Objective
The purpose of this study was to examine whether women with gestational diabetes mellitus (GDM) and their offspring have reduced endothelial progenitor cell subsets and vascular reactivity.
Study Design
Women with GDM, healthy control subjects, and their infants participated. Maternal blood and cord blood were assessed for colony-forming unit–endothelial cells and endothelial progenitor cell subsets with the use of polychromatic flow cytometry. Cord blood endothelial colony-forming cells were enumerated. Vascular reactivity was tested by laser Doppler imaging.
Results
Women with GDM had fewer CD34, CD133, CD45, and CD31 cells (circulating progenitor cells [CPCs]) at 24-32 weeks’ gestation and 1-2 days after delivery, compared with control subjects. No differences were detected in colony-forming unit–endothelial cells or colony-forming unit–endothelial cells. In control subjects, CPCs were higher in the third trimester, compared with the postpartum period. Cord blood from GDM pregnancies had reduced CPCs. Vascular reactivity was not different between GDM and control subjects.
Conclusion
The normal physiologic increase in CPCs during pregnancy is impaired in women with GDM, which may contribute to endothelial dysfunction and GDM-associated morbidities.
Maternal vascular remodeling occurs throughout pregnancy to support increasing fetal metabolic demands. The endothelium has a major role in sustaining vascular health. Cellular and molecular processes that maintain endothelial function require a complex interplay between endothelial cells and subpopulations of circulating cells, termed endothelial progenitor cells (EPCs). In nonpregnant adults, reduced EPCs correlate with increased vascular disease risk. EPC dysfunction is speculated to contribute to the pathogenesis of several gestational disorders that include preeclampsia and gestational diabetes mellitus (GDM). However, the few studies that have assessed EPCs during pregnancy reported conflicting data because of the variety of assays that were used to quantitate EPCs. Moreover, since these studies were conducted, the definition of an EPC has evolved because most methods that were used previously have identified hematopoietic progenitors, not endothelial progenitors.
The most common techniques to quantitate EPCs are colony assays and flow cytometry. Colony-forming unit endothelial cells (CFU-ECs) are hematopoietic progenitors that facilitate angiogenesis; endothelial colony-forming cells (ECFCs) are progenitors with vessel-forming capacity. Flow cytometry detects the simultaneous expression of several antigens on individual cells and is used to examine cell subsets that are involved in vascular homeostasis. Specifically, circulating progenitor cells (CPCs) coexpress CD34, CD133, CD45, and CD31 (CD34+CD133+CD45+CD31+). CPCs are hematopoietic progenitors with robust angiogenesis-facilitating functions. Importantly, CPCs are being used as markers of vascular disease risk and to evaluate antiangiogenic therapies for cancer. Therefore, we questioned whether pregnant women with GDM and their offspring have reduced CFU-ECs and CPCs.
Materials and Methods
Women with GDM and healthy pregnant control subjects were recruited through obstetric clinics and advertisements. Women were studied from 24-32 weeks’ gestation according to the obstetricians’ best estimate of gestation, 1-2 postpartum days, and 6-8 postpartum weeks. Newborn infants were studied at 1-2 days of age. GDM was defined according to the American College of Obstetrics and Gynecology guidelines. Exclusion criteria included type 1 or 2 diabetes mellitus; illnesses known to affect glucose metabolism (ie, Cushing syndrome, polycystic ovarian syndrome); the use of medications that affect glucose metabolism (ie, dexamethasone); multiple gestation; cardiovascular disease; history of preeclampsia, and women who carried fetuses with chromosomal abnormalities. Additionally, delivery at <35 weeks’ gestation was an exclusion criterion for mother and infant postpartum visits and cord blood studies, although data that were obtained during the third-trimester visit were included in all analyses. Historical and clinical data were obtained at each visit. Maternal blood was collected for glycosylated hemoglobin (HgA1C), fructosamine, CFU-EC assays, and polychromatic flow cytometry (PFC). Cord blood was used for CFU-EC assays, ECFC assays, and PFC. The Institutional Review Board at the Indiana University School of Medicine approved all protocols; informed consent was obtained from all women.
Mononuclear cell preparation
Blood was collected in citrate cell preparation tubes (BD Biosciences, Franklin Lakes, NJ) and processed in our angiogenesis and endothelial progenitor cell core. Tubes were centrifuged at 1600 g for 30 minutes, and mononuclear cells were collected.
CFU-EC assay
CFU-ECs were assessed with the use of the EndoCult Kit (StemCell Technologies, Vancouver, BC, Canada), as described elsewhere. Briefly, mononuclear cells were plated in complete EndoCult media (Cambrex, Walkersville, MD) on fibronectin-coated tissue culture plates (BD Biosciences). After 48 hours, nonadherent cells were replated on fibronectin-coated tissue culture plates for 3 days; colonies were scored in a blinded fashion.
ECFC assay
ECFCs were enumerated as described elsewhere. Briefly, tissue culture plates that had been precoated with collagen (BD Biosciences) were seeded with mononuclear cells. The medium was changed daily. Colonies appeared at 5-8 days and were identified as circumscribed monolayers of cobblestone-appearing cells. All samples were scored in a blinded fashion.
PFC
PFC uses advanced analytic methods to enumerate rare cell subpopulations and represents a superior technique, compared with conventional flow cytometric approaches. Accurate measurement of infrequent cell populations with PFC is accomplished by including critical controls to enhance data acquisition, reproducibility, and analysis. Mononuclear cells were stained with directly conjugated monoclonal antibodies: CD31 fluoroscein isothiocyanate (BD Pharmingen, San Diego, CA), CD34 phycoerythrin (BD Pharmingen), CD133 allophycocyanin (Miltenyi Biotec, Auburn, CA), CD45 Allophycocyanin-AlexaFluor 750 (Invitrogen, Carlsbad, CA), the amine reactive viability dye, ViViD (Invitrogen); CD41a (BD Pharmingen) and CD235a (R&D Systems, Minneapolis, MN) both conjugated to Pacific Blue (Invitrogen) for platelet and red blood cell exclusion, respectively. Fluorescence minus 1 control samples were prepared as negative gating controls, and anti-mouse immunoglobulin CompBeads (BD Biosciences, Bedford, MA) were stained with antibodies as single-color compensation controls, as described elsewhere. CPCs that coexpress CD34, CD133, CD45, and CD31 (CD34+CD133+CD45+CD31+) and circulating endothelial cells (CECs) that express CD34 and CD31, but not CD133 and CD45 (CD34+CD133–CD45–CD31+), were analyzed on a Becton Dickinson LSR II cytometer with FlowJo software (version 8.7.3; Tree Star, Inc, Ashland, OR). CD133 negative nonangiogenic progenitors were enumerated (CD34+CD133–CD45+CD31+) to calculate a ratio of CPCs to nonangiogenic progenitors (nonCPCs), as described elsewhere. At least 500,000 events were collected. CPC data are reported as percent live cells in a low side scatter and low forward scatter gate. No differences were detected in total events that were evaluated between control and GDM samples. Analyses were conducted by the angiogenesis and endothelial progenitor cell core personnel who were blinded to all clinical data.
Vascular reactivity
Endothelial function in women (24-32 weeks’ gestation) and newborn infants (1-2 days of age) was evaluated by iontophoresis of acetylcholine to the skin and measurement of blood flow by laser Doppler imaging, as described elsewhere. A probe that was connected to a laser Doppler imager (wavelength 780 nm; Periflux 5001; Perimed, Stockholm, Sweden) was attached to the forearm. The probe temperature was 32°C during all tests. Basal skin perfusion was measured for 2 minutes, then acetylcholine was introduced by iontophoresis for 20 seconds at 60-second intervals and repeated 6 times. The laser Doppler output is expressed in perfusion units (PU) of output voltage (1 perfusion unit = 10 mV). The percent change in perfusion was calculated by the division of perfusion unit after each acetylcholine stimulation by basal perfusion unit × 100. Subjects were fasting for 2-3 hours.
Statistical methods
Unless otherwise stated, data were given as mean ± SEM. Subjects’ characteristics between control and GDM groups were compared with the use of 2-sample independent t tests for continuous outcomes and either chi-square or Fisher’s exact tests for discrete outcomes. Repeated-measures analyses of variance with appropriate transformation were used to analyze CPC and CEC data. The models compared outcomes at different time periods between the groups and within the groups. To adjust for prepregnancy body mass index (BMI) in comparing 2 groups at each time period, the model included prepregnancy BMI. Areas under curve were computed to analyze vascular reactivity, and Exact Wilcoxon 2-sample rank sum tests were applied to compare groups. Spearman rank correlations were used for all correlation analyses.
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