Yale University
Yale University
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Yale Medicine Thesis Digital Library
School of Medicine
January 2019
Inhibition Of The Akt1-Mtorc1 Axis Alters Venous Remodeling To
Inhibition Of The Akt1-Mtorc1 Axis Alters Venous Remodeling To
Improve Arteriovenous Fistula Patency
Improve Arteriovenous Fistula Patency
Arash Fereydooni
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Recommended Citation
Recommended Citation
Fereydooni, Arash, “Inhibition Of The Akt1-Mtorc1 Axis Alters Venous Remodeling To Improve
Arteriovenous Fistula Patency” (2019). Yale Medicine Thesis Digital Library. 3899.
https://elischolar.library.yale.edu/ymtdl/3899
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Inhibition of the Akt1-mTORC1 Axis Alters Venous
Remodeling to Improve Arteriovenous Fistula Patency
A Thesis Submitted to the
Yale University School of Medicine
in Partial Fulfillment of the Requirements for the
Degree of Doctor of Medicine and
Master of Health Sciences
By
Arash Fereydooni
2020
Abstract
Arteriovenous fistulae (AVF) are the most common access created for
hemodialysis, but up to 60% do not sustain dialysis within a year, suggesting a need to
improve AVF maturation and patency. In a mouse AVF model, Akt1 regulates fistula wall
thickness and diameter. We hypothesized that inhibition of the Akt1-mTORC1 axis alters
venous remodeling to improve AVF patency. Daily intraperitoneal injections of
rapamycin reduced AVF wall thickness with no change in diameter. Rapamycin
decreased smooth muscle cell (SMC) and macrophage proliferation; rapamycin also
reduced both M1 and M2 type macrophages. AVF in mice treated with rapamycin had
reduced Akt1 and mTORC1 but not mTORC2 phosphorylation. Depletion of
macrophages with clodronate-containing liposomes was also associated with reduced
AVF wall thickness and both M1- and M2-type macrophages; however, AVF patency was
reduced. Rapamycin was associated with improved long-term patency, enhanced early
AVF remodeling and sustained reduction of SMC proliferation. These results suggest
that rapamycin improves AVF patency by reducing early inflammation and wall
thickening while attenuating the Akt1-mTORC1 signaling pathway in SMC and
macrophages. Macrophages are associated with AVF wall thickening and M2-type
macrophages may play a mechanistic role in AVF maturation. Rapamycin is a potential
translational strategy to improve AVF patency.
Acknowledgements
I am eternally indebted to my incredible mentor, Professor Alan Dardik, for his
constant support and insight; he has served as an inspiring role model and showed me
what it means to be a successful surgeon-scientist. He has invested in my career and
given me opportunities I did not deserve. I am grateful to my colleagues at Dardik Lab
for their help, particularly Dr. Jolanta Gorecka for her teamwork and willingness serve as
a valuable sounding board.
I would like to also thank my clinical mentors, Dr. Cassius Ochoa Chaar and Dr.
Naiem Nassiri, for showing me what it means to be excellent academic surgeons, to
deliver the best comprehensive care to our patients, and not to be afraid to push the
envelope and advance the field of vascular surgery. Drs. Julia Chen, Christine Deyholos,
Anand Brahmandam, Robert Botta, Jason Chin and Kristine Orion, I sincerely appreciate
your teaching, mentorship and friendship. Dr. Raul Guzman, thank you for your
leadership, support and encouragement.
I would like to thank the Howard Hughes Medical Institute, the Society for
Vascular Surgery and the American Heart Association for funding my research at Dardik
Lab. I would also like to thank the Office of Student Research for their support with my
research endeavors throughout medical school.
Most importantly, my journey to become a surgeon-scientist would not be
possible without the sacrifices of my parents, Alireza and Naimeh, who unrooted their
lives and immigrated to the United States ten years ago to provide my sisters and me
with better educational opportunities. This work is dedicated to them.
Table of Contents
1. Introduction……………………………………………………………………………………………………………1
1.1. Poor Clinical Outcomes in Arteriovenous Fistulae Utilization……………………….1
1.2. Mechanisms of Fistula Maturation and Failure…………………………………………….1
1.3. Akt1 signaling in AVF maturation…………………………………………………………………4
2. Statement of Purpose and Aims……………………………………………………………………………..6
2.1. Statement of Purpose
2.2. Aims
3. Methods…………………………………………………………………………………………………………………7
3.1. Study Approval…………………………………………………………………………………………….7
3.2. Infrarenal aorto-caval fistula………………………………………………………………………..7
3.3. Confirmation of fistula patency and measurement of fistula dilation…………..7
3.4. Histology.…………………………………………………………………………………………………….8
3.5. Immunohistochemistry and Immunofluorescence……………………………………….8
3.6. Western Blot.……………………………………………………………………………………….……10
3.7. Rapamycin and clodronate treatment…………………………………………………..……11
3.8. Adenovirus treatment………………………………………………………………………………..12
3.9. Statistics.……………………………….…………………………………………………………………..12
4. Results………………………………………………………………………………………………………………….13
4.1. Reduced AVF wall thickness, extracellular matrix deposition, SMC and
macrophages with rapamycin…………………………………………………..………………………13
4.2. Reduced M1- and M2-type macrophages with rapamycin…………………………15
4.3. Reduced Akt1 and mTORC1 but not mTORC2 phosphorylation with
rapamycin…………………………………………………..………………………………….…………………17
4.4. Macrophage depletion is associated with reduced AVF wall thickness and
patency …………………………………………………..………………………………….……………………24
4.5. Rapamycin treatment is associated with reduced AVF wall thickness but
increased AVF patency..…………………………..………………………………….……………………26
4.6. Rapamycin enhances early AVF remodeling to improve patency……………….27
5. Discussion…………………………………………………………………………………………………………….31
6. Conclusion…………………………………………………………………………………………………………….36
7. References……………………………………………………………………………………………………………37
8. Appendix………………………………………………………………………………………………………………42
1
1. Introduction
1.1 Poor Clinical Outcomes in Arteriovenous Fistulae Utilization
Veins are frequently exposed to arterial environment by surgeons when creating
arteriovenous fistulae (AVF) for hemodialysis access in end-stage renal disease (ESRD).
With over half a million people affected by ESRD in the United States and a mortality of
approximately 88,000 people each year, the incidence of ESRD requiring therapy is over
100,000 new cases a year.1 An AVF, which joins a vein directly to the artery is the
preferred mode of hemodialysis access with demonstrated superior long-term results
compared to prosthetic grafts and catheter access.2 Despite the known superiority, AVF
are still far from perfect; they must mature, e.g. dilate, thicken and increase flow prior
to use. However AVF can fail to mature in ~30% of cases3 and even if matured correctly,
primary AVF failure occurs in ~35-40% in just the first year.4 These poor clinical results of
AVF reflect our imperfect understanding of how the vein adapts to the arterial
environment and clearly shows that our knowledge gap creates an unmet medical need
for novel approaches to enhance venous adaptation.4-6 The Society of Vascular Surgery
recently published enhancing AVF maturation and durability as one of its highest and
most critical clinical research priorities.7
1.2. Mechanisms of Fistula Maturation and Failure
Following AVF creation, the vein is exposed to a high flow and shear stress, low
pressure arterial environment, leading to “maturation” of both the arterial inflow and
venous outflow segments – a process necessary to sustain the high flow rates required
2
for a successful dialysis session. Adaptation of the vein to the increased flow and shear
stress requires dilation and outward remodeling of the venous wall. This process is
accomplished by a delicate balance of extracellular matrix (ECM) remodeling,
inflammation, growth factor secretion, and cell adhesion molecule upregulation in all
three layers of the venous wall.8-11
During fistula maturation, the ECM of the venous limb exhibits changes as an
adaptive response to the “arterialized” environment.12 These changes can be
categorized in to three temporal phases; early phase (breakdown), transition phase
(reorganize) and late phase (rebuild). The early phase is characterized by an increased
ratio of matrix metalloproteinase (MMP) to tissue inhibitor of metalloproteinase (TIMP),
which results in degradation of collagen and elastin scaffolds, allowing for easier cell
migration during the transition and late phases. Reorganization of scaffolds and
rebuilding of the ECM with larger non-collagenous and glycoproteins such as fibronectin
occur after the breakdown phase to allow for complete fistula maturation.13
While ECM degradation is regulated by MMP, its deposition is modulated by
transforming growth factor-β (TGF-β).14 Diverse cell types in the venous wall, such as
endothelial cells (EC), smooth muscle cells (SMC), and inflammatory cells produce TGF-β
and its expression is upregulated during both early and late phases of AVF maturation.
While local inflammation of the vessel wall is necessary for successful fistula maturation,
elevated systemic inflammatory markers predict fistula failure.9,15 Locally, macrophages
and T-cells play an important role in AVF maturation, with maturation being promoted
by M2 type macrophage and a lack of T cell activity resulting in AVF maturation failure.
3
Furthermore, presence of CD4+ T-cells in mature AVF coincides with the presence of
macrophages, and the absence of mature T-cells results in reduced macrophage
infiltration.16,17 Systemic inflammation has been shown to negatively correlate with AVF
maturation, and higher levels of C-relative protein increase the risk of AVF failure.
Further, prednisolone, a drug with anti-inflammatory properties, enhances venous
outward remodeling.18 Use of paclitaxel, a chemotherapeutic and immunosuppressive
agent, during drug-coated balloon angioplasty leads to inhibition of neointimal
hyperplasia (NIH) and has shown encouraging 6-month patency rates.19-21 However,
increased infection rates have become a major concern for paclitaxel use in AVF.22
Successful AVF maturation relies on venous wall thickening and outward
remodeling in order to support flow rates required for successful hemodialysis. AVF
failure occurs via 2 distinct mechanisms; early fistula failure occurs secondary to lack of
outward remodeling or wall thickening, while late failure occurs as a result of
development of NIH and impaired outward remodeling in a previously functional
conduit.23 Unfortunately primary maturation and patency rates of AVF remain low. Up
to 60% of AVF fail to mature by 5 months after creation, and literature shows primary
patency rates of 60% at 1 year and 51% at 2 years, with secondary patency rates of 71%
at 1 year and 64% at 2 years.5,24,25 Factors such as diabetes mellitus, peripheral vascular
disease, congestive heart failure, and older age are poor prognostic factors for
successful AVF placement.26 Furthermore, studies have demonstrated prolonged
maturation time, decreased patency, and increased early thrombosis of AVF in female
patients, differences not accounted for by smaller vein size in females.27-29
4
1.3. Akt1 signaling in AVF maturation
Erythropoietin-producing hepatocellular carcinoma (Eph) receptors with ephrins,
their ligands, play an essential role in vascular development and determine arterial
versus venous identities.30,31 Eph receptor activation leads to downstream signaling via
the PI3K-Akt pathway, resulting in cell migration and proliferation, functions critical for
venous remodeling.32,33 Specifically, Eph-B4 modulates adaptation and AVF maturation
with distinct patterns of altered vessel identity.34-36 During successful AVF maturation,
the venous limb gains expression of ephrin-B2 and has increased Eph-B4 expression,
relative to control veins, suggesting acquisition of dual arterial-venous identity.12
Although the route of ephrin-B2 signaling during AVF maturation remains unknown, it
must be membrane bound and circulating endothelial progenitor cells can be a source.37
In vivo, Eph-B4 activation attenuates Akt1 phosphorylation leading to reduced
venous wall thickening, reduced outward remodeling and improved long-term patency
rates. This was corroborated with constitutively active-Akt1 studies which lead to
increased venous wall thickening and dominant negative-Akt1 studies which lead to
reduced outward remodeling.36 Therefore, it is proposed that Eph-B4 can regulate
venous remodeling via an Akt1-mediated mechanism.36 Moreover, Akt1 expression is
upregulated during venous remodeling, both during vein graft adaptation,38 as well as
during AVF maturation, a consistent response to two different hemodynamic
environments;36 during AVF maturation, Akt1 regulates both venous wall thickening as
well as dilation.36 Mammalian target of rapamycin (mTOR) is a key regulatory protein
that integrates signals from several pathways including the Akt1 pathway to modulate
5
inflammation and coordinate cell growth and proliferation, all of which occur during
venous remodeling.39 Rapamycin, an mTOR inhibitor, is currently used for human
therapy to prevent NIH by reducing proliferation and migration of smooth muscle
cells.40,41
6
2. Statement of Purpose and Aims
2.1. Statement of Purpose
Since rapamycin inhibits Akt1 signaling, and Akt1 mediates venous remodeling,
we hypothesize that inhibition of the Akt1-mTORC1 axis in macrophages with rapamycin
alters venous adaptive remodeling in AVF.
2.2. Aims
Specific Aim I: Determine the effects of rapamycin as a downstream inhibitor of Akt1
signaling on AVF patency
Specific Aim II: Determine the effects of macrophage depletion on AVF maturation
7
3. Methods
3.1. Study Approval
All animal experiments were performed in strict compliance with federal
guidelines and with approval from the Yale University IACUC.
3.2. Infrarenal aorto-caval fistula
Mice used for this study were wild type C57BL6/J. Mice were 9–12 weeks of age
when the infrarenal aorto-caval fistulae were created as previously described;42,43 only
male mice were studied since female sex is the only predictor of non-maturation of
human AVF in some studies.44 Briefly, AVF were created by needle puncture from the
aorta into the inferior vena cava (IVC) using a 25G needle. Visualization of pulsatile
arterial blood flow in the IVC was assessed as a technically successful creation of AVF.
Following surgery, all animals were monitored daily and evaluated weekly by a
veterinarian for changes in health status.
3.3. Confirmation of fistula patency and measurement of fistula dilation
Doppler ultrasound (40 MHz; Vevo770 High Resolution Imaging System; Visual
Sonics Inc., Toronto, Ontario, Canada) was used to confirm the patency of the AVF and
to measure the diameter of the vessels as previously described.42,43 Doppler ultrasound
was performed prior to operation (day 0 values) and serially post-operatively. Increased
end-diastolic flow through the aorta and a high velocity pulsatile flow within the IVC
confirmed the presence of an AVF during post-operative studies. Patency was again
8
confirmed at time of AVF harvest by direct visualization of pulsatile arterial blood flow
into the IVC, and in all cases correlated with the ultrasound findings.
3.4. Histology
After euthanasia, the circulatory system was flushed under pressure with PBS
followed by 10% formalin and the AVF was harvested en bloc. The tissue was then
embedded in paraffin and cut in 5 μm cross sections. Hematoxylin and eosin staining
was performed for all samples. Elastin Van Gieson (EVG) staining was used to measure
intima-media thickness in 5 μm cross sections of the IVC using sections obtained 100-
200 µm cranial to the fistula. Four equidistant points around the IVC and opposite the
aortic wall were averaged in each cross section to obtain the mean AVF outer wall
thickness. Additional unstained cross sections in this same region were used for
immunofluorescence microscopy.
3.5. Immunohistochemistry and Immunofluorescence
Tissue sections were de-paraffined using xylene and a graded series of alcohols.
Sections were heated in citric acid buffer (pH 6.0) at 100 °C for 10 min for antigen
retrieval. The sections were blocked with 5% bovine serum albumin PBS containing
0.05% Triton X-100 (T-PBS) for 1h at room temperature prior to incubation overnight at
4 °C with the primary antibodies diluted in T-PBS. All the primary antibodies have been
listed in the Table 1. Sections were then treated with secondary antibodies at room
temperature for 1h using goat anti-rabbit Alexa Fluor 488 (Life Technologies), donkey
9
anti-goat Alexa-Fluor-488 (Life Technologies), or donkey anti-rabbit Alexa-Fluor-568 (Life
Technologies). Sections were stained with Slow Fade® Gold Antifade Mount with DAPI
(Life Technologies) and coverslip was applied. Digital fluorescence images were
captured and intensity of immunoreactive signal was measured using Image J software
(NIH, Bethesda, Maryland). Intensity of the merge signal was determined by applying a
color threshold selective for the appropriate signal.
Table 1. List of Antibodies
Target antigen
Vendor or Source
Catalog #
Cleaved caspase-3
Cell Signaling
9664
proliferating cell nuclear antigen
Dako
M0879
Collagen I
Novus Biologicals
NB600-408
Collagen III
Novus Biologicals
NB600-594
fibronectin
Abcam
ab2413
CD68
Bio-Rad
MCA1957
iNOS
Cell Signaling
2977S
interleukin-10
Abcam
ab9969
TNFa
Abcam
ab9635
CD206
Bio-Rad
MCA2235T
VECAM1
Abcam
ab134047
ICAM1
R&D Systems
AF796-SP
Phospho-Akt1
Cell Signaling
9018
10
Akt1
Cell Signaling
2967
Phospho-mTOR (Ser2481)
Cell Signaling
2974
Phospho-mTOR (Ser2448)
Cell Signaling
2971
phospho-4EBP1
Cell Signaling
2855
4EBP1
Cell Signaling
9452
phospho-70SK1
Abcam
17464
70S6K1
Cell Signaling
9202
phospho-PKCα
Abcam
23513
PKCα
Cell Signaling
2056
Phospho-SGK1
Thermo Fischer
44-1260G
SGK1
Abcam
59337
Alpha-actin
Dako
M0851
GAPDH
Cell Signaling
2118
3.6. Western Blot
The venous limb of the AVF was harvested and treated with RIPA lysis buffer
containing protease inhibitors. Equal amounts of protein were loaded and run in SDS-
PAGE followed by Western blot analysis. Protein expression was probed with the
antibodies listed in Table 1.
Membranes were developed using Western Lightning Plus ECL reagent
(PerkinElmer). Membranes were stripped with Restore Western Blot Stripping Buffer
(Pierce Biotechnology) and then re-probed. Band densitometry was performed using
11
Image J and was normalized to GAPDH or the ratio of phosphorylated to total protein
was calculated.
3.7. Rapamycin and clodronate treatment
Intraperitoneal (IP) injections of rapamycin (100 µg; #553212, Sigma Aldrich)
were delivered every 24h beginning on the day of operation and continued throughout
the study period. In mice treated with adenovirus containing constitutively active Akt1,
250 µg of rapamycin was used. The control group received an equal volume injection of
vehicle (DMSO) as control. In the adventitial delivery group, pluronic gel was used to
deliver 100 µg of rapamycin to the adventitia of the venous AVF wall of at the time of
surgery.
Intraperitoneal injections of clodronate-containing liposomes (0.5 mg/Kg; CLD-
8909, Encapsula Nano Sciences) were delivered every 72hr beginning on postoperative
day 1 and continued throughout the study period. The control group received an equal
volume injection of vehicle (PBS). Intraperitoneal injections of 20 µg Ephrin-B2/Fc (R&D)
were delivered 24h prior to AVF creation and every 48h thereafter. Control mice
received an equal volume injection of vehicle (PBS) as control.
3.8. Adenovirus treatment
Infrarenal aorto-caval AVF were created as described above. After unclamping
and confirming fistula flow, 1·106 copies of commercially available vectors (Vector
Biolabs, Malvern, PA) containing either constitutively active Akt1 adenovirus (Myr-HA-
12
Akt1), or a control virus (WT-HA-Akt1) were applied to the AVF adventitial surface in a
25% w/v pluronic gel. The HA reporter tag in these vectors were used for
immunofluorescent confirmation of virus delivery. After visual confirmation that the
pluronic gel mixture had solidified, the abdomen was closed as described above.
3.9. Statistics
Data are represented as mean value ±SEM. All data were analyzed using Prism 8
software (GraphPad Software, Inc., La Jolla, CA). The Shapiro-Wilk test was performed to
analyze normality and the F test was performed to evaluate homogeneity of variances.
For two-group comparisons with normally distributed data, the unpaired Student’s t test
was used for data with equal variances among groups and the unpaired Student’s t test
with Welch correction was used for data with unequal variances. For multiple group
comparisons with normally distributed data, the one-way ANOVA followed by the
Sidak’s post-hoc test was used. Patency outcomes were analyzed with the use of
Kaplan–Meier curves to display the distribution of occlusion events detected over time.
P values < 0.05 were considered significant.
13
4. RESULTS
4.1. Reduced AVF wall thickness, extracellular matrix deposition, SMC and
macrophages with rapamycin
To determine the effects of mTOR signaling during venous remodeling such as
occurs during AVF maturation, we used a mouse model of AVF that recapitulates human
AVF maturation.43 Aortocaval fistulae were created as previously described and
afterwards mice received daily intraperitoneal (IP) injections of rapamycin (100 µg) or
vehicle alone; in mice treated with rapamycin, rapamycin was detectable in serum
without any systemic signs of immunosuppression or toxicity (Supplemental Figure 1A).
The IVC of sham-operated and fistula of control-treated and rapamycin-treated mice
were harvested and analyzed on postoperative days 3, 7 and 21 (Supplemental Figure
1B). Compared to sham-operated mice, control AVF showed wall thickening that was
reduced in AVF treated with rapamycin (Fig. 1A and B; Supplemental Figure 1C and D);
however, there was no significant difference in the dilation of the IVC (Fig. 1C) or the
aorta (Supplemental Figure 1E), as well as immunoreactivity of p-eNOS-ICAM dual-
positive cells (Fig. 1D; Supplemental Figure 1F), between rapamycin-treated and control
groups. Since rapamycin treatment reduced AVF wall thickening, we determined the
effect of rapamycin on components of the AVF wall including several extracellular
matrix (ECM) proteins as well as endothelial cells (EC),45,46 smooth muscle cells
(SMC),36,47,48 and macrophages.47,49,50 There was reduced immunoreactivity
of collagen I, collagen III, and fibronectin in the AVF wall of rapamycin-treated mice,
compared to control mice (Fig. 1E and F; Supplemental Figure 1G). There were fewer
14
Figure 1. Reduced AVF wall thickness, extracellular matrix deposition, SMC and macrophages with
rapamycin. (A) Representative photomicrographs showing AVF wall thickness in mice treated with
rapamycin vs. control (day 21). Scale bar, 25 µm. L, lumen. (B) Bar graph showing AVF wall thickness in
mice treated with rapamycin vs. control; p<0.0001 (ANOVA); *, p<0.0001 (Sidak’s post hoc); n=5-9. (C)
Line graph showing relative AVF diameter in mice treated with rapamycin vs. control; normalized to day 0;
p=0.534 (ANOVA); n=6. (D) Bar graphs showing quantification of dual IF after control or rapamycin
treatment at days 3, 7, 21, normalized to sham. p-eNOS-ICAM1: p<0.1383 (ANOVA); n=4-6. (E)
Photomicrographs showing representative of extracellular matrix immunoreactive signals in control or
rapamycin treated groups (day 7). Collagen I or III (red) and fibronectin (green). (F) Bar graphs showing
quantification of IF, normalized to sham. Collagen I: p<0.0001 (ANOVA); *, p=0.0006, day 7; *, p<0.0001,
day 21 (post hoc); n=4. Collagen III: p<0.0001 (ANOVA); *, p=0.0122, day 7; *, p<0.0001, day 21 (post
hoc); n=4. Fibronectin: p<0.0001 (ANOVA); *, p<0.0001 (post hoc); n=5. (G) Bar graphs showing number of
ICAM-1+, α-actin+ or CD68+ cells in AVF after control or rapamycin treatment. ICAM-1: p=0.7455 (ANOVA).
n=5. α-actin: p<0.0001 (ANOVA). *, p<0.0002, day 3; *, p<0.0001, day 7; *, p<0.0001, day 21 (post hoc);
n=5. CD68: p<0.0001 (ANOVA). *, p<0.0001, days 3 and 7; *, p=0.0463, day 21 (post hoc); n=5. (H)
Photomicrographs showing representative IF of PCNA (red) merged with ICAM, α-actin or CD68 (green),
and DAPI (blue) in AVF of control vs rapamycin treated mice (day 7); L, lumen; scale bar, 25 μm. White
arrowheads indicate merged signal. (I) Bar graphs showing percentage of dual positive cells. PCNA-ICAM:
p=0.4137 (ANOVA). n=4-5. PCNA-α-actin: p<0.0001 (ANOVA). *, p<0.0001, day 3; *, p=0.0011, day 7 (post
hoc); n=4-5. PCNA-CD68: p<0.0001 (ANOVA). *, p=0.0002, day 3; *, p=0.0023, day 7 (post hoc); n=4-5. (J)
Photomicrographs showing representative IF of cleaved caspase-3 (red) merged with ICAM, α-actin or
CD68 (green), and DAPI (blue) in AVF of control or rapamycin treated mice (day 7); L, lumen; scale bar,
25 μm. White arrowheads indicate merged signal. (K) Bar graphs showing percentage of dual positive
cells. Cleaved caspase-3-ICAM: p=0.08777 (ANOVA); n=4-5. Middle graph, cleaved caspase-3-α-actin:
p=0.1266 (ANOVA). n=4-5. Right graph, cleaved caspase-3-CD68: p=0.2663 (ANOVA); n=4-5.
15
numbers of α-actin-positive cells and CD68-positive cells, without any change in
numbers of intercellular adhesion molecule-1 (ICAM-1)-positive cells, in the AVF of
rapamycin-treated mice compared to control mice, consistent with reduced numbers of
SMC and macrophages but not EC with rapamycin treatment (Fig. 1G; Supplemental
Figure 1H). The reduced number of α-actin-positive cells and CD68-positive cells with
rapamycin treatment was associated with reduced PCNA immunoreactivity (Fig. 1H and
I; Supplemental Figure 1I); however, there was no increase in cleaved caspase-3
immunoreactivity with rapamycin treatment (Fig. 1J and K; Supplemental Figure 1J).
These data suggest that the reduced AVF wall thickening with rapamycin treatment is
associated with less SMC and macrophage proliferation.
4.2. Reduced M1- and M2-type macrophages with rapamycin
Since rapamycin treatment was associated with reduced macrophage
proliferation (Fig. 1), we determined whether rapamycin had differential effects on
macrophage subpopulations. The wall of the rapamycin-treated AVF showed decreased
iNOS and TNF-a immunoreactive protein, markers of M1-type macrophages, as well as
decreased IL-10 and CD206 protein, markers of M2-type macrophages, at both days 3
and 7 (Fig. 2A and B). Rapamycin-treated AVF also showed reduced immunoreactivity of
CD68-iNOS dual-positive cells as well as CD68-TNF-a dual-positive cells in the adventitia
(Fig. 2C and D; Supplemental Figure 2A); there was also reduced immunoreactivity of
CD68-IL-10 dual-positive cells as well as CD68-CD206 dual-positive cells in the adventitia,
at both days 3 and 7 (Fig. 2E and F; Supplemental Figure 2B). Rapamycin treatment was
16
Figure 2. Reduced M1- and M2-type macrophages with rapamycin. (A) Representative Western blot
showing iNOS, TNF-α, IL-10 and CD206 protein expression levels in AVF treated with rapamycin or
control (day 3 and 7). (B) Graphs showing densitometry measurements of iNOS, TNF-α, IL-10 and
CD206 expression in the AVF after control or rapamycin treatment, normalized to GAPDH. iNOS:
p=0.0011 (ANOVA). *, p=0.0241, day 3; *, p=0.0054, day 7 (post hoc); n= 2-3. TNF-α: *p=0.0020
(ANOVA). *, p=0.0223, day 3; *, p=0.0250, day 7 (post hoc); n= 2-3. IL-10: *p<0.0001 (ANOVA). *,
p=0.0011, day 3; *, p=0.0006, day 7 (post hoc); n= 2-3. CD206: p=0.0013 (ANOVA). *, p=0.0126, day
3; *, p=0.0200, day 7 (post hoc); n= 2-3. (C) Photomicrographs showing representative dual IF for
CD68 (red) and iNOS (green, top row) or CD68 (red) and TNF-a (green, bottom row) in AVF after
control or rapamycin treatment (day 7). Scale bar, 25 μm. L, lumen. (D) Bar graphs showing
quantification of dual IF after control or rapamycin treatment. Left graph, iNOS-CD68: p<0.0001
(ANOVA). *, p=0.0006, day 3; *, p=0.0004, day 7; *, p=0.0073, day 21 (post hoc); n=5. Right graph,
TNF-a-CD68: p<0.0001 (ANOVA). *, p<0.0001, day 3; *, p<0.0001, day 7 (post hoc); n=5. (E)
Photomicrographs showing representative dual IF for CD68 (red) and IL-10 (green, top row) and CD68
(red) and CD206 (green, bottom row) in control or rapamycin treated AVF (day 7). (F) Bar graphs
showing quantification of dual IF after control or rapamycin treatment (day 7). Left graph, IL-10-
CD68: p<0.0001 (ANOVA). *, p<0.0001, day 3; *, p<0.0001, day 7 (post hoc); n=5. CD206-CD68:
p<0.0001 (ANOVA). *, p<0.0001, day 3; *, p<0.0001, day 7 (post hoc); n=5. (G) Photomicrograph of
representative of CD45+ cells in control or rapamycin treated mice AVF (day 7). (H) Bar graph
showing number of CD45 immunoreactive cells in AVF after control vs rapamycin treatment;
p<0.0001 (ANOVA); *, p<0.0001, day 3; *, p=0.0020, day 7; *, p=0.2110, day 21 (post hoc); n=5. (I)
Representative photomicrographs showing VCAM-1 (top row) and ICAM-1 (bottom row) IF in AVF
after control or rapamycin treatment (day 7). (J) Bar graphs showing relative quantification of VCAM-
1 and ICAM-1 intensity in AVF, normalized to sham vessels. VCAM-1: p=0.3162 (ANOVA); n=6. ICAM-
1: p=0.9280 (ANOVA); n=4-6. Data represent mean ± SEM.
17
also associated with fewer number of leukocyte common antigen (CD45)
immunoreactive cells (Fig. 2G and H; Supplemental Figure 2C), but there was no
difference in immunoreactivity of vascular cell adhesion molecule-1 (VCAM-1) or ICAM-1
(Fig. 2I and J; Supplemental Fig. 2D). These data suggest that rapamycin is associated
with reduced immunoreactivity of both M1-type and M2-type macrophages as well as
fewer leukocytes during AVF remodeling.
4.3. Reduced Akt1 and mTORC1 but not mTORC2 phosphorylation with rapamycin
Since mTOR binds to either the Raptor regulatory subunit to form mTORC1, a
downstream target of Akt1,51 or to the Rictor regulatory subunit to form mTORC2,52 an
upstream regulator of Akt1,39 we next determined whether rapamycin altered the
phosphorylation of either of these complexes during AVF remodeling. Rapamycin was
associated with reduced numbers of p-Akt1 immunoreactive cells (days 7 and 21) and p-
mTORC1 immunoreactive cells (days 3 and 7), but there was no difference in the
numbers of p-mTORC2 immunoreactive cells (Fig. 3A and B; Supplemental Figure 3A).
Similarly, mice treated with rapamycin had decreased expression of phosphorylated
Akt1 and phosphorylated mTORC1, with no significant change in expression of
phosphorylated mTORC2, in the AVF wall (days 3-21; Fig. 3C and D). Reduced Akt1 and
mTORC1 phosphorylation with rapamycin was similarly reduced in both p-Akt1-α-actin
dual-positive cells as well as p-mTORC1-α-actin dual-positive cells (Fig. 3E and F;
Supplemental Figure 3D); immunoreactivity was also reduced with rapamycin
treatment in p-Akt1-CD68 dual-positive cells as well as p-mTORC1-CD68 dual-positive
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Figure 3. Reduced Akt1 and mTORC1 but not mTORC2 phosphorylation with rapamycin. (A)
Photomicrographs showing representative IF of p-Akt1+ (top, red), p-mTORC1+ (middle, red) and
p-mTORC2+ (bottom, red) cells in control or rapamycin treated mice AVF (day 7). Scale bar,
25μm. L, lumen. (B) Bar graphs showing number of p-Akt1+, p-mTORC1+ and p-mTORC2 + cells in
AVF after rapamycin or control treatment. p-Akt-1: *, p<0.0001 (ANOVA); *, p<0.0001, day 7; *,
p =0.0105, day 21 (post hoc); n=4-5. p-mTORC1: p<0.0001 (ANOVA); *, p<0.0001, day 3; *,
p<0.0001, day 7 (post hoc); n=4-5. p-mTORC2: p=0.2870 (ANOVA); n=4-5. (C) Representative
Western blot showing Akt1, mTORC1, mTORC2 phosphorylation level after control vs rapamycin
treatment. (D) Graphs showing densitometry measurement of Akt1, mTORC1 and mTORC2
phosphorylation. p-Akt1: tAkt1, p=0.0002 (ANOVA); *, p=0.0110, day 7; *, p=0.0359, day 21
(post hoc); n=3. p-mTORC1: tmTORC1, p=0.0004 (ANOVA); *, p=0.0157, day 3; *, p=0.0192, day
7; *, p=0.0366, day 21 (post hoc); n=3. p-mTORC2: tmTORC2: P=0.9893 (ANOVA); n = 3. (E)
Photomicrographs showing representative IF of dually-positive α-actin (green) and p-Akt1 (red,
first row) or p-mTORC1 (red, second row) in AVF after control or rapamycin treatment (day 7). (F)
Bar graphs showing quantification of dual IF after control vs rapamycin treatment. P-Akt1-α-
actin: p<0.0001 (ANOVA); *, p=0.0002, day 7; *, p=0.0017, day 21 (post hoc); n=4-5. p-mTORC1-
α-actin: p<0.0001 (ANOVA); *, p=0.0136, day 7; *, p<0.0001, day 21 (post hoc); n=4. (G)
Photomicrographs showing representative dual IF for CD68 (green) and p-Akt1 (red, top row) or
p-mTORC1 (red, bottom row) in AVF after control or rapamycin treatment (day 7). (H) Bar graphs
showing quantification of dual IF after control vs rapamycin treatment. p-Akt1-CD68: p<0.0001
(ANOVA); *, p=0.0013, day 7; *, p=0.0183, day 21 (post hoc); n=4-5. p-mTORC1-CD68: p<0.0001
(ANOVA); *, p<0.0001, day 3; *, p<0.0001, day 7 (post hoc); n=4-5. Data represent mean ± SEM.
19
cells (Fig. 3G and H; Supplemental Figure 3E). However, there was no significant
difference in immunoreactivity of p-Akt1-ICAM dual-positive cells or p-mTORC1-ICAM
dual-positive cells with rapamycin treatment (Supplemental Figure 3B and C). These
data suggest that rapamycin is associated with less Akt1-mTORC1 signaling, in both SMC
and macrophages, during AVF remodeling.
Since these data show that rapamycin reduces mTORC1, but not mTORC2,
phosphorylation (Fig. 3), we evaluated the phosphorylation of P70S6K and 4EBP1,
downstream targets of mTORC1.53 There were significantly fewer number of cells that
were immunoreactive for p-P70S6K1 or p-4EBP1 in the AVF of mice treated with
rapamycin compared to control (Fig. 4A; Supplemental Figure 4A); however, there was
no effect on the number of cells that were immunoreactive for p-PKCα or p-SGK1,
downstream targets of mTORC2 (Fig. 4B; Supplemental Figure 4B). Similarly, AVF
treated with rapamycin had significantly decreased expression of phosphorylated
P70S6K and 4EBP1 (Fig. 4C and D), but no significant change in expression of
phosphorylated PKCα or SGK1 (Fig. 4E; Supplemental Figure 4C). These results suggest
that rapamycin regulates the mTORC1, but not mTORC2 pathway, during venous
remodeling.
The AVF of mice treated with rapamycin similarly showed decreased
immunoreactivity of p-P70S6K-α-actin dual-positive cells and p-4EBP1-α-actin dual-
positive cells (Fig. 4F and G; Supplemental Figure 4D); rapamycin-treated AVF also
showed decreased immunoreactivity of p-P70S6K-CD68 dual-positive cells and p-4EBP1-
CD68 dual-positive cells (Fig. 4H and I; Supplemental Figure 4E). However, there was no
