Embed Size (px)
Citation preview
Author's Accepted Manuscript
Bariatric Surgery Improves the Circulating Numbersand Biological Activity of Late Outgrowth Endothe-lial Progenitor Cells
William O. Richards M.D., Kelley Bose Prutzman R.N, B.S.N., Martha F. O’Hea B. Sc., Jonathon P.Audia Ph.D., Diego F. Alvarez M.D. Ph.D.
PII: S1550-7289(14)00195-6DOI: http://dx.doi.org/10.1016/j.soard.2014.04.025Reference: SOARD1991
To appear in: Surgery for Obesity and Related Diseases
Cite this article as: William O. Richards M.D., Kelley Bose Prutzman R.N, B.S.N.,Martha F. O’Hea B. Sc., Jonathon P. Audia Ph.D., Diego F. Alvarez M.D. Ph.D.,Bariatric Surgery Improves the Circulating Numbers and Biological Activity of LateOutgrowth Endothelial Progenitor Cells, Surgery for Obesity and Related Diseases, http://dx.doi.org/10.1016/j.soard.2014.04.025
This is a PDF file of an unedited manuscript that has been accepted for publication. As aservice to our customers we are providing this early version of the manuscript. Themanuscript will undergo copyediting, typesetting, and review of the resulting galley proofbefore it is published in its final citable form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers that applyto the journal pertain.
www.elsevier.com/locate/buildenv
Bariatric Surgery Improves the Circulating Numbers and Biological Activity of Late
Outgrowth Endothelial Progenitor Cells
William O. Richards, M.D.1, Kelley Bose Prutzman, R.N, B.S.N.
1, Martha F. O’Hea, B. Sc.
1,
Jonathon P. Audia, Ph.D.2,3,4,
*, Diego F. Alvarez, M.D. Ph.D.4,5,6,
*
Departments of Surgery1, Microbiology and Immunology
2, the Laboratory of Molecular
Biology3, the Center for Lung Biology
4, Internal Medicine
5, and Pharmacology
6, University of
South Alabama College of Medicine, Mobile AL, 36688
* Corresponding authors:
Jonathon P. Audia: Laboratory of Molecular Biology, Department of Microbiology and
Immunology, University of South Alabama College of Medicine, Mobile, AL 36688. Tel 251-
460-6929. Fax 251-460-7269. E-mail: [email protected]
Diego F. Alvarez: Departments of Internal Medicine and Pharmacology, University of South
Alabama College of Medicine, Mobile, AL 36688. Tel 251-460-6392. Fax 251-460-7452. E-
mail: [email protected]
Funding Sources: ASMBS grant 2010 (to W. O. Richards)
Running Title: Bariatric surgery and late outgrowth EPCs
Acknowledgments
The authors acknowledge the superb contributions of the staff of the Department of Surgery,
University of South Alabama. This study was funded by a grant from the American Society for
Metabolic and Bariatric Surgery (ASMBS) (awarded in 2010 to WOR).
Disclosures
Drs. William Richards, Jonathon P. Audia, Diego F. Alvarez and Ms. Kelley Bose Prutzman and
Ms. Martha F. O’Hea have no conflicts of interest or financial ties to disclose.
Abstract
Background: While the salutary effects of bariatric surgery as a treatment for excess body
weight and type 2 diabetes are established, there is scant evidence for effects on other
contributors to cardiovascular diseases such as repair of endothelial dysfunction.
Objectives: This study evaluates outcomes of bariatric surgery on late outgrowth endothelial
progenitor cells (LOEPCs), a cell phenotype essential for endothelial repair.
Setting: University of South Alabama Medical Center, Mobile, Alabama.
Methods: Subjects with a body mass index > 35 kg/m2 and type 2 diabetes were enrolled into
either medical or bariatric surgical arms. Primary outcomes included analysis of isolated
LOEPCs from peripheral blood for growth, function, and mitochondrial respiration. Plasma was
used for metabolic profiling.
Results: Medical arm subjects showed no improvement in any of the parameters tested.
Bariatric surgical arm subjects showed a 24% reduction in body mass index as early as 3-months
post-intervention and resolution of type 2 diabetes at 24-months post-intervention (HbA1c–31%
reduction; fasting glucose–29% reduction). Bariatric surgery increased the numbers of LOEPCs
8-fold and increased LOEPC network formation 3-fold at 24-months post-intervention. The
increased numbers and activity of LOEPCs in the bariatric surgical arm correlated with
improvements in body mass index, insulin and triglyceride levels only at 24-month post-
intervention. LOEPC mitochondrial respiration displayed a trend towards improvement when
compared to baseline as evidenced by an increase (36%) at 24 months in the bariatric arm.
Conclusions: Bariatric surgery increases LOEPC levels and activity, which correlates with
weight loss- and improved metabolic profile at 24-months post-intervention.
Key Words: Bariatric surgery, endothelial progenitor cells, sleeve gastrectomy, Roux-En-Y
gastric bypass, mitochondrial respiration, late outgrowth EPCs
Introduction
Cardiovascular diseases are leading causes of mortality that often manifest with secondary
complications that further diminish quality of life. Studies initiated in the 1960’s aimed at
delineating risk factors associated with cardiovascular disease unveiled a link to obesity [1]
.
Importantly, obesity-related metabolic/inflammatory perturbations that drive type 2 diabetes,
hyper-triglyceridemia, hyper-cholesterolemia, hypo-high density lipoproteinemia, and more
recently insulin resistance, are now renowned as seminal players in cardiovascular diseases [2, 3]
.
Recognizing that obesity is a burgeoning epidemic with a continuing upward trend, strategies to
reduce excess body weight and re-establish metabolic/inflammatory homeostasis are an
immediate priority. In particular, bariatric surgery has emerged as an important intervention and
is becoming widely used to treat obesity-related co-morbidities such as type 2 diabetes and
insulin resistance [4-6]
. However, other salutary effects of bariatric surgery beyond treatment of
excess body weight, type 2 diabetes, and insulin resistance are poorly understood. Herein, we
have explored the relationship between bariatric surgery and levels/biological activity of
circulating endothelial progenitor cells (EPCs) that are associated with positive outcomes in
subjects with cardiovascular diseases.
Endothelial cells form barriers that limit the movement of fluids, solutes, and gases between the
blood and underlying tissues as well as regulate vasoreactivity. Traditional concepts hold that in
response to acute insults such as infection, damaged portions of the endothelial barrier are
thought to be restored via recruitment of circulating EPCs which directly proliferate and repair
the endothelial barrier and/or indirectly signal to resident endothelial cells to proliferate and
repair the endothelial barrier [7-9]
. However, in response to chronic insults such as prolonged
exposure to the cadre of metabolic risk factors associated with obesity, damaged portions of the
endothelial barrier are not readily restored. Instead, decreased generation of nitric oxide and
other vaso-relaxing agents, and development of atherosclerosis typify chronic endothelial barrier
injury [10, 11]
. Experimental evidence suggests that loss of EPCs and impairment of EPC activity
are, at least in part, culprits that explain chronically diminished endothelial reparative potential
[12-15]. Indeed, subjects with low circulating levels of EPCs who suffer from chronic diseases
such as type 2 diabetes and atherosclerosis are prone to rapid deterioration [16, 17]
. Conversely,
subjects with high circulating levels of EPCs who undergo myocardial infarction show increased
functional reserve [18]
.
As part of their direct reparative capacity, EPCs possess distinct characteristics such as rapid
proliferation, the ability to migrate, form networks and, ultimately generate de novo vessels
(vasculogenesis) in response to damage [19, 20]
. EPCs are believed to arise in adults from the bone
marrow, from the intima of both microvascular- and conduit-blood vessels [8, 19, 21, 22]
. Despite
these distinct origins, primary circulating EPCs are typically isolated from the mononuclear
fraction of peripheral blood. However, not all cells in the mononuclear fraction identified as
circulating EPCs by using conventional staining and flow cytometry approaches possess
vasculogenic capacity [20]
. Evidence suggests that circulating EPCs in the mononuclear fraction
display at least two distinct growth phenotypes in vitro that correlate with vasculogenic capacity.
Isolation of EPCs by flow cytometry using selective surface markers yields cells that display
rapid outgrowth within one week post-isolation (early outgrowth EPCs, EOEPCs) [20, 21]
. While
EOEPCs do not directly exhibit vasculogenic capacity, they do exhibit the ability to restore
endothelial function via signaling to resident endothelial cells. The second EPC growth
phenotype isolated from the mononuclear fraction displays an outgrowth lag and is not observed
until 4-6 weeks post-isolation (late outgrowth EPCs, LOEPCs) [20, 23]
. The LOEPCs directly
exhibit vasculogenic capacity. To date, the majority of studies examining roles for circulating
EPCs in obesity-related cardiovascular diseases have focused on using EOPECs with only
relatively few studies employing LOEPCs. Therefore, the goal of the present study was to
examine the effects of bariatric surgery on the levels and activity of LOEPCs in cohorts of
subjects undergoing either medical intervention or bariatric surgery to resolve excess body
weight and type 2 diabetes.
Methods
Study subjects
The study protocol was approved by the University of South Alabama institutional review board
(no. 10-131) and the study conducted according to the principles of the Declaration of Helsinki.
The study is included in the database of national registries (NCT01213940). Written informed
consent was obtained from all subjects.
The study enrolled subjects between the ages of 18 to 65 who fulfilled the National Institutes of
Health criteria for bariatric surgery [body mass index (BMI) ≥ 35 kg/m2]. Study enrollees also
possessed glycosylated hemoglobin levels (HGA1c) > 6.5 and a diagnosis of type 2 diabetes as
determined by a fasting glycemia > 126 mg/dL or currently treated with any pharmacological
hypo-glycemic.
Study design
Six subjects were enrolled in a medical arm and underwent for six months nutritional and
physical activity modifications aimed at reducing caloric intake and increasing caloric
expenditure, respectively. This arm incorporated the standard 6-month physician directed weight
loss program required for insurance coverage qualification for bariatric surgery. Each patient
was asked to participate in 7 consecutive visits in a 6-month period that focused on lifestyle
modification. Caloric restriction aimed at 1200-1500 calories per day depending on the patient’s
BMI and the exercise program started with modest targets of walking 20 minutes per day (if
physically able) to other forms of exercise ranging from swimming to weight lifting 3 times per
week. Each patient’s program was individually tailored.
Fourteen subjects were enrolled in a bariatric surgical arm and underwent either laparoscopic
Roux-en-Y gastric bypass or laparoscopic sleeve gastrectomy. Roux-en-Y gastric bypass was
fashioned in an antecolic manner with an end-to-side gastrojejunal anastomosis of 21 mm. Roux
limb was measured at 100-150 cm and adjusted to BMI. Sleeve gastrectomy was constructed
using a 34 French bougie starting at 4 cm proximal to the pylorus. Subjects were discharged
from the hospital when tolerated a liquid diet. There were no surgical complications and no
mortality during the study.
Blood chemistry measurements
Peripheral blood was obtained from each subject as previously described [6]. Samples from
subjects in the medical study arm were obtained at baseline, 3-, and 6-months post-enrollment.
Samples from subjects in the bariatric surgical arm were obtained at baseline (day of the
surgery), 3-, 6-, and 24-months post-surgery. Subjects were instructed to fast for at least 12-
hours prior to sample collection. Blood chemistry analysis included measurements of glucose,
HbA1c, insulin, and a lipid profile. HOMA-IR score (homeostasis model assessment-estimated
insulin resistance) was calculated and subjects with a score > 2.6 were considered insulin
resistant.
EPC isolation
Following collection of the mononuclear/platelet fraction from peripheral blood [6]
, cells were
collected aseptically and stained with trypan blue to allow for enumeration of live/dead cells
using a Countess Automated Cell Counter (Invitrogen Life Technologies). The entire
mononuclear fraction was suspended into endothelial growth two medium (EGM-2, Lonza) and
cells seeded at ~5 x 106 live cells/well into a 35 mm culture dish pre-treated with human
collagen-I. On average, each subject sample yielded 2-4 culture dishes of cells. All cultured
cells were incubated at 37 °C in a humidified incubator (5% CO2 and 21% O2). At 24-hours
post-seeding, the culture medium containing all non-adhering cells was transferred into a new 35
mm culture dish pre-treated with fibronectin, a more adhesive substratum, to allow for
enrichment of EOEPCs. The cells that had adhered to the original collagen-I-coated dishes at
24-hours post-seeding were supplemented with fresh EGM-2 medium for enrichment of
LOEPCs. Subsequently, EGM-2 medium for all culture conditions were replaced bi-weekly.
EOEPCs and LOEPCs were assessed from fibronectin-coated plates at 1-week- and from
collagen-I coated plates at 4-6-weeks- post-seeding, respectively. In all cases, for each patient, at
each time point, cells were independently isolated and assayed. Cells from multiple patients
were never pooled.
Enumeration of circulating LOEPCs
At the end of 4-6-weeks of incubation on collagen-I-coated culture dishes, the total number of
LOEPC colonies was counted using phase-contrast light microscopy and data normalized to the
number of viable mononuclear cells initially seeded.
Activity of circulating LOEPCs
Individual LOEPCs (primary cells, passage 1) were enumerated as described above, suspended
in EGM-2 medium, and 5 x 104 cells seeded into Matrigel-coated 95 mm
2 wells (48-well cluster
plate). Networks were visualized using phase contrast light microscopy and counted at 8-hours
post-seeding.
Assessment of LOEPC mitochondrial oxygen consumption
LOEPC mitochondrial respiration was determined as an index of cellular fitness. Mitochondrial
respiration was measured as previously described [6]
. Briefly, LOEPCs were enumerated as
described above and suspended into DMEM medium (Invitrogen Life Technologies). The linear
rate of oxygen consumption (mitochondrial respiration) was measured by placing LOEPCs into
each chamber of an Oroboros O2K polarographic high-resolution respirometer at 37 °C under
continuous stirring. Oxygen flux is reported as IO2 in pmol/s/mL/106 cells. Basal rates of IO2
were measured after cells had achieved a steady state. Maximal rates of IO2 were determined by
titration of the cell permeable protonophore carbonyl cyanide 4-(trifluoromethoxy)
phenylhydrazone (FCCP).
Statistical analyses
Data are reported as mean ± standard error. GraphPad Prism v4.3 was used for all analyses.
Comparison between baseline and 24-month samples was performed by unpaired t-test.
Comparison among baseline and all time points was performed by one-way ANOVA, followed
by a Newman-Keuls post-hoc test. Linear regression was used to establish correlation between
two parameters and the coefficient of determination, R2, reported to indicate their linear variance.
Differences with a p value of < 0.05 were considered significant.
Results
Subject demographics and the effects of medical and bariatric surgical interventions on
biometrics and blood chemistry analyses
A total of 20 subjects meeting the criteria specified were enrolled in the study (4 men and 16
women). The average ages of the male and female cohorts were 50- and 44-years, respectively
(combined average of 47-years). Six and 14 subjects were enrolled into the medical- (2 males
and 4 females) and bariatric surgical- (2 males and 12 females) arms, respectively. The average
BMIs of the male and female cohorts at the time of enrollment in any arm of the study were 49.6
kg/m2 (range 39.4–69.4 kg/m
2) and 43.9 kg/m
2 (range 38.4–73.3 kg/m
2), respectively (average of
46.1 kg/m2), differences that were not statistically significant.
The average excess BMI weight lost (%EBMIL) in the medical arm was 0.2% (from 44.1 to 44.0
kg/m2) and 4.1% (from 44.1 to 42.3 kg/m
2) at 3- and 6-months after enrollment, respectively.
Compared to baseline (time of enrollment), %EBMIL was not significant. The average
%EBMIL in the bariatric surgical arm was 24.1% (from 46.6 to 35.4 kg/m2), 25.1% (from 46.6
to 34.9 kg/m2), and 38.2% (from 46.6 to 28.8 kg/m
2) at 3-, 6-, and 24-months after enrollment,
respectively. Compared to baseline (time of surgery), %EBMIL was significant at all-time
points (p < 0.0001 by one-way ANOVA).
All blood chemistry analyses (metabolic profiling) in the medical arm showed no significant
improvement compared to baseline at any of the time points analyzed (Fig. 1). Indeed, these
results are in agreement with the substantial evidence showing that long-term medical treatment
is ineffective in sustaining weight loss and does not reduce cardiovascular mortality [24-26]. In
contrast, subjects in the bariatric surgical arm showed significant improvement in HbA1c levels
(p = 0.0008), triglycerides (p = 0.03), and HDL (p = 0.008) at all-time points compared to
baseline (by one-way ANOVA). In addition, subjects showed significant improvement in fasting
glucose (p = 0.005), insulin (p = 0.04), HOMA-IR (p = 0.02), and LDL (p = 0.04) when baseline
values were compared to 24-months (by unpaired t-test). Together these data suggest that
perhaps caloric restriction to a degree unfeasible without bariatric surgery leads to significant
improvement in metabolic profiles.
Effect of bariatric surgery on circulating LOEPC numbers and biological activity
Circulating EPCs display two distinct populations based on growth phenotype in vitro that
correlate with vasculogenic capacity. EOEPCs display rapid outgrowth and a distinct cellular
morphology (Fig. 2A, upper panels) but do not directly exhibit vasculogenic capacity [8, 20, 21]
.
Conversely, LOEPCs display an outgrowth lag and a distinct cellular morphology (Fig. 2A,
bottom panels), and directly exhibit vasculogenic capacity [8, 20, 23]
. We were able to identify
EOEPCs from all subjects enrolled in the medical arm of the study but intriguingly, were able to
identify LOEPCs only from a single subject at baseline and, 3- and 6-months post-intervention
(one colony at each time point). This result is consistent with reports indicating that metabolic
disorders adversely impact the homeostatic levels of circulating EPCs [13, 14, 17]
.
As observed in the medical arm, we identified EOEPCs from all subjects in the bariatric surgical
arm. In contrast to medical intervention, bariatric surgery resulted in a time-dependent increase
in the number of circulating LOEPCs with a significant increase above baseline at 24-months
(Fig. 2B, p = 0.006 by one-way ANOVA). To determine whether bariatric intervention affects
the vasculogenic capacity of isolated LOEPCs, we examined their ability to generate networks
when seeded onto Matrigel in vitro. At each time point, 50,000 cells were seeded and networks
measured at 8-hours post seeding. The numbers of LOEPCs networks increased in a time-
dependent manner post-bariatric surgery with a significant increase above baseline at 24-months
(Fig. 2C, p = 0.02 by one-way ANOVA). Together these data indicate that not only bariatric
surgery improves overall numbers of circulating LOEPCs but also improves their biological
function. Moreover, these data suggest that perhaps caloric restriction to a degree unfeasible
without bariatric surgery leads to significant improvement in LOEPC number and function.
Correlating LOEPC numbers and biological activity to subject biometrics and metabolic
profiling
A linear regression analysis was performed to determine which, if any, of the subject biometric
and metabolic parameters that improved upon bariatric surgery correlated with increased
circulating numbers and biological activity of LOEPCs. The top panels of Figure 3 show a
significant linear relationship between LOEPC numbers and BMI, insulin, and triglycerides (p <
0.05). The bottom panels of Figure 3 show a linear relationship between LOEPC network
formation and BMI, insulin, and triglycerides (although not statistically significant). None of the
other metabolic parameters tested gave a significant linear correlation. Together, these data
suggest that improvements in BMI, insulin, and triglycerides may be prognostic of a subject’s
ability to repair metabolic dysregulation-induced damage to the endothelium.
Finally, to interrogate whether the energetic state of the LOEPCs might account for the
differences in biological activity observed post-bariatric surgery, we measured cellular
respiration as an indicator of mitochondrial fitness [27]
. In these assays a basal respiration rate
(IO2 in pmol/s/mL/106 cells) was first measured followed by serial titration of FCCP, a cell
permeable protonophore that uncouples the electron transport system and allows for the maximal
rate of respiration [6, 27]
. The basal IO2 from LOEPCs isolated at baseline, 6-, and 24-months
post-bariatric surgery were 19.0 ± 3.5, 17.1 ± 4.7, and 25.8 ± 8.2, respectively. The maximal IO2
from LOEPCs isolated at baseline, 6-, and 24-months post-bariatric surgery were 20.5 ± 5.1, 20.2
± 3.8, and 29.6 ± 6.6, respectively. Although the data were not significant, there is a trend
towards increase in both basal and maximal LOEPC mitochondrial respiration at 24-months
post-bariatric surgery.
Discussion
Our study is the first to demonstrate that bariatric surgery improves number and function of
LOEPCs in patients undergoing treatment for excess body weight. Moreover, patients
undergoing medical treatments for excess body weight did not show improvements in LOEPC
number and function. These data is significant because LOEPCs play a direct role in vascular
endothelial repair [7, 8]
. The strength of our study which followed each patient over a 24-month
time period, is assessment of LOEPCS by functional characterization rather than by using
immunophenotyping which yields heterogeneous EPC populations, not all of which display
direct vascular repair capacity [7, 8]
. Despite the importance of LOEPCs in vascular repair, there
has been no direct assessment of LOEPCs in patients treated for excess body weight with or
without bariatric surgery. In addition, our study is the first to examine mitochondrial respiration
as a potential mechanism explaining the improvements in LOEPC number and function post-
bariatric surgery and parallels studies showing that EPCs from patients with pulmonary
hypertension display defective mitochondrial respiration [28]
. Limitations of the study include the
small number of patients enrolled in both the medical- and surgery-study arms. Although
EOEPCs do not directly repair the vascular endothelium, they have been shown to play indirect
vascular reparative roles, thus, future studies to compare EOEPC and LOEPC total numbers in
patients treated for excess body weight will be of interest.
The morbid synergism exerted by excess body weight, type 2 diabetes, cardiovascular diseases,
and other body weight-related inflammatory and metabolic disorders are prominent social,
health, and economic threats [29]
. Currently, bariatric surgery represents the front line treatment
to mitigate body weight related co-morbidities [30]
. The complex nature relating excess body
weight-related disorders to damage and repair at the cellular level represents a significant barrier
towards our understanding of these related stressors. Inflammatory and metabolic insults inflict
damage to endothelial cells and elicit stress responses that direct cellular repair [31]
. Intriguingly,
from all metabolic parameters assessed, only fasting glucose and insulin mirrored the time course
displayed by LOEPCs, whereas body weight reduction was observed as early as three months
post-bariatric surgery. Together these data may suggest that body weight reduction alone is not
an overall indicator of patient’s prognosis.
Our study also recapitulates the previous observations that subjects with excess body weight and
type 2 diabetes have impaired numbers and function of LOEPCs [14]
. The results underscore the
need for long-term follow up studies aimed at elucidating the mechanism(s) increasing LOEPC
levels and activity and lay the basis for their prospective usage as a cell-based therapy, which has
yielded positive outcomes in several ischemic animal models and in subjects with peripheral
arterial disease [13, 32]
. EOEPCs and LOEPCs are rare populations of circulating cells
incriminated in endothelial barrier function restoration [8, 9, 20]
. Importantly, EOEPCs and
LOEPCs are susceptible to injury by the same stressors that damage the native endothelium,
which may partially explain the poor endothelial repair in subjects with excess body weight and
related inflammatory and metabolic disorders [13, 17, 33]
. Indeed, in this study we were unable to
isolate significant numbers of LOEPCs from subjects pre-intervention or in the medical arm and
only bariatric surgical intervention yielded a time-dependent increase in circulating LOEPC
numbers and biological activity.
Our observations are supported by model studies showing that in diabetic mice, recovery from
hind limb ischemia is defective due to lower levels of circulating EPCs which impairs de novo
vessel formation [34]
. Mechanisms that regulate the total levels of circulating EPCs include cell
mobilization from their niches, and loss by programmed cell death. In subjects with type 2
diabetes, decreased mobilization of total EPCs and total EPC programmed cell death are linked
to accumulation of toxic N-linked glycation end-products [14, 33]
. While mechanism(s) involved
in decreased EPC mobilization are poorly understood, the mechanisms of EPC programmed cell
death, are associated with N-linked glycation product-mediated increases in intracellular reactive
oxygen species and mitochondrial caspase-signaling cascades [33]
. The mechanism(s) by which
bariatric surgery, results in salutary EPC effects including increase in its activity remains to be
determined.
Based upon the present study examining the salutary effects of bariatric surgery in subjects with
excess body weight and type 2 diabetes, an intriguing trend has emerged. For metabolic
parameters such as BMI, HbA1c, triglycerides, and HDL the window for improvement appears to
open as early as 3-months post-surgery. In our previous study on the effects of bariatric surgery
in subjects with excess body weight but without type 2 diabetes, only HDL showed significant
improvement in the 3-month window [6]. Intriguingly, improvements resulting from bariatric
surgery may involve resolution of inflammation as evidenced by a reduction in the activation
levels of the sentinel inflammatory regulator caspase-1 [6]
. Furthermore, mitochondrial
respiration in both circulating monocytes and skeletal muscle (an indicator of cellular fitness)
also improved in the 3-month window [6]
. The current study of LOEPCs in subjects with excess
body weight and type 2 diabetes has revealed a late phase in our emerging model describing the
temporal dynamics of subject response to bariatric surgery. Highlighting this late phase are the
data describing the resolution of type 2 diabetes and insulin resistance which parallel the
observed increases in circulating LOEPC numbers and biological activity. Further studies are
required to reveal mechanisms involved in improving LOEPC number and function post-
bariatric surgery.
References
1. Kannel WB, LeBauer EJ, Dawber TR, McNamara PM. Relation of body weight to
development of coronary heart disease. The Framingham study. Circulation. 1967;35:734-
44.
2. Adams KF, Schatzkin A, Harris TB et al. Overweight, obesity, and mortality in a large
prospective cohort of persons 50 to 71 years old. N Engl J Med. 2006;355:763-78.
3. Sjostrom L, Lindroos AK, Peltonen M et al. Lifestyle, diabetes, and cardiovascular risk
factors 10 years after bariatric surgery. N Engl J Med. 2004;351:2683-93.
4. Dixon JB, O'Brien PE, Playfair J et al. Adjustable gastric banding and conventional therapy
for type 2 diabetes: a randomized controlled trial. JAMA. 2008;299:316-23.
5. Kim S, Richards WO. Long-term follow-up of the metabolic profiles in obese patients with
type 2 diabetes mellitus after Roux-en-Y gastric bypass. Ann Surg. 2010;251:1049-55.
6. Nijhawan S, Richards W, O'Hea MF, Audia JP, Alvarez DF. Bariatric surgery rapidly
improves mitochondrial respiration in morbidly obese patients. Surg Endosc. 2013
;27:4569-73
7. Dimmeler S, Zeiher AM. Vascular repair by circulating endothelial progenitor cells: the
missing link in atherosclerosis? J Mol Med (Berl). 2004;82:671-7.
8. Yoder MC. Defining human endothelial progenitor cells. J Thromb Haemost. 2009;7 Suppl
1:49-52.
9. Yoon CH, Hur J, Park KW et al. Synergistic neovascularization by mixed transplantation
of Early Endothelial Progenitor Cells and Late Outgrowth Endothelial Cells: The role of
angiogenic cytokines and matrix metalloproteinases. Circulation. 2005;112:1618-27.
10. Hink U, Li H, Mollnau H et al. Mechanisms underlying endothelial dysfunction in diabetes
mellitus. Circ Res. 2001;88:E14-E22.
11. Meigs JB, Hu FB, Rifai N, Manson JE. Biomarkers of endothelial dysfunction and risk of
type 2 diabetes mellitus. JAMA. 2004;291:1978-86.
12. Bellows CF, Zhang Y, Simmons PJ, Khalsa AS, Kolonin MG. Influence of BMI on level of
circulating progenitor cells. Obesity (Silver Spring). 2011;19:1722-6.
13. Caballero S, Sengupta N, Afzal A et al. Ischemic vascular damage can be repaired by
healthy, but not diabetic, endothelial progenitor cells. Diabetes. 2007;56:960-7.
14. Chen MC, Sheu JJ, Wang PW et al. Complications impaired endothelial progenitor cell
function in Type 2 diabetic patients with or without critical leg ischaemia: implication for
impaired neovascularization in diabetes. Diabet Med. 2009;26:134-41.
15. McGuire TR, Brusnahan SK, Bilek LD et al. Inflammation associated with obesity:
relationship with blood and bone marrow endothelial cells. Obesity (Silver Spring).
2011;19:2130-6.
16. Cubbon RM, Kahn MB, Wheatcroft SB. Effects of insulin resistance on endothelial
progenitor cells and vascular repair. Clin Sci (Lond). 2009;117:173-90.
17. van AJ, Moser J, Lexis CP et al. Type 2 diabetes mellitus is associated with an imbalance
in circulating endothelial and smooth muscle progenitor cell numbers. Diabetologia.
2012;55:2501-12.
18. Voo S, Eggermann J, Dunaeva M, Ramakers-van OC, Waltenberger J. Enhanced functional
response of CD133+ circulating progenitor cells in patients early after acute myocardial
infarction. Eur Heart J. 2008;29:241-50.
19. Alvarez DF, Huang L, King JA et al. Lung microvascular endothelium is enriched with
progenitor cells that exhibit vasculogenic capacity. Am J Physiol Lung Cell Mol Physiol.
2008;294:L419-L430.
20. Yoder MC, Mead LE, Prater D et al. Redefining endothelial progenitor cells via clonal
analysis and hematopoietic stem/progenitor cell principals. Blood. 2007;109:1801-9.
21. Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells
for angiogenesis. Science. 1997;275:964-7.
22. Ingram DA, Mead LE, Moore DB et al. Vessel wall-derived endothelial cells rapidly
proliferate because they contain a complete hierarchy of endothelial progenitor cells.
Blood. 2005;105:2783-6.
23. Lin Y, Weisdorf DJ, Solovey A, Hebbel RP. Origins of circulating endothelial cells and
endothelial outgrowth from blood. J Clin Invest. 2000;105:71-7.
24. Adams TD, Gress RE, Smith SC et al. Long-term mortality after gastric bypass surgery. N
Engl J Med. 2007;357:753-61.
25. Carlsson LM, Peltonen M, Ahlin S et al. Bariatric surgery and prevention of type 2 diabetes
in Swedish obese subjects. N Engl J Med. 2012;367:695-704.
26. Schauer PR, Kashyap SR, Wolski K et al. Bariatric surgery versus intensive medical
therapy in obese patients with diabetes. N Engl J Med. 2012;366:1567-76.
27. Haller T, Ortner M, Gnaiger E. A respirometer for investigating oxidative cell metabolism:
toward optimization of respiratory studies. Anal Biochem. 1994;218:338-42.
28. Xu W, Koeck T, Lara AR et al. Alterations of cellular bioenergetics in pulmonary artery
endothelial cells. XU2007. Proc Natl Acad Sci U S A. 2007;104:1342-7.
29. Wang YC, McPherson K, Marsh T, Gortmaker SL, Brown M. Health and economic burden
of the projected obesity trends in the USA and the UK. Lancet. 2011;378:815-25.
30. Schauer PR, Bhatt DL, Kirwan JP et al. Bariatric Surgery versus Intensive Medical
Therapy for Diabetes - 3-Year Outcomes. N Engl J Med. 2014.
31. Aird WC. The role of the endothelium in severe sepsis and multiple organ dysfunction
syndrome. Blood. 2003;101:3765-77.
32. Kamihata H, Matsubara H, Nishiue T et al. Improvement of collateral perfusion and
regional function by implantation of peripheral blood mononuclear cells into ischemic
hibernating myocardium. Arterioscler Thromb Vasc Biol. 2002;22:1804-10.
33. Chen J, Song M, Yu S et al. Advanced glycation endproducts alter functions and promote
apoptosis in endothelial progenitor cells through receptor for advanced glycation
endproducts mediate overpression of cell oxidant stress. Mol Cell Biochem. 2010;335:137-
46.
34. Yan J, Tie G, Park B et al. Recovery from hind limb ischemia is less effective in type 2
than in type 1 diabetic mice: roles of endothelial nitric oxide synthase and endothelial
progenitor cells. J Vasc Surg. 2009;50:1412-22.
Figure Legends
FIGURE 1. Subject blood metabolic profiling. Blood was collected from subjects pre- and post-
intervention at the times shown in each panel. Blood was assayed for glycosylated hemoglobin
(HbA1c), fasting glucose, insulin, homeostasis model assessment-estimated insulin resistance
(HOMA-IR), triglycerides, total cholesterol, low-density lipoprotein (LDL), and high-density
lipoprotein (HDL). Data are expressed as means ± standard error. All reported p values in the
figures were generated by one-way ANOVA. Asterisk (*) denotes a significant difference
compared to baseline (time = 0) where p < 0.05 by one-way ANOVA. Hash mark (#) denotes
significant difference compared to baseline (time = 0) where p < 0.05 by unpaired t-test. Dotted
lines represent normal laboratory values. Subjects enrolled in the bariatric surgical arm
displayed significant improvements in HbA1c, triglycerides, and HDL levels at 24-months with
such improvements exhibited as early as three months post-surgery. Importantly, all other
parameters tested in this bariatric surgical arm were significantly different from baseline only at
24-months post-intervention. Subjects enrolled in the medical arm did not display significant
improvements in any of the parameters tested.
FIGURE 2. Isolation, enumeration of circulating levels, and biological activity of LOEPCs
isolated from patients in the bariatric surgery arm. (A). Blood was collected from subjects pre-
and post-intervention and EOEPCs and LOEPCs were enriched from the mononuclear fraction as
described in the Methods section. Upper panel depict images from EOEPCs expanding for 1-
week on fibronectin-coated culture dishes for comparison. Left and middle upper panels depict
an image (10X magnification) of EOEPCs expanding and defining a colony forming units/foci,
respectively. Note the presence of outwardly migrating cells away from the original colony
focus (middle upper panel). Right upper panel depicts an image (40X magnification) of
EOEPCs that were subcultured and seeded onto fresh fibronectin-coated culture dishes. Note the
canonical elongated fusiform shape of the cells that occurs upon subculture. Bottom panels
depict images from LOEPCs expanding for 4-6 weeks on collagen-I-coated culture dishes. Left
and middle bottom panels depict an image (10X magnification) of LOEPCs expanding and
defining a colony forming units/foci, respectively. Note the presence of outwardly migrating
cells away from the original colony focus. Right bottom panel depicts an image (40X
magnification) of a confluent LOEPC monolayer at 4-weeks post-seeding. Note the canonical
oval-round shape of the cells. (B) The total number of LOEPC colonies was counted at the time
points shown and normalized per 107 total cells in the mononuclear fraction. Data are expressed
as means ± standard error. Asterisk (*) denotes a significant difference compared to baseline
(time = 0) where p < 0.05 by one-way ANOVA. (C). Blood was collected from subjects pre-
and post-intervention and LOEPCs were enriched from the mononuclear fraction, harvested, and
seeded onto Matrigel-coated culture dishes as described in the Methods section. The total
number of LOEPC networks was counted as an index of biological function at the time points
shown. Data are expressed as means ± standard error. Asterisk (*) denotes a significant
difference compared to baseline (time = 0) where p < 0.05 by one-way ANOVA.
FIGURE 3. LOEPC and metabolic profiling correlation analyses at 24-months post-bariatric
surgery. Selected results from the LOEPC analyses were directly compared to the metabolic
analyses by linear regression. R2 and p values are reported in each panel. The top panels show
LOEPC number correlations to BMI, insulin, and triglycerides where p < 0.05 and the bottom
panels show the corresponding LOEPC network formation correlations which display correlative
R2 values but were not statistically significant.
