), the infrarenal abdominal aorta of rabbits
nisms by which it may produce its biological effects underwent balloon injury at week 2, as demonstrated
on atheroma in vivo. A recent meta-analysis of ran- previously (1).
domized trials demonstrated the potential for pacli- INTRAVASCULAR IMAGING OF PB AND PLAQUE
taxel DCBs to increase all-cause mortality (6,7); in INFLAMMATION. I V U S . IVUS images of the abdom-
contrast, several other PTX DCB studies have not inal aorta were acquired with a 40-MHz clinical
shown any differences in overall mortality (8–10). catheter by automated 0.5-mm/s pull back (iLab2,
Therefore, additional studies investigating the effects Polaris2 Software Imaging System, Boston Scientific,
of PTX DCBs on atherosclerosis could provide greater Marlborough, Massachusetts). Imaging commenced
understanding of their vascular safety profile. from the abdominal aorta at the level of the lowest
This study evaluated the effects of PTX DCB-PTA renal down to the common iliac artery. IVUS was
on inflammation and lesion progression in rabbit performed at 6 weeks both pre- and post-angioplasty
atherosclerosis in vivo and compared the results to and at the terminal 10-week imaging session.
animals treated with conventional PTA or sham-PTA.
IVUS IMAGING QUANTITATIVE ANALYSIS. IVUS
In addition to assessing plaque atheroma volume and
datasets at 6 and 10 weeks were manually co-registered
plaque burden (PB) changes by serial intravascular
to each other using fiducial landmarks, including side
ultrasound (IVUS), we assessed changes in lesion
branches and the known pull back rates of the imaging
inflammation using serial intravascular near-infrared
systems, and then analyzed by manual segmentation
fluorescenceoptical coherence tomography (NIRF-
(OsiriX, Geneva, Switzerland; and Fiji imaging v2.0.0,
OCT) molecularstructural imaging of inflammatory
National Institute of Health, University of Wisconsin,
protease activity (11,12). To further elucidate mecha-
Madison, Wisconsin) (15), in accordance with expert
nisms underlying our in vivo findings, we performed
consensus IVUS recommendations (16). The cross-
histological and molecular assays of resected lesions
sectional area (CSA) of the external elastic membrane
and investigated the effects of PTX on human aortic
(EEM) and vessel lumen were measured every 0.4 mm
smooth muscle cells in vitro.
from axial IVUS images for each rabbit at 6 and
METHODS 10 weeks, across the area that had undergone balloon
treatment (total 40 mm length, 200 images per animal
STUDY DESIGN. All animal studies were approved by [100 images per timepoint]). These measurements
the Institutional Animal Care and Use Committee permitted calculation of the atheroma CSA and PB) for
of Massachusetts General Hospital (Protocol each IVUS slice. All obtained cross-sectional slices
#2013N000015). Figure 1 shows the overview of the were analyzed and included in the image analysis re-
in vivo serial imaging study of rabbit atherosclerosis sults. On a per-slice basis (every 0.4 mm), atheroma
progression and DCB intervention or control subjects. progression was quantified as the change in IVUS PB
EXPERIMENTAL ATHEROSCLEROSIS LESION GENERATION. (D PB) between 6 and 10 weeks. The Supplemental
Lesions were generated in rabbit aortas using balloon Appendix section provides the formulas used for
injury in cholesterol-fed animals (Figure 1) as previ- NIRF and IVUS quantitative analysis.
ously performed (13,14). After baseline, 6-week sur- NIRF plaque inflammation measurements. Twenty-four
vival intravascular NIRF-OCT, and IVUS imaging, hours before NIRF-OCT imaging sessions, rabbits
rabbits randomly received 1 of 3 treatments: PTX received the protease NIRF imaging agent ProS-
DCB-PTA (n ¼ 10; IN.PACT Admiral 4.0  40 mm, ense750 VM110 intravenously (a quenched sensor
Medtronic, Santa Rosa, California; 3.5 m g/mm 2 of engineered to generate fluorescence following pro-
drug-coated concentration with urea excipient); teolytic activation by cathepsins B, L, or S; Perki-
conventional PTA (n ¼ 10; INVATEC Admiral Xtreme nElmer, Waltham, Massachusetts) (11). Automated
4.0  40 mm, Medtronic); or sham-PTA (n ¼ 5), reconstruction of dual-modal NIRF-OCT pull backs
688 Chowdhury et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 7, 2020

Paclitaxel-DCB Angioplasty Suppresses Inflammatory Plaque Progression JULY 2020:685–95

F I G U R E 1 Experimental Study Design

All 25 rabbits received 1.0% high-cholesterol diet (HCD) 2 weeks before aorta balloon injury, and for 4 weeks thereafter, followed by normal
chow for the final 4 weeks. At 4 weeks after balloon injury, rabbits were intravenously injected with ProSense VM110 (400 nmol/kg). Rabbits
underwent in vivo multimodal survival near-infrared fluorescenceoptical coherence tomography (NIRF-OCT), intravascular ultrasound
(IVUS), and x-ray angiography 24 h later. Then animals randomly underwent drug-coated balloon percutaneous transluminal angioplasty
(DCB-PTA) (n ¼ 10), PTA (n ¼ 10), or sham-PTA (n ¼ 5) therapy. Four weeks later at week 10, multimodal imaging NIRF-OCT and IVUS
imaging were repeated, followed by sacrificed and ex vivo fluorescence imaging, as well as RNA and histopathological analysis. FRI ¼
fluorescence reflectance imaging; IHC ¼ immunohistochemistry; qPCR¼ quantitative polymerase chain reaction.

was performed, as previously described (17). Quanti- PTA segment (Fogarty balloon injured and then
tative NIRF data were generated using co-registered staged experimental angioplasty 4 weeks later)
OCT images to correct the NIRF signal according to (Figure 1); and 3) the balloon-injured region (Fogarty
the distance between the catheter and the lumen balloon injury only). The remaining tissue was snap
surface, and expressed as both the NIRF concentra- frozen using dry ice and 2-methylbutane, then placed
tion (nM) averaged over all the slices that included in optimal cutting temperature compound medium

the plaque and the average NIRF concentration per and stored at 80 C for later sectioning
OCT slice at 0.4 mm intervals (11). The change in NIRF and histopathology.
concentration ( D NIRF, in nM) on a per slice basis was IN VITRO STUDIES. Primary human smooth muscle
calculated as the difference between the NIRF con- cells from healthy donors were purchased from Cell
centration at 6 and 10 weeks. Applications Inc (Cat #354K-05a, San Diego, Califor-
Readers (M.M.C., K.S., M.S.A.) were blinded to the nia). Human smooth muscle cells were incubated
group assignment of each animal for all IVUS and with lipopolysaccharide 100 ng/ml in vitro for 24 h
NIRF image analyses. and then incubated with graded concentrations
MOLECULAR ANALYSIS. Following sacrifice, rabbit of PTX up to 1  105 M for 90 min. Cells then un-
aortas were perfused with cold saline and harvested. derwent RNA extraction and quantitative polymerase
Aortas were then processed, measured, and chain reaction analysis as detailed in the
sectioned. Sections were created every 0.5 cm, with Supplemental Appendix.
selected tissue from 3 regions stored in RNAlater for
subsequent RNA analysis. The 3 regions included: 1) STATISTICAL ANALYSIS AND REPRODUCIBILITY.
the normal aorta (no Fogarty balloon injury); 2) the Continuous variables were reported mean  SD,
JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 7, 2020 Chowdhury et al. 689
JULY 2020:685–95 Paclitaxel-DCB Angioplasty Suppresses Inflammatory Plaque Progression

F I G U R E 2 Representative NIRF Inflammation and IVUS Plaque Burden Imaging of DCB-PTA, PTA, and Sham-PTA Groups

2D NIRF MAP ID NIRF PLOT 2D IVUS AXIAL MAP ID IVUS PLOT

IVUS Plaque Burden (%)

50
6 wk 10 wk

NIRF Concentration (nM)

400
6 wk 10 wk
DCB- 6-week
PTA

10-week 0
0 0 mm 40
0 mm 40

B 6 wk 10 wk
NIRF Concentration (nM) 400

IVUS Plaque Burden (%)

6 wk 10 wk 50
6-week
PTA

10-week
0 0
0 40
mm 0 mm 40
NIRF Concentration (nM)

6 wk 10 wk 6 wk 10 wk
C 400

IVUS Plaque Burden (%)

50
6-week
Sham-
PTA

10-week
0
0 40 0
mm 0 mm 40

0nM 150nM

Depiction of 2D NIRF maps at 6-week and 10-week timepoints after injection (24 h prior) with ProSense VM110, comparing (A) DCB-PTA, (B) PTA, and (C) Sham-PTA
representative subjects. The area subject to angioplasty is illustrated by the red dotted box (2D NIRF map). The accompanying 1D plots show the NIRF concentration
and plaque burden by IVUS at both timepoints. Inset graphs demonstrate 6-week (blue) and 10-week (red) keys. IVUS white scale bar, 1 mm. Complete intravascular
imaging data is presented in Supplemental Figures S1 to S3. 2D ¼ 2-dimensional; 1D ¼ 1-dimensional; other Abbreviations as in Figure 1.

median (25th, 75th percentiles), or median inter- of the linear regression assumptions were performed.
quartile range (IQR), as appropriate. Normality was A log-transformation to the D NIRF as an outcome
assessed visually using histograms and Q-Q plots, and variable was performed to improve the model fit.
formally using the Shapiro-Wilk test. The nonpara- Findings are reported as a percentage change derived
metric Kruskal-Wallis test compared the differences from geometric mean ratios. For all regression exper-
among DCB-PTA, PTA, and sham-PTA groups, and the iments, sensitivity analyses were performed using
Mann Whitney U test compared differences between serum cholesterol and C-reactive protein levels at
DCB-PTA and PTA groups. Regression analyses using endpoint (week 10), instead of baseline. We also
simple and multiple linear regression models were examined correlations between D NIRF and other
adjusted for baseline (week 6) serum cholesterol, IVUS-derived metrics using Pearson’s correlation. For
C-reactive protein, minimum lumen area, and the histological and mRNA analyses, comparisons be-
respective plaque parameters (baseline percent tween groups were made using the Kruskal-Wallis
atheroma volume [PAV], total atheroma volume H test, with a post hoc analysis using Dunn’s test
[TAV], and PB). For each model, formal statistical tests of multiple comparisons. The p values were
690 Chowdhury et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 7, 2020

Paclitaxel-DCB Angioplasty Suppresses Inflammatory Plaque Progression JULY 2020:685–95

10 receiving PTX DCB-PTA, 10 receiving uncoated

T A B L E 1 Lesion Anatomical and Inflammation Measures, and Cholesterol and CRP Levels
for DCB-PTA, PTA, and Sham-PTA Groups
PTA, and 5 rabbits receiving sham-PTA. All animals
survived until the planned sacrifice at 10 weeks and
DCB-PTA PTA Sham-PTA
(n ¼ 10) (n ¼ 10) (n ¼ 5) p Value
entered the analysis.
Total atheroma volume (mm3) Serial IVUS images of lesion anatomy and NIRF-
6 weeks 165.6 (31.0) 145.6 (21.7) 137.0 (16.4) 0.348 OCT images of inflammatory cathepsin protease
10 weeks 142.1 (45.1) 237.5 (32.0) 302.8 (164.3) <0.001* activity were acquired for all 3 groups, with repre-
D Change 19.7 (4.6) 98.0 (64.7) 183.6 (155.5) <0.001* sentative 2-dimensional (2D) NIRF maps, 1D NIRF
Percent atheroma volume (%) plots, 2D IVUS images, and 1D IVUS plots (Figure 2).
6 weeks 16.0 (4.4) 14.3 (5.1) 12.8 (5.1) 0.136
Complete rabbit data showcasing 2D NIRF maps,
10 weeks 14.0 (4.3) 21.1 (3.7) 19.5 (6.6) 0.001†
quantitative 1D plots, and corresponding 1D IVUS PB
D Change 3.0 (4.6) 5.7 (4.1) 9.6 (1.4) 0.001†
Average plaque burden (%)
plots are presented in Supplemental Figures S1 to S3.
6 weeks 16.2 (5.5) 14.2 (4.9) 12.9 (5.2) 0.179
EFFECTS OF DCB-PTA, PTA, OR SHAM-PTA OF
10 weeks 14.6 (5.3) 19.9 (4.2) 19.7 (6.3) 0.009†
ATHEROSCLEROSIS BURDEN AND INFLAMMATION.
D Change -2.5 (7.6) 5.8 (4.0) 9.8 (1.0) 0.004†
2 In all rabbits at both 6 and 10 weeks, IVUS frames
Minimal lumen area (mm )
6 weeks 5.5 (2.1) 6.3 (1.9) 6.6 (1.7) 0.600 were analyzed every 0.4 mm over each 40 mm an-
10 weeks 7.0 (1.3) 7.9 (1.9) 7.5 (3.0) 0.379 gioplasty balloon length (100 image slices per rabbit
D Change 1.1 (3.1) 0.7 (2.0) 2.6 (3.9) 0.944 per time point; a total of 5,000 IVUS images un-
Percent area stenosis, average (%) derwent assessment of lesion plaque area, lumen
6 weeks 26.9 (6.9) 23.4 (4.5) 27.6 (9.1) 0.417 area, and vessel wall area). The total lesion plaque
10 weeks 21.1 (5.6) 21.9 (7.4) 27.6 (10.4) 0.392
volume and burden was calculated over the angio-
D Change 5.2 (4.0) 1.2 (2.1) 0.0 (0.5) <0.001*
plasty region for each rabbit. The baseline 6-week,
NIRF inflammation, average (nM)
6 weeks 17.8 (10.2) 19.6 (3.9) 17.6 (10.1) 0.818
follow-up 10 week, and change in plaque characteris-
10 weeks 40.2 (43.5) 63.4 (45.7) 63.5 (57.9) 0.092 tics, cholesterol, and C-reactive protein measurements
D Change 18.3 (43.8) 44.6 (35.5) 48.4 (47.6) 0.100 are presented in Table 1. At the week 6 baseline, the 3
Total cholesterol (mg/dl) groups exhibited similar plaque structural measures,
6 weeks 1,654.0 (691.0) 2,153.0 (867.0) 1,967.0 (79.0) 0.304 including PAV, TAV, and PB (p ¼ 0.348, p ¼ 0.136, and
10 weeks 439.5 (310.0) 572.5 (634.0) 214.0 (133.0) 0.051
p ¼ 0.179, respectively). Similarly, the 3 groups had
D Change 1,082.0 1,620.0 1,668.0 0.394
(382.0) (759.0) (756.0)
comparable baseline NIRF lesion inflammation
CRP (ng/ml) (Table 1) (p ¼ 0.818). The average percentage area ste-
6 weeks 19.4 (8.0) 26.8 (12.6) 9.6 (6.4) 0.017‡ nosis for each group (DCB-PTA, PTA, and sham-PTA)
10 weeks 22.7 (29.7) 24.6 (51.9) 10.5 (7.7) 0.038‡ was 20.8%, 22.1%, and 24.8%, respectively, which
D Change 0.4 (31.1) 1.7 (6.6) 1.7 (6.3) 0.650 indicated the presence of mildly stenotic plaques at
baseline 6-week imaging.
Values are median (interquartile range). Plaque area measurement obtained using intravascular ultrasound
(IVUS). All 6-week imaging measures were obtained from IVUS or near-infrared fluorescence-optical coherence At follow-up assessment at 10 weeks (4 weeks after
tomography (NIRF-OCT) images collected before drug-coated balloon percutaneous transluminal angioplasty
(DCB-PTA), PTA, or sham-PTA. *p < 0.001; †p < 0.01; ‡p < 0.05.
baseline imaging), DCB-PTA induced several benefits
CRP ¼ C-reactive protein. on experimental lesions, in comparison to the PTA
and sham-PTA groups (Table 1). Analyzing changes in
lesion anatomical features, DCB-PTA significantly
reduced lesion progression, and moreover, induced
2-sided, and a p value of <0.05 was considered statis-
lesion regression (DCB-PTA, PTA, sham-PTA, respec-
tically significant. Analyses were performed using
tively): AV (19.7 mm3 [4.6], 98.0 mm 3 [64.7],
Stata/IC version 15.0 (StataCorp, College Sta-
183.6mm 3 [155.5]; p < 0.001); DPAV (3.0% [4.6],
tion, Texas).
5.7% [4.1], 9.6% [1.4]; p ¼ 0.001); D average PB (2.5%
For the purposes of direct statistical analysis,
[7.6], 5.8% [4.0], 9.8% [1.0]; p ¼ 0.004). DCB-PTA also
comparative data were analyzed between the DCB-
significantly reduced lesion percent area stenosis by
PTA and PTA groups, specifically with reference to
5.2% (p < 0.001 vs. the other groups). The minimal
regression analyses. Sham-PTA data are available in
luminal area also increased over time in all groups. In
the Supplemental Appendix.
addition, DCB-PTA significantly reduced the pro-
RESULTS gression of lesion inflammation compared with the
PTA group (average NIRF cathepsin activity: 18.3 nM
A total of 25 rabbits with aortic lesions (15 male, 10 [43.8] vs. 44.6 nM [35.5]; p¼0.028) (Supplemental
female) were randomized in the study (Figure 1), with Table 1).
JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 7, 2020 Chowdhury et al. 691
JULY 2020:685–95 Paclitaxel-DCB Angioplasty Suppresses Inflammatory Plaque Progression

T A B L E 2 Assessment of DCB-PTA Versus PTA on Plaque Progression

DCB-PTA vs. PTA (Unadjusted) DCB-PTA vs. PTA (*Adjusted)

Estimate 95% Confidence Intervals p Value Estimate 95% Confidence Intervals p Value

D Total atheroma volume (mm3) 1,14.5 152.4 to 76.6 <0.001† 114.0 159.6 to 68.4 <0.001†
D Percent atheroma volume (%) 8.5 12.7 to 4.4 <0.001† 8.2 11.7 to 4.7 <0.001†
D Average plaque burden (%) 7.3 12.1 to 2.5 0.005‡ 7.0 11.6 to 2.4 0.006‡

Unadjusted and adjusted estimates comparing the changes in total atheroma value, percent atheroma value and plaque burden in DCB-PTA vs. PTA (measured as D change)
derived from generalized linear regression models with 95% confidence intervals. *Adjusted for baseline cholesterol, C-reactive protein, and minimal lumen area, and cor-
responding atheroma parameter at baseline (6 weeks). †p < 0.001; ‡p < 0.01.
Abbreviations as in Table 1.

On multivariable analyses adjusted for baseline reproduced a key feature of inflammatory athero-
cholesterol levels, C-reactive protein, minimal lumen sclerosis rather than homogeneous smooth muscle
area, and baseline IVUS parameters (TAV, PAV, and cellrich neointimal hyperplasia characteristic of
PB), DCB-PTA remained significantly more effective typical stent restenosis. Following immunohisto-
in reducing TAV, PAV, and average PB compared with chemical staining with the RAM-11 rabbit macrophage
PTA, in which all 3 measures increased (p < 0.006 for marker, and the smooth muscle cell smooth muscle
each comparison) (Table 2). Similarly, after adjust- actin marker, DCB-PTAtreated lesion sections dis-
ment for baseline cholesterol level, C-reactive protein played significantly lower RAM11þ macrophage and
level, minimal lumen area, and baseline NIRF smooth muscle actin markerþ area than PTA- or
inflammation, treatment with DCB-PTA significantly sham-PTA-treated regions (45 to 50 sections analyzed
reduced the average plaque NIRF inflammatory per group for RAM11; p < 0.001 [Figures 3A and 3B],
cathepsin activity signal by 48.1% (95% confidence and 25 to 30 sections analyzed per group for smooth
interval: 69.45 to 12.0; p ¼ 0.018) (Table 3) muscle actin; p < 0.001 [Figures 3C and 3D]).
compared with the PTA group. To investigate the effects of DCB-PTA on RNA
For all regression models, sensitivity analyses us- transcripts, a subgroup of resected aortic lesions from
ing endpoint week 10 levels of C-reactive protein, every rabbit underwent RNA extraction and quanti-
cholesterol, and minimum lumen area produced the fication of cathepsin subtype (B, L, S), along with
same inference (statistical files in Supplemental tumor necrosis factora and interleukin-1b gene
Appendix). expression. Lesions treated with DCB-PTA exhibited
RELATIONSHIP BETWEEN PLAQUE INFLAMMATION significantly reduced expression of all measured
AND PB OVER TIME. To further assess the relation- transcripts (p < 0.001) compared with lesions from
ship between changes in lesion inflammation and the PTA and sham-PTA groups, which corroborated
changes in PB after angioplasty, we examined the
association between the change in NIRF concentra-
tion and change in IVUS measures of lesion for each
T A B L E 3 Assessment of DCB-PTA Versus PTA on NIRF Plaque Inflammation,
slice by pooling data for both angioplasty groups and Relationships Between Plaque NIRF Inflammation and Plaque Progression
(DCB-PTA plus PTA groups). Across all pooled slices,
Unadjusted and Adjusted Regression Estimates Comparing the Percentage Change
the D NIRF lesion inflammation correlated positively Difference in D NIRF inflammation Between DCB-PTA vs. PTA (measured as D change)
with D TAV, D PAV, and D PB (r ¼ 0.50, r ¼ 0.49, and % Change (95% CI) p Value
r ¼ 0.46, respectively; p ¼ 0.02; p ¼ 0.03, and D NIRF (unadjusted) 52.4 (72.7 to 16.1) 0.013†
p ¼ 0.04, respectively) (Table 3) from week 6 D NIRF (*adjusted) 48.1 (69.4 to 12.0) 0.018†
to week 10.
Pearson Correlation Coefficients for D NIRF vs. IVUS-Derived Metrics (measured as D)
EFFECTS OF DCB-PTA, PTA, AND SHAM-PTA ON rp Value p Value
PLAQUE MACROPHAGE AND SMOOTH MUSCLE CELL D NIRF vs. D TAV 0.50 0.006‡
CONTENT, AND RNA TRANSCRIPTS IN VIVO AND IN D NIRF vs. D PAV 0.49 0.030†
VITRO. To gain mechanistic insight into the effects of D NIRF vs. D PB 0.46 0.041†
PTX on the artery wall, a subgroup of rabbit lesions
*Adjusted for baseline cholesterol, baseline CRP, baseline minimal lumen area, and corresponding
from PTX DCB-PTA, PTA, and sham-PTA animals un- atheroma parameter, at baseline (6 weeks). †p < 0.05; ‡p < 0.01.
derwent histopathological assessment after the week CI ¼ confidence interval; IVUS ¼ intravascular ultrasound; PAV ¼ percent atheroma volume;
PB ¼ plaque burden; rp ¼ reflecting Pearson; TAV ¼ total atheroma volume; other abbreviations
10 sacrifice. The presence of lesional macrophages as in Table 1.
(Figure 3A) supported findings that lesions
692 Chowdhury et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 7, 2020

Paclitaxel-DCB Angioplasty Suppresses Inflammatory Plaque Progression JULY 2020:685–95

F I G U R E 3 Comparison of Atheroma Macrophage and Smooth Muscle Cell Presence, and Plaque Cathepsin RNA Expression in DCB-PTA, PTA, and Sham-PTA
Subjects, as Well as in Vitro Assessment of the Effects of Paclitaxel on Cathepsin Expression in Human Aortic Smooth Muscle Cells

Lesion sections from representative DCB-PTA, PTA, and sham-PTA animals underwent (A and B) RAM-11 macrophage (n ¼ 143 sections) and (C and D) alpha-actin
smooth muscle cell (n ¼ 79 sections) immunohistochemical detection. The percent positively stained area in lesions was assessed; balloon dots are actual values per
section; bars demonstrate median (25th and 75th percentiles). (E) Gene expression normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) (housekeeping gene), displayed using comparative Ct method (2-DDCt). mRNA expression of cathepsin genes measured by quantitative polymerase chain
reaction. Verification of expression demonstrated by immunoblotting of cathepsin B, after protein extraction. Statistical comparisons made using Kruskal-Wallis H
test, with a post hoc analysis using Dunn’s test of multiple comparisons. (F) In vitro human aortic smooth muscle cells were incubated with graded concentrations of
paclitaxel, and then underwent RNA extraction to assess cathepsin transcripts. n ¼ 4, bars demonstrate median (25th and 75th percentiles). ***p < 0.001. Abbreviations
as in Figure 1.

in vivo findings that showed dampened inflammation expression of cathepsins B, L, and S mRNA transcripts
following DCB-PTA (Figure 3E). in aortic smooth muscle cells, in graded fashion
(p < 0.001) (Figure 3F).
PTX-BASED EFFECTS ON CATHEPSIN PROTEASE
TRANSCRIPT EXPRESSION IN VITRO. To ascertain DISCUSSION
whether the PTX resident in DCBs could directly exert
anti-inflammatory effects relevant to diminished Treatment with PTX DCB-PTA, but not conventional
atheroma progression, primary human aortic smooth PTA or sham-PTA, reduced both lesion inflammation
muscle cells were stimulated with lipopolysaccharide and lesion progression in experimental atheroscle-
in vitro, and then incubated with graded concentra- rosis in rabbits. These findings provided new experi-
tions of PTX for 90 min. Cells were then processed for mental evidence that PTX-based DCB treatment
mRNA analysis (quantitative polymerase chain reac- favorably affected experimental atherosclerosis,
tion; see the Supplemental Appendix). PTX reduced which would have implications for the safety of DCBs,
JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 7, 2020 Chowdhury et al. 693
JULY 2020:685–95 Paclitaxel-DCB Angioplasty Suppresses Inflammatory Plaque Progression

as well as the ability of DCBs to serve as a regional stent healing (11,12,27,28). A recent first-in-human
endovascular therapy for atherosclerosis. clinical study in patients with coronary artery dis-
Inflammation drives atherosclerosis progression ease demonstrated the safety and feasibility of
(18), and antiinterleukin-1b mediated suppression of intracoronary NIRF-OCT imaging to detect coronary
inflammation can reduce cardiovascular events in plaque 633-nmexcited NIR autofluorescence, a po-
patients (19). The present serial intravascular in vivo tential measure of intraplaque hemorrhage or oxida-
NIRF-OCT molecular imaging study revealed that tive stressinduced lipid byproducts (29). The
DCB-PTA significantly reduced inflammatory prote- present study thus provided a clinical foundation for
ase activity implicated in lesion progression NIRF inflammation imaging using ProSense VM110
compared with PTA or sham-PTA, in experimental (Perkin Elmer, Waltham, Massachusetts) (VM110 in
atherosclerosis (20). Macrophages and smooth muscle Detection of Microscopic Tumors: A Phase I Study;
cells and their products, including cathepsin pro- NCT03286062) or LUM015 (Lumicell, Inc., Wellesley,
teases, contribute to the initiation, progression, and Massachusetts), a similar NIRF cathepsin
complications of atherosclerosis (21). PTX treatment proteasesensitive agent (30), which were recently
of human primary aortic smooth muscle cells further evaluated in patients. Both NIRF-OCT catheter tech-
demonstrated a dose-dependent reduction in nology and imaging agents are positioned to enable
cathepsin mRNA transcripts. The present findings clinical NIRF inflammation imaging trials of patients
indicated that PTX exerted anti-inflammatory effects with atherosclerosis in the near future.
that underlay the observed favorable PTX DCB-PTA This study provided new experimental evidence
effects on experimental atherosclerosis. that PTX DCB-PTA could mitigate lesion inflammation
From a lesional volume perspective, PTX DCB and progression and could therefore serve safely as a
therapy induced experimental lesion regression (de- regional strategy for treating atherosclerosis. Potential
creases in TAV, PAV, average PB, and percent area clinical extensions of DCB-PTA could consider treat-
stenosis) (Table 1) in contrast to PTA or sham-PTA ment of flanking atheroma around culprit plaques in
treatment. Mechanisms underlying this observation patients already undergoing coronary or peripheral
likely include PTX-based anti-inflammatory effects as endovascular treatment. However, before extending
supported by a moderate correlation between lesion the application of PTX-DCBs, the current controversy
inflammation and lesions progression in these ex- surrounding PTX-DCBs (6–10) requires resolution; the
periments (Table 3). Additional mechanisms might endovascular community eagerly awaits further ana-
include favorable remodeling effects of the balloon lyses and new clinical and experimental data. Recent
angioplasty itself independent of PTX, because the data on the late mortality signal with DCBs remain
PTA alone group also exhibited reduced lesion pro- inconclusive; however, the long-term risks of these
gression compared with the sham-PTA group. For devices need to be balanced against their established
example, treatment of experimental atherosclerosis and sustained efficacy in improving limb-related out-
with bare metal stents stabilized rabbit plaques by comes in patient with femoropopliteal PAD.
reducing macrophage content (22,23).
Although the present rabbit model of atheroscle- STUDY LIMITATIONS. Balloon injury and severe
rosis (13,14) included a component of restenosis pa- hyperlipidemia to induce experimental atheroscle-
thology following endovascular injury, the rosis in rabbits does not fully recapitulate human
experimental pathology recapitulated several fea- atherosclerosis complexity but does reproduce some
tures of human atherosclerosis, such as the presence pathological features of human plaques. In addition,
of plaque macrophages (Figure 3). Moreover, although the model we used enabled the study of DCBs in
other animal endovascular studies of PTX clearly atherosclerosis in arteries of a similar caliber to hu-
demonstrated inhibition of neointimal proliferation man coronary and peripheral-sized vessels. Further-
after balloon injury (24–26), the present study pro- more, the investigated experimental lesions induced
vided new insights into the effects of DCBs on by balloon injury in this study did not fully recapit-
experimental lesions with features of atherosclerosis, ulate human atherosclerosis, and therefore, addi-
which demonstrated inhibition of both lesion tional DCB studies in clinical subjects are required. A
inflammation and lesion progression. future area of investigation regarding the durability
The ability to image inflammation in coronary and of DCB treatment for preventing atheroma progres-
peripheral arterysized vessels using high-resolution sion in more advanced lesions and for the longer term
intravascular NIRF molecular imaging offers a trans- are questions that exceeded the model and scope of
lational approach to understanding the mechanisms this study. Although the present study required 2
of clinical atherosclerosis progression and impaired intravascular imaging approaches (NIRF-OCT and
694 Chowdhury et al. JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 7, 2020

Paclitaxel-DCB Angioplasty Suppresses Inflammatory Plaque Progression JULY 2020:685–95

IVUS), further development of an integrated NIRF- PERSPECTIVES

IVUS (28) imaging system might provide a single
intravascular imaging approach for future athero-
COMPETENCY IN MEDICAL KNOWLEDGE: PTX
sclerosis studies.
DCBs reduce peripheral and coronary artery resteno-
CONCLUSIONS sis, but their effects on atherosclerosis have not been
fully characterized. There is recent concern regarding
PTX DCB-PTA reduced lesion inflammation and lesion
their safety profile.
progression in experimental rabbit atherosclerosis in
contrast to PTA or sham-PTA. These preclinical find- TRANSLATIONAL OUTLOOK 1: PTX DCB-PTA re-
ings supported the vascular safety and efficacy of duces preclinical lesion inflammation and lesion pro-
PTX-DCB angioplasty. gression in contrast to PTA or sham-PTA, as
ACKNOWLEDGMENT The authors thank Dr. Amy demonstrated in vivo using translatable intravascular
Mikkola, MGH Comparative Medicine Clinical Pa- molecularstructural imaging technology.
thology Laboratory, for assay of serum cholesterol
levels. TRANSLATIONAL OUTLOOK 2: Following resolu-
tion of current PTX DCB safety concerns, the present
ADDRESS FOR CORRESPONDENCE: Dr. Farouc Jaffer
study supports evaluating paclitaxel DCBs as a
or Dr. Gary Tearney, Massachusetts General Hospital, regional endovascular therapy to stabilize athero-
Cardiovascular Research Center, Simches Research sclerosis and improve long-term vascular outcomes in
Building, Room 3206, Boston, Massachusetts 02114. patients with PAD and coronary artery disease.
E-mail: [email protected] OR tearney@mgh.
harvard.edu.

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