Page 234 - Haematologica - Vol. 105 n. 6 - June 2020

C. Han et al.
Taken together, these results demonstrate that enhancing EV clearance prevented pcEV-induced hypertension, vascu- lar leakage, and hypercoagulation.
Placenta-derived extracellular vesicles induced vasoconstriction
We used three complementary experiments to investi- gate how pcEV induce hypertension. First, pcEV increased the wall tension of the carotid artery (Figure 4A). The vasoconstriction was relaxed to the baseline level upon removal of pcEV and was induced again after reincubation with pcEV. Second, pcEV triggered the calcium influx of cultured smooth-muscle cells in a dose-dependent manner (Figure 4B). Third, a single-dose infusion of pcEV but not PBS induced a rapid and dose-dependent reduction of cerebral blood flow (Figure 4C-F). The reduction was tran- sient, and the blood flow quickly recovered to either the pre-infusion level (Figure 4C) or a persistently low level (Figure 4D). This pcEV-induced reduction in blood flow was also detected in mice infused with total EV purified from plasma of patients with preeclampsia (Figure 4G). Lactadherin (400 μg/kg) given together with pcEV did not prevent the reduction of blood flow (Figure 4H) but signif- icantly shortened its duration (Figure 4I). These data demonstrate that pcEV directly induced vasoconstriction by mobilizing intracellular calcium, resulting in the sys- temic reduction in microvascular blood flow.
Discussion
We have investigated the role of pcEV in the pathogen- esis of preeclampsia by examining plasma samples from preeclamptic women, studying mouse models, and con- ducting in vitro experiments. Consistent with previous reports,10,11 we detected pcEV in blood samples of women in the late stage of pregnancy, but pcEV levels were sig- nificantly higher in patients with preeclampsia (Figure 1A) and rapidly returned to baseline during the postpar- tum period (Figure 1B). These clinical findings led us to study the activity of pcEV in new mouse models. These new mouse models offered advantages over previously used models because they did not require surgery, genetic manipulation, or pharmacological interventions. We made several novel observations that collectively define a causal role of pcEV in the pathogenesis of preeclampsia and a means to prevent it.
First, pregnant mice developed hypertension and pro- teinuria only after intravenous infusion of pcEV from injured placenta (Figures 1H), suggesting that preeclamp- sia can be induced either by a high level of circulating pcEV or by pcEV derived from injured placenta. Our results support the latter possibility because pcEV from injured placenta caused a preeclampsia-like condition in non-pregnant mice at a significantly lower number of 1×107 vesicles/mouse (Figure 2A, B) than the 3.8±0.9x107 vesicles/mouse found in pregnant mice (Figure 1F). These pcEV from injured placenta were similar in size and syn- cytin expression to those found in pregnant mice, but expressed a significantly highly level of anionic phospho- lipids recognized by annexin V. These anionic phospho- lipids were strongly procoagulant and could also disrupt the integrity of the endothelium. In addition, syncytiotro- phoblast EV from women with preeclampsia carry less endothelial nitric oxide synthase compared to those from
women with normal pregnancies25 and thus have a reduced synthesis of the vasodilating factor nitric oxide. The fast development of hypertension in non-pregnant mice infused with pcEV (30 min after infusion) (Figures 2 and 3) is likely caused by the quick infusion of a large quantity of pcEV (< 5 min). In contrast, pcEV are probably released gradually during pregnancy and induce hyper- tension when they reach a critical threshold level in the circulation over a longer period of time.
Second, pcEV disrupted the endothelial barrier in vitro, especially in the presence of platelets to induce vascular leakage (Figure 2D), which could be responsible for pcEV- induced proteinuria (Figures 1I and 2C). The synergistic activity between pcEV and platelets is consistent with a recent study showing that EV activate maternal platelets to promote inflammation and a preeclampsia-like patholo- gy,32 but how platelets enhance pcEV-induced endothelial injury remains to be investigated. Nevertheless, the pcEV- induced endothelial injury not only results in vascular leak- age, but could also contribute to the development of hyper- tension by impairing the endothelium-dependent vasodila- tion machinery (e.g., endothelial nitric oxide synthase – nitric oxide pathway)30,33 and allowing pcEV to act directly on smooth muscle to trigger calcium-dependent vasocon- striction (Figure 4). Several calcium signaling pathways are involved in regulating smooth-muscle contractility,34,35 but the molecule(s) on pcEV that triggers one or all of these pathways remains to be identified. This vasoconstrictive activity is unlikely to be limited to pcEV because EV from injured endothelial cells also induce hypertension in preg- nant mice in a platelet-dependent manner.32 The pcEV- induced vasoconstriction reduced blood flow (Figure 4C-F), potentially resulting in tissue ischemia that further propa- gates injury to the placenta and the endothelium.
Third, pcEV induced a systemic hypercoagulable state defined by shortened clotting time, platelet activation, and the expression of procoagulant PS (Online Supplementary Figure S4). This hypercoagulable state has been widely reported in patients with preeclampsia5,36,37 and causes extensive fibrin deposition in glomerular ves- sels (Figure 2J-L). The fibrin deposition could potentially induce vascular leakage and increase rigidity of the vessel wall, contributing to pcEV-induced renal dysfunction (i.e., proteinuria) and hypertension, respectively.38 Surprisingly, fibrin deposition may be organ-specific because it was extensively detected in the kidney, but minimally in the liver vasculature (Online Supplementary Figure S6). Instead, focal tissue necrosis and infiltration of inflammatory cells were detected in the liver of pcEV-infused mice, but the cause of this tissue injury remains to be identified. Nevertheless, our findings are consistent with those of a recent cohort study, which showed that plasma samples from preeclamptic patients had elevated levels of EV from activated platelets and leukocytes as well as tissue factor- bearing EV, as compared to women with normal pregnan- cies.39 However, another study found no association between preeclampsia and plasma levels of anionic phos- pholipid-expressing EV.40
Finally, we have recently shown that lactadherin given intravenously promotes the phagocytosis of EV by cou- pling the vesicles to macrophages in the liver through their PS- and integrin-binding domains.28 Here, we further show that lactadherin given intravenously also promotes the phagocytosis of pcEV (Figure 3A, B) and prevents pcEV-induced hypertension, proteinuria, and coagulation
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