Vascular Endothelial Cell Dysfunction in Preeclampsia

Editors' comment : It was not until the early 1980s that physicians and scientists gained an appreciation of the physiological importance of the endothelium, the simple unicellular layer lining the luminal surface of blood vessels. Indeed, in Chesley's first, single-authored edition of this text, the reference to this term was as it related to the “endotheliosis” lesion of the renal glomerulus. We now recognize that endothelial cells are critical sensors of the milieu interieur and potent regulators of vascular tone, organ perfusion, and ischemia. The “endothelial hypothesis” of preeclampsia etiology provides for a convergence of several factors thought to play fundamental roles in its pathogenesis: leukocytes, platelets, cytokines, fatty acids, oxygen free radicals, placental microvesicles, cell-free DNA fragments, “antiangiogenic” factors, and autoantibodies are all considered. Moreover, a variety of therapeutic interventions have been conceived based on the principle of endothelial cell protection, and clinical trials emanating from these concepts are reviewed in the chapter.

Introduction

The clinical manifestations of preeclampsia–eclampsia have been recognized since antiquity, but the pathophysiology of this syndrome remained completely obscure for nearly two millennia. Beginning in the mid-19th century, abnormal renal handling of nitrogen and water, referred to as that time as “dropsy,” was first reported among eclamptic women. Recognition that these signs were a manifestation of endothelial cell injury took another 90 years. Renal glomerular capillary endotheliosis, swelling of endothelial cytoplasm, and obliteration of endothelial fenestrae were originally observed in 1924 by Mayer and described further by Bell and later refined by Spargo et al. The latter investigator is credited for introducing the terminology “glomerular capillary endotheliosis” as a characteristic feature of preeclampsia. Since this time, research has focused extensively on understanding the mechanisms for endothelial cell dysfunction as a key pathophysiologic feature for women with preeclampsia.

Endothelial cells are strategically positioned at the interface between circulating blood and vascular smooth muscle or the extravascular space where they occupy a surface area of more than 1000 m ² . These cells are able to secrete a variety of signaling molecules directly into the circulation, potentially reaching every cell in the body. In turn, endothelial cells are themselves targets for cellular and soluble plasma constituents (e.g., cytokines, lipoproteins, platelets, leukocytes, placental microvesicles, antibodies, and other circulating factors). Endothelial cells modulate vascular tone, coagulation, permeability, and the targeting and trafficking of immune cells. Under normal, physiological conditions, the endothelium maintains homeostatic balance. Hemostasis is maintained in equilibrium by procoagulant and anticoagulant influences and endothelial tight junctions control vascular permeability.

Endothelial cell dysfunction or “activation” is a term used to define an altered state of endothelial cell differentiation, typically induced as a result of cytokine stimulation. It represents an inflammatory response to sublethal injury of these cells and is proposed to play a major role in the pathophysiology of atherosclerosis. Dysfunction of the maternal endothelium is believed to result in vasospasm, microthrombosis, and vascular permeability that lead to the classical signs and symptoms observed in women with preeclampsia.

In this chapter, we will present evidence in support of the hypothesis that a variety of proinflammatory and vasoactive circulating factors act upon the maternal vascular endothelium to create endothelial oxidative stress, which is a common downstream converging mechanism in the clinical manifestations of preeclampsia. Findings relevant to endothelial cell biology in preeclampsia will be reviewed in this chapter in five parts. In Part I, we review the evidence favoring endothelial cell dysfunction as a pivotal hypothesis for the pathophysiology of preeclampsia. Part II reviews the concept that circulating factors causally induce disturbances in maternal endothelial cell function. Part III proposes that prooxidant principles lead to a common convergence that creates an oxidative stress environment within maternal endothelia of preeclamptic women. Part IV discusses clinical trials that have been focused on improving vascular function. Finally, in Part V we conclude with a summary of the findings, speculations about future diagnostic and therapeutic approaches to preeclampsia, and some suggestions for the direction of new investigations into the mechanisms of this enigmatic condition.

Part I: Endothelial Cell Dysfunction and Preeclampsia

Over the past decade, the attention of scholars of preeclampsia has focused on impaired trophoblast invasion, uteroplacental ischemia, immune system activation, oxidative stress, and generalized maternal endothelial cell activation or injury as major mechanistic factors in its pathogenesis. It is likely that several etiologies underlie the development of this syndrome, but these pathogenetic mechanisms are shared.

As described in Chapter 5 by Burton, Cindrova-Davies, Yung, and Taylor, failure of trophoblast differentiation, invasion, and vascular remodeling of the placental bed are believed to be critical initiating factors in the development of preeclampsia and reduced angiogenic factor production and action ( Chapter 9 by Karumanchi et al.). If we imagine the placental bed as the root of the disease process, this might be a good place to begin to look for evidence of endothelial cell dysfunction. Indeed, the endothelium lining the uterine spiral arterioles, which normally undergoes denudation and replacement by invading endovascular cytotrophoblasts, may manifest the first pathological changes associated with preeclampsia. ^, Other vascular manifestations have been also noted at this site. Acute atherosis, as manifested by endothelial cell vacuolization, myointimal proliferation, and foam cell infiltration of the tunica media, has been described in placentas from women with preeclampsia. More recently, Johnsen et al. demonstrated that preeclamptic pregnancies with specific genetic combinations between fetal HLA and maternal killer immunoglobin-like receptors (KIRs) had a 4.5 times greater likelihood to develop acute atherosis in spiral arteries in the decidua basalis.

Concentrations of freely circulating endothelial cells, markers of vascular injury, have been reported to be more than fourfold higher in women with preeclampsia compared to normal pregnant controls, suggesting that endothelial dysfunction extends beyond the placental vascular bed and the glomerular endothelium in preeclampsia. Indeed, studies using flow-mediated dilation, which is the gold standard for noninvasive assessment of nitric-oxide-dependent endothelial function in humans, demonstrated impaired dilatory responses to reactive hyperemia in maternal brachial arteries of patients with preeclampsia. Endothelial cell dysfunction in preeclampsia may result from a variety of circulating factors, including many discussed in other chapters such as angiogenic factors, and metabolic and inflammatory mediators.

Endothelial activation is manifest biochemically by the synthesis and secretion of variety of endothelial cell products including nitric oxide, prostanoids, endothelin-1, platelet-derived growth factor, fibronectin, selectins, and other molecules that influence vascular tone and remodeling. ^, When these factors are elaborated in response to acute mechanical or biochemical endothelial cell damage, they facilitate efficient wound healing. However, when activated by a chronic pathological process, such as preeclampsia whereby the homeostasis of vascular relaxing factors (e.g., nitric oxide) are perturbed, these responses can create a vicious cycle of vasospasm, microthrombosis, and disruption of vascular integrity, creating serious disturbances in vascular function, tissue perfusion, and blood pressure regulation. Cerebral ischemia, edema and convulsions, pleural effusion and ascites, and hepatic dysfunction, in addition to the classical proteinuria and peripheral edema reflecting renal and subcutaneous vasculatures, respectively, are evidence of the many maternal vascular beds at risk. Endothelial cells within these compromised vessels not only lose their normal constitutive, homeostatic functions but also acquire new pathological properties (e.g., vasoconstrictor and procoagulant production) that increase vascular resistance and exacerbate ischemia.

In this section, we review evidence that supports the concept that one of the most important targets of circulating “toxins” in preeclampsia is the maternal vascular endothelium that ultimately leads to maternal vascular oxidative stress and dysfunction.

Circulating Markers of Endothelial Cell Activation

Procoagulant Proteins and Plasminogen Activators

Hypercoagulability increases in normal pregnancy and is exaggerated in preeclamptic pregnancies, in which a maternal hypercoagulable state results in intravascular coagulation, microthrombosis in several organs including the placenta, with further impairment of the uteroplacental circulation. The clinical significance of this problem is reviewed in Chapter 16 , but hypercoagulability also underlies the principle of endothelial cell dysfunction in preeclampsia. Excessive fibrin deposition in the placenta was reported by Kitzmiller and Benirschke, suggesting that a disordered balance of placental coagulation and fibrinolysis may play a role in the activation of hemostasis. Roberts et al. posited that the hypercoagulable state was due, in part, to diffuse endothelial cell activation. The reduced expression of several relevant endothelial-cell-associated anticoagulant proteins has been shown in preeclampsia, including antithrombin III, protein C, and protein S. The significance of these proteins as markers of maternal endothelial cell dysfunction is reviewed below.

Resistance to activated protein C is an inherited mutation of the coagulation factor V gene. The presence of the factor V Leiden mutation predisposes to thromboembolic events, and its prevalence is increased in women with severe, early onset preeclampsia compared to women with normal pregnancies. ^, In addition, in women with severe, early-onset preeclampsia, 25% had evidence of functional protein S deficiency, 18% demonstrated hyperhomocysteinemia, and 29% had detectable anticardiolipin IgG and IgM antibodies. ^,

Increased endothelial expression of other procoagulant proteins, including tissue factor, von Willebrand factor, platelet-activating factor, β-thromboglobulin, cellular fibronectin, and thrombomodulin, also has been reported. The latter two endothelial cell markers have been shown to differentiate preeclampsia from other forms of hypertension in pregnancy. ^, Similarly, inhibitors of fibrinolytic or antithrombotic proteins appear to play a role in the imbalance of the coagulation cascade. Plasminogen activator inhibitor type 1, whose synthesis during pregnancy is predominantly placental in origin, and tissue-type plasminogen activator were observed to be increased in the plasma of women with preeclamptic pregnancies. Increased decidual and amniotic fluid concentrations of tissue-type plasminogen activator also have been reported in preeclampsia. Circulating levels of tissue-type plasminogen activator, thrombomodulin, and fibronectin were found to correlate directly with severity of the syndrome. ^,

Endothelial Cell Adhesion Molecules

In addition to reduced anticoagulant synthesis, endothelial cells express extracellular matrix glycoproteins with procoagulant activities as a response to injury or activation. Two examples are fibronectin and von Willebrand factor. Both of these proteins are predominantly localized to the abluminal extracellular matrix of human endothelium, but as discussed below these can be actively secreted from endothelial cells under conditions of cellular activation. In addition, exposed fibronectin is stimulatory for neutrophil attachment and has important pathophysiological consequences.

Elevated concentrations of fibronectin in women with preeclampsia have been recognized for decades and have been confirmed in other clinical conditions associated with endothelial cell dysfunction. A specific cellular fibronectin isoform (cFN) is nonhepatic in origin and almost exclusively localized to the vascular endothelium has been of particular interest. It can be distinguished at the protein level by two extra domains (ED-A and ED-B) generated by differential mRNA splicing in endothelial cells. While it is a major component of the endothelial extracellular matrix, cFN is normally only a minor component of circulating fibronectin and thus is an accurate marker of endothelial cell injury.

Lockwood et al. showed that ED-A fibronectin levels were increased in a cross-sectional study of women who later developed preeclampsia. Taylor et al. used a monoclonal antibody that recognizes a conformational epitope near the ED-B region of cFN and showed in a prospective, longitudinal study that plasma cFN concentrations were increased as early as the second trimester in women destined to develop preeclampsia but the analyte was not elevated in women developing transient hypertension without proteinuria.

The concentrations of other endothelial cell adhesion molecules, including vascular cell adhesion molecule 1 (VCAM-1), P-selectin, E-selectin, and platelet endothelial cell adhesion molecule 1 (PECAM-1), were elevated in serum from women with preeclampsia. Concentrations of intercellular adhesion molecule 1 (ICAM-1) and VCAM-1 also appeared to have some value as predictors of preeclampsia because they were elevated 3–15 weeks before the onset of clinical symptoms. However, these findings were not observed as early as 11–13 weeks of gestation in women who later developed preeclampsia. Collectively, these data suggest that serum concentrations of endothelial cell adhesion molecules are increased in preeclampsia, providing evidence of generalized endothelial cell dysfunction, but their predictive potential may be limited.

Mitogenic Activities and Growth Factors

Endothelial cells also respond to injury or activation with the release or secretion of mitogenic proteins or peptides. With acute vascular trauma, this response teleologically encourages the proliferation of vascular smooth muscle, allowing vessel remodeling and repair. However, in illnesses such as atherosclerosis or preeclampsia, mitogenic factors cause reduced blood flow by promoting vessel wall hypertrophy. Indeed, plasma from women with preeclampsia collected prior to delivery had more mitogenic activity than plasma from the same women obtained 48 h postpartum. By contrast, plasma from normal pregnant women had essentially the same mitogenic activity before and after delivery. These “mitogenic indices” were elevated from as early as the first trimester of pregnancy, compared to women who proceeded to have normal pregnancy outcomes. Other studies indicate that the mitogenic activity is protease-, heat-, and acid-labile and has an apparent molecular mass of ~150,000 Da. To our knowledge the following hypothesis has not been tested directly, but activating angiotensin II type 1 receptor (AT1R) autoantibodies, which are present in women with preeclampsia as discussed in detail below, would be expected to have the identical biochemical characteristics since myocardial fibroblasts are induced to undergo mitogenesis through activation of the AT1R.

Based on the highly apparent molecular mass of the mitogenic activator(s) discovered above, the plasma activity was postulated to be attributable to insulin-like growth factor binding protein complexes. However, most studies failed to show differences in circulating concentrations of these complexes in preeclampsia. In a longitudinal study comparing 20 primipara who developed preeclampsia with 20 matched, normal pregnant controls, de Groot et al. found that midtrimester maternal plasma insulin-like growth factor binding protein-1 concentrations were significantly reduced in the group of women who developed preeclampsia approximately 20 weeks later. They interpreted their data as reflecting reduced trophoblastic invasion of the maternal decidual stroma and decreased vascular deportation of insulin-like growth factor binding protein-1 in pregnancies that resulted in preeclampsia. This observation was confirmed by Hietala et al., but it should be noted that some reports of third trimester maternal serum insulin-like growth factor binding protein-1 show elevations in women with active, symptomatic severe preeclampsia. ^,

The growth factor binding protein that to date has had the greatest impact on the preeclampsia field is the soluble receptor of vascular endothelial growth factor (VEGF), namely soluble fms-like tyrosine kinase-1 (sFlt-1). The history and role of this glycoprotein are presented comprehensively in Chapter 9 by Karumanchi, Rana, and Taylor, and the role of sFlt-1 in preeclampsia-associated endothelial dysfunction is discussed in detail later in this chapter. Briefly, it is now broadly established that elevated sFlt-1 concentrations in women with preeclampsia are associated with decreased circulating levels of free VEGF and free placental growth factor (PlGF), blocking their angiogenic signaling in the microvasculature. Overall, many of the biomarkers of endothelial cell activation described above are also functional mediators of endothelial cell dysfunction, present in the circulation of women with manifest preeclampsia.

Part II: Circulating Factors Induce Endothelial Cell Dysfunction

Efforts to identify the putative circulating toxic factors responsible for endothelial cell dysfunction in preeclampsia have been ongoing for the past decade with disappointing results. The effort continues, but while specific candidates have been identified, most data suggest that multiple molecular species account for endothelial injury. Because of their direct contact with the vascular endothelial cell monolayer in vivo, a variety of plasma constituents are likely candidates for endothelial cell activation in preeclampsia. Formed blood elements (e.g., platelets and neutrophils), placental membrane microvesicles, soluble proteins (e.g., autoantibodies and cytokines), lipids, cell-free nucleic acids, cytokines, and matrix metalloproteinases (MMPs) have all been identified in the plasma of women with preeclampsia. In this section, we systematically discuss some of these postulated mediators of endothelial cell dysfunction in preeclampsia.

Formed Elements in Blood as Activators of Endothelium

Platelets

Clinical manifestation of abnormal platelet function includes hypercoagulability, as reviewed in Chapter 16 . In vitro evidence of abnormal platelet aggregation in women with preeclampsia suggests that this phenomenon could lead to endothelial cell activation. Early retrospective cohort studies of women with renal disease in pregnancy indicate that the incidence of preeclampsia was significantly less common in women treated prophylactically with anticoagulants or antiplatelet drugs, such as heparin or aspirin, respectively. Subsequently, the efficacy of aspirin treatment was questioned because of inconsistent findings among numerous randomized trials. This inconsistency, however, was due to large heterogeneity in experimental design and participant selection among studies.

The platelet-specific nitric oxide donor, S -nitrosoglutathione, was administered to 10 small cohorts of women with early onset preeclampsia at 21–33 weeks of gestation. Significant, dose-dependent reductions in mean arterial pressure, uterine artery resistance indices, and proteinuria were observed. Interestingly, S -nitrosoglutathione improved indices of vascular tone and arterial stiffness before affecting maternal blood pressure. Platelet activation, as measured by P-selectin expression, also was found to be decreased by S -nitrosoglutathione treatment. Thus, activated platelets may be one of the circulating factors in preeclampsia that mediate maternal endothelial cell activation, and platelet-specific nitric oxide donors may prove beneficial in the management of severe preeclampsia. ^,

Neutrophils

Circulating leukocytes bind to selectins on the surface of activated endothelial cells and modify the function of the vascular intima. Neutrophil activation was studied in 20 patients with eclampsia and 10 patients with preeclampsia and compared to 10 normotensive controls. Plasma levels of neutrophil elastase, a protein secreted by activated neutrophils and macrophages during inflammation, were increased significantly in eclamptic and preeclamptic women. Elastase values in cases of eclampsia were highly correlated with mean blood pressure, serum endothelin-1 levels, and endothelial cytotoxicity, measured by fura-2 release from human umbilical vein endothelial cell (HUVEC) cultures. Lactoferrin also has been used as an indicator of neutrophil activation in normal and preeclamptic pregnancies. A comparative study between 40 normal and 42 women with preeclampsia in the third trimester of pregnancy demonstrated that predelivery ratios of lactoferrin per neutrophil were higher in the preeclamptic than in the normal pregnant cohort.

These and other data suggest that neutrophil activation plays a role in preeclampsia. Using whole blood cytometric analysis, Studena et al. observed an increase in leukocyte surface antigen expression in normal pregnancy relative to nonpregnant women and a further increase in activation antigen expression in cells from preeclamptic pregnancies. The findings support the hypothesis of Redman and Sargent that vascular inflammation plays a primary role. This hypothesis is discussed in detail in Chapter 7 by Redman, Robertson, and Davidge. By contrast, eosinophils do not appear to have a compelling effect in preeclampsia.

Activated monocytes and neutrophils release the hemoprotein myeloperoxidase. Interestingly, myeloperoxidase induces low-density lipoprotein oxidation, activates metalloproteinases, and oxidatively consumes endothelium-derived nitric oxide, which are all reported to be involved in the vascular pathophysiology of preeclampsia (see sections below). Gandley et al. showed that myeloperoxidase levels are significantly increased in the circulation and placenta of women with preeclampsia, and they speculate that myeloperoxidase contributes to oxidative stress in the endothelium and placenta of women with preeclampsia.

Activation of neutrophils leads to the formation of neutrophil extracellular traps (NETs), which are DNA-containing structures that trap and kill circulating pathogenic microorganisms, contributing to the development of a proper innate immune response. However, excessive production of NETs is associated with chronic inflammatory conditions such as cardiovascular disease. Expression of NETs is increased in placentas from patients with preeclampsia. In addition, production of NETs is greater in the maternal circulation of women with preeclampsia compared to healthy pregnant women. Incubation of isolated neutrophils from normal individuals with plasma from women with preeclampsia potentiated the formation of NETs through the action of cellular components, such as microparticles, rather than through plasma-soluble proteins. Thus, it is possible that NETs are one of the mechanisms by which neutrophils are associated with the pathophysiology of preeclampsia.

Placental Membrane Microvesicles

Extracellular vesicles are membrane particles that are constituently released by all tissues, including the placenta. Placental extracellular vesicles are primarily derived from the syncytiotrophoblast, and therefore, they are referred as syncytiotrophoblast extracellular vesicles (STBEV). STBEVs have been shown to play a role in normal ^, and complicated pregnancies, including preeclampsia. A detailed discussion about the origins, cargo, and functional role of STBEVs in preeclampsia is provided in Chapter 8 by Redman, Vatish, and Davidge.

STBEVs have been studied for their vasoactive properties and specifically for their effects on the maternal vascular endothelium. Early studies showed that STBEVs inhibited proliferation and induced extensive disruption of the honeycomb structure of the endothelium. ^, Others have shown that in addition to reduced endothelial cell growth, STBEVs also increased apoptosis of endothelial cells in vitro and reduced vascular reactivity to acetylcholine, a measure of endothelial function, in subcutaneous adipose tissue arteries from pregnant women. In addition, STBEVs from women with preeclampsia reduced nitric-oxide-mediated vasodilation in isolated uterine arteries from pregnant rats.

It remains unclear exactly how STBEVs affect endothelial function. It is likely that the cargo of STBEVs determines their vasoactive effects on the vascular endothelium. A recent study reported greater expression and activity of neprilysin in STBEV isolated from plasma of women with preeclampsia compared to normal pregnant women. Neprilysin is a membrane-bound metalloprotease that cleaves and inactivates endogenous vasoactive peptides and has been implicated in cardiovascular disease. Through these properties, it is possible that neprilysin also mediates, at least in part, the adverse effects of STBEV on the maternal vascular endothelium. Others have shown that STBEVs may change the transcriptome of the target cells, resulting in functional changes. Cronqvist et al. demonstrated that STBEVs can be internalized by human coronary endothelial cells and transfer placenta-specific miRNAs into the endoplasmic reticulum and the mitochondria of the target cells, resulting in downregulation of preeclampsia-associated genes, such as fms-related tyrosine kinase 1 (FLT-1) gene. Thus, it is likely that extracellular vesicles derived from a dysfunctional preeclamptic placenta contribute to generalized maternal endothelial dysfunction by transferring materials with antiangiogenic and provasoconstrictor properties.

Endothelial Progenitor Cells (EPCs)

Recent research highlights the potential role of endothelial progenitor cells (EPCs) in the pathology of preeclampsia. EPCs encompass two distinct types of cells, circulating angiogenic cells and endothelial colony forming cells, both of which are involved in de novo vessel formation and repair. Endothelial colony forming cells are highly proliferative and differentiate into mature endothelial cells at the site of vessel formation, while circulating angiogenic cells are hematopoietic and promote migration and proliferation of endothelial colony forming cells via the release of paracrine factors (reviewed in Sipos ). A decline in circulating EPCs is associated with endothelial dysfunction and cardiovascular disease. Compared to normal pregnancies, in which the concentration of circulating EPCs increases with gestational age, ^, women with preeclampsia have significantly reduced numbers of EPCs. It has been suggested that limited bioavailability of nitric oxide, which is required for mobilization of EPCs, and an increase in antiangiogenic factors in preeclampsia, may contribute to endothelial dysfunction. Interestingly, diminished levels of EPCs persist in the circulation of preeclamptic mothers postpartum and are suggested to be associated with long-term cardiovascular risk. EPCs from pregnancies complicated with early-onset preeclampsia (symptoms before 34 weeks of gestation) showed increased cellular senescence. This was mediated by increased expression of high-temperature requirement factor A4, a placenta-specific serine protease with increased concentrations in the circulation of women with preeclampsia. Thus, it is possible that the preeclamptic microenvironment (i.e., reduced nitric oxide bioavailability, increased circulating high-temperature requirement factor A4) promotes EPC senescence, contributing to endothelial dysfunction, impairing endothelial repair, and increasing risk of long-term cardiovascular disease.

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