In the present study, we report several novel findings regarding the role of MSC in preeclampsia. First, our results demonstrate that viability and function of MSC harvested from adipose tissue at the time of delivery are impaired in preeclamptic compared to normotensive pregnancies. In contrast, the migration capacity of MSC was increased, possibly secondary to MCP-1 upregulation—a potent trophic factor for MSC—in neighboring fat. Second, we show that a pro-inflammatory adipose tissue milieu, as demonstrated by upregulation of TNF-alpha, is associated with upregulation of SASP components in PE-MSC compared to NP-MSC. The mechanistic link between inflammation and upregulation of SASP components was confirmed by experiments showing the upregulation of IL-6, IL-8, and MCP-1 in control MSC after exposure to TNF-alpha. Third, our results indicate decreased pro-angiogenic potential of PE-MSC and, fourth, provide evidence that this is due, at least in part, to their senescence, as treatment with dasatinib both decreases senescent MSC burden and results in improved MSC angiogenic potential. Taken together, our data suggest that the pro-inflammatory milieu of the abdominal tissue—where MSC reside—is associated with MSC senescence and both a decrease in MSC-mediated angiogenic effects and an increase in SASP components, the latter further contributing to the vicious cycle of inflammation → senescence → anti-angiogenesis. By implicating MSC senescence in the pro-inflammatory and anti-angiogenic mechanisms of preeclampsia, our study opens new avenues for preeclampsia treatment, such as autologous stem cell transplantation. If MSC function and viability appear suboptimal due to senescence, pre-intervention testing and pre-conditioning with senolytic agents may be considered. The process of senescence is critical for embryogenesis and, therefore senolytics are contraindicated during pregnancy. However, their therapeutic use in non-pregnant women could be considered for prevention of preeclampsia in patients with previous unsuccessful pregnancies due to preeclampsia and its complications. This approach would be facilitated by the mechanism of action of senolytics: these agents are effective when administered intermittently, and a single dose (or a short duration of treatment) would result in a decrease in senescent cell burden after the affected pregnancy and prior to planning the next one. Given their short elimination half-lives, the risk of adverse effects for future pregnancies would be minimal. In addition, senolytics could be considered during the post-reproductive years in women with a history of preeclampsia, who may experience persistent increase in senescent cell burden, potentially leading to increased risks for metabolic syndrome, an accelerated aging-like state, or multimorbidity. Of note, adverse effects of dasatinib are rare, appear only after prolonged administration of the drug, and are usually reversible after a dose interruption. We present here a proof of principal study showing a potential benefit of senescent cell clearance in preeclampsia. One alternative approach would be to use, in place of senolytics, senomorphic agents (those that that attenuate the SASP, such as metformin) [13], which are safe for use even in pregnancy. Finally, for these patients, the continued development of new senolytic drugs with less toxicity, which is underway, will be of particular importance.
Stem cells play fundamental roles in the repair and self-renewal of tissues throughout life. Tissue injury activates regenerative mechanisms that promote repair by recruiting local resident stem cells, bone marrow-derived hematopoietic progenitor/stem cells, or MSC. MSC are multipotent cells that have been identified in almost all tissues, including kidney and placenta [5]. MSC have a potent modulatory effect and may contribute to the state of Th2 polarization and immune tolerance in pregnancy, by either a direct inhibitory effect on the proliferation of Th1 cells or by shifting a Th1 to a Th2 phenotype. MSC also exhibit pro-angiogenic [2,3,4] and anti-inflammatory effects through downregulation of TNF-alpha and stimulation of IL-10 [5]. In pregnancy, MSC can traffic through the placenta, in a process triggered by fetal VEGF, and may be responsible for fetal microchimerism in normal pregnancy [30]. In addition, placental MSC reside in a peri-vascular niche in the developing placenta [31], with emerging evidence suggesting that they play an important role in placental development by contributing to vasculogenesis and angiogenesis. A study of the differential expression of microRNAs in decidua-derived MSC from severe preeclampsia and normal pregnancies indicated that angiogenesis, response to hypoxia, apoptosis, the TGF-beta receptor signaling pathway, cell migration, and immune response, were regulated by increased MSC microRNAs in patients with preeclampsia [32]. Taken together, MSC play an important role in the regulation of placentation in normal pregnancy. In turn, MSC dysregulation may contribute to the pathophysiology of preeclampsia. However, their function in preeclampsia with respect to their potency for attenuating inflammation and repairing vascular injuries has not yet been studied. Our study is the first to provide data supporting the notion that MSC are dysregulated in preeclampsia, and linking senescence of MSC to the anti-angiogenic state, one of the hallmarks of vascular injury in preeclampsia.
Data presented in this study need to be interpreted in the context of the current state of knowledge of preeclampsia pathophysiology and the widely accepted concept that maternal disease is caused by pro-inflammatory and anti-angiogenic mediators that are released by ischemic placenta. Previous studies have shown accelerated placental aging and increased placental senescence in preeclamptic placentas [7]. We postulate that, once established, increased placental senescent cell burden persists, with affected cells acquiring a SASP secretome consisting of circulating inflammatory cytokines and reactive oxygen species, ultimately leading to maternal vascular and tissue injury. Furthermore, we showed that senescence can spread from cell to cell [14], suggesting the possibility that senescent cells in the placenta could cause other cells to become senescent elsewhere in the mother, potentially establishing a reservoir of these cells. Therefore, the SASP secretome of placental origin may be one of the missing links between placental ischemia and maternal disease in preeclampsia. In future experiments, we will compare MSC in “placental” vs. “maternal” forms of preeclampsia, the former clinically characterized by early (≤ 34 weeks of gestation) and severe disease, placental changes of ischemia and infarction, and consequent intrauterine growth restriction; the latter described by preexisting maternal disease (hypertension, diabetes mellitus), late onset (> 34 weeks of gestation) with the absence of ischemic placental changes, and normal intrauterine fetal growth. We postulate that differential degrees of senescent cell burden may, indeed, contribute to the differences in clinical presentations between these disease subtypes.
Preeclampsia is commonly viewed as a vascular disease of pregnancy. Of note, reduction in capillary density in the skin has been reported in association with preeclampsia, both prior to the clinical onset of the disease [33] and at the time of diagnosis [34]. We postulated that MSC senescence and impaired angiogenesis may lead to sustained vascular injury and rarefaction. Indeed, the MSC from preeclamptic pregnancies showed increased staining for SABG, a marker of senescence. The mechanistic link between MSC dysfunction and senescence was studied using a senolytic agent as a means of rescuing functional phenotypes. Senolytic agents promote selective apoptosis in senescent, but not normal cells, by transiently disabling their pro-survival pathways [13, 35]. The resultant decrease in senescent cell number and SASP inhibition have been shown to delay or alleviate age- and disease-related adverse phenotypes, as well as to improve established vascular disease in aged and hypercholesterolemic mice [36, 37]. In the current study, the mechanistic link between MSC dysfunction and senescence was studied using dasatinib, a tyrosine kinase inhibitor that specifically targets senescent MSC. The following outcomes of dasatinib treatment of MSC support the role of MSC senescence in preeclampsia. First, senescent MSC burden decreased after treatment. Second, the number of apoptotic MSC in preeclampsia increased, as expected to occur with senolytic agents, which target survival pathways in senescent cells and cause their apoptosis. Third, the angiogenic potential of PE-MSC significantly improved after treatment. It is noteworthy that downregulation of SASP components occurred in both NP-MSC and PE-MSC. It has been widely accepted that even normal pregnancy is associated with systemic inflammation, which is further exaggerated in preeclamptic pregnancies [38]. Consequently, down-regulation of the SASP in both groups can be attributed to anti-inflammatory effects of dasatinib. However, the downregulation of the SASP was associated with downregulation of p16 in only PE-MSC, but not NP-MSC. Furthermore, only PE-MSC, but not NP-MSC, demonstrated improved angiogenic potential after treatment. Taken together with the results of SABG staining, these data indicate that improved PE-MSC angiogenic potential was achieved through a decrease in senescent cell burden.
A notable limitation of our study is its small sample size, which did not allow for the characterization of MSC viability and function across the spectrum of severity (mild vs. severe) and presentations (early vs. late) of preeclampsia; these will be addressed in ongoing studies in our laboratory. Also, only a single time point was studied. While the mechanistic link between inflammation and upregulation of SASP components was confirmed by experiments showing the upregulation of IL-6, IL-8, and MCP1 in control MSC after exposure to TNF-alpha, the observed differences between PE-MSC and NP-MSC could be secondary to hypertension and differences in gestational age. Although it would be reasonable to examine endometrial MSC in preeclampsia for purposes of studying placental physiology, we opted for abdominal fat MSC for several reasons. First, the characteristics of MSC residing in the different organs are similar [12], suggesting that, in a given subject, the functional status of diverse MSC is comparable. Second, if autologous stem cell transplant is to be considered as a potential therapy for preeclampsia, fat tissue is readily accessible and abundantly available. Characterization of adipose tissue-derived MSC is critical to advance toward this goal. Third, this approach would allow for longitudinal examination of MSC obtained at the time of delivery to those collected postpartum in future studies. Collection of endometrial MSC would be feasible only at the time of delivery, e.g., during C-section.
Despite these limitations, our study reveals novel insights involving MSC senescence in preeclampsia, which may open new venues for preeclampsia research and novel treatment strategies. While stem cell therapies to treat placental disorders may seem like a far-fetched concept [5], stem cells are explored for therapeutic use and have been found to be effective in a broad spectrum of disease entities [39]. This study implies, however, that for autologous administration, new therapeutic approaches may be needed to restore MSC viability and function. Additional research is also required to characterize MSC viability and function across the spectrum of severity and presentations of preeclampsia.
