Our aim was to evaluate the long-term effects of preeclampsia on vascular function in a mouse model induced by sFlt-1 overexpression. CD-1 mice at day 8 of gestation were injected via the tail vein with adenovirus carrying sFlt1 (AdsFlt1), adenovirus carrying the murine IgG2α Fc fragment as the adenovirus control (AdmFc), or saline. Vascular function in the mothers was investigated 6–8 mo after delivery by recording blood pressure (BP) by telemetry (AdsFlt1 n = 8, AdmFc n = 6, saline n = 4) and exploring carotid artery reactivity in a wire myograph (AdsFlt1 n = 6, AdmFc n = 8, saline n = 4). sFlt-1 blood levels at 6–8 mo postpartum had returned to low levels and were comparable between the three groups (P = 0.808). There was no statistically significant difference in BP (P = 0.067) or vascular reactivity between the three groups of postpartum mice (phenylephrine P = 0.079, thromboxane P = 0.979, serotonin P = 0.659, acetylcholine P = 0.795, sodium nitroprusside P = 0.728, isoproterenol P = 0.370). Our results indicate that in a mouse model overexpression of sFlt-1 does not lead to increased in BP and altered vascular function in the absence of the pregnancy and has no long-term effect on BP and vascular function in the postpartum mothers. Our findings favor the hypothesis that increased cardiovascular diseases in women with history of preeclampsia are likely the result of preexisting risk factors common to preeclampsia and cardiovascular diseases.
- vascular reactivity
- blood pressure
cardiovascular diseases (CVD) are the leading cause of death in women in the United States (23) and worldwide. Epidemiological studies have demonstrated that preeclampsia identifies mothers at an increased risk for development of hypertension, coronary heart disease, stroke, and diabetes later in life (2, 7, 21, 24). The relative risk is highest (range 2.6–8.1) among the women with most severe manifestations of preeclampsia or those accompanied with preterm delivery and/or intrauterine growth restriction. In case of women with milder forms of preeclampsia, the relative risk may range from 1.9–2.2 (16).
Recent meta-analysis of papers published between 1960 and December 2006 calculated that the relative risks (95% confidence intervals) for hypertension were 3.70 (2.70 to 5.05) after 14.1-yr weighted mean follow-up, for ischemic heart disease 2.16 (1.86 to 2.52) after 11.7 yr, for stroke 1.81 (1.45 to 2.27) after 10.4 yr, and for venous thromboembolism 1.79 (1.37 to 2.33) after 4.7 yr compared with women who did not develop preeclampsia (4). It has also been reported that women with a history of preeclampsia had significantly higher systolic and diastolic blood pressure, body mass index (BMI), cholesterol, and triglyceride levels than women with no history of preeclampsia and normal pregnancy, even after adjusting for smoking, chronic hypertension before pregnancy, and a BMI above 30; those changes occur at least 3 mo after delivery and after ending lactation (12). These reports suggest that one of the most serious complications of pregnancy, preeclampsia, is associated with an increased risk of maternal CVD later in life. It might be argued, however, that the pregnancy challenge to the maternal vasculature could be considered as a “stress” test (26). In the majority of individuals metabolic and inflammatory changes are absorbed by physiological buffers. However, in women who develop preeclampsia, a phenotype may exist whereby the inflammatory and metabolic response to pregnancy is exaggerated and buffering mechanisms are inadequate. Thus preeclampsia may serve as early marker for increased risk of early cardiovascular events in relatively young-age women, and recognition that preeclampsia may unmask those apparently healthy but CVD-predisposed women could offer an important opportunity for prevention.
Preeclampsia is a syndrome unique to pregnancy and is characterized by hypertension, proteinuria, edema, and other systemic manifestations of end-organ damage. It occurs in 4–5% of all pregnancies and is a leading cause of maternal morbidity and mortality (15, 20, 28). Recent reports support a role for abnormal angiogenesis in the development of preeclampsia (13, 14, 22). In a prospective cohort, Levine and colleagues (9) found that soluble fms-like tyrosine kinase 1 (sFlt-1) levels in women who develop preeclampsia rise above those in women who remain normotensive. This occurs several weeks before the onset of the clinical syndrome. The sFlt-1 is the soluble form of the vascular endothelial growth factor (VEGF) receptor 1, which binds VEGF and other angiogenic factors in the circulation, thereby decreasing their action. As further evidence for the role of dysregulation of angiogenesis in the causation of preeclampsia, Maynard et al. (13) reported a preeclampsia-like condition in rats with overexpression of sFlt1 induced by transfection with an adenovirus carrying sFlt-1 (AdsFlt1-induced preeclampsia). Our group has successfully reproduced this animal model in mice and further refined it by using telemetric transducers to measure blood pressure in unrestrained conscious animals. As well, we have characterized the vascular, hematological, hepatic, placental, and renal changes in response to overexpression of sFlt-1 during pregnancy, thus providing substantial evidence for study of preeclampsia and its consequences in the mice (10, 11).
The objective of the present study was to assess whether preeclampsia itself may induce irreversible vascular changes accompanied with increased blood pressure in the mother later in life. For this purpose, a mouse model of sFlt1-induced preeclampsia was utilized and vascular responses to a number dilatory and constrictor agonists in vitro and blood pressure in vivo was studied 6–8 mo after pregnancy.
MATERIALS AND METHODS
The study protocol was approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch, Galveston, Texas. Pregnant CD-1 mice at day 6 of gestation were purchased from Charles River (Wilmington, MA). The mice were maintained in the animal care facility at the University of Texas Medical Branch. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch. The mice were housed separately in temperature- and humidity-controlled quarters with constant light-dark cycles of 12:12 h. They were provided with food and water ad libitum. Surgical procedures were performed according to the Animal Care and Use Committee guidelines with anesthesia using ketamine (Ketalar; Parke-Davis, Morris Plains, NJ) and xylazine (Gemini; Rugby, Rockville Center, NY). The animals were killed by CO2 inhalation according to the Animal Care and Use Committee and the American Veterinary Medical Association guidelines.
At day 8 of gestation, pregnant CD-1 mice were randomly divided into three groups and injected via the tail vein with adenovirus carrying sFlt1 (AdsFlt1, 109 plaque-forming units in 100 μl), adenovirus carrying the murine IgG2α Fc fragment as the adenovirus control (AdmFc, 109 plaque-forming units in 100 μl), or saline (100 μl). The procedure to produce AdsFlt1 and AdmFc is described elsewhere (11). Pregnant mice were allowed to deliver. Pups were weaned from their respective mothers 3 wk after delivery. Experiments were performed in mice six to eight mo postpartum. If human lifespan is about 80 yr and mice live about for 2 yr when in captivity, then 6 mo in mouse would be an equivalent of about 20 yr in human. Animal care, feeding, and other conditions were the same for all three groups of animals.
In vivo blood pressure measurements.
After anesthesia with a mixture of ketamine (Ketalar, Parke-Davis, Morris Plains, NJ) and xylazine (Gemini, Rugby, Rockville Center, NY), a vertical midline skin incision along the neck was made and the submaxillary glands were gently separated. The left common carotid artery was carefully isolated. Then the catheter was introduced into the carotid artery through a small incision in the vessel wall and the body of the transducer [PA-C10 model, Data Systems International (DSI), Overland Park, KS] was secured in a subcutaneous pouch along the animal's right flank through the same ventral neck incision. The neck incision was closed with 6-0 silk. Mice were kept warm on a heating pad and monitored closely until full recovery from anesthesia. Recording of blood pressure began 72 h after surgical implantation of the pressure transducer and was continuously monitored for 7 consecutive days by use of RLA 1020 telemetry receivers (DSI), BCM consolidation matrix (DSI), and an adapter, where the signal was demultiplexed. This output subsequently was band-pass filtered and amplified. The information was fed to data acquisition and recording system, Dataquest software (A.R.T.3.1; Gartner Dataquest, Stamford, CT). Then the mice were euthanized, and the carotid arteries were isolated for in vitro vascular reactivity studies.
Vascular reactivity studies.
Two-millimeter segments of right carotid arteries were mounted in a wire myograph (model 410A, J.P. Trading, Aarhus, Denmark) with 25-μm tungsten wires. The preparations were bathed in Krebs solution maintained at 37°C, pH ∼7.4. A mixture of 95% O2 and 5% CO2−6 mmol/l) in vessels precontracted with phenylephrine (10−6 or 3 × 10−6 mmol/l) was determined. Only arteries demonstrating a substantial response to acetylcholine (more than 70–80% of relaxation) and constriction to high potassium were used for experimental studies.
After 1 h of equilibration, relaxant responses to the endothelium-dependent vasorelaxant acetylcholine (10−10–10−5 mmol/l), the endothelium-independent vasorelaxant sodium nitroprusside (10−10–10−5 mmol/l) and the β-adrenoreceptor agonist isoproterenol (10−10–10−5 mmol/l) were obtained after precontraction of the vessels with phenylephrine (10−7–10−6 mmol/l). In addition, contractile responses to the α1-adrenergic agonist phenylephrine (10−10–10−5 mmol/l), thromboxane A2 mimetic U46619 (10−10–10−5 mmol/l), and 5-hydroxytryptamine or serotonin (10−10–10−5 mmol/l) were assessed.
Blood sFlt-1 measurements.
Blood was collected via heart puncture at the time of euthanasia and was spun down. The sFlt-1 level in the blood was measured by using mouse soluble VEGF R1 immunoassay (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Blood pressure data (AdsFlt1 n = 8, AdmFc n = 6, saline n = 4) obtained from the telemetry system were plotted as mean values over a 24-h period, expressed as means ± SE, and analyzed by one-way ANOVA (P < 0.05).
In the vascular reactivity studies (AdsFlt1 n = 6, AdmFc n = 8, saline n = 4), results are expressed as means ± SE; n represents the number of mice used in each experiment. KCl was used as a reference to calculate the percent of contraction achieved by the contractile agents studied, whereas phenylephrine precontraction was used to obtain the percentage of relaxation induced by the vasorelaxant agents. Concentration-response curves were constructed. The area under the concentration response curve and means at different concentrations were compared by one-way ANOVA (P < 0.05).
The sFlt-1 levels (AdsFlt-1 n = 10, AdmFc n = 7, or saline n = 4) were calculated with a standard curve that was derived from known concentrations of the recombinant protein. Comparisons between groups were made using one-way ANOVA (P < 0.05).
Drugs and solutions.
Krebs solution was composed of 119 mmol/l NaCl, 4.7 mmol/l KCl, 1.2 mmol/l KH2PO4, 25 mmol/l NaHCO3, 1.2 mmol/l MgCl2, 2.5 mmol/l CaCl2, 0.026 mmol/l EDTA, and 11.5 mmol/l glucose. KCl, acetylcholine hydrochloride, phenylephrine hydrochloride, isoproterenol, serotonin hydrochloride, and sodium nitroprusside were obtained from Sigma-Aldrich (St. Louis, MO), and U46619 was obtained from Cayman Chemical (Ann Arbor, MI). All drug dilutions were with Krebs solution.
There was no statistically significant difference in the levels of sFlt1 between the AdsFlt1, AdmFc, or saline pretreatment groups (P = 0.808). The levels in all three groups were in the normal range for mice (1.1.8–7.1 ng/ml) (Fig. 1). Low levels of sFlt1 are in agreement with studies demonstrating that expression of the recombinant virus by use of adenoviral technology has been efficient but transient, which could be association with inflammatory processes in the liver (27). Also, studies in postpartum mothers with preeclampsia show a rapid decrease in sFlt1 levels after delivery (8, 25).
There was no statistically significant difference in mean blood pressure in mice injected with AdsFlt1, AdmFc, or saline during pregnancy (Fig. 2, P = 0.067).
All relaxation agents tested produced a significant relaxation of the carotid artery with no differences between AdsFlt1, AdmFc, and saline groups (Fig. 3, only acetylcholine shown). There was no statistical difference in responses to contractile agents between postpartum AdsFlt1, AdmFc, and saline-pretreated mice (Fig. 4, only responses to thromboxane are shown). P values for vascular reactivity experiments were as follows: phenylephrine P = 0.079, thromboxane P = 0.979, serotonin P = 0.659, acetylcholine P = 0.795, sodium nitroprusside P = 0.728, isoproterenol P = 0.370.
In this study, we explored the effects of sFlt-1-induced preeclampsia on long-term maternal cardiovascular function. Our results suggest that overexpression of sFlt-1 does not lead to increased blood pressure and altered vascular function in the absence of pregnancy. Overexpression of sFlt-1 during pregnancy also has no long-term effect on blood pressure and vascular function in the postpartum mothers. This is the first-ever experimental study that explores the impact of preeclampsia on long-term maternal vascular function and suggests that preeclampsia by itself in animal model is not associated with long-term effect on blood pressure control and vascular function in mothers with history of preeclampsia. This study has, however, to be interpreted within caution. The results are limited to just one age group, and food intake and weight gain after pregnancy were not controlled. Moreover, the vascular studies were performed in conduit arteries, whereas it cannot be excluded that some abnormalities may initially occur within the resistance vasculature without the effect on blood pressure due to the compensatory enhancement or changes in ratio of endothelium-derived factors that confer endothelium-dependent dilatation.
In human studies, Chambers et al. (6) demonstrated impaired endothelium-dependent dilatation to flow in the brachial artery in women with a history of preeclampsia (the earliest testing was 3 mo postdelivery with a median of 3 yr) compared with women who had a normal pregnancy; however, the findings could not be explained by differences in established risk factors. Ramsay et al. (17) reported that microvascular function, as reflected by reduced laser Doppler imaging responses to acetylcholine iontophoresis in the circulation of the skin, is impaired in women 15–25 yr following a pregnancy affected by preeclampsia. However, other studies exists that show that microvascular responses to acetylcholine are upregulated one year after preeclampsia (5). Thus whether preeclampsia is a cause or just an exacerbation of a preexisting condition needs further research. Preeclampsia is unlikely to be associated with a single causative factor, and the condition shares common pathogenic features with cardiovascular diseases, including alterations in insulin sensitivity, hypertriglyceridemia, and a proinflammatory state. Therefore, currently the most appreciated suggestion is that women with a previous history of preeclampsia have increased cardiovascular risk because of exacerbation of underlying maternal risk factors; our study employing an animal model without preexisting maternal predisposition followed by normal vascular function and blood pressure postpartum would favor such a suggestion.
In human pregnancy, support that maternal constitutional factors contribute to the onset of preeclampsia continue to accumulate (3). Endothelial dysfunction, which is associated with insulin resistance, may be a predisposing vascular mechanism for both coronary heart disease and preeclampsia. Savvidou et al. (19) demonstrated that mean flow-mediated dilation in pregnant women who eventually developed preeclampsia was 3.58%, compared with 8.59% in women with normal outcome. This impairment is as great as that observed in patients with CVD risk factors that are known to induce endothelial dysfunction. Moreover, low flow-mediated dilation predated the development of preeclampsia by 10 wk. Intriguingly, a recent study by Sattar et al. suggested that the elevated risk of coronary heart disease in women with previous preeclampsia is not explicable on the basis of conventional risk factors and that subtle alterations in novel risk factors (e.g., ICAM-1, VCAM-1, and HbA1c) may play a significant role (18). In that report, risk (as assessed on the basis of the lipid profile) would not have identified these women as being at risk for cardiovascular disease, stressing the need to identify causes of preeclampsia that may also be associated with long-term adverse cardiovascular outcomes in women.
In 2003, an underlying cardiovascular abnormality was the cause of death in 483,842 women (1). This translates into a death rate of 308.8 per 100,000. That same year, the prevalence of CVD in women was 38.2 million (23). Data from the Framingham Heart Study indicate that the lifetime risk for CVD is more than 1 in 2 for women at age 40 (23). From 1970 to 2003, discharges for women diagnosed with coronary heart disease increased by 40% (1). Clearly, women remain at high risk for CVD. Therefore, further basic research into the mechanisms involved in the development of CVD in women is warranted. The presence of gender differences in cardiovascular diseases is well established. Therefore, pregnancy may well be a screening test for long-term cardiovascular health and disease. Studying the long-term implications of adverse pregnancy outcomes may help unmask novel mechanisms of disease in women and provide evidence for effective preventive strategies.
Although our animal model to study the consequences of hypertensive disorders in pregnancy on later cardiovascular health has not provided the conclusive results, further studies are warranted and possibilities exist to test whether preexisting maternal factors or acquired postpartum risk factors may alter CVD function toward increased blood pressure with altered vascular function in such animal model. Such studies, if performed, could be optimal to target impaired pathways and modification of risk profile for later cardiovascular disease in women with adverse pregnancy outcome.
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