1) Determine differences in EPC and SMPC availability in the peripheral blood of T2DM patients with or without coronary artery disease (CAD) or peripheral artery disease (PAD) as well as in age- and sex-matched non-diabetic subjects with or without…
ID
Source
Brief title
Condition
- Coronary artery disorders
- Diabetic complications
- Arteriosclerosis, stenosis, vascular insufficiency and necrosis
Synonym
Research involving
Sponsors and support
Intervention
Outcome measures
Primary outcome
From the peripheral blood obtained PBMCs will be isolated using Ficoll density
gradient centrifugation. Isolated cells will then be subjected to:
1) flowcytometric analyses:
These phenotypic analyses allows quantification circulating endothelial and
smooth muscle progenitor cells (EPCs and
SMPCs, respectively) in the peripheral blood of diabetic and non-diabetic
subjects and correlate frequencies of these
specific cell subsets to the presence of cardiovascular disease.
2) flowcytometric cell sorting:
These analyses allow purification of specific EPC and SMPC subsets which
will be used for more in-depth functional and
molecular analyses as described below.
3) in vitro EPC and SMPC culture:
These analyses allow functional studies on EPCs and SMPCs like tube
formation, anti-thrombotic activity, differentiation
into mature ECs and SMCs, SMC contractility, and matrix production.
4) molecular analyses:
In order to gain insight into the molecular pathways involved in aberrant
EPC and SMPC function as determined under
point 3, mRNA and proteins will be isolated from purified cells susbsets
and/or after expansion/differentiation in vitro.
Isolated mRNA and proteins will then be subjected to gene expression
analysis (qRT-PCR and microarray analysis
[Illumina platform] and protein expression analysis (Western-blotting and
kinome profiling [PepChip platform]),
respectively.
5) histological analyses
From a subpopulation of patients with moderate to severe peripheral artery
disease the endarterectomy specimen will be collected and
processed for histological and moelcular biological analyses. Results of
these analyses will be correlated with the in vitro generated data on
cultured cells using the methods described above (1 to 4).
Collectively, the results of these analyses will contribute to the
determination of the level of vascular progenitor cell dysfunction in T2D and
to the identification of novel molecular targets for therapeutic intervention
aiming at modulating vascular progenitor cell frequency and function in order
to attenuate development of cardiovascular disease and restenosis in T2DM.
Secondary outcome
n.a.
Background summary
Individuals with Type 2 diabetes mellitus (T2DM) have increased rates and
severity of cardiovascular disease (peripheral artery disease [PAD] and
coronary artery disease [CAD]) and restenosis. The pathogenetic mechanism(s)
underlying increased cardiovascular disease and restenosis in T2DM is largely
unknown and adequate treatment strategies are lacking. We hypothesize that a
disturbed balance in vascular progenitor cells involved in maintenance of
endothelial integrity (i.e. endothelial progenitor cells [EPCs]), and
atherogenesis and neointima formation (i.e. smooth muscle progenitor cells
[SMPCs]) is responsible for increased susceptibility for cardiovascular disease
and restenosis in T2DM. Mechanistic understanding of the molecular pathways
involved in aberrant vascular progenitor cell function in T2DM is anticipated
to be a key requirement for the rational design of interventions aiming at
enhancing EPC-mediated vascular repair (i.e. promoting reendothelialization and
collateralization in the ischemic vascular bed) and reducing SMPC-mediated
atherogenesis and development of restenosis in T2DM. The current research
proposal is a next logical step to unravel these processes by performing
numerical (flowcytometry and in vitro cell culture) and functional (in vitro
cell culture and gene- and kinome profiling) analyses on vascular progenitor
cells. To this end, vascular progenitor cells will be obtained from Type 2
diabetics with or without cardiovascular disease as well as from non-diabetics
with or without cardiovascular disease.
Study objective
1) Determine differences in EPC and SMPC availability in the peripheral blood
of T2DM patients with or without coronary artery disease (CAD) or peripheral
artery disease (PAD) as well as in age- and sex-matched non-diabetic subjects
with or without CAD or PAD.
2) Determine molecular and functional differences in EPCs and SMPCs between
these groups of patients.
These objectives will contribute to the identification of novel molecular
targets for therapeutic intervention aiming at modulating vascular progenitor
cell frequency and function in order to attenuate development of cardiovascular
disease and restenosis in T2DM.
Study design
For this study six groups of patients will be included as summarized below:
Group 1: T2DM, + PAD, - CAD
Group 2: T2DM, - PAD, + CAD
Group 3: T2DM, - PAD, - CAD
Group 4: no T2DM, + PAD, - CAD
Group 5: no T2DM, - PAD, + CAD
Group 6: no T2DM, - PAD, - CAD
On the first next planned visit to the outpatient clinic of the Dept. Internal
Medicine, Dept. Vascular Surgery or Cardiology of the UMCG, six extra tubes
(total 60 ml) of peripheral blood will be obtained, in addition to the
laboratory assessments (including HbA1c and lipid profile), which are routinely
performed at each visit at the outpatient clinic. Peripheral blood samples will
be collected in 6x 10ml EDTA vacutainer tubes (K3E 15% 0.12 ml).
Since not all anticipated analyses can be performed simultaneously and because
of limitations in obtainable cell numbers, patients will be requested to donate
60 ml of peripheral blood during two consecutive visits to the outpatient
clinic. During the total duration of the study, each patient will donate 2x
60ml = 120 ml blood.
From a subgroup of patients included in group 1 (n=10) and 4 (n=10) (i.e. PAD
patients with- or without T2DM, respectively) that will undergo endovascular
surgery because of moderate to severe claudication (stage IIb-IV Fontaine
classification) the atherosclerotic plaque will be collected. Prior to
endarterectomy, 60 ml of peripheral blood will be collected. Immediately
following endarterectomy the plaque will be divided into two parts: one part
will be snap frozen, the other half will be formalin-fixed. Plaques will be
used for histological analyses as well as gene/protein expression analyses.
Study burden and risks
The nature of the research is such that participation entails no inherent risk
for the subject. The extent of the burden is relatively low (donation of 2x 60
ml peripheral blood during regular visits at the outpatient clinic of the UMCG)
or donation of 60 ml blood prior to endovascular surgery. Healthy control
subjects will come to our center specifically for the purpose of this study and
will therefore receive a modest compensation. Participants of the study will
not have immediate benefit from the outcome of the study.
Hanzeplein 1
PO Box 30.001 9700 RB Groningen
NL
Hanzeplein 1
PO Box 30.001 9700 RB Groningen
NL
Listed location countries
Age
Inclusion criteria
Patients eligible for participation are diagnosed with T2DM (duration 10-15 yrs) and divided into diabetic patients with PAD, with CAD or without cardiovascular disease. Patients eligible for participation in the various control groups are patients without a history of T2DM but with PAD or CAD, respectively. In addition, healthy individuals who have neither a history of diabetes, cardiovascular disease, inflammatory or autoimmune disease, nor obesity nor any other chronic disease are will be included.;Definitions PAD and CAD:;Peripheral artery disease (PAD)
PAD is defined by the presence of carotid stenosis or lower extremity atherosclerosis obliterans. Severity of atherosclerosis of carotid vessels will be assessed by ultrasonography as a continuous variable. Diagnosis of atherosclerotic involvement of the lower extremities will be assessed by history of claudication or rest pain, bilateral pulses examination (dorsal pedal, posterior tibial, popliteal, and femoral arteries), ultrasonography performed bilaterally at levels of popliteal and femoral arteries, and, eventually, angiography. Patients are then classified according to the Leriche/Fontaine clinical classification of lower limb atherosclerosis obliterans.;Coronary artery disease (CAD)
CAD is defined as previous myocardial infarction, resting ECG indicative of past MI, positive ECG at exercise stress test, echocardiography stress-test positive for inducible ischemia, evidence of significant coronary artery stenose on angiography with or without typical chest pain. ;Participants allocated to the different groups will be matched for:
- gender
- age
- smoking
- BMI
- arterial hypertension
- HbA1c (diabetics)
- lipid profile
Exclusion criteria
Predefined exclusion criteria for T2DM include diagnosis of T1DM and presence of any of the following (self-reported) medical conditions:
- microvascular disease (diabetic retinopathy and nephropathy)
- auto-immune diseases
- current or prior cancers
- acute or chonic infection
- recent surgery or vascular intervention
- age >80yrs
- recent myocardial infarction
- hemodialysis
- immunosuppression
- any other unrelated disease;Exclusion criteria for non-diabetics include:
- diagnosis of T1DM or T2DM
- auto-immune diseases
- current or prior cancers
- acute or chonic infection
- recent surgery or vascular intervention
- age >80yrs
- recent myocardial infarction
- hemodialysis
- immunosuppression
- any other unrelated disease
Design
Recruitment
Followed up by the following (possibly more current) registration
No registrations found.
Other (possibly less up-to-date) registrations in this register
No registrations found.
In other registers
| Register | ID |
|---|---|
| CCMO | NL25792.042.08 |