Preeclampsia complicates 3–7% of all pregnancies worldwide and is a major cause of maternal, fetal, and neonatal mortality and morbidity. This multisystem disorder is characterized by the onset of hypertension and proteinuria in the second half of pregnancy.1 Women who develop preeclampsia are at increased risk of hypertension, cardiovascular mortality, and stroke in later life.2 Several cardiovascular risk factors have been identified with increased risk of preeclampsia such as essential hypertension, obesity, and diabetes mellitus. The mechanism(s) by which these disorders are associated with preeclampsia are not fully understood, but impaired tissue perfusion and microcirculatory abnormalities have been implicated in the pathogenesis.3 We have previously reported that women who developed preeclampsia had reduction in the density of capillaries (ie, rarefaction) at the time of diagnosis when compared with women with normal pregnancy and healthy nonpregnant control participants.4
We therefore explored whether capillary rarefaction precedes the onset of preeclampsia and could play a role in its pathogenesis. We prospectively studied changes in functional and structural capillary densities from early pregnancy until 15 weeks postpartum. We also studied changes in angiogenic and antiangiogenic factors including vascular endothelial growth factor (VEGF) receptor 2, soluble Endoglin, and soluble fms-like tyrosine kinase 1, because there is increasing evidence for a possible role for antiangiogenic factors in the pathogenesis of preeclampsia5 and the induction of capillary rarefaction and hypertension.6
MATERIALS AND METHODS
The study was performed at St George's Hospital, London, and was approved by the Wandsworth Research Ethics Committee. Written informed consent was obtained from all participants. We recruited 322 consecutive white women with singleton pregnancies at 11–16 weeks of gestation. Women with multifetal pregnancies, pre-existing medical disorders (other than essential hypertension), or who were on medication (other than vitamins or folic acid supplementation) were excluded. At entry to the study, 298 women were normotensive, 13 had had previous preeclampsia, and 11 had a history of mild untreated essential hypertension. During the study, 17 women were lost to follow-up or their pregnancies terminated because of severe fetal anomaly and were excluded (Table 1). Participants were studied at five predetermined consecutive visits: 11–16 weeks, 20–24 weeks, 27–32 weeks, 34–38 weeks, and 5–15 weeks postpartum. Gestational age was determined by ultrasonography dating in the first trimester. Preeclampsia was defined as new onset of raised blood pressure greater than 140/90 mm Hg on two separate occasions at least 4 hours apart accompanied by proteinuria defined as 300 mg or greater per 24 hours or urinary protein:creatinine ratio greater than 30 mg/mmol, or 2+ or greater on urine dipstick.7 For individuals with pre-existing hypertension, preeclampsia was defined as new-onset proteinuria of 300 mg or greater per 24 hours or urinary protein and creatinine ratio greater than 30 mg/mmol, or 2+ or greater on urine dipstick.
Blood pressure was measured in the same arm throughout the study using the semiautomated digital oscillometric device Omron HEM907. Three sitting and two standing blood pressure measurements were obtained at 1-minute intervals using appropriate cuff size.
Skin capillary density was measured according to a standardized technique as described previously.8–10 The skin of the dorsum of the middle phalanx of the left hand was examined. Four microscopic fields (0.66 mm2 each) centered on an ink spot were studied to reduce selection bias. These four fields were recorded continuously for 5 minutes each to permit detection of intermittently perfused capillaries. Microscopic images were obtained using the CapiScope system CAM1, and the number of capillaries was counted manually online using computer software.4 Basal capillary density represents the number of capillaries that are “open” or “functioning” at the time of measurement and was calculated as the mean of the four microscopic fields. It is suggested that capillaries, especially in the skin, work on a “rota system,” ie, some are perfused, whereas others are shut down. This would seem likely for the skin where the blood supply far exceeds the nutrient requirements of the tissue. Thus, the maximal capillary density, also referred to as the “structural” or “anatomical” capillary density, is comprised of the functioning capillaries plus those capillaries that “open up.” We have previously shown that venous congestion allows visualization of the maximal number of skin capillaries during intravital video microscopy, exceeding that seen with postocclusive reactive hyperemia.8 In the present study, we determined maximal capillary density by applying a miniature blood pressure cuff to the base of the left middle finger and inflating the cuff and maintaining the pressure at 60 mm Hg for 2 minutes. The capillaries were counted in one of the four microscopic fields chosen at random.8
In a subgroup of 31 women with normal pregnancy outcome and seven patients with preeclampsia, we measured the levels of VEGF receptor 2, soluble Endoglin, and soluble fms-like tyrosine kinase 1 at 11–16 weeks, 27–32 weeks, and 34–38 weeks of gestation using commercially available enzyme-linked immunosorbent assay kits. We used the DVR100B kits for soluble fms-like tyrosine kinase measurement, DVR200 kits for VEGF receptor 2 measurement, and DNDG00 kits for s-Endoglin. Blood was collected in 5-mL plain tubes, centrifuged at 2,000 rpm at 4°C, and serum was separated and stored at −80°C in 500-μL aliquots until the time of analysis. All assays were performed in duplicate by a single investigator (V.N.).
One-way analysis of variance was performed for comparisons among groups at baseline and when significant differences were found, the Bonferroni correction was carried out to examine where the significance lay. For the longitudinal changes in the measured variables including data on systolic and diastolic blood pressure, basal and maximal capillary density, angiogenic, and antiangiogenic factors, we used analysis of variance for repeated measures (general linear model) with the Bonferroni adjustment for multiple comparisons within participants. For these variables, each measurement at 20–24 weeks, 27–32 weeks, 34–38 weeks, and 5–15 weeks postpartum was paired with measurements at baseline (11–16 weeks). We used independent samples t test to compare measurements “between participants.” Correlations between changes in angiogenic and antiangiogenic factors and changes in capillary density were made with Pearson's correlation. Statistical significance was declared when the P value was <.05. All statistical analysis was carried out using IBM SPSS 19.
Table 1 shows the baseline characteristics, at entry to the study, of the 305 women who completed the study. At the end of pregnancy, 272 women had a normal pregnancy, 16 developed preeclampsia, eight had hypertension (either essential or gestational), and nine pregnancies were complicated by intrauterine growth restriction without preeclampsia or hypertension. Table 2 shows the baseline characteristics of study individuals according to their pregnancy outcomes. Of the 13 women with a history of previous preeclampsia, four (30.8%) developed preeclampsia in the current pregnancy and of the 10 women with hypertension, three (30%) developed preeclampsia. At the baseline visit (11–16 weeks), both systolic and diastolic blood pressure were significantly higher, albeit within the normal range, in women who later on developed preeclampsia compared with women with normal pregnancy (106/62±9/9 mm Hg compared with 113/68±12/11 mm Hg, P=.002 and P=.035, respectively). As mentioned earlier, three of the women who later developed preeclampsia had a history of mild untreated hypertension and four had a history of previous preeclampsia. Table 3 shows longitudinal changes in systolic and diastolic blood pressure, basal capillary density, maximal capillary density, and angiogenic and antiangiogenic factors in normal pregnancy and in women who developed preeclampsia from the first trimester through four planned visits to 5–15 weeks postpartum.
There was a significant progressive increase in both systolic and diastolic blood pressure in normal pregnancy (P=.001 and P<.001, respectively). Compared with baseline, there was a nonsignificant increase in diastolic blood pressure at 20–24 weeks and 27–32 weeks of gestation. However, the subsequent changes were significant at 34–38 weeks (P<.001) and at 5–15 weeks postpartum (P<.001) (Table 3; Fig. 1A and B).
Compared with baseline at 11–16 weeks of gestation, the increase in systolic and diastolic blood pressure was nonsignificant at all visits in women who developed preeclampsia (Table 3; Fig. 1A and B). It is important to stress here that the blood pressure changes analyzed are those taken at the fixed predetermined visits (27–32 weeks and 34–38 weeks of gestation), which do not reflect the higher readings when preeclampsia was actually diagnosed, because then these women did not attend for their designated visits. This introduces the problem of missing data and analysis of blood pressure taken either before the onset of preeclampsia, or in the puerperium when the blood pressure had normalized. This explains the apparent lack of a significant rise in systolic and diastolic blood pressure in a group of women who nevertheless developed preeclampsia.
Compared with the baseline visit at 11–16 weeks of gestation, the earliest statistically significant fall in mean basal capillary density occurred at 34–38 weeks of gestation (P=.009). At the postpartum visit, the changes were not statistically significant. The earliest statistically significant decrease in maximal capillary density occurred at 34–38 weeks of gestation (P=.017). At the 5–15 weeks postpartum visit, the mean decrease was not statistically significant. (Table 3; Fig. 2A and B).
The mean onset of preeclampsia was at 35.6±4.8 weeks of gestation (range. 27.0–41.5 weeks). The changes in basal capillary density were not significant throughout pregnancy or in the postpartum visit. However, when compared with the baseline visit, there was a significant reduction in maximal capillary density (ie, structural rarefaction) at the 20- to 24-week visit (mean change 7.0 capillaries per field; 95% confidence interval −12.6 to −1.4; P=.015), 7–21 weeks before the onset of preeclampsia. There was further significant rarefaction at 27–32 weeks of gestation (P=.009) and at 34–38 weeks of gestation (P<.001) (Fig. 2B). In contrast to normal pregnancy, significant reduction in maximal capillary density persisted in the postpartum visit.
The changes in basal capillary density in women who went on to develop preeclampsia were not significant compared with women who had a normal pregnancy. We could not rule out the possibility that we have not seen significant differences because of the small number of participants with preeclampsia. However, maximal capillary density was significantly lower at 27–32 weeks of gestation (P=.003), at 34–38 weeks of gestation (P=.03), and at the postpartum visit (P=.02).
In normal pregnancy, serum VEGF receptor 2 levels increased significantly at 27–32 weeks of gestation (P=.02) and then reduced at 34–38 weeks of gestation but remained significantly higher compared with the first visit (P=.04). In preeclampsia, the increases from baseline in VEGF receptor 2 levels at 27–32 weeks of gestation and at 34–38 weeks of gestation were not statistically significant (Table 3).
The increase from baseline in serum fms-like tyrosine kinase 1 at 27–32 weeks and 34–38 weeks of gestation was not significant when compared with the first visit in normal pregnancy and in women who developed preeclampsia. In normal pregnancy, serum soluble Endoglin did not change significantly from baseline to 27–32 weeks of gestation but increased significantly at 34–38 weeks of gestation (P=.04). The changes in soluble Endoglin in pre-eclamptic pregnancies were not significant. Again we could not rule out the possibility that we have not seen significant differences at other visits because of the small number of participants who developed preeclampsia. There was no significant difference in VEGF receptor 2, fms-like tyrosine kinase 1, and soluble Endoglin levels between the two groups at 11–16 weeks and 27–32 weeks of gestation. At 34–38 weeks of gestation, VEGF receptor 2 levels were significantly lower (P=.05), whereas fms-like tyrosine kinase 1 and soluble Endoglin levels were significantly higher (P<.001 and P=.004, respectively) in women who went on to develop preeclampsia. The change in soluble Endoglin from 11–16 weeks to 27–32 weeks of gestation was significantly related with the change in maximal capillary density in the same period (Pearson correlation coefficient −0.45, P=.02), indicating that the higher the increase in soluble Endoglin, the greater the loss of structural capillary density. There was no correlation between levels of fms-like tyrosine kinase 1 or VEGF receptor 2 and other capillary measurements (P=.154 and P=.108, respectively).
This study shows that significant structural capillary rarefaction precedes the onset of preeclampsia. Women who went on to develop preeclampsia had a significant (P=.015) 9% reduction in their structural capillary density by 20–24 weeks of gestation, which progressed to 17% by 27–32 weeks of gestation and reached a nadir of 20% by 34–38 weeks of gestation. Our observations suggest that the earlier and more pronounced capillary rarefaction may play a role in the pathophysiology of preeclampsia. Our findings therefore not only corroborate our previous report that capillary rarefaction is associated with preeclampsia,4 but have advanced our knowledge in establishing the temporal relationship that structural capillary rarefaction precedes preeclampsia. These findings further support the concept of widespread maternal microcirculatory abnormalities that precede the onset of preeclampsia.11 We further substantiate previous studies which suggest that preeclampsia is an exaggerated antiangiogenic state12 with the structural capillary rarefaction and the resultant loss of endothelial surface and reduction of nitric oxide.13
Although the pathophysiological basis of preeclampsia remains uncertain, the consistent histopathologic lesion is defective and inadequate cytotrophoblastic invasion of spiral arterioles.14 In response to the hypoxia exaggerated by capillary rarefaction, the placenta secretes soluble factors into the maternal vasculature, which in turn induce widespread endothelial dysfunction and the clinical features of preeclampsia.15 One of the factors thought to be important is the naturally occurring antiangiogenic molecule soluble fms-like tyrosine kinase-1, also known as soluble VEGF receptor 1,16 which has been shown to be produced in large amounts by placental trophoblasts in preeclampsia17 and released into the maternal circulation.18,19 It acts as a potent antiangiogenic molecule by binding circulating VEGF and placental growth factor.20 Increased antiangiogenic activity in response to placental ischemia may seem contradictory, because teleologically one would expect increased angiogenic activity not least because VEGF, a proangiogenic molecule, is also upregulated by hypoxia.21 Cytotrophoblasts, in contrast to endothelial cells, respond in opposite ways to hypoxia in terms of angiogenic balance because they induce excess production of fms-like tyrosine kinase 1 and a deficiency of VEGF.17 However, the situation is undoubtedly complex and other factors may well play a role too. For instance, we have previously reported that placental catecholamine release is increased in preeclampsia,22 and we postulated that in response to hypoxia, the placenta releases a signal (catecholamine) recognizable by the maternal vasculature to raise blood pressure and improve perfusion of the fetoplacental unit and that what starts as a physiological response becomes pathologic because the rise in blood pressure fails to correct the hypoxia.22
An additional remarkable finding in our study was that although the reduction in capillary density recovered to baseline levels within the puerperium in women who remained normotensive, in those who developed preeclampsia, there was no resolution of the capillary rarefaction. This observation appears to be consistent with the reports of long-term risk of endothelial dysfunction, hypertension, and cardiovascular disease in women who develop preeclampsia23,24 and the observations that capillary rarefaction is a hallmark of essential hypertension.25,26 The strong familial predisposition to preeclampsia, essential hypertension, and atherosclerosis suggests that there may be common risk factors, pathways, or both for these disorders.27 The mechanisms and consequences of microvascular rarefaction are not fully understood but could possibly increase peripheral vascular resistance either directly through the reduction of the total cross-sectional area of the terminal vasculature or indirectly through inducing renal ischemia and stimulation of the renin–angiotensin system. Microvascular rarefaction is not confined to the skin but is a widespread phenomenon affecting several tissues in hypertensive individuals including the myocardium, the kidneys, and the brain.10,28,29 In support for a possible role of microvascular rarefaction in increasing blood pressure is the current realization that angiogenesis inhibitor drugs induce hypertension in a significant proportion of patients and this increase in blood pressure can be related to or even be explained by capillary rarefaction and alteration in endothelial function in the whole systemic vascular network.30 More recently it has been shown in preclinical studies that these drugs can cause a preeclampsia-like syndrome further supporting a role for the capillary microcirculation in the pathogenesis of this syndrome.31
We acknowledge the limitations of our study that include the relatively small number of women who developed preeclampsia and the fewer numbers of observations as a prospective study progresses. However, the small number of women who developed preeclampsia represented 4% of our study population, a reflection of the prevalence of preeclampsia (3–7%) in populations worldwide. A longitudinal study design overcomes the issue of small numbers because the variability between individuals, which would require large numbers to compensate in a cross-sectional study, is minimized by repeated measurements in a given individual. In addition, this longitudinal study design is arguably the only effective way of examining temporal relationships in observational studies.
In conclusion, significant structural capillary rarefaction precedes the onset of preeclampsia and may therefore play a role in the pathogenesis of this disease. Our results substantiate the concept of widespread maternal microcirculatory abnormalities that precede the onset of preeclampsia.
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