[final version of the paper that was accepted for publication]
ELEVATED BLOOD PRESSURE IN PRETERM-BORN OFFSPRING ASSOCIATES WITH
A DISTINCT ANTIANGIOGENIC STATE AND MICROVASCULAR ABNORMALITIES
IN ADULT LIFE
Short title: Preterm Birth, Angiogenesis and Blood Pressure
1
Adam J Lewandowski BSc (Hons), DPhil,
1
Esther F Davis MBBS, DPhil,
1
Grace Yu PhD,
1
Janet E
Digby PhD,
1
Henry Boardman BSc, MBBS,
1
Polly Whitworth RGN,
2
Atul Singhal FRCP,
2
Alan
Lucas FMedSci, 3 Kenny McCormick FRCPCH, 4 Angela C Shore PhD, 1 Paul Leeson PhD FRCP
1
Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford,
Oxford, UK.
2
MRC Childhood Nutrition Research Centre, Institute of Child Health, University College London,
London, UK.
3
Department of Paediatrics, John Radcliffe Hospital, Oxford, UK.
4 Vascular Medicine, NIHR Exeter Clinical Research Facility and University of Exeter Medical School,
Exeter, UK.
Address for correspondence:
Professor Paul Leeson, Oxford Cardiovascular Clinical Research Facility, Division of Cardiovascular
Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford.
OX39DU. e-mail: paul.leeson@cardiov.ox.ac.uk. Tel:+44(0)1865572846, Fax:+44(0)1865572840
Manuscript ID#: HYPE201404662R2
1
ABSTRACT
Preterm-born individuals have elevated blood pressure. We tested the hypothesis that this associates with an enhanced antiangiogenic circulating profile and that this association is mediated by variations in capillary density. We studied 204 adults at age 25 years (range: 20-30 years) of which
102 had been followed prospectively since very preterm birth (mean gestational age 30.3±2.5weeks) and 102 were born term to uncomplicated pregnancies. A panel of circulating biomarkers, including soluble endoglin (sENG) and soluble fms-like tyrosine kinase-1 (sFlt-1), were compared between groups and related to perinatal history and adult cardiovascular risk. Associations with cardiovascular phenotype were studied in 90 individuals who had undergone detailed assessment of microvascular, macrovascular and cardiac structure and function. Preterm-born individuals had elevations in sENG (5.64±1.03 vs 4.06±0.85ng/mL, P <0.001) and sFlt-1 (88.1±19.0 vs
73.0±15.3pg/mL, P <0.001) compared to term-born individuals, proportional to elevations in resting and ambulatory blood pressure, as well as degree of prematurity ( P <0.05). Maternal hypertensive pregnancy disorder associated with additional increases in sFlt-1 ( P =0.002). Other circulating biomarkers, including those of inflammation and endothelial activation, were not related to blood pressure. There was a specific graded association between sENG and degree of functional and structural capillary rarefaction ( P =0.002 and P <0.001) and, in multivariable analysis, capillary density mediated associations between sENG and blood pressure. Preterm-born individuals exhibit an enhanced antiangiogenic state in adult life that specifically relates to elevations in blood pressure.
The association appears to be mediated through capillary rarefaction and is independent of other cardiovascular structural and functional differences in the offspring.
Key words: Preterm birth, prematurity, angiogenesis, capillaries, microcirculation, blood pressure, gestational hypertension
2
INTRODUCTION
Significant advances in perinatal care have resulted in individuals born very preterm now routinely
However, their early cardiovascular growth and development has occurred
under unusual physiological conditions. Detailed imaging has identified that, as adults, they have a unique preterm cardiovascular phenotype, characterized by disproportionate reductions in cardiac and vascular size,
3-9 which extends to involve the microvasculature.
10
Microvascular rarefaction is a major determinant of increased vascular resistance and associated with
the development of hypertension.
Indeed, pharmacological manipulation of microvascular
development through pathways that include soluble endoglin (sENG), soluble fms-like tyrosine
kinase-1 (sFlt-1) and vascular endothelial growth factor (VEGF) can induce hypertension.
occurs pathologically during onset of pregnancy complications that result in maternal hypertension,
in which capillary rarefaction is linked with increases in placental-derived sENG and s-FLt-1.
Umbilical cord blood analysis shows that antiangiogenic factors are also increased in the fetal circulation in complicated pregnancies.
17
Intriguingly, cord sera from preterm infants impairs vasculogenesis in vitro ,
18
and their circulating endothelial colony forming cells express an antiangiogenic phenotype.
19
Individuals born very preterm have higher blood pressure throughout
6-8, 20-22 and these observations raise the possibility that an antiangiogenic milieu,
potentially triggered by or related to the causes of preterm birth, underlies microvascular and blood pressure variation in preterm-born individuals. We hypothesized that these pathways are relevant to cardiovascular risk later in life, and therefore, we measured a panel of circulating angiogenic markers in adults who had been born very preterm, studied how levels vary with perinatal factors, and determined whether they are related to adult cardiovascular risk. We then identified features of the preterm cardiovascular phenotype that mediated associations between angiogenic factors and blood pressure.
3
METHODS
Study Population
We have prospectively followed individuals born preterm between 1982 and 1985 since recruitment at birth to randomized feeding regimes.
3-7
Of the initial 926 subjects (birthweight <1850g), 240 agreed to be recontacted, of which 102 agreed to attend Oxford for detailed cardiovascular phenotyping at age 23 to 28 years.
3-7
Compared with the original cohort, those followed to adulthood had similar perinatal characteristics (Table S1) except for a small 70-g (5%) difference in birth weight, accounted for by a marginally shorter gestational age, such that birthweight Z score does not differ. For the current study, 102 young adults born term to uncomplicated pregnancies with similar age and sex distribution to the preterm-born young adults were recruited via advertisement to undergo identical investigations. Blood sampling, anthropometry, blood pressure measurements, questionnaires and cardiovascular assessments (cardiac and macrovascular assessments) were undertaken in all participants. During an interim review, the ‘microvascular-angiogenesis’ hypothesis was developed, and we amended the protocol to include ambulatory blood pressure and microvascular assessment in 30 of the 102 preterm-born adults and 60 of the 102 term-born adults
(Microvascular Substudy). All data was coded with subject and study-specific IDs (e.g. EVS001) to ensure anonymity and blinded analysis. The study was registered with ClinicalTrials.gov
(NCT01487824) and the initial study protocol, as well as the subsequent amendment, was approved by the relevant ethics committee (Oxfordshire Research Ethics Committee A: 06/Q1604/118). All participants provided signed informed consent.
Main study measures
Perinatal data and cardiovascular risk factors
Details of perinatal data collection, cardiovascular measures and other data collected in young adulthood at the 23 to 28 year follow up study have previously been reported.
3-7
In young adulthood,
4
blood pressure was measured as the average of the last two of three brachial blood pressure recordings from an automatic digital monitor (HEM-705CP, OMRON, Japan) with a cuff placed on the left arm after participants had rested supine for 10 minutes.
3-7
Anthropometry included height,
weight and skinfold thickness. Medical and lifestyle information was collected by questionnaire.
Blood samples were drawn following a 12 hour overnight fast, centrifuged, separated within 30 minutes and stored at -80 o
C. Fasting lipid and metabolic profiles, including C-reactive protein, were measured at the Oxford Hospital Biochemistry Laboratory using routine clinical quality validated assays.
Circulating biomarkers of angiogenesis, inflammation and endothelial activation
We quantified serum circulating VEGF-A (VEGF
165
), sFlt-1, PlGF, and sENG concentrations with commercial enzyme-linked immunosorbent assays (ELISAs) (Quantikine, R&D Systems Europe,
Abingdon, UK). Soluble E-selectin (sE-selectin), soluble vascular cell adhesion molecule-1
(sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured using
Millipore MILLIPLEXMAP kits based on the Luminex xMAP platform (Millipore Corporation,
Billerica, MA, USA). Full technical details are in the Online Supplemental File.
Additional cardiovascular measures
Methods for previously reported cardiac and macrovascular measures are in the Online Supplemental
File.
3-5
Arterial stiffness was assessed by carotid-femoral pulse wave velocity (PWV) using applanation tonometry (SphygmoCor Analysis System, Australia) and, in addition, Cardio-Ankle
Vascular Index 25 (Vasera, Fukuda Denshi, Japan) to derive a blood pressure-independent measure of arterial stiffness. Endothelial function was quantified as brachial artery flow-mediated dilatation
24
.
Cardiovascular magnetic resonance on a Siemens 1.5T Sonata scanner was used to quantify cardiac mass and function,
5
as well as thoracic and abdominal aortic size.
5
Microvascular substudy measures
Structural and functional capillary density
Intravital video capillaroscopy with a Leica Stereo Microscope at 200x magnification and Schott
LED light (wavelength 450nm to 610nm, consistent with hemoglobin absorption wavelength) was used to assess microvascular structure and function on the dorsal surface of the middle phalanx of the left hand in six adjacent image fields at baseline and following venous occlusion. A Bosch
Dinlon CCD Analogue Camera relayed images to a Norban Vista black and white high-resolution monitor which were recorded on a Sony DVO-1000MD DVD recorder. Calibration was performed with a 1.0 mm glass stage graticule (Knight Optical UK) and images analysed with with Image
ProPlus. Full details of patient preparation, data acquisition and image analysis are in the Online
Supplemental File.
Ambulatory Blood Pressure
24-hour blood pressure monitoring was performed on the left arm using an automatic digital monitor
(A&D Instruments Ltd., Japan). Details of device fitting and patient instructions are provided in the
Online Supplemental File.
Statistical Analysis
SPSS Version 22 was used for statistical analysis. Variable normality was assessed visually from normality curves and by Shapiro-Wilk test. Comparison between groups for continuous variables was performed by two-sided, independent-samples student t -test for normally distributed data and
Mann Whitney and Kruskal-Wallis tests for skewed data. Categorical variables were compared by
Chi-Square test. Linear regression models were performed using a Forced Entry Method. Pearson correlations (r) were used for bivariate associations and unstandardized regression coefficients (B) or standardized regression coefficients (β) were used for bivariable and multivariable regression
6
models. 102 subjects per group provided 80% power at P =0.05 to identify a 0.38SD difference between groups. For subgroup analysis, 30 preterm-born and 60 term-born subjects provided 80% power at P =0.05 to identify a 0.62SD difference between groups. Comparisons were adjusted for age and sex. Results are presented as Mean±Standard Deviation. Mediation analyses were performed to determine potential causal pathways between capillary density, sENG, and 24 hour systolic blood pressure levels in preterm-born adults. Bivariable regression analyses were first performed using a forced entry method followed by multivariable regression analyses with 24 hour systolic blood pressure as the dependent variable, and sENG and capillary density as independent variables. P values <0.05 were considered statistically significant.
RESULTS
Study Population
Demographic and risk factor characteristics of the preterm- and term-born adults are presented in
Table S2, with corresponding information limited to the microvascular substudy group in Table 1.
There were no significant differences in number of smokers, personal and family medical history, or lifestyle factors such as socioeconomic status, physical activity or diet between preterm- and termborn adults. However, preterm-born adults had a distinct metabolic profile and higher brachial blood pressure.
3-7
In the microvascular substudy, a 10mmHg higher systolic blood pressure was evident on
24-hour ambulatory blood pressure monitors during both awake and sleep periods in preterm-born adults (Table 1). Of the 102 preterm-born young adults that took part in the study, 47 were delivered by prelabour cesarian section or induced (iatrogenic), of which 29 were born to hypertensive pregnancies. The remaining 55 preterm births were spontaneous (idiopathic).
7
Preterm Birth and Circulating Levels of Antiangiogenic and Inflammatory Biomarkers
Preterm-born adults had increased sFlt-1 and sENG levels compared to term-born individuals
( P <0.001) (Figure 1 and Table 2). There were no differences in VEGF-A levels ( P= 0.36) or CRP between groups ( P =0.17), but sICAM-1 and sVCAM-1 levels were significantly elevated in those born preterm ( P <0.001) (Table 2). PlGF and sE-selectin levels were not detectable. In bivariable regression analyses, there was an inverse graded relation between degree of prematurity, assessed as gestational age, and sENG and sFlt-1, with a positive relation to sICAM-1 ( P <0.05). There were no graded differences with sVCAM-1 (Table S3).
To understand whether preterm birth per se or other perinatal factors accounted for these associations, we used bivariable regression analyses to identify other potentially relevant associations (Table S3). No perinatal factors apart from gestational age predicted sENG levels.
Gestational hypertension during the indicated pregnancy was associated with higher offspring sFLT1 levels but no differences in other circulating markers (B=13.5 pg/mL/category, P =0.002, where pregnancy-induced hypertension was a dichotomous categorical variable with 1=normotensive pregnancy and 2=preeclamptic pregnancy). Figure 1, Panel B, demonstrates sFlt-1 levels in those born preterm due to maternal hypertension was 97.7±15.9 pg/mL compared to 84.2±18.9 pg/mL in those born preterm to normotensive mothers ( P =0.002). sFlt-1 levels in those with a normotensive mother were still significantly elevated compared to term-born young adults ( P <0.001). However, this difference between preterms born to a hypertensive versus normotensive pregnancy was not apparent in sENG levels (5.79±1.04 vs 5.58±1.04 ng/mL,
P =0.37), which were significantly higher than in term-born young adults ( P <0.001) (Figure 1, Panel A). APGAR Score at 5 minutes also related to sFlt-1 levels (B=-1.86 pg/mL/point, P =0.05) and to sICAM-1 in young adulthood (B=-14.3 ng/mL/point, P =0.01).
8
In multivariable regression models, which included gestational age, birthweight, five minute
APGAR score, gestational hypertension, and antenatal glucocorticoid exposure, gestational age remained an independent predictor for sENG (B=-0.15 ng/mL/gestational week, 95% confidence interval: -0.27 to -0.031, P =0.008) but none of the other circulating biomarkers (Table S4). For sFLT-1, being born to a pregnancy complicated by hypertension was the main independent predictor of adult levels (B=14.7 pg/mL/category, 95% confidence interval: 6.21 to 23.2, P =0.001) and for sICAM-1, the association was with lower five minute APGAR scores (B=-21.8 ng/mL/category,
95% confidence interval: -32.4 to -11.2, P <0.001).
Adult Preterm Cardiovascular Risk Factors and Circulating Biomarkers
We then determined whether levels of these circulating markers were related to variations in cardiovascular risk factors in adult life. Both sENG and sFlt-1 positively related to adult systolic, diastolic and mean arterial pressures in preterm-born young adults (Table S5), and these associations were replicated with ambulatory blood pressure measures in the microvascular study subgroup
(association between 24-hour systolic blood pressure and sENG was B=3.48 ng/mL/mmHg, P =0.007 and for sFlt-1 was B=0.10 pg/mL/mmHg, P =0.005). Figure 2 illustrates the step increase in systolic blood pressure across tertiles of sENG and sFlt-1. sVCAM-1 was positively related to insulin
( P =0.001), with trends for association with body mass index ( P =0.09) and waist-to-hip ratio
( P =0.07) (Table S5).
Capillary Rarefaction, Circulating Biomarkers and Increased Blood Pressure
We proceeded to analyses focused on circulating factors related to blood pressure. We tested the hypothesis that these factors would also relate to features of the cardiovascular phenotype previously reported to differ in preterm-born offspring, including cardiac and vascular structure and function, as well as capillary rarefaction. sENG was not related to aortic size or stiffness, cardiac structure or
9
function, or endothelial responses, either in the full cohort or those who took part in the microvascular substudy (Table S6). However, there were striking inverse associations between sENG and both functional (β=-0.57, P =0.002) and structural (β=-0.65, P <0.001) capillary density in those born preterm. Preterm-born individuals had significant reductions in these microvascular parameters compared to full term-born individuals (baseline: 93.5±10.6 vs 105.2±10.6 capillaries/mm
2
, P <0.001; venous occlusion: 101.8±12.7 vs 115.9±11.8 capillaries/mm
2
, P <0.001)
(Figure 3), in whom there were no associations between angiogenic factors and microvascular parameters. Although sFLT-1 was related to sENG and blood pressure, it was not associated with any of the measured cardiovascular parameters (Table S6 and Figure 4). Although sex differences did not exist in sFlt-1 or sENG levels in the preterm group, interestingly, preterm-born males were associated with a significant reduction in both functional and structural capillary density compared to preterm-born females ( P =0.04 and P =0.01). Though numbers were small (n=15 males, n=15 females), this was consistent with the elevation in systolic blood pressure observed in males born preterm (125.3±9.6 vs 117.9±10.8,
P <0.001).
We then performed a mediation analysis within the preterm group to determine relative hierarchies of blood pressure, capillary rarefaction and sENG in their co-associations (Figure 4). In bivariable analyses, functional and structural capillary rarefaction were each inversely related to both resting systolic blood pressure ( P =0.05 and P =0.04) and 24-hour blood pressure measures (B=-0.42 capillaries/mm
2
/mmHg, P <0.001 and B=-0.32 capillaries/mm
2
/mmHg, P =0.001). In multivariable regression, controlling for sENG levels did not alter the association between structural or functional capillary density and 24-hour systolic blood pressure (β=-0.48, P =0.05), but sENG was no longer a significant predictor of blood pressure (β=0.25, P =0.28). From this we concluded associations between sENG levels and systolic blood pressure are mediated by capillary density.
10
DISCUSSION
Preterm-born individuals have increased sFlt-1, sENG, sVCAM-1 and sICAM-1 levels in young adulthood and, of these, variation in sENG and s-FLT1 are proportional to both resting and ambulatory blood pressure levels. Of particular interest are the associations between sENG and blood pressure, which appear to be specifically related to degree of prematurity, rather than other perinatal factors, and mediated by differences in microvascular structure and function. sFlt-1 levels were largely related to pregnancy-induced hypertension as a cause for preterm birth, and we were not able to identify any associated variations in cardiovascular phenotype. sICAM-1 appeared to be more closely related to infant condition at birth rather than prematurity per se , and seemed to be relevant to metabolic function in later life. This latter association requires further investigation, but for the current work, we focused on developing our mechanistic understanding of hypertension related to preterm birth.
A reduced number of capillaries, or ability to recruit capillaries, is considered a key factor underlying increased peripheral vascular resistance and is known to be a characteristic finding in patients with
Experimental elevation in blood pressure can directly lead to capillary
rarefaction, most likely through an impact of increased generation of endothelial-derived reactive
oxygen species on microvascular integrity.
Capillary rarefaction can also antedate the onset of
hypertension, as reduced capillary density is observed in normotensive individuals at high risk of developing hypertension.
12
However, capillary rarefaction is not a generalized observation in those predisposed to hypertension. Whereas reduced capillary number has been identified as a specific vascular abnormality in young adults with a family predisposition, 30 individuals born with low birthweight do not consistently demonstrate changes in capillary density.
31
We also observed a lack of association between birthweight and capillary density in our preterm study. The finding of reduced capillary density relating to preterm birth per se is more established, even when blood
11
pressure differences are still relatively modest.
Therefore, it is plausible that capillary rarefaction
is a primary event in preterm-born individuals that is established very early in postnatal life.
The second and third trimesters of pregnancy are periods of rapid fetal growth with accelerated organ development, which requires a quickly expanding capillary network. Premature exposure of an early third trimester fetal circulation to postnatal circulatory physiology in animal models and humans is known to have an adverse impact on development, leading to disproportionately small cardiac and
In addition, maternal factors linked with preterm birth may be of relevance.
Preterm birth is a pathophysiological event, often associated with combinations of stress, hypoxia,
et al.
demonstrated that the gene profile of endothelial colony forming cells isolated from the mononuclear cell fraction obtained from cord blood, following preterm birth, exhibit a programmed shift towards increased expression of genes with antiangiogenic
Physiologically, this was sufficient for both the cells and the cord sera to impair
angiogenesis in vitro.
19
D’Souza et al.
found that infants born later in gestation following maternal preeclampsia, a condition characterized by elevated placental-derived antiangiogenic factors in both
maternal and fetal circulations,
had capillary rarefaction evident at birth.
40
These observations raise the possibility that the circulating angiogenic state is both altered and directly relevant to vasculogensis in the offspring both pre and postnatally. Although we found the association with gestational age was independent of birthweight Z score, we did not have accurate measures of fetal growth restriction or placental dysfunction in our available dataset. Iatrogenic preterm delivery, which occurs in 30% of preeclamptic pregnancies, is often indicated due to fetal growth problems, as a consequence of placental dysfunction, and it remains possible that placental dysfunction may be a determinant or modifier, either in whole or in part, of some features of the long-term changes in angiogenic factors and the microvasculature we have identified.
41
There is significant heterogeneity in the clinical background of premature delivery including incidence of infection, preterm premature
12
rupture of the membrane and iatrogenic preterm delivery. In future studies, it will be important to establish whether the microvascular abnormalities are established postnatally due to the hemodynamic changes associated with preterm birth, and therefore of relevance to all those born preterm, or whether they vary with the prenatal clinical history. If the latter, larger studies with a range of pregnancy complications will be of value to establish whether it is possible to stratify risk of hypertension following preterm birth based on the clinical background.
We were interested to know whether there is any long-term relevance of these pathways to later cardiovascular risk. The only report of angiogenic profile in offspring of complicated pregnancies comes from Kvehaugen et al.,
42
who studied children aged five to eight years born to pregnancies complicated by hypertension, but with a high rate of preterm birth. They found no difference in sENG or sFLT-1 levels, despite differences in the mother. However, there were also no differences in blood pressure between groups, and we found a graded association between antiangiogenic factors and blood pressure. In addition, there is evidence to suggest that associations may become more evident with age. Bonamy et al.
studied slightly older children between 8 and 12 years of age and found a 6.94% reduction in resting (functional) capillary density and a 5.27% reduction in capillary density post venous occlusion (structural) 10 compared to the 11.1% to 12.2% reduction in functional and structural capillary density compared to term-born controls at age 25 to 28 years. By this point, both blood pressure and elevations in sENG are evident and proportional to the reductions in capillary density. Furthermore, mediation analysis suggested a pivotal role of the microcirculation in the links between antiangiogenic profile and blood pressure in preterm-born individuals. Studies focused on this pathway will help further dissect these associations and could identify interventions of specific benefit for cardiovascular risk reduction in preterm-born offspring. While our capillary density sex differences in the preterm group are of interest, larger study groups will be required to
13
further explore these findings and to determine whether this is due to pathophysiological sex differences of prematurity.
A strength of our study was the detailed cardiovascular phenotyping of the cohort. This allowed us to study the importance of all the key reported features of the preterm cardiovascular phenotype, from heart to capillary, to changes in blood pressure in each individual. Differences in aortic and cardiac size did not appear relevant to blood pressure variation in individuals born preterm. However, it might be expected that these differences will adversely affect the response of the individual to risk factors, and it is possible myocardial microvascular responses also differ. In preterm animal models, a higher incidence of heart failure has been observed in response to hypertension.
43
As the microvascular hypothesis was included as an amendment to the study protocol, not all individuals underwent these investigations. Although the overall characteristics of this subgroup were similar to the full cohort, the reduced size did mean we had insufficient subjects born to hypertensive pregnancies to study microvascular differences in this group separately. It is possible that pregnancyinduced hypertension is associated with an additional impact on capillary density in the offspring in view of their dual elevation in sENG and sFLT-1. This, and associations between the other markers and metabolic derangements, will require investigation in further, larger study cohorts.
PERSPECTIVES
This is the first demonstration of a heightened antiangiogenic state in later life in individuals who were born preterm. These findings may indicate a potential mechanistic link between an imbalance in proangiogenic and antiangiogenic factors, capillary rarefaction, and blood pressure elevation in preterm-born young adults. Whether this pathway provides a modifiable target for future prevention strategies to delay the development of hypertension in individuals born preterm is of significant future interest.
14
ACKNOWLEDGMENTS
We are grateful to the volunteers who participated in these studies and all those who have previously contributed to data collection.
SOURCES OF FUNDING
This work was supported by grants to Professor Leeson from the British Heart Foundation
(FS/06/024 and FS/11/65/28865). Dr Lewandowski was supported by the Commonwealth
Scholarship and Fellowship Program. Additional grants were received from the National Institute for
Health Research Oxford Biomedical Research Centre and Oxford British Heart Foundation Centre for Research Excellence. Previous cohort follow-up was supported by the Medical Research Council.
CONFLICTS OF INTEREST/DISCLOSURES
None.
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NOVELTY AND SIGNIFICANCE
1.
WHAT IS NEW?
Preterm-born individuals have increased sFlt-1, sENG, sVCAM-1 and sICAM-1 levels in young adulthood.
Variation in sENG and s-FLT1 are proportional to both resting and ambulatory blood pressure levels.
The associations between sENG and blood pressure appear to be specifically related to degree of prematurity, rather than other perinatal factors, and mediated by differences in microvascular structure and function.
sFlt-1 levels were largely related to pregnancy-induced hypertension as a cause for preterm birth.
2.
WHAT IS RELEVANT?
These findings may indicate a potential mechanistic link between an imbalance in proangiogenic and antiangiogenic factors, capillary rarefaction, and blood pressure elevation in preterm-born young adults.
SUMMARY
This is the first demonstration of a heightened antiangiogenic state in later life in individuals who were born preterm. These findings implicate a link between an imbalance in proangiogenic and antiangiogenic factors, capillary rarefaction and elevated blood pressure in preterm-born young adults that may represent a novel target for intervention
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FIGURE LEGENDS
Figure 1 – A, sENG was increased in preterm-born young adults (PTYA, blue) compared to termborn adults (YAT, green). B, sFlt-1 in those born preterm to hypertensive pregnancy (Hyp, darker blue) were 97.7±15.9pg/mL compared to 84.2±18.9pg/mL in those born preterm to normotensive mothers (Norm, lighter blue) which was still significantly higher than sFlt-1 levels in term-born adults (YAT, green; 73.0±15.3pg/mL).
Figure 2 –Systolic blood pressure in preterm-born adults increased with each sENG (left, 117.4±8.6 vs 121.7±9.7 vs 127.8±10.4mmHg) and sFlt-1 (right, 118.9±8.5 vs 121.1±9.8 vs 126.7±12.0mmHg,
P =0.006) tertile (1 to 3, lowest to highest).
Figure 3 – Functional (baseline) and structural (venous occlusion) capillary density was reduced in preterm-born adults (PTYA, blue) (93.5±10.6capillaries/mm
2
and 101.8±12.7capillaries/mm
2
, respectively) compared to term-born adults (YAT, green; 105.2±10.6capillaries/mm
2
, P <0.001 and
115.9±11.8capillaries/mm
2
).
Figure 4 – Mediation analysis to identify potential mechanism through which increased antiangiogenic levels and capillary rarefaction relate to systolic blood pressure. Solid lines indicate significant relationships, while dotted lines indicate non-significant relationships. Bivariable regression demonstrated sENG related to 24 hour systolic blood pressure and reduced functional
(baseline, BO) and structural (venous occlusion, VO) capillary density. sFlt-1 positively associated to systolic blood pressure but not capillary densities. Both baseline and structural capillary density inversely related to systolic blood pressure. In multivariable regression, associations between sENG and systolic blood pressure were diminished when functional or structural capillary densities were included, consistent with density being a relationship mediator (black arrows).
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TABLES
Table 1: Characteristics of Microvascular Study Cohort
Parameters Preterm-Born
Young Adults
(n=30)
Demographics &
Anthropometrics
Gestational Age (weeks)
Birthweight (grams)
Age (years)
Males, n (%)
BMI (kg/m 2 )
30.5±2.7
1295.6±304.5
26.6±1.0
15 (50.0)
26.3±7.2
Term-Born
Young Adults
(n=60)
39.6±0.8
3411.2±319.0
26.2±1.9
30 (50.0)
23.0±3.3
P-Value
<0.001
<0.001
0.37
>0.99
0.006
Smokers, n (%)
Biochemistry
5 (16.7) 10 (16.7) >0.99
Total Cholesterol (mmol/L)
HDL-C (mmol/L)
LDL-C (mmol/L)
Triglycerides (mmol/L)
Glucose (mmol/L)
4.79±0.77
1.47±0.38
2.69±0.82
1.10±0.64
4.87±0.35
55.1±34.8
4.23±0.86
1.46±0.42
2.39±0.67
0.86±0.33
4.60±0.31
36.0±18.0
0.009
0.85
0.12
0.03
0.001
Insulin (pmol/L) 0.002
Full 24-Hour Blood Pressure
(mmHg)
Systolic
Diastolic
Mean Arterial Pressure
123.3±6.4
69.6±4.3
87.3±4.1
53.7±6.9
112.8±7.5
67.1±4.9
82.1±5.2
45.7±5.8
<0.001
0.02
<0.001
Pulse Pressure
Awake Blood Pressure (mmHg)
<0.001
Systolic
Diastolic
Mean Arterial Pressure
126.3±6.5
72.9±5.2
90.5±4.4
53.4±7.6
116.6±8.3
70.3±5.9
85.7±6.1
46.3±6.1
<0.001
0.05
0.001
Pulse Pressure
Sleep Blood Pressure (mmHg)
<0.001
Systolic
Diastolic
Mean Arterial Pressure
110.3±9.2
59.0±6.9
75.6±7.0
51.4±7.4
101.1±8.1
56.4±4.7
71.0±5.0
44.7±7.0
<0.001
0.04
0.001
Pulse Pressure <0.001
Values as Mean±Standard Deviation unless stated otherwise. P-values were adjusted for age and sex. BMI indicates body mass index; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol.
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Table 2: Circulating Angiogenic, Antiangiogenic and Inflammatory Markers
Circulating Biomarkers Preterm-Born
Young Adults
(n=102)
Term-Born
Young Adults
(n=102)
Angiogenic & Antiangiogenic
Markers
VEGF-A (pg/mL) sENG (ng/mL)
139.5±71.3
5.64±1.03
88.1±19.0
148.5±65.9
4.06±0.85
73.0±15.3 sFlt-1 (pg/mL)
Inflammatory Markers
CRP (mmol/L) sICAM-1 (ng/mL)
2.27±4.02
121.3±10.9
1187.8±271.3
1.53±3.35
112.9±10.1
864.4±155.4 sVCAM-1 (ng/mL)
P-Value
0.36
<0.001
<0.001
0.17
<0.001
<0.001
Values as Mean±Standard Deviation. P-values were adjusted for age and sex. VEGF-A indicates vascular endothelial growth factor A; sENG, soluble endoglin; sFlt-1, soluble fms-like tyrosine kinase-1; CRP, C-reactive protein; sICAM-1, soluble intercellular adhesion molecule-
1; sVCAM-1, soluble vascular cell adhesion molecule-1. Placental growth factor (PlGF) and soluble E-selectin (sE-selectin) were not detectable.
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