Postpartum cardiomyopathy (PPCM) is a disease of unknown etiology and exposes women to high risk of mortality after delivery. Here, we show that female mice with a cardiomyocyte-specific deletion of stat3 develop PPCM. In these mice, cardiac cathepsin D (CD) expression and activity is enhanced and associated with the generation of a cleaved antiangiogenic and proapoptotic 16 kDa form of the nursing hormone prolactin. Treatment with bromocriptine, an inhibitor of prolactin secretion, prevents the development of PPCM, whereas forced myocardial generation of 16 kDa prolactin impairs the cardiac capillary network and function, thereby recapitulating the cardiac phenotype of PPCM. Myocardial STAT3 protein levels are reduced and serum levels of activated CD and 16 kDa prolactin are elevated in PPCM patients. Thus, a biologically active derivative of the pregnancy hormone prolactin mediates PPCM, implying that inhibition of prolactin release may represent a novel therapeutic strategy for PPCM.

Peripartum cardiomyopathy (PPCM) is a life-threatening pregnancy-associated cardiomyopathy in previously healthy women. Although PPCM is driven in part by the 16-kDa N-terminal prolactin fragment (16K PRL), the underlying molecular mechanisms are poorly understood. We found that 16K PRL induced microRNA-146a (miR146a) expression in ECs, which attenuated angiogenesis through downregulation of NRAS. 16K PRL stimulated the release of miR-146a-loaded exosomes from ECs. The exosomes were absorbed by cardiomyocytes, increasing miR146a levels, which resulted in a subsequent decrease in metabolic activity and decreased expression of Erbb4, Notch1, and Irak1. Mice with cardiomyocyte-restricted Stat3 knockout (CKO mice) exhibited a PPCM-like phenotype and displayed increased cardiac miR-146a expression with coincident downregulation of Erbb4, Nras, Notch1, and Irak1. Blocking miR-146a with locked nucleic acids or antago-miRs attenuated PPCM in CKO mice without interrupting full-length prolactin signaling, as indicated by normal nursing activities. Finally, miR-146a was elevated in the plasma and hearts of PPCM patients, but not in patients with dilated cardiomyopathy. These results demonstrate that miR-146a is a downstream-mediator of 16K PRL that could potentially serve as a biomarker and therapeutic target for PPCM.

Peripartum cardiomyopathy (PPCM) is a life-threatening heart disease developing towards the end of pregnancy or in the months following delivery in previously healthy women in terms of cardiac disease. Enhanced oxidative stress and the subsequent cleavage of the nursing hormone Prolactin into an anti-angiogenic 16 kDa subfragment emerged as a potential causal factor of the disease. We established a prospective registry with confirmed PPCM present in 115 patients (mean baseline left ventricular ejection fraction, LVEF: 27 ± 9 %). Follow-up data (6 ± 3 months) showed LVEF improvement in 85 % and full recovery in 47 % while 15 % failed to recover with death in 2 % of patients. A positive family history of cardiomyopathy was present in 16.5 %. Pregnancy-associated hypertension was associated with a better outcome while a baseline LVEF ≤ 25 % was associated with a worse outcome. A high recovery rate (96 %) was observed in patients obtaining combination therapy with beta-blocker, angiotensin-converting enzyme (ACE) inhibitors/angiotensin-receptor-blockers (ARBs) and bromocriptine. Increased serum levels of Cathepsin D, the enzyme that generates 16 kDa Prolactin, miR-146a, a direct target of 16 kDa Prolactin, N-terminal-pro-brain-natriuretic peptide (NT-proBNP) and asymmetric dimethylarginine (ADMA) emerged as biomarkers for PPCM. In conclusion, low baseline LVEF is a predictor for poor outcome while pregnancy-induced hypertensive disorders are associated with a better outcome in this European PPCM cohort. The high recovery rate in this collective is associated with a treatment concept using beta-blockers, ACE inhibitors/ARBs and bromocriptine. Increased levels of Cathepsin D activity, miR-146a and ADMA in serum of PPCM patients support the pathophysiological role of 16 kDa Prolactin for PPCM and may be used as a specific diagnostic marker profile.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-013-0366-9) contains supplementary material, which is available to authorized users.

Background-Peripartum cardiomyopathy (PPCM) is a potentially life-threatening heart disease that occurs in previously healthy women. We identified prolactin, mainly its 16-kDa angiostatic and proapoptotic form, as a key factor in PPCM pathophysiology. Previous reports suggest that bromocriptine may have beneficial effects in women with acute onset of PPCM. Methods and Results-A prospective, single-center, randomized, open-label, proof-of-concept pilot study of women with newly diagnosed PPCM receiving standard care (PPCM-Std; nϭ10) versus standard care plus bromocriptine for 8 weeks (PPCM-Br, nϭ10) was conducted. Because mothers receiving bromocriptine could not breast-feed, the 6-month outcome of their children (nϭ21) was studied as a secondary end point. Blinded clinical, hemodynamic, and echocardiographic assessments were performed at baseline and 6 months after diagnosis. Cardiac magnetic resonance imaging was performed 4 to 6 weeks after diagnosis in PPCM-Br patients. There were no significant differences in baseline characteristics, including serum 16-kDa prolactin levels and cathepsin D activity, between the 2 study groups. PPCM-Br patients displayed greater recovery of left ventricular ejection fraction (27% to 58%; Pϭ0.012) compared with PPCM-Std patients (27% to 36%) at 6 months. One patient in the PPCM-Br group died compared with 4 patients in the PPCM-Std group. Significantly fewer PPCM-Br patients (nϭ1, 10%) experienced the composite end point of poor outcome defined as death, New York Heart Association functional class III/IV, or left ventricular ejection fraction Ͻ35% at 6 months compared with the PPCM-Std patients (nϭ8, 80%; Pϭ0.006). Cardiac magnetic resonance imaging revealed no intracavitary thrombi. Infants of mothers in both groups showed normal growth and survival. Conclusions-In this trial, the addition of bromocriptine to standard heart failure therapy appeared to improve left ventricular ejection fraction and a composite clinical outcome in women with acute severe PPCM, although the number of patients studied was small and the results cannot be considered definitive. Larger-scale multicenter and blinded studies are in progress to test this strategy more robustly. (Circulation. 2010;121:1465-1473.)Key Words: cardiomyopathy Ⅲ heart failure Ⅲ hormones Ⅲ parturition Ⅲ pregnancy P eripartum cardiomyopathy (PPCM) is characterized by new onset of heart failure between 1 month before and 5 months after delivery in previously healthy women. 1 The clinical presentation and management of PPCM and its outcome have been reviewed recently. 1,2 Only 23% to 54% of patients show recovery of cardiac function within 6 months. [3][4][5][6] Investigation of a large cohort of PPCM patients demonstrated that this condition is associated with a proinflammatory response, as evidenced by elevated plasma levels of tumor necrosis factor-␣, Fas-Apo-1, interleukin-6, and C-reactive protein (CRP). 5,7,8 Editorial see p 1463 Clinical Perspective on p 1473We recently reported that enhanced oxidative stress in a mou...

The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model relies on laser injury to perforate Bruch's membrane, resulting in sub-retinal blood vessel recruitment from the choroid. By recapitulating the main features of the exudative form of human AMD, this assay has served as the backbone for testing antiangiogenic therapies. This standardized protocol can be applied to transgenic mice and can include treatments with drugs, recombinant proteins, antibodies, adenovirus and pre-miR to aid in the search for new molecular regulators and the identification of novel targets for innovative treatments. This robust assay requires 7-14 days to complete, depending on the treatment applied and whether immunostaining is performed. This protocol includes details of 2 how to induce CNV, including laser induction, lesion excision, processing plus different approaches to quantify neoformed vasculature.

Angiogenesis, the process of development of a new microvasculature, is regulated by a balance of positive and negative factors. We show both in vivo and in vitro that the members of the human prolactin͞growth hormone family, i.e., human prolactin, human growth hormone, human placental lactogen, and human growth hormone variant are angiogenic whereas their respective 16-kDa N-terminal fragments are antiangiogenic. The opposite actions are regulated in part via activation or inhibition of mitogen-activated protein kinase signaling pathway. In addition, the N-terminal fragments stimulate expression of type 1 plasminogen activator inhibitor whereas the intact molecules have no effect, an observation consistent with the fragments acting via separate receptors. The concept that a single molecule encodes both angiogenic and antiangiogenic peptides represents an efficient model for regulating the balance of positive and negative factors controlling angiogenesis. This hypothesis has potential physiological importance for the control of the vascular connection between the fetal and maternal circulations in the placenta, where human prolactin, human placental lactogen, and human growth hormone variant are expressed.Prolactin (PRL), growth hormone (GH), and placental lactogen (PL) are homologous protein hormones believed to have arisen from a common ancestral gene (1). PRL participates in the regulation of reproduction, osmoregulation, and immunomodulation (2, 3) whereas GH is involved in regulating growth and morphogenesis (4). Human (h) GHs, unlike other mammalian GHs, bind to the PRL receptor and thus display PRL-like activity; however, hPRL does not bind to the hGH receptor (5). PRL and GH are produced mainly by the anterior pituitary in all vertebrates. PRL is expressed also in lymphocytes and in the decidua (6). The human placenta expresses two structural homologs of hGH, hPL and a variant of hGH (hGH-V) (7). hGH-V rather than pituitary hGH is believed to regulate maternal metabolism during the second half of pregnancy. hPL is somatotropic in fetal tissues and contributes to stimulating mammary cell proliferation (8). Rodent placentas express and secrete several proteins whose biological activities are more PRL-like than GH-like; these include proliferin (PLF) and a proliferin-related peptide (PRP) (9).Members of the PRL͞GH family and derived peptides have been reported to both stimulate and inhibit angiogenesis. PLF expressed during the first half of pregnancy in the mouse is angiogenic whereas PRP expressed later in gestation is antiangiogenic. These findings suggest that PLF and PRP might play a role in initiating and stopping placental neovascularization (9). Human GH was reported to be angiogenic in vitro (10) whereas both bovine and chicken GH were shown to be angiogenic in vivo (11). We have shown that the 16-kDa N-terminal fragments (16K) of rat PRL and hPRL are antiangiogenic both in vitro (12) and in vivo (13). Rat PRL is cleaved by cathepsin D (14) to yield a 16-kDa N-terminal fragment and a 7-kDa C...

BackgroundMicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the control of angiogenesis renders them very attractive in the development of new approaches for restoring the angiogenic balance. Whereas miRNA-21 has been demonstrated to be highly expressed in endothelial cells, the potential function of this miRNA in angiogenesis has never been investigated.Methodology/Principal FindingsWe first observed in endothelial cells a negative regulation of miR-21 expression by serum and bFGF, two pro-angiogenic factors. Then using in vitro angiogenic assays, we observed that miR-21 acts as a negative modulator of angiogenesis. miR-21 overexpression reduced endothelial cell proliferation, migration and the ability of these cells to form tubes whereas miR-21 inhibition using a LNA-anti-miR led to opposite effects. Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration. Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells. RhoB silencing impairs endothelial cell migration and tubulogenesis, thus providing a possible mechanism for miR-21 to inhibit angiogenesis. Finally, the therapeutic potential of miR-21 as an angiogenesis inhibitor was demonstrated in vivo in a mouse model of choroidal neovascularization.Conclusions/SignificanceOur results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB.

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