Amelioration of glucose control mobilizes circulating pericyte progenitor cells in type 2 diabetic patients with microangiopathy.

Abstract

Chronic diabetic complications result from an imbalance between vascular damage and regeneration. Several circulating lineage-committed progenitor cells have been implicated, but no data are available on pericyte progenitor cells (PPCs). Based on the evidence that PPCs increase in cancer patients after chemotherapy, we explored whether circulating PPC levels are affected by glucose control in type 2 diabetic patients, in relation to the presence of chronic complications. We enumerated peripheral blood PPCs as Syto16+CD45-CD31-CD140b+ events by flow cytometry at baseline and after 3 and 6 months of glucose control by means of add-on basal insulin therapy on top of oral agents in 38 poorly controlled type 2 diabetic patients. We found that, in patients with microangiopathy (n = 23), the level of circulating PPCs increased about 2 fold after 3 months and then returned to baseline at 6 months. In patients without microangiopathy (control group, n = 15), PPCs remained fairly stable during the whole study period. No relationship was found between change in PPCs and macroangiopathy (either peripheral, coronary, or cerebrovascular). We conclude that glucose control transiently mobilizes PPCs diabetic patients with microangiopathy. Increase in PPCs may represent a vasoregenerative event or may be a consequence of ameliorated glucose control on microvascular lesions.

Full text

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Hindawi Publishing Corporation
Experimental Diabetes Research
Volume 2012, Article ID 274363, 8 pages
doi:10.1155/2012/274363
Research Article
Ameliorationof GlucoseControl Mobilizes
CirculatingPericyteProgenitor CellsinType2
DiabeticPatients withMicroangiopathy
GianPaolo Fadini,1,2PatriziaMancuso,3Francesco Bertolini,3
Saulade Kreutzenberg,1and Angelo Avogaro1,2
1Department of Clinical and Experimental Medicine, University of Padova, 35128 Padova, Italy
2Venetian Institute of Molecular Medicine, 35129 Padova, Italy
3European Institute of Oncology, 20141 Milan, Italy
Correspondence should be addressed to Gian Paolo Fadini, gianpaolofadini@hotmail.com
Received 5 December 2011; Accepted 20 December 2011
Academic Editor: Paolo Madeddu
Copyright © 2012 Gian Paolo Fadini et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Chronic diabetic complications result from an imbalance between vascular damage and regeneration. Several circulating lineage-
committed progenitor cells have been implicated, but no data are available on pericyte progenitor cells (PPCs). Based on the
evidence that PPCs increase in cancer patients after chemotherapy, we explored whether circulating PPC levels are affected by
glucose control in type 2 diabetic patients, in relation to the presence of chronic complications. We enumerated peripheral blood
PPCs as Syto16+CD45−CD31−CD140b+ events by flow cytometry at baseline and after 3 and 6 months of glucose control by
means of add-on basal insulin therapy on top of oral agents in 38 poorly controlled type 2 diabetic patients. We found that, in
patients with microangiopathy (n = 23), the level of circulating PPCs increased about 2 fold after 3 months and then returned
to baseline at 6 months. In patients without microangiopathy (control group, n = 15), PPCs remained fairly stable during the
whole study period. No relationship was found between change in PPCs and macroangiopathy (either peripheral, coronary, or
cerebrovascular). We conclude that glucose control transiently mobilizes PPCs diabetic patients with microangiopathy. Increase in
PPCs may represent a vasoregenerative event or may be a consequence of ameliorated glucose control on microvascular lesions.
1.Introduction
Chronic diabetic complications are thought to result from
the detrimental effects exerted by hyperglycemia and associ-
atedmetabolicabnormalitiesonvascularstructureandfunc-
tion [1]. Moreover, in recent years, it became apparent that
vascular regeneration is impaired in diabetes, at least in part
through pauperization of bone-marrow-derived progenitors
[2]. Indeed, evidences accumulated to support the existence
of circulating progenitors for several phenotypes not limited
to the hematopoietic lineages and potentially important for
the cardiovascular system [3]. Thus, endothelial [4], smooth
muscle [5], osteoblast [6] and, possibly, cardiomyocyte pro-
genitor cells [7] from the bloodstream have been described.
These cells may have various protective or detrimental
effects on vascular structure and function, although their
quantitative contribution to cardiovascular biology is far
from being definitely elucidated [3]. In parallel, the presence
of mature circulating endothelial cells (CECs) is meant
to represent an epiphenomenon of the ongoing vascular
damage, as these cells are passively released from the vessel
wall [8]. Importantly, most these cells have been implicated
in the setting of diabetes and its chronic complication,
suggesting a multifaceted contribution of blood-derived
cells in the complex pathophysiology of diabetic micro-
and macroangiopathy. These include reduced EPCs [9] and
cardiomyocyte differentiation [10], increased generation of
smooth muscle progenitor cells [11] and procalcific cells [6],
paralleled by a high concentration of circulating shed CECs
[12]. Very recent data suggest the existence of circulating

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2 Experimental Diabetes Research
pericyte progenitor cells (PPCs) [13]. Pericytes provide a
variety of functions, such as capillary blood flow regulation,
clearance and phagocytosis of cellular debris, and regulation
of vascular permeability. Importantly, pericytes stabilize
and monitor the maturation of endothelial cells by direct
communication between the cell membrane and paracrine
signaling [14]. They are recruited through the PDGF-B and
PDGFR-Beta signaling, while PDGFR-Beta deficient mice
display extensive vascular leakage, hemorrhage, and edema
due to a defect of capillary coverage by pericytes [15, 16].
Interestingly, PPCs were found to be increased in patients
and mice with malignant tumors and also increased after
chemotherapy [13].
We hypothesized that PPCs play a role in the setting of
diabetic microangiopathy. Based on this background and on
the proposed role for PPC in response to cancer therapy, in
this study we explored whether glucose control affects levels
of circulating PPCs in type 2 diabetic patients, in relation to
microvascular complications.
2.MaterialsandMethods
2.1. Patients. The study was approved by the Ethic commit-
tee of the University Hospital of Padova (protocol no. 1584P)
and is registered in http://clinicaltrials.gov/ (NCT00699686).
It was conducted in accordance with the Declaration of
Helsinki and all patients provided written informed consent.
Briefly, this was a trial of optimization of glucose control
in type 2 diabetic patients poorly controlled on oral agents,
with addition of basal insulin on top of their ongoing
antihyperglycemic regimen. Insulin glargine and insulin
detemir were compared in a randomized cross-over fashion
during a 3+3 month period. The study design and clinical
characteristics of the study population have been previously
described [17]. The primary aim was to detect differences
in the change of endothelial progenitor cells (EPCs) and
circulating progenitor cells (CECs) levels in the bloodstream
between the 2 insulin regimens. Out of a total of 42 patients,
21 were randomized to receive insulin glargine for 3 months
and then insulin detemir for 3 months without washout,
and 21 patients were randomized to the detemir-glargine
treatment sequence. As a result of the study, we found that
optimization of glucose control per se reduced CECs and
other markers of endothelial damage, and increased EPCs,
as markers of endothelial regeneration [17]. There was no
difference in the effects of glargine versus detemir in terms
of markers of endothelial damage and regeneration. This
allowed us to consider the 2 insulin regimens and a single
type of treatment. In parallel to EPCs and CECs, we also
quantified circulating pericyte progenitor cells (PPCs) to
evaluate the effects of glucose control on this cell type. PPC
analysis was carried out in 38 patients and was unsuccessful
in 4, due to technical reasons. Inclusion criteria were T2D
withHbA1c>7.0%onoralagents,age40–80andpresenceof
macroangiopathy (either coronary, peripheral or cerebrovas-
cular artery disease). Exclusion criteria were T1D, acute
hyperglycaemia, use of glitazones, DPP-4 inhibitors, cancer,
any acute disease or infection, recent (within 3 months)
surgery or cardiovascular intervention, serum creatinine
>2.0mg/dL, advanced liver disease, inability to provide
informedconsent,andpregnancy/lactation.Allpatientswere
characterized with anthropometric measures, evaluation
of concomitant risk factors, diabetic complications and
medications, as described elsewhere. Briefly, retinopathy was
defined by a digital funduscopic examination as any degree
of retinopathy according to the Early Treatment Diabetic
Retinopathy Study (ETDRS) Research Group classification
[18]. Nephropathy was defined by measuring urinary albu-
min/creatinineratioon3differentsamplesandtheestimated
glomerular filtration rate according to the MDRD equation
[19]. Neuropathy was defined according to classical symp-
toms and signs, eventually confirmed by electromyography.
2.2. Flow Cytometry. Analysis was performed on frozen
peripheral blood mononuclear cells according to a standard-
ized protocol. PPCs were evaluated by six-color flow cytom-
etry following an approach recently validated in our labora-
tory for the enumeration of CECs with some modifications
[13].PPCsweredefined asSyto16+CD45−CD31−CD140b+
events. The nuclear staining Syto16 was used to discrim-
inate between nucleated cells, platelets, and cell debris.
The panel of monoclonal antibodies used included anti-
CD45 (to exclude hematopoietic cells), anti-CD31 (an EC
differentiation marker), and anti-CD140b (PDGFR-Beta).
All antibodies were from Becton Dickinson (BD, Mountain
View,CA).Cellsuspensionswereevaluatedaftercellrecovery
by a FACSCanto (BD). After acquisition of at least 1 ×
106cells per blood sample, analyses were considered as
informativewhenadequatenumbersofcells(i.e.,>100)were
collected in the PPC enumeration gates. PPCs were defined
as nucleated cells, negative for the hematopoietic marker
CD45 and the EC marker CD31 and positive for CD140b.
The gating strategy is illustrated in Figure 1. This definition
identifies circulating cells not belonging to either leukocyte
populations (CD45-neg) or shed endothelial cells (CD31-
neg) and expressing the pericyte marker CD140b (PDGFR-
Beta).
2.3. Statistical Analysis. Data are expressed as mean ±
standard error for continuous variables or as percentages
for categorical variables. Comparisons between two groups
wereperformedusingtwo-tailStudent’st-testforcontinuous
variables or the chi-square test for categorical variables. To
assess changes of PPC levels over time, we used the analysis
of variance (ANOVA) for repeated measures with post-hoc
paired t-tests. Statistical significance was accepted at P <
0.05.
3.Results
3.1. Patient Characteristics and Effects of Glucose Control.
The characteristics of the 38 patients included in the study
and divided by the presence/absence of microangiopathy
are resumed in Table 1. Microangiopathy was defined as
the presence of anyone among retinopathy, nephropathy
(micro- or macroalbuminuria with or without renal failure),
andneuropathy.Besidesretinopathy,microalbuminuria,and

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Experimental Diabetes Research3
250
200
150
100
50
102
103
104
105
CD45-APC
SSC
(a)
0
103
104
105
0
103
104
105
Syto16-FITC
CD31-PE Cy7
(b)
102
102
103
104
105
103
CD140b-PE
104
105
CD31-PE Cy7
Q1Q2
Q3Q4
(c)
102
102
103
104
105
103
104
105
CD140b-PE
CD31-PE Cy7
Q1 Q2
Q3 Q4
(d)
Figure 1: The gating strategy for enumeration of circulating PPCs. (a) Peripheral blood mononuclear cells were first gated into the CD45-
negative fraction to exclude hematopoietic cells. (b) The total Syto16+ population of nucleated cells was selected to avoid inclusion of
contaminating red cells, platelets, and debris in the analysis. (c, d) The resulting population was analyzed for expression of CD31 and
CD140b. Panel (c) shows a case with low baseline PPCs (Syto16+CD45−CD31−CD140b+ cells), while (d) shows the same case 3 months
after initiation of glucose control.
neuropathy, differences between the two groups regarded
lower HDL cholesterol levels and higher incidence of periph-
eral arterial disease (PAD) in patients with microangiopathy.
These patients were subjected to optimization of glucose
control for 6 months by means of addition of a basal insulin
therapyaccordingtoaprotocoldescribedelsewhere.Asthere
were no differences between glargine and detemir in the
effects on endothelial markers of damage and regeneration,
these treatment regimens were considered altogether as a
single intervention. On average, HbA1c dropped from 8.8 ±
0.2% to 7.2±0.1% (P < 0.001) indicating good optimization
of glucose control, and 17 patients (45% of total) reached a
HbA1clevelof7.0%oflower.Betweenthetwogroups(w/wo
microangiopathy), there were no differences in baseline
HbA1c levels or achieved HbA1c during the intensification
protocol at 3 months (no microangiopathy 7.4 ± 0.2;
microangiopathy 7.2 ± 0.1; P = 0.30) and 6 months (no
microangiopathy 7.3 ± 0.2; microangiopathy 7.1 ± 0.1; P =
0.17; Figure 2(a)).
3.2. Changes in PPC Levels during Optimization of Glu-
cose Control. Circulating PPCs were measured at baseline,
3 months and 6 months. In the entire study population of 38
subjects, there was a trend toward increased PPC levels at 3
monthsversusbaseline,whichwasnotstatisticallysignificant
(P = 0.29).TherewerenodifferencesinPPClevelsaccording
totypeofinsulinused(P = 0.74intheanalysisforcross-over
design). When patients were divided according to the pres-
ence or absence of microangiopathy, we found that PPC level
remained unchanged during the entire course of the study

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4 Experimental Diabetes Research
Table 1: Patients characteristics. P values are shown for paired Student’s t-test or the chi-square test as appropriate. ACEi/ARB denotes
angiotensin-converting enzyme inhibitors or angiotensin receptor blockers.
Characteristic
Without
microangiopathy
With
microangiopathy
P
Number 2315—
Age (years)62.7 ±2.4
68.8
67.2 ±1.2
81.8
0.08
Sex male (%)
BMI (kg/m2)
0.36
29.5 ±1.3
103.5 ±3.3
9.0 ±0.3
27.2 ±0.6
99.8 ±2.2
8.6 ±0.2
0.10
Waist (cm)
0.35
Baseline HbA1c (%)
0.19
Concomitant risk factors
Total cholesterol (mg/dL) 183.1 ±6.5
51.3 ±2.9
105.6 ±5.2
131.1 ±14.5
0.0
176.8 ±9.7
43.2 ±2.1
97.1 ±8.5
181.8 ±25.2
13.6
0.61
HDL cholesterol (mg/dL)
0.026
LDL cholesterol (mg/dL)
0.43
Triglycerides (mg/dL)
0.11
Smoking habit (%)0.13
Hypertension (%) 93.881.80.29
Complications
Retinopathy (%)0.0 45.5
<0.01
<0.01
<0.01
0.03
Microalbuminuria (%) 0.0 50.0
Neuropathy (%)0.0 40.9
Peripheral arterial disease (%)6.3 36.4
Coronary artery disease (%)18.827.30.55
Cerebrovascular disease (%)75.077.2 0.82
Medications
Metformin (%)93.8 81.8 0.29
Sulphonylureas (%) 68.868.2 0.97
Aspirin (%) 68.8 86.30.19
Statin (%) 56.368.2 0.46
ACEi/ARBs (%) 87.5 63.60.10
Other antihypertensives (%) 68.863.60.75
in patients without microangiopathy, while it significantly
increased at 3 months only in patients with microangiopathy
(P = 0.01 using post-ANOVA t-test; Figure 2(b)). Among
the 3 different types of microangiopathy that were con-
sidered, presence of micro-/macroalbuminuria (Figure 2(c))
and neuropathy (Figure 2(d)) were associated with PPC
increase at 3 months, while retinopathy was not significantly
discriminative of patients that increase PPC levels during
the glucose control protocol (Figure 2(e)). Interestingly, in
all cases, PPC levels returned to baseline at 6 months. As
a control experiment, we also divided patients according to
the presence/absence of PAD, which was more prevalent in
patients with microangiopathy, and found that there was no
differences in the trend of PPC levels over time in the two
groups of patients (Figure 2(f)). The same was for coronary
and cerebrovascular disease, which showed no correlation
with PPC levels over time (not shown). Concentration of
HDL cholesterol was not associated with change in PPC
levels during the study (not shown).
4.Discussion
In this study, we found that in type 2 diabetic patients
with microangiopathy glucose control is associated with a
transient increase in circulating PPC levels.
Mounting evidence suggeststhat multilineage circulating
progenitor cells have a variety of implications in diabetes
and its complications. After endothelial progenitor cells
(EPCs), smooth muscle progenitors, osteoblast precursors,
and cardiomyocyte progenitors [4–6, 20], recent data now
suggest the existence of circulating pericyte progenitor cells
(PPCs) [13]. These cells have been identified and isolated
from human or murine peripheral blood and reside in
the nonhematopoietic (CD45-neg) compartment, and are
distinct from CECs as they lack endothelial antigens (CD31-
neg), but express the typical pericyte marker CD140b
(PDGFR-Beta). This antigenic phenotype supports the per-
icytic origin, while electron microscopy confirmed their
progenitor-like morphology, with high nucleus/cytoplasm

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Experimental Diabetes Research5
10
9
8
7
6
5
4
3
2
1
0
No micro (n = 15)
Micro (n = 23)
∗
∗
∗
∗
HbA1c (%)
(a)
40
35
30
25
20
15
10
5
0
No micro (n = 15)
Any micro (n = 23)
∗
Pericyte progenitor cells (/106PBMCs)
(b)
40
35
30
25
20
15
10
5
0
∗
ACR <30 (n = 27)
ACR >30 (n = 11)
Pericyte progenitor cells (/106PBMCs)
(c)
40
35
30
25
20
15
10
5
0
No neuro (n = 27)
Neuro (n = 11)
∗
Pericyte progenitor cells (/106PBMCs)
(d)
40
35
30
25
20
15
10
5
0
No retino (n = 26)Retino (n = 12)
Baseline
3 months
6 months
Pericyte progenitor cells (/106PBMCs)
(e)
40
35
30
25
20
15
10
5
0
No PAD (n = 28) PAD (n = 10)
Baseline
3 months
6 months
Pericyte progenitor cells (/106PBMCs)
(f)
Figure 2: Effects of glucose control on HbA1c and PPCs. (a) There were no differences in HbA1c levels in patients with and without
microangiopathy during time (∗P < 0.05 versus baseline). (b) Increase in PPC levels was seen only in patients with microangiopathy
(ANOVA P < 0.05;∗post hoc P < 0.05). (c–f) Patients were divided according to the presence of micro-/macroalbuminuria, neuropathy,
retinopathy, and peripheral arterial disease (PAD): a significant PPCs increase was detected in patients with urinary albumin-creatinine ratio
(ACR) >30mg/g and in the presence of neuropathy (ANOVA P < 0.05;∗post hoc P < 0.05).

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6 Experimental Diabetes Research
ratio, rough endoplasmic reticulum cisterns and centrioles
and absence of Weibel-Palade bodies typical of CECs [13].
In the setting of diabetic complications, pericytes may
play an important role. Pericytes are an important com-
ponent of the neurovascular unit both in the central and
peripheral nervous system and may intervene in the patho-
genesis of peripheral neuropathy, through the modulation
of vasa nervorum [21]. Moreover, glomerular mesangial
cells, which play a central role in the pathobiology of
diabetic nephropathy [22], are specialized pericytes [23].
Finally, pericyte loss is one of the earliest features of diabetic
retinopathy and the consequent defective endothelial cover-
age of retinal capillaries favors microaneurysmatic dilation
and increased permeability [24]. Therefore, the study of
PPCs may have important implications in the setting of
diabetic microvascular complications.
Interestingly,PPCswerefoundtobeincreasedinpatients
and mice with malignant tumors and also increased after
chemotherapy [13]. Therefore, we analyzed whether the
level of circulating PPCs is influenced by optimization of
glycemic control in type 2 diabetic patients in relation to the
presence of microangiopathy. In a cohort of 38 patients in
which HbA1c was drastically reduced by insulin therapy, a
significant increase in PPC level at 3 months was detected
only in the presence of microangiopathy. Of note, this
increase was transient, as cell counts returned to baseline at
6 months. Importantly, the PPC increase occurred during
the first 3 month period, when HbA1c dropped markedly
and then stabilized for the subsequent 3 months, suggesting
that glucose control was the driver of PPC increase. We
found that nephropathy and neuropathy were associated
with PPC mobilization, while retinopathy was not. This
is probably due to the fact that most patients had mild
nonproliferative retinopathy and that a stratification for
retinopathy severity was impossible, as groups of patients
were too small. Moreover, the systemic levels of PPCs may
not reflect processes ongoing within the central nervous
system.
There are several potential implications of our present
findings. First, it is possible that glucose control induces a
mobilization of bone-marrow-derived PPCs, as previously
shown for EPCs [25]. These cells would then function to
stabilizebloodvesselsandcountertheprogressionofdiabetic
microangiopathy. However, the study of GFP+ bone marrow
chimeric mice suggests that murine PPCs are derived from
peripheral tissues and not from the bone marrow [13].
Therefore, non-bone-marrow sources of these regenerative
cells should be postulated [26]. The Madeddu’s laboratory
has clearly demonstrated that PPCs can be isolated from the
saphenous vein and display potent cardiovascular regenera-
tive activity [27, 28]. At present, we can only speculate on
the mechanisms that induce PPC mobilization: it has been
previously documented that circulating progenitor cells are
recruited from the bloodstream to the perivascular space
through the SDF-1/CXCR4 axis, whence they are mobilized
by VEGF [29]. As insulin has been reported to stimulate
VEGF and to interact with PDGFR (CD140b) signaling
[30, 31], these growth factors may be important. A transient
release of tissue PPCs induced by glucose control may also
reflect regression of pathologic vascular structures in organs
hit by diabetic microangiopathy, just as it happens in cancer
chemotherapy. Regression of microvascular lesions owing to
lower oxidative stress and inflammation achieved by glucose
control [32] may also be responsible for passive mobilization
of these cells from tissues to the bloodstream. Alternatively,
the transient PPCs increase may be related to the worsening
of diabetic microangiopathy that is sometimes induced by
rapid glucose control [33, 34]. Unfortunately, owing to the
relatively short duration of our study, it is impossible to
determine whether the increase in PPC was associated with a
favorable or unfavorable evolution of microangiopathy.
This study has other limitations, including the relatively
small sample size and, importantly, the incomplete char-
acterization of circulating PPCs. Indeed, it must be noted
thatcogentdemonstrationsthattheseSyto16+CD45–CD31–
CD140b+ cells truly belong to the pericyte lineage and act
as progenitors are still missing for the following reasons.
First, there is no surface antigen that can unequivocally
identify pericyte lineage cells. Second, we found a small
degree of coexpression of the pericyte marker NG2 [35] by
circulating CD140b cells (not shown), suggesting that the
pericytic phenotype of PPCs is incomplete. Additionally, it
is not clear how PPCs are related to CD34+CD140b+ cells,
whichSchoberetal.identifiedasperivascularsmoothmuscle
progenitors cells related to the severity of cardiac allograft
vasculopathy[36].Finally,selectivecultureofPPCsisneeded
to test their phenotype, proliferative potential, and function
in vitro and in vivo.
5.Conclusions
Despite these drawbacks, the interpretation of our results
lends to several intriguing speculations on the pathophysi-
ology of diabetic microangiopathy and response to therapy.
The mobilization of PPCs induced by amelioration of
glucose control deserves a special attention in relation to the
evolution of microangiopathy over time. Further studies are
required to reach a better characterization of PPCs and to
understand their relationships with diabetic complications.
Acknowledgment
This study was supported by a European Foundation for the
Study of Diabetes (EFSD)/AstraZeneca Young Investigator
Award grant to G. P. Fadini.
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