Trophoblast- and Vascular Smooth Muscle Cell-Derived MMP-12 Mediates Elastolysis during Uterine Spiral Artery Remodeling

During the first trimester of pregnancy, the uterine spiral arteries are remodeled, creating heavily dilated conduits that lack maternal vasomotor control but allow the placenta to meet an increasing requirement for nutrients and oxygen. To effect permanent vasodilatation, the internal elastic lamina and medial elastin fibers must be degraded. In this study, we sought to identify the elastolytic proteases involved in this process. Primary first-trimester cytotrophoblasts (CTBs) derived from the placenta exhibited intracellular and membrane-associated elastase activity; membrane-associated activity was primarily attributable to matrix metalloproteinases (MMP). Indeed, Affymetrix microarray analysis and immunocytochemistry implicated MMP-12 (macrophage metalloelastase) as a key mediator of elastolysis. Cultured human aortic smooth muscle cells (HASMCs) exhibited constitutive membrane-associated elastase activity and inducible intracellular elastase activity; these cells also expressed MMP-12 protein. Moreover, a specific inhibitor of MMP-12 significantly reduced CTB- and HASMC-mediated elastolysis in vitro, to 31.7 ± 10.9% and 23.3 ± 8.7% of control levels, respectively. MMP-12 is expressed by both interstitial and endovascular trophoblasts in the first-trimester placental bed and by vascular SMCs (VSMCs) in remodeling spiral arteries. Perfusion of isolated spiral artery segments with CTB-conditioned medium stimulated MMP-12 expression in medial VSMCs. Our data support a model in which trophoblasts and VSMCs use MMP-12 cooperatively to degrade elastin during vascular remodeling in pregnancy, with the localized release of elastin peptides and CTB-derived factors amplifying elastin catabolism.Transformation of the uterine spiral arteries during the first 20 weeks of gestation ensures that a constant supply of blood is delivered to the developing placenta, at an optimal rate of flow.^1–3 This allows the placenta to meet an increasing requirement for nutrients and oxygen and enables the developing fetus to attain its growth potential. The remodeling process leads to vessel dilatation, loss of spirality, and decreased vasoactivity, allowing a nonpulsatile low-pressure supply of blood to be delivered to placental villi at the maternofetal interface. Early alterations in arterial structure include endothelial vacuolation, hypertrophy of vascular smooth muscle cells (VSMCs), and disruption of medial smooth muscle layers, which occur in the absence of fetal-derived trophoblast and correlate with perivascular accumulation of macrophages and uterine natural killer (uNK) cells.^4,5 After colonization of the uterine decidua and myometrium by extravillous cytotrophoblast (EVT), endothelial cells and VSMCs are lost from the arterial wall and replaced by trophoblast embedded in a fibrinoid matrix. Remodeling is regulated in a spatial and temporal manner, such that the successive steps of trophoblast adherence, intravasation, fibrinoid deposition, and mural incorporation are effected without any loss in vessel integrity. A complex and highly orchestrated combination of vascular cell apoptosis, dedifferentiation, and matrix breakdown is probably required to achieve this alteration in vessel wall structure.^5–9Two distinct populations of EVT originate from anchoring placental villi and contribute to vessel transformation.^10,11 Interstitial EVT invade the uterine wall, migrating through the decidua and myometrium to adopt a perivascular position. Endovascular EVT enter the lumen of the spiral arteries and migrate as far as the first third of the myometrium, colonizing the arterial wall from within. Impaired arterial remodeling is distinguished by shallow EVT invasion, decreased numbers of EVT, and the persistence of muscular, narrow-bore arteries, and is associated with second trimester miscarriage,¹² preterm labor,¹³ pre-eclampsia,¹⁴ and fetal growth restriction.¹⁵To effect a permanent increase in vessel diameter it is crucial that elastin fibers within each artery are catabolized, eliminating their capacity for stretch and recoil. Myometrial segments of the spiral arteries possess an internal elastic lamina (IEL), and the musculo-elastic media of both decidual and myometrial arteries is rich in elastic fibers.^16,17 During pregnancy, EVT traverse the IEL during mural incorporation,¹⁸ thus it is highly likely that they possess elastase activity: indeed, first-trimester EVT synthesize and secrete the elastolytic proteases matrix metalloproteinase-2 (MMP-2), MMP-7, MMP-9, cathepsin B, and cathepsin L.^19,20 Although both uNK cells and macrophages produce enzymes capable of elastolysis,⁵ uNK cells are not abundant in myometrium,²¹ and elastin breakdown is associated with the presence of endovascular EVT¹⁷ rather than macrophages.²² Previous studies have demonstrated that the availability of nitric oxide (NO) can influence protease expression and activity,^23–26 and we have shown NO to be an important regulator of trophoblast function.^27–29 As dysregulation of NO production has been implicated in the pathogenesis of pre-eclampsia and intrauterine growth restriction (IUGR),^30–32 NO availability may regulate the process of arterial remodeling by controlling trophoblast elastolysis.Rodent models of atherosclerosis have highlighted a role for VSMC-derived cathepsins as mediators of IEL breakdown during lesion formation,³³ demonstrating that the arterial wall may be a potential source of elastases. Similarly, caspase-2, −3, and −7 derived from apoptotic VSMCs have been implicated as mediators of elastin breakdown.³⁴ Thus, during the process of spiral artery transformation, resident VSMCs may also be stimulated to produce elastase(s) in response to pregnancy hormones, trophoblast invasion, or soluble factors released by cells within the placental bed. In this study we have investigated the origin and identity of the proteases involved in mediating elastin breakdown during spiral artery remodeling.