Am J Hypertens 22(9): 1014-1019

PMC: PMC2830891

PMID: 19498340

Maternal Endothelial Progenitor Colony-Forming Units With Macrophage Characteristics Are Reduced in Preeclampsia

BACKGROUND

Endothelial progenitor cells (EPCs) provide paracrine support to the vascular endothelium and may also replace damaged or senescent endothelial cells. Low numbers of endothelial progenitor colony-forming units (CFU-ECs) in culture are a predictive biomarker of vascular disease. We hypothesized that the number of CFU-ECs derived from maternal blood are decreased in women with preeclampsia compared to normal pregnancy.

METHODS

Primigravid women with singleton normal (n = 12) or preeclamptic (n = 12) pregnancies were studied during the third trimester. The culture assay was performed using a pre-plating step to eliminate mature endothelial cells and nonprogenitor cells; colonies per well were counted and further characterized.

RESULTS

Colony numbers were fourfold lower on average in preeclampsia compared to control samples (P < 0.005). A majority of the cells comprising individual colonies were positive for both endothelial (Ulex europaeus lectin staining and acetylated low-density lipoprotein (LDL) uptake) and monocyte/macrophage (CD45, CD14, CD115) characteristics. The SRY gene was detected in CFU-ECs derived from umbilical cord blood samples from male fetuses but not in CFU-ECs from peripheral blood of mothers with male fetuses. Maternal plasma concentrations of the antiangiogenic factor, soluble fms-like tyrosine kinase-1 (sFlt-1) were elevated (P < 0.0001) whereas placental growth factor (PlGF) was reduced (P < 0.01) in women with preeclampsia, but these factors did not correlate with CFU-EC counts.

CONCLUSIONS

CFU-ECs derived from culture of peripheral blood mononuclear cells, a correlate of cardiovascular risk in nonpregnancy populations, are rarified in women with preeclampsia compared to normal pregnancy. PCR analysis is consistent with a maternal origin of these cells.

METHODS

Study population

The Magee-Womens Hospital institutional review board approved the study. Written, informed consent was obtained from all study subjects who were recruited at the time of presentation to Magee-Womens Hospital for prenatal care. The study groups consisted of 12 primigravid women with uncomplicated pregnancy (controls) and 12 primigravid women with preeclampsia. All subjects were in the third trimester of pregnancy.

Preeclampsia was defined using the criteria of de novo hypertension and proteinuria arising after the 20th week of gestation, and hyperuricemia, with reversal of hypertension and proteinuria after delivery.²⁷ Gestational hypertension was defined as systolic blood pressure >140 mm Hg or diastolic blood pressure >90 mm Hg arising after 20 weeks’ gestation in a previously normotensive woman. Proteinuria was defined as >300 mg of protein in a 24-h urine collection, 1+ or greater protein on dipstick of a catheterized urine sample, or 2+ or greater urine protein on dipstick of a voided urine sample, or a random urine protein-to-creatinine ratio >0.3. Hyperuricemia was defined as >1 s.d. above normal for the given gestational age (at term >5.5 mg/dl (3.3 mmol/l)).

Controls remained normotensive and nonproteinuric during pregnancy, and were delivered at term of infants of appropriate weight for gestational age. Patients with chronic hypertension, diabetes, renal disease, or other significant pre-existing metabolic disorders, with a recent history of illicit drug use or cigarette smoking, or with multifetal gestation were excluded.

Four additional women with uncomplicated singleton pregnancies scheduled to undergo cesarean delivery, plus one adult male laboratory volunteer, provided blood samples to probe for the SRY locus. Maternal blood samples from the four women were obtained preoperatively; umbilical cord fetal blood from the same patients was obtained immediately following delivery. Two women delivered male infants; two delivered female infants.

Isolation peripheral blood cells

A volume of 20–40 ml of peripheral venous blood was withdrawn from the antecubital vein into sterile tubes containing 4 mmol/l potassium-ethylenediaminetetraacetic acid and processed within 2 h of collection. The plasma was separated from blood cells by centrifugation at 1,500g and stored at −70 °C without thaw until further analysis. After removal of plasma, the blood cells were diluted back to original volume with Dulbecco’s phosphate buffered saline containing 1.4 mg/ml disodium ethylenediaminetetraacetic acid, and then further diluted 1:1 with Dulbecco’s phosphate buffered saline plus 2% fetal bovine serum (Lonza Group, Walkersville, MD). The sample was overlaid onto Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ) and centrifuged at 740g for 40 min. The cells in the peripheral blood mononuclear cell fraction were collected, washed in Dulbecco’s phosphate buffered saline plus 2% fetal bovine serum, and counted in a hemocytometer. More than 95% of the isolated cells excluded trypan blue in all cases, indicating viability.

CFU assay

CFUs were quantified using a pre-plating step to remove more rapidly adhering mature endothelial cells and non-progenitor cells.⁵ The EndoCult Liquid Medium kit (StemCell Technologies, Vancouver, British Columbia, Canada) was used according to the manufacturer’s protocol. The isolated cells were resuspended in EndoCult medium and seeded at a density of 5 × 10^{cells per fibronectin-coated well of 6-well plates (BD Biosciences, Bedford, MA). After 48 h of culture at 37 °C in an atmosphere of 5% CO}₂, the nonadherent cells were collected and replated in EndoCult media at 1 × 10^{cells/well in 24-well fibronectin-coated plates (BD Biosciences). CFU numbers became maximal after an additional 72 h of culture, and at that time were counted by light microscopy. Only colonies consisting of round cells centrally with spindle-shaped cells (length at least three times greater than width) radiating from the central core were counted. CFUs/well were averaged from a minimum of four wells per patient. To assess reproducibility of the assay, we initially counted CFUs from two blood samples, obtained 72 h apart, from each of six different normotensive women. The intraclass correlation coefficient was 0.92 (}r^{= 0.85,}P < 0.001).

Genomic PCR for the detection of SRY and rhodopsingene

Blood samples were processed for the CFU assay as described above. Following formation of CFUs, wells were washed twice with phosphate buffered saline and the adherent cells were scraped from the each well and used for PCR. Genomic DNA was extracted using QIAmp DNA blood mini kit (Qiagen, Valencia, CA). PCR primers were synthesized at DNA Synthesis Facility of the University of Pittsburgh. Primer sequences for SRY amplifications were 5′-CTAGACCGCAGAGGCGCCAT-3′ (forward), 5′-TAGTACCCACGCCTGCTCCGG-3′ (reverse).²⁸ PCRs (50 μl) contained 0.5 μg genomic DNA- and SRY gene-specific primers (0.5 μmol/l each) and were performed using Tfl DNA polymerase (Epicentre, Madison, WI). Conditions were as follows: denaturation at 94 °C for 5 min, 1 cycle, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s with another extension cycle at 72 °C for 5 min (Genie Thermal Cycler; Techne, Burlington, NJ). The PCR product (242 bp) was electrophoresed on a 2% agarose gel and the ethidium bromide gel was photographed using Polaroid film and a UV transilluminator (UVP, Upland, CA). As a control for loading, a 133 bp region of exon 5 of the rhodopsin gene was amplified using primers, 5′-GACTCAAGCCTCTTGCCTTC-3′ (forward) and 5′-CACAGAGTCCTAGGCAGGTC-3′ (reverse).

Uptake of acetylated LDL and surface lectin staining

Lectin cell surface staining, an endothelial-lineage characteristic, and cytoplasmic accumulation of acetylated low-density lipoprotein (LDL), characteristic of endothelial-lineage cells and macrophages, were assessed.⁵10 Cells were incubated for 4 h with 10 μg/ml of 1,1′-dioctadecyl-3,3,3,3′ (β-tetramethylindocarbocyanine per-chlorate (DiI)-labeled acetylated LDL (DiI-Ac-LDL; Biomedical Technologies, Stoughton, MA), fixed in 2% paraformaldehyde for 20 min, and then counterstained for 1 h with 10 μg/ml of fluorescein isothiocyanate-labeled Ulex europaeus agglutinin I (lectin; Sigma-Aldrich, St Louis, MO). Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen, Eugene, OR). Lectin and DiI-Ac-LDL-doubly positive cells were determined as a percentage of total cells in 1–3 CFUs from each of three women with normal pregnancy and three with preeclampsia. Human umbilical vein endothelial cells (Lonza Group), seeded at passage 4–6 onto fibronectin-coated culture slides (BD Biosciences), were used as a positive control for DiI-Ac-LDL uptake and lectin staining.

Immunophenotyping

CFUs were fixed in 2% paraformaldehyde for 20 min, blocked with Image-iT FX signal enhancer (Invitrogen) and incubated overnight with 1–10 μg/ml of primary or isotype control antibodies, in phosphate buffered saline at 4 °C. Cells were incubated with secondary antibody for 2 h at room temperature and counterstained with DAPI. We used rabbit antihuman CD115 (#CBL776; Chemicon, Billerica, MA), and murine monoclonal antibodies against human CD14 (#555396; BD Pharmingen, San Diego, CA), CD45 (#555480; BD Pharmingen), CD3 (#555337; BD Pharmingen) and von Willebrand factor (#555849; BD Pharmingen). To detect von Willebrand factor, the cells were first permeabilized with 0.1% Triton X-100 (Sigma) for 20 min. Rabbit antihuman IgG G1 (#08–6199; Invitrogen, Camarillo, CA) or mouse antihuman IgG (#555784; BD Pharmingen) were used as isotype controls, as appropriate. Secondary antibodies included goat antirabbit IgG(H+L) antibody conjugated to Alexa Fluor 568 (#{"type":"entrez-nucleotide","attrs":{"text":"A11036","term_id":"492396","term_text":"A11036"}}A11036; Invitrogen) or goat antimouse IgG(H+L) antibody directly conjugated to Alexa Fluor 568 (#{"type":"entrez-nucleotide","attrs":{"text":"A11031","term_id":"489249","term_text":"A11031"}}A11031; Invitrogen) or Alexa Fluor 594 (#A11005; Invitrogen). Human umbilical vein endothelial cells were used as a positive control for von Willebrand factor staining.

Indian ink uptake

The phagocytosis capability of total cells of CFUs from three women with normal pregnancy and three women with preeclampsia was evaluated. At the peak of colony formation, 5 μl/ml of Indian ink (Scientific Device Laboratory, Des Plaines, IL) was added to the wells and incubated for 4 h then washed with phosphate buffered saline. The percentage of cells positive for intracellular Indian ink granules was determined by light microscopy.²⁹ Human umbilical vein endothelial cells served as a negative control.

sFlt-1 and PlGF

Enzyme-linked immunosorbent assays for human sFlt-1 and free PlGF were performed in duplicate with the use of commercial kits (R&D Systems, Minneapolis, MN). These assays were previously validated for pregnancy samples in our laboratory.³⁰ Interassay and intra-assay coefficients of variation, respectively, were 7.4 and 3.2% for sFlt-1, and 11.2 and 5.4% for PlGF.

Statistical analysis

Due to departure of CFU counts and plasma sFlt-1 and PlGF concentrations from normal distribution, these variables were compared by the Mann-Whitney U-test. Clinical continuous variables were compared by unpaired t-test. Fisher’s exact test was used to compare categorical demographic variables. Spearman’s rank correlation coefficient ρ (r_s) was calculated to assess the correlation of CFU counts with plasma sFlt-1 and PlGF concentrations. P values <0.05 were considered statistically significant.

Study population

Isolation peripheral blood cells

CFU assay

Genomic PCR for the detection of SRY and rhodopsingene

Uptake of acetylated LDL and surface lectin staining

Immunophenotyping

Indian ink uptake

sFlt-1 and PlGF

Statistical analysis

RESULTS

Patient clinical characteristics

Patient characteristics are summarized in Table 1. The groups did not differ by gestational age at the time of blood sampling, age, pre-pregnancy body mass index, or early gestational blood pressures. By definition, patients with preeclampsia had higher predelivery mean systolic and diastolic blood pressures. Patients with preeclampsia were often delivered at an earlier mean gestational age. Gestational age at the time of venipuncture, however, was not different.

Table 1

Characteristics of study subjects

	Control (n = 12)	Preeclampsia (n = 12)	P value
Maternal age (years)	27 ± 5	28 ± 6	0.88
BMI	25.9 ± 5.2	27.4 ± 6.3	0.54
Gestational weeks at venipuncture	36.0 ± 3.6	34.0 ± 3.7	0.16
Gestational weeks at delivery	39.0 ± 2.0	34.3 ± 3.9	<0.005
SBP prior to 20 weeks’ gestation	112± 10	114 ± 8	0.61
DBP prior to 20 weeks’ gestation	68 ± 6	71 ± 8	0.36
SBP at delivery	122± 13	154 ± 10	<0.0001
DBP at delivery	74 ± 9	97 ± 10	<0.0001
Serum uric acid (mg/dl)	4.7 ± 1.1	6.2 ± 1.2	<0.05
Birthweight (g)	3,409 ± 596	2,033 ± 905	<0.001
% Black race^a	8% (1/12)	42% (5/12)	0.16
Proteinuria^,^b	0% (0/12)	100% (12/12)	<0.0001

Values are listed as mean ± s.d. or percentage (number/total). BMI, pre-pregnancy body mass index (kg/m^{); DBP, diastolic blood pressure; SBP, systolic blood pressure.}

^{Comparison by Fisher’s exact test.}

^{Proteinuria as defined in Methods.}

CFU assay

As shown in Figure 1, the number of CFUs per well was approximately fourfold lower in preeclampsia compared to uncomplicated pregnancy controls (median 1.7 (range 0–14.5) vs. 8.6 (range 2–44); P < 0.005). The difference remained significant when the analysis was restricted to samples obtained before the onset of labor (n = 7 preeclamptic: median CFUs 3.0 (range 0–14.5); n = 11 controls: median 9.3 (range 2–44); P < 0.05). Five of 12 women were of black race in the preeclampsia group compared to 1 of 12 in the control group (Table 1). CFU numbers remained different by group when restricted to Caucasians (preeclamptic: median CFUs 0.7 (range 0–14.5); control: median 9.3 (range 2–44); P < 0.05).

An external file that holds a picture, illustration, etc.
Object name is nihms172969f1.jpg

Figure 1

Scatterplot of colony-forming unit counts during the third trimester. Each symbol corresponds to average values per culture well from a different individual.

Detection of SRY and rhodopsin gene

PCR showed SRY positivity (presence of a Y chromosome) in CFU cells from cord blood of all male infants and from peripheral blood from the adult male. In contrast, no SRY band was detected from maternal blood CFUs (regardless of fetal gender) or CFUs from cord blood of female infants (Figure 2).

An external file that holds a picture, illustration, etc.
Object name is nihms172969f2.jpg

Figure 2

No evidence of fetal endothelial progenitor cells (EPCs) in maternal blood by genomic PCR for SRY gene. Reactions contained 0.5 μg DNA- and SRY-specific primers. PCR product (242 bp) was electrophoresed on 2% agarose. (a) Lane 1, 100 bp DNA ladder; lane 2, EPCs from adult male; lanes 3, 5, 7 and 9, EPCs from maternal blood; lanes 4 and 8, umbilical cord blood EPCs from male fetus (positive control); lanes 6 and 10: umbilical cord blood EPCs from female fetus. (b) The 133 bp region of rhodopsin gene (RDP) was amplified as a control for loading.

Immunophenotyping and Indian ink uptake

Both the CFU central and radial cells were >90% positive for DiI-Ac-LDL uptake and lectin surface staining (Figure 3), and this did not appear to differ by pregnancy outcome. The cells comprising CFUs were likewise >90% positive for the hematopoietic surface antigen CD45, the monocyte/macrophage antigen CD14, and macrophage antigen CD115 (Figure 3). CFUs were weakly and variably positive for von Willebrand factor (mean % positive cells ± s.e.m.: 65 ± 23). The central cluster cells were >90% CD3^{but the peripherally radiating cells were CD3}^{. A majority of both central and radial cells showed accumulation of India ink granules (mean % positive cells ± s.e.m.: 54% ± 15), consistent with macrophage phagocytic activity.}

An external file that holds a picture, illustration, etc.
Object name is nihms172969f3.jpg

Figure 3

Characteristics of colony-forming units (CFUs) in culture derived from maternal blood samples (original magnification ×100), representative of three patients with normal pregnancy and three with preeclampsia. (a) Brightfield image of CFU in culture. (b and c) Same CFU as a, showing accumulation of Dil-acLDL (red) and FITC-conjugated Ulex lectin (green), respectively. (d) Brightfield image of CFU. (e) Same CFU as d, showing overlay of DAPI nuclear staining (blue) and CD45 (red). (f) Control for e and g, showing overlay of DAPI staining and absence of immunoreactivity to mouse antihuman IgG isotype control followed by goat antimouse Alexa Fluor 568 secondary antibody. (g) Overlay of DAPI and CD14 (red). (h) Overlay of DAPI and CD115 (red). (i) Control for h, showing overlay of DAPI staining and absence of immunoreactivity to rabbit antihuman IgG G1 plus goat antirabbit Alexa Fluor 568 secondary antibody.

Plasma sFlt-1 and PlGF

The median plasma sFlt-1 concentration was elevated in women with preeclampsia compared to controls (28,141 pg/ml (range 9,156–68,912) vs. 4,805 pg/ml (range 1,462–11,372); P < 0.0001). Plasma-free PlGF was lower in women with preeclampsia compared to controls (median 48 (range 3–128) vs. median 148 (53–628); P < 0.005). CFU counts did not correlate with plasma sFlt-1 or PlGF values among either preeclamptics (sFlt-1: r_s = 0.11, P = 0.70; PlGF: r_s = −0.27, P = 0.36) or controls (sFlt-1: r_s = 0.24, P = 0.40; PlGF: r_s = −0.31, P = 0.27).

Patient clinical characteristics

Table 1

Characteristics of study subjects

	Control (n = 12)	Preeclampsia (n = 12)	P value
Maternal age (years)	27 ± 5	28 ± 6	0.88
BMI	25.9 ± 5.2	27.4 ± 6.3	0.54
Gestational weeks at venipuncture	36.0 ± 3.6	34.0 ± 3.7	0.16
Gestational weeks at delivery	39.0 ± 2.0	34.3 ± 3.9	<0.005
SBP prior to 20 weeks’ gestation	112± 10	114 ± 8	0.61
DBP prior to 20 weeks’ gestation	68 ± 6	71 ± 8	0.36
SBP at delivery	122± 13	154 ± 10	<0.0001
DBP at delivery	74 ± 9	97 ± 10	<0.0001
Serum uric acid (mg/dl)	4.7 ± 1.1	6.2 ± 1.2	<0.05
Birthweight (g)	3,409 ± 596	2,033 ± 905	<0.001
% Black race^a	8% (1/12)	42% (5/12)	0.16
Proteinuria^,^b	0% (0/12)	100% (12/12)	<0.0001

Values are listed as mean ± s.d. or percentage (number/total). BMI, pre-pregnancy body mass index (kg/m^{); DBP, diastolic blood pressure; SBP, systolic blood pressure.}

^{Comparison by Fisher’s exact test.}

^{Proteinuria as defined in Methods.}

CFU assay

Figure 1

Scatterplot of colony-forming unit counts during the third trimester. Each symbol corresponds to average values per culture well from a different individual.

Detection of SRY and rhodopsin gene

Figure 2

Immunophenotyping and Indian ink uptake

Figure 3

Plasma sFlt-1 and PlGF

DISCUSSION

Optimal function of the vascular endothelium requires complex interactions between the endothelial cells and cells circulating in the peripheral blood. We report that the number of progenitor CFUs, derived from equivalent numbers of peripheral blood mononuclear cells in culture, is fourfold lower in women with preeclampsia compared to women with uncomplicated pregnancies during the third trimester. Both endothelial- and monocytic/macrophage-like characteristics of the CFUs were observed, consistent with CFUs in the nonpregnancy setting.⁶9162529 Sugawara et al. reported that CFU counts increase with gestational age during normal human pregnancy, correlating with plasma estrogen, and later reported decreased numbers of CFUs and higher senescence values of CFU cells from women with preeclampsia compared to normal pregnancy.³¹32 Their data may not be strictly comparable to ours because they incubated the cells from the peripheral blood mononuclear fraction in endothelial cell growth-specific media for 7 days, and without a pre-plating step to exclude more rapidly adhering endothelial cells and other nonprogenitor cells.

Putative EPCs quantified by flow cytometry correlate poorly with CFUs in culture, suggesting that these assays are measuring different populations of circulating cells.¹¹ One flow cytometry study did not find the quantity of circulating EPCs to differ between preeclampsia and normal pregnancy.³³ However, the enumeration was based upon CD133^/CD34^{/ VEGFR-2}^{(triple) positive cells, recently suggested as consisting of a rarer subtype of CD45}^{primitive hematopoietic progenitor.}³⁴ CD133^/CD34^/VEGFR-2^{or CD133}^/VEGFR-2^{cells in umbilical cord (fetal) blood are reportedly decreased in preeclampsia compared to normal pregnancy.}³⁵36 The preeclampsia cord blood CD133^/VEGFR-2^{cells were inversely correlated with preeclampsia cord blood concentrations of the antiangiogenic factor sFlt-1 (refs.}³⁵36).

As expected,²⁶ sFlt-1 and free PlGF values were significantly higher and lower, respectively, in maternal plasma from women with preeclampsia compared to normal pregnancy in our study. These variables, however, did not correlate with CFU counts in either pregnancy group. If both CFU deficiency and sFlt-1 play a role in the pathogenesis of preeclampsia, it is possible that they act by divergent mechanisms. Within each group, CFUs also did not correlate with any of the continuous variables listed in Table 1 (data not shown).

A relatively rare EPC subtype termed late-outgrowth, “endothelial colony-forming cells” (ECFCs) can be obtained from culture of blood cells on collagen in endothelial-specific media; ECFC colonies that are visually indistinguishable from (but more proliferative than) endothelial cells appear at day 7–14 of culture and can be clonally isolated and expanded.⁶91637 Unlike CFUs, ECFCs are distinct from hematopoietic cells, being primarily CD45^{and CD14}^{, and possess}de novo tubule forming ability in vitro.⁹ Importantly, when CFU-derived cells and ECFCs are co-injected into mice with hind-limb ischemia vascular damage there is synergistic vascular repair/neovascularization, beyond that achieved by injection of the same total number of either cell type alone.²⁵38 CFU cells may enhance the survival, proliferation, and angiogenic function of EPCs and mature endothelial cells primarily by secreting proangiogenic cytokines in paracrine fashion.⁴2538

Gussin et al. reported that circulating cells from ~25 ml of peripheral blood yielded late-outgrowth, endothelial-like cells in 8 of 13 normal pregnant subjects but in none of 10 nonpregnant controls.³⁹ In this study, fluorescence in situ hybridization showed only X-chromosome-specific signals and no Y-chromosome-specific signals in the late-outgrowth cells. They later showed no SRY products consistent with male cells in late-outgrowth cultures from women carrying male fetuses.⁴⁰ We found no evidence for the SRY gene in any of our CFU cultures from women carrying male fetuses, suggesting that CFU cells in the maternal circulation are likewise of maternal origin, at least predominantly so.

Deficiency of the circulating cells responsible for assembly of CFUs in culture could represent a primary source of endothelial dysfunction in vivo by lessening endothelial repair capacity. Alternatively, CFU quantity may merely be another marker of vascular dysfunction or inflammation in women affected by preeclampsia. Longitudinal studies will be necessary to determine whether diminution of circulating CFUs and other angiogenic cells relative to normal pregnancy heralds development of the preeclampsia syndrome.

Acknowledgments

This study was supported in part by National Institutes of Health grants HD049453 (C.A.H.), MO1-RR000056 and 1 UL1 RR024153-01, and the Pennsylvania Department of Health (Research Formula Fund). The Pennsylvania Department of Health specifically disclaims responsibility for analyses, interpretations or conclusions.

^{Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA}

^{Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA}

^{Department of Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA}

^{Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA}

^{Center for Vascular Remodeling and Regeneration, University of Pittsburgh, Pittsburgh, Pennsylvania, USA}

Correspondence: Carl A. Hubel (ude.cmpu@aclebuh)

Abstract

BACKGROUND

METHODS

RESULTS

CONCLUSIONS

Abstract

The cardiovascular adaptation to pregnancy involves a complex maternal response to the presence of the growing conceptus, including alterations in vascular endothelial cells that contribute to a profound fall in peripheral vascular resistance. There is extensive evidence that maternal endothelial cell dysfunction underlies the hypertension, proteinuria and multi-organ damage that occurs during preeclampsia. The mechanism of altered endothelial homeostasis in the pathophysiology of preeclampsia, however, remains poorly understood.¹2

Endothelial progenitor cells (EPCs) are a heterogeneous group of circulating cells derived from bone marrow as well as nonmarrow sites. These cells are thought to play a role in vascular remodeling and maintenance of the adult endothelium by releasing growth factors that contribute to paracrine support and repair of the endothelial lining, and in some cases by physically incorporating into regenerating endothelium.³9 Since the first report of EPCs by Asahara et al.,¹⁰ both flow cytometry and cell culture-based approaches have been developed to distinguish and quantify different subpopulations of circulating EPCs.⁷911

The culture of peripheral blood mononuclear cells under certain conditions favors the formation of discrete colonies termed endothelial colony-forming units (CFU-ECs or CFUs) that are characterized by a central core of rounded cells with multiple spindle-shaped cells radiating from the central cluster.⁵91012 CFUs are derived from hematopoietic stem cells and exhibit monocyte/macrophage characteristics while expressing some endothelial-lineage markers. Although they remain under the classification umbrella of EPCs in the literature, the cells that comprise these CFUs do not differentiate into endothelial cells or assemble into vascular networks in vitro.⁶9

Hill et al.⁵ observed a direct relationship between the number of CFUs and brachial artery endothelium-dependent vasodilatory function in individuals with varying degrees of cardiovascular risk but without history of cardiovascular disease. In this study, CFU numbers were a stronger predictor of flow-mediated brachial reactivity than conventional (Framingham) risk factors. The concentration of CFUs in culture is decreased in nonpregnant patients with type 1 diabetes, chronic obstructive pulmonary disease, congestive heart failure, acute stroke, rheumatoid arthritis, and in overweight and obese compared to normal weight adults.¹³18 Circulating EPCs are reportedly reduced in patients with type 2 diabetes and further reduced with concomitant peripheral vascular disease.¹⁹ Reports regarding EPCs, including CFUs, in both peripheral vascular disease and essential hypertension, however, have been inconsistent.²⁰24

CFU-derived cells have been shown to improve blood low recovery and capillary density in animal models of hind-limb ischemia or myocardial ischemia.⁶25 Data further suggest that they synergize with other EPC subpopulations to increase vascular repair and protect against endothelial disease.⁶25

We tested the hypothesis that CFUs obtained from maternal peripheral blood are decreased in women with preeclampsia compared to normal pregnancy. We evaluated CFU endothelial and monocyte/macrophage characteristics in the setting of pregnancy and whether fetal cells could be detected among the cells comprising CFUs. Given the importance of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in mobilization of EPCs from bone marrrow,⁸ we also hypothesized that CFU counts would correlate positively with plasma concentrations of free PlGF and correlate inversely with soluble fms-like tyrosine kinase-1 (sFlt-1; also known as soluble VEGF receptor-1), an antiangiogenic factor increased in most women with preeclampsia that binds and thus neutralizes VEGF and PlGF.²⁶

Footnotes

Disclosure: The authors declared no conflict of interest.

Footnotes

References

1. Roberts JMEndothelial dysfunction in preeclampsia. Semin Reprod Endocrinol. 1998;16:5–15.[PubMed][Google Scholar]
2. Ness RB, Sibai BMShared and disparate components of the pathophysiologies of fetal growth restriction and preeclampsia. Am J Obstet Gynecol. 2006;195:40–49.[PubMed][Google Scholar]
3. Urbich C, Heeschen C, Aicher A, Dernbach E, Zeiher AM, Dimmeler SRelevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells. Circulation. 2003;108:2511–2516.[PubMed][Google Scholar]
4. Urbich C, Aicher A, Heeschen C, Dernbach E, Hofmann WK, Zeiher AM, Dimmeler SSoluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells. J Mol Cell Cardiol. 2005;39:733–742.[PubMed][Google Scholar]
5. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel TCirculating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003;348:593–600.[PubMed][Google Scholar]
6. Hur J, Yoon CH, Kim HS, Choi JH, Kang HJ, Hwang KK, Oh BH, Lee MM, Park YBCharacterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol. 2004;24:288–293.[PubMed][Google Scholar]
7. Barber CL, Iruela-Arispe MLThe ever-elusive endothelial progenitor cell: identities, functions and clinical implications. Pediatr Res. 2006;59:26R–32R.[PubMed][Google Scholar]
8. Schatteman GC, Dunnwald M, Jiao CBiology of bone marrow-derived endothelial cell precursors. Am J Physiol Heart Circ Physiol. 2007;292:H1–18.[PubMed][Google Scholar]
9. Hirschi KK, Ingram DA, Yoder MCAssessing identity, phenotype, and fate of endothelial progenitor cells. Arterioscler Thromb Vasc Biol. 2008;28:1584–1595.[Google Scholar]
10. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JMIsolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964–967.[PubMed][Google Scholar]
11. George J, Shmilovich H, Deutsch V, Miller H, Keren G, Roth AComparative analysis of methods for assessment of circulating endothelial progenitor cells. Tissue Eng. 2006;12:331–335.[PubMed][Google Scholar]
12. Hur J, Yang HM, Yoon CH, Lee CS, Park KW, Kim JH, Kim TY, Kim JY, Kang HJ, Chae IH, Oh BH, Park YB, Kim HSIdentification of a novel role of T cells in postnatal vasculogenesis: characterization of endothelial progenitor cell colonies. Circulation. 2007;116:1671–1682.[PubMed][Google Scholar]
13. Loomans CJ, de Koning EJ, Staal FJ, Rookmaaker MB, Verseyden C, de Boor HC, Verhaar MC, Braam B, Rabelink TJ, van Zonneveld AJEndothelial progenitor cell dysfucntion: a novel concept in the pathogenesis of vascular complications of type 1 diabetes. Diabetes. 2004;53:195–199.[PubMed][Google Scholar]
14. Grisar J, Aletaha D, Steiner CW, Kapral T, Steiner S, Seidinger D, Weigel G, Schwarzinger I, Wolozcszuk W, Steiner G, Smolen JSDepletion of endothelial progenitor cells in the peripheral blood of patients with rheumatoid arthritis. Circulation. 2005;111:204–211.[PubMed][Google Scholar]
15. Grisar J, Aletaha D, Steiner CW, Kapral T, Steiner S, Saemann M, Schwarzinger I, Buranyi B, Steiner G, Smolen JSEndothelial progenitor cells in active rheumatoid arthritis: effects of tumour necrosis factor and glucocorticoid therapy. Ann Rheum, Dis. 2007;66:1284–1288.[Google Scholar]
16. Prater DN, Case J, Ingram DA, Yoder MCWorking hypothesis to redefine endothelial progenitor cells. Leukemia. 2007;21:1141–1149.[PubMed][Google Scholar]
17. MacEneaney OJ, Kushner EJ, Van Guilder GP, Greiner JJ, Stauffer BL, DeSouza CAEndothelial progenitor cell number and colony-forming capacity in overweight and obese adults. Int J Obes (Lond) 2009;33:219–225.[Google Scholar]
18. Chu K, Jung KH, Lee ST, Park HK, Sinn DI, Kim JM, Kim DH, Kim JH, Kim SJ, Song EC, Kim M, Lee SK, Roh JKCirculating endothelial progenitor cells as a new marker of endothelial dysfunction or repair in acute stroke. Stroke. 2008;39:1441–1447.[PubMed][Google Scholar]
19. Fadini GP, Miorin M, Facco M, Bonamico S, Baesso I, Grego F, Menegolo M, de Kreutzenberg SV, Tiengo A, Agostini C, Avogaro ACirculating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Coll Cardiol. 2005;45:1449–1457.[PubMed][Google Scholar]
20. Shaffer RG, Greene S, Arshi A, Supple G, Bantly A, Moore JS, Parmacek MS, Mohler ER., 3rd Effect of acute exercise on endothelial progenitor cells in patients with peripheral arterial disease. Vasc Med. 2006;11:219–226.[PubMed]
21. Delva P, De Marchi S, Prior M, Degan M, Lechi A, Trettene M, Arosio EEndothelial progenitor cells in patients with severe peripheral arterial disease. Endothelium. 2008;15:246–253.[PubMed][Google Scholar]
22. Imanishi T, Moriwaki C, Hano T, Nishio IEndothelial progenitor cell senescence is accelerated in both experimental hypertensive rats and patients with essential hypertension. J Hypertens. 2005;23:1831–1837.[PubMed][Google Scholar]
23. Oliveras A, Soler MJ, Martinez-Estrada OM, Vazquez S, Marco-Feliu D, Vila JS, Vilaró S, Lloveras JEndothelial progenitor cells are reduced in refractory hypertension. J Hum Hypertens. 2008;22:183–190.[PubMed][Google Scholar]
24. Delva P, Degan M, Vallerio P, Arosio E, Minuz P, Amen G, Di Chio M, Lechi AEndothelial progenitor cells in patients with essential hypertension. J Hypertens. 2007;25:127–132.[PubMed][Google Scholar]
25. Yoon CH, Hur J, Park KW, Kim JH, Lee CS, Oh IY, Kim TY, Cho HJ, Kang HJ, Chae IH, Yang HK, Oh BH, Park YB, Kim HSSynergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases. Circulation. 2005;112:1618–1627.[PubMed][Google Scholar]
26. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SACirculating angiogenic factors and the risk of preeclampsia. N Engl J Med. 2004;350:672–683.[PubMed][Google Scholar]
27. Chesley LCHypertension in pregnancy: definitions, familial factor, and remote prognosis. Kidney Int. 1980;18:234–240.[PubMed][Google Scholar]
28. Mendes JR, Strufaldi MW, Delcelo R, Moisés RC, Vieira JG, Kasamatsu TS, Galera MF, Andrade JA, Verreschi ITY-chromosome identification by PCR and gonadal histopathology in Turner’s syndrome without overt Y-mosaicism. Clin Endocrinol (Oxf) 1999;50:19–26.[PubMed][Google Scholar]
29. Zhang SJ, Zhang H, Wei YJ, Su WJ, Liao ZK, Hou M, Zhou JY, Hu SSAdult endothelial progenitor cells from human peripheral blood maintain monocyte/macrophage function throughout in vitro culture. Cell Res. 2006;16:577–584.[PubMed][Google Scholar]
30. Shibata E, Rajakumar A, Powers RW, Larkin RW, Gilmour C, Bodnar LM, Crombleholme WR, Ness RB, Roberts JM, Hubel CASoluble fms-like tyrosine kinase 1 is increased in preeclampsia but not in normotensive pregnancies with small-for-gestational-age neonates: relationship to circulating placental growth factor. J Clin Endocrinol Metab. 2005;90:4895–4903.[PubMed][Google Scholar]
31. Sugawara J, Mitsui-Saito M, Hoshiai T, Hayashi C, Kimura Y, Okamura KCirculating endothelial progenitor cells during human pregnancy. J Clin Endocrinol Metab. 2005;90:1845–1848.[PubMed][Google Scholar]
32. Sugawara J, Mitsui-Saito M, Hayashi C, Hoshiai T, Senoo M, Chisaka H, Yaegashi N, Okamura KDecrease and senescence of endothelial progenitor cells in patients with preeclampsia. J Clin Endocrinol Metab. 2005;90:5329–5332.[PubMed][Google Scholar]
33. Matsubara K, Abe E, Matsubara Y, Kameda K, Ito MCirculating endothelial progenitor cells during normal pregnancy and pre-eclampsia. Am J Reprod Immunol. 2006;56:79–85.[PubMed][Google Scholar]
34. Case J, Mead LE, Bessler WK, Prater D, White HA, Saadatzadeh MR, Bhavsar JR, Yoder MC, Haneline LS, Ingram DAHuman CD34+AC133+VEGFR-2+ cells are not endothelial progenitor cells but distinct, primitive hematopoietic progenitors. Exp Hematol. 2007;35:1109–1118.[PubMed][Google Scholar]
35. Xia L, Zhou XP, Zhu JH, Xie XD, Zhang H, Wang XX, Chen JZ, Jian SDecrease and dysfunction of endothelial progenitor cells in umbilical cord blood with maternal pre-eclampsia. J Obstet Gynaecol Res. 2007;33:465–474.[PubMed][Google Scholar]
36. Hwang HS, Maeng YS, Park YW, Koos BJ, Kwon YG, Kim YHIncreased senescence and reduced functional ability of fetal endothelial progenitor cells in pregnancies complicated by preeclampsia without intrauterine growth restriction. Am J Obstet Gynecol. 2008;199:259.e1–259.e7.[PubMed][Google Scholar]
37. Mead LE, Prater D, Yoder MC, Ingram DAIsolation and characterization of endothelial progenitor cells from human blood. Curr Protoc Stem Cell Biol. 2008;Chapter 2(Unit 2C.1)[PubMed][Google Scholar]
38. Chade AR, Zhu X, Lavi R, Krier JD, Pislaru S, Simari RD, Napoli C, Lerman A, Lerman LOEndothelial progenitor cells restore renal function in chronic experimental renovascular disease. Circulation. 2009;119:547–557.[Google Scholar]
39. Gussin HA, Bischoff FZ, Hoffman R, Elias SEndothelial precursor cells in the peripheral blood of pregnant women. J Soc Gynecol Investig. 2002;9:357–361.[PubMed][Google Scholar]
40. Gussin HA, Sharma AK, Elias SCulture of endothelial cells isolated from maternal blood using anti-CD105 and CD133. Prenat Diagn. 2004;24:189–193.[PubMed][Google Scholar]