Patent Description:
Soluble fms-like tyrosine kinase-<NUM> (sFlt-<NUM> or sVEGF-<NUM>) is a splice variant of VEGF receptor <NUM> (Flt-<NUM>) and acts as a receptor of vascular endothelial growth factor (VEGF) (Kendall et al. However, sFlt-<NUM> lacks the transmembrane and cytoplasmic domains of the receptor, and thereby blunts the VEGF signalling pathway by binding to the free circulating VEGF (Wu et al. The abnormal sFlt-<NUM> expression was identified in many pathological conditions, including different type of cancers, liver cirrhosis, diabetes and peripheral arterial disease (Bando et al. , <NUM>; Ebos et al. , <NUM>; Lamszus et al. , <NUM>; Jaroszewicz et al. , <NUM>; Blann et al. , <NUM>; Findley et al.

Among these diseases, the role of sFlt-<NUM> is best characterized in the pathogenesis of preeclampsia. Preeclampsia is a maternal medical condition clinically defined as hypertension, proteinuria and renal damage in pregnant women. Currently preeclampsia is the major cause of maternal and perinatal mortality and morbidity (Steegers et al. , <NUM>) and affects <NUM>% to <NUM>% of all pregnancies and women worldwide (Sibai et al. The pathogenesis of preeclampsia is complex, but it has been suggested that the endothelial dysfunction and resulted abnormal angiogenesis, a process which new blood vessels form from existed ones, in the placenta underlie the clinical manifestation of this medical condition (Hladunewich et al. , <NUM>; Silasi et al. , <NUM>; Maynard et al. The excessive expression of sFlt-<NUM> in the plasma and placenta of preeclamptic women was identified and the plasma level of sFlt-<NUM> could be used as a potential clinical marker of preeclampsia (Stepan et al. Moreover, the elevated sFlt-<NUM> was thought to be the main cause of preeclampsia (Maynard et al. , <NUM>; Powe et al. Our previous data demonstrated that Heme oxygenase <NUM> (Hmoxl), a protective enzyme, negatively regulates sFlt-<NUM> release, whereas the VEGF growth factors promotes sFlt-<NUM> expression (Cudmore et al. , <NUM>; Ahmad et al. Based on these findings, our and others started pilot trials to use statin, a potent Hmoxl inducer, to prevent the preeclampsia in high risk women (Costantine et al. However, the molecular mechanism of how Hmoxl or VEGF regulates sFlt-<NUM> is still unknown.

MicroRNA is a <NUM>-<NUM>-nt small non-coding RNA which binds to <NUM>'-UTR of the mRNA of their target genes with a partially complement manner, and thus leads to the translational repression of target genes (Bartel <NUM>; Lewis et al. , <NUM>; Williams <NUM>). Recently, microRNAs are shown to be important in the regulation of many developmental, physiological and pathological processes (Williams <NUM>; He and Hannon, <NUM>). Microarray profiling and further quantitative PCR analysis have revealed that microRNAs are differently expressed in the placenta of preeclamptic and normal pregnant women (Pineles et al. , <NUM>; Zhu et al. , <NUM>; Hu et al. , <NUM>; Mayor-Lynn et al. , <NUM>; Enquobahrie et al. , <NUM>; Noack et al. Furthermore, the interaction of microRNA and Hmox1 in stem cell differentiation, lung carcinoma and oxidant injury was reported (Kozakowska et al. , <NUM>; Skrzypek et al. , <NUM>; Hou et al. Thus, we hypothesized that Hmoxl may regulate sFlt-<NUM> release via microRNAs and the dysregulation of microRNAs in placenta contributes to the preeclamptic pathogenesis. Utilizing a qPCR based microarray, our present study identified two microRNAs, miR-<NUM> and miR-374b, which can be regulated by hemin, a potent Hmoxl inducer. Furthermore, these microRNAs were demonstrated to be regulated by statin, Hmoxl and VEGF which also regulate sFlt-<NUM> expression. Moreover, sFlt-<NUM> was proved to be the direct target of these microRNAs and the regulation of sFlt-<NUM> by Hmoxl was conducted by these microRNAs. Most importantly, these microRNAs are decreased in the preeclampsia patients and RuPP preeclamptic model. The negative correlation of these microRNAs and blood pressure in animal model was also discovered. Our study provides the novel molecular mechanism of sFlt-<NUM> regulation and these microRNAs may serve as the new therapeutic targets of sFlt-<NUM> related diseases.

MicroRNA-<NUM> is a member of broadly conserved miR-<NUM>/<NUM> family. It has been shown to regulate epigenesis by targeting DNMT1 (Xiang et al. , <NUM>; Ji et al. , <NUM>; Huang et al. , <NUM>; Braconi et al. , <NUM>), inhibits cancer cell proliferation and adhesion (Zhou et al. , <NUM>; Mancini et al. , <NUM>) and increases cell cytolysis (Zhu et al.

Further study confirmed its anti-tumor effect, and hence the down-regulation of miR-<NUM> expression by hypermethylation in various cancer types (Hiroki et al. , <NUM>; Tsuruta et al. , <NUM>; Braconi et al. , <NUM>; Chen et al. , <NUM>; Huang et al. , <NUM>; Wang et al. , <NUM>; Stumpel et al. , <NUM>; Kitano et al. , <NUM>; Zhou et al. Most importantly, it has been shown to be up-regulated in the placenta of preeclampsia patients (Zhu et al. Furthermore, miR-<NUM> reduces tumor cell angiogenesis (Zheng et al. , <NUM>; Xu et al. , <NUM>) and down-regulates expression of important pregnancy-related gene (HLA-G) (Manaster et al.

Since miR-<NUM> inhibits cancer cell proliferation and angiogenesis and also implicated in the preeclampsia by microarray profiling, we hypothesized that miR-<NUM> in the preeclampsia regulates endothelial function to contribute to the pathogenesis of preeclampsia. In the current study, we confirmed the up-regulation of miR-<NUM> in the placenta of different gestational placentas, more precisely defined preeclampsia patients as well as in the animal preeclampsia models, demonstrated the increase of miR-<NUM> expression under hypoxia and inflammatory condition. Further study revealed the decrease of PlGF expression, endothelial cell adhesion and angiogenic abilities upon overexpression of miR-<NUM>. Moreover, the intraperitoneal injection of virus expressing miR-<NUM> caused devascularization in the placenta and restricted fetal growth. Furthermore, we identified a novel target of miR-<NUM>, ITGA5, in both endothelial cells and mouse models and proved the negative correlation of miR-<NUM> and ITGA5 in the preeclampsia patient placental tissue. The identification of miR-<NUM> and its target, ITGA5, in the pathogenesis of preeclampsia will contribute to the understanding of this medical condition and may offer novel therapeutic targets.

MicroRNA-<NUM> is a member of the broadly conserved miR-<NUM>/<NUM> super family. It has been shown to be a tumor suppressor and suppresses cancer cell proliferation, migration, invasion and angiogenesis by targeting various downstream factors (Amer et al. , <NUM>; Zhao et al. , <NUM>; Jain et al. , <NUM>; Guo et al. , <NUM>; Wang et al. , <NUM> a and b; Yang et al. , <NUM>; Luo et al. , <NUM>; Fu et al. , <NUM>; Wang et al. Furthermore, miR-<NUM> regulates insulin signalling pathway and is implicated in the type <NUM> diabetes as well as diabetic associated renal injury and retinopathy (Chen et al. , <NUM>; Mortuza et al. , <NUM>; Yang et al. , <NUM>; Ortega et al. , <NUM>; Herrera et al. , <NUM>; Guo et al. Moreover, miR-<NUM> is also associated with acute myocardial infarction and cardiac hypertrophy by inhibiting cell cycle in cardiomyocytes (Long et al. , <NUM>; You et al. , <NUM>; van Rooij et al. , <NUM>; Busk and Cirera, <NUM>; Porrello et al. Interestingly, miR-<NUM> has been recently demonstrated to regulate aortic extracellular matrix and can be used as biomarker for deep vein thrombosis (Zampetaki et al. , <NUM>; Qin et al. Further studies revealed its role in the pregnancy and associated complications. The expression of miR-<NUM> was significantly up-regulated in third trimester human placentas compared to first trimester (Gu et al. More importantly, the expression of miR-<NUM> in the preeclamptic placenta is different compare to the normal pregnant placenta, implying the possible role of mir-<NUM> in the pathogenesis of preeclampsia (Xu et al. , <NUM>; Zhu et al. , <NUM>; Hu et al.

Endothelial nitric oxide synthase (eNOS or NOS3) is an enzyme that breaks down L-arginin to generate nitric oxide (NO) gas in endothelial cells and plays a key role in the vascular endothelium (Huang, <NUM>). eNOS produces low concentration of NO which offers protection to the endothelial function and integrity and loss of eNOS causes a variety of diseases including preeclampsia (Albrecht et al. , <NUM>; Förstermann and Münzel, <NUM>; Fatini et al. Moreover, eNOS/NO has been identified as a key mediator of neocascularization (Duda et al. , <NUM>) and VEGF fails to angiogenesis in eNOS-/- mouse (Lin and Sessa, <NUM>). However the regulatory mechanisms of eNOS expression are largely unknown. So far, only one microRNA, miR-<NUM>, was reported to directly target eNOS in endothelial cells (Sun et al.

Since miR-<NUM> inhibits cancer cell proliferation and angiogenesis and also implicated in the preeclampsia by microarray profiling, we hypothesized that miR-<NUM> negatively regulates endothelial function to contribute to the pathogenesis of preeclampsia. In the current study, we confirmed the up-regulation of miR-<NUM> in the placenta of different gestational placentas, obesity pregnant women, more precisely defined preeclampsia patients as well as in the animal preeclampsia models, demonstrated the increase of miR-<NUM> expression under inflammatory (or incombination with hypoxia) condition. Further study revealed the decrease of P1GF expression, endothelial cell proliferation, viability, adhesive and angiogenic abilities upon overexpression of miR-<NUM>. Furthermore, we identified eNOS as a novel target of miR-<NUM> and overexpression of eNOSS1177D rescued miR-<NUM> mediated suppression of angiogenesis in endothelial cells. Giving the importance of eNOS in the pathogenesis of preeclampsia, the identification of miR-<NUM> will contribute to the current understanding of this medical condition and may offer novel therapeutic targets.

The invention provides an miRNA modulator or a combination thereof for use in the treatment of preeclampsia and/or fetal growth restriction, wherein
the modulator is an miRNA inhibitor is a nucleic acid molecule substantially complementary to the miRNA to allow a duplex to form and is selected from an inhibitor of miR-<NUM> or miR-<NUM>, or a combination thereof.

miR-<NUM>, miR-374b, miR-<NUM> and miR-<NUM> and inhibitors of them are generally known in the art and indeed are commercially available from, for example, Qiagen Ltd, Manchester, United Kingdom.

Typically inhibitors are nucleic acid molecules substantially complementary to the miRNA to allow a duplex to form. For example, one or two bases may not be complementary, but still allow duplex formation to occur.

The term "functional fragments" is intended to mean fragments of miRNA (or inhibitors of miRNA) retaining the same biological activity as the native miRNA or its inhibitor. The native miRNA or its inhibitors may also have additional sequences of nucleic acids, such as <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> nucleic acids added to the <NUM>' or <NUM>' end of the miRNA sequence or its complementary sequence as appropriate. To increase the stability or achieve better efficiency, the native miRNA or its inhibitors may be modified chemically, for example pegylated. The activity is typically the ability to treat one or more symptoms of preeclampsia. The term "homologue" is intended to mean a miRNA having the same activity as the miRNA, such as the regulation of the same gene(s) as the miRNA. For example, it is known that animal miRNAs are able to recognise target mRNAs by using as little as <NUM>-<NUM> nucleotides (the seed region) at the <NUM>' end of the miRNA. Hence, typically such homologues are typically miRNA having at least <NUM> to <NUM>, or at least <NUM> to <NUM> nucleotides at the <NUM>' end which are identical to those of the native miRNA. The miRNA may be a miRNA mimic. Such mimics are chemically synthesized dsRNA which mimic endogenous miRNAs after transfection into cells.

miRNA may be inhibited by inhibitors of miRNA. For example, single stranded inhibitors of siRNA, such as inhibitors of miR-<NUM> or mirR-<NUM> may be used. Moreover, the inhibitors of miRNA, functional fragments or homologues thereof, may be cloned into an expressing plasmid or packed into adenovirus, AAV or Lentivirus and injected into the organism by intravenously (IV), intramuscularly (IM) or subcutaneously (SC) administration.

The invention will now be described by way of example only with reference to the following figures:.

Recombinant growth factors, vascular endothelial growth factor A (VEGF-A), VEGF-E were purchased from RELIATech (Brauschweig, Germany). Hemin and pravastatin were purchase from Sigma-Aldrich (USA). M199 medium was purchased from Invitrogen (Paisley, UK). Quantitative PCR primer against miR-<NUM> and mir-374b, qScript™ microRNA cDNA and Synthesis PerfeCTa® SYBR® Green SuperMix Kit were purchased from Quanta Biosciences.

Institutional Ethics Committee approved the placental tissue collection and written informed consent was obtained. All women were followed prospectively from enrolment until delivery. Human placental tissues were collected from women with pregnancy complicated by preeclampsia (N=<NUM>) and Intrauterine growth restriction (IUGR, N=<NUM>), and from normotensive pregnant women (N=<NUM>). The placental tissues collected were further used for quantitative PCR and western blot. Preeclampsia was defined as blood pressure > <NUM>/<NUM> Hg on at least two consecutive measurements and maternal proteinuria of at least <NUM>/<NUM> and IUGR was defined as a foetus with estimated weight below the 10th percentile for its gestational age and abdominal circumference below the <NUM>. 5th percentile.

Placenta samples of RUPP preeclamptic model were a gift from Dr. Fergus McCarthy (Cork University Maternity Hospital, Wilton, Cork, Ireland). The experimental procedure and the characterization of rat undergone RUPP surgery were described previously (McCarthy et al.

Human umbilical vein endothelial cells (HUVECs) were isolated and cultured in M199 medium as described previously (Bussolati et al, <NUM>). Experiments were performed on third or fourth passage HUVEC.

MicroRNA expression profiling in HUVEC cells stimulated with hemin was determined using a qPCR based array system. HUVEC cells were treated with <NUM> Hemin for <NUM> hours and the total RNA was isolated for Human miFinder RT<NUM> miRNA PCR Array (Qiagen, Cat. The PCR procedure and result analysis were performed according to manufacturer's instruction.

Chemically synthesized double-stranded microRNA mimic and single-stranded inhibitor against miR-<NUM> (mi-<NUM> and anti-<NUM>) and miR-374b (mi-374b and anti-374b) were purchased from Qiagen and the efficiency was tested by Qpcr (<FIG>).

HUVECs were trypsinized, and <NUM>×<NUM><NUM> cells were electroporated with ≈<NUM> ug mimic-<NUM> or mimic-374b, ≈<NUM> ug anti-<NUM> or anti-<NUM> or control molecules using electroporation (Amaxa GmbH, Cologne, Germany) as described previously (Cudmore et al.

The recombinant, replication-deficient adenovirus-encoding rat HO-<NUM> (AdHO-<NUM>) was used as described previously (Cudmore et al.

The siRNA against Hmoxl was described previously (Cudmore et al.

Sample preparation and real-time quantitative PCR was performed as described previously (Cudmore et al.

Enzyme-linked immunosorbent assay (ELISA) kits for human sFlt-<NUM> was obtained from R&D Systems and performed according to the manufacturer's specifications.

The plasmid containing <NUM>'UTR of sFlt-<NUM>, pmiR-Flt1, was ordered from GeneCopoeia (Cat No. HmiT054531-MT01, MD, USA). The firefly luciferase cDNA was fused with <NUM>'UTR of sFlt-<NUM> and the control renilla luciferase gene was driven by CMV promoter in the same plasmid. The mutant plasmids containing miR-<NUM> and miR-374b binding sites, pmiR-Flt1-M1 and pmiR-Flt1-M2, were generated using site directed mutagenesis technique. The binding site of miR-<NUM> was mutated from "TTTGTAGCATTGTCATCACTCCT" to "TTTGTCGACGGATAGAGAAT". The two binding sites of miR-374b were mutated from "GTCAAAATAGATTATTATAA" to "GTCAAGAGCAAGGCGCA" and from "TACAATATTTGTACTATTATAT" to "TACAATATTTAGACGCGCT".

For microRNA target assay, HEK293 cells were transfected with pmiR-ITGA5 or pmiR-ITGA5M together with mimic-con or mimic-<NUM>. After overnight incubation, the relative firefly luciferase activity was measured and normalized to the renilla activity according to the manufacture's protocol of Dual-Luciferase® Reporter Assay System (E1910, Promega).

The direct targets of miR-<NUM> and miR-374b were predicted using online programs, microRNA. org (http://www. org/microrna/home. do), PicTar (http://pictar. mdc-berlin. de/) and Target Scan (http://www. targetscan.

All data are expressed as mean + S. Statistical comparisons were performed using Student's t-Test or Mann-Whitney U test. Statistical significance was set at a value of p<<NUM>.

Our previous study showed that the release of sFlt-<NUM> in endothelial cells can be regulated by vascular endothelial growth factor (VEGF) and heme oxygenase <NUM> (Hmoxl) (Cudmore et al. , <NUM>; Ahmad et al. However the molecular mechanisms underlie the sFlt-<NUM> regulation is unclear. Thus we hypothesized that microRNAs may be regulated in the downstream of VEGF or Hmoxl and directly target sFlt-<NUM> mRNA translation. The pilot study confirmed our previous observation that VEGF-E significantly induced sFlt-<NUM> release from endothelial cells (<FIG>), while statin, the potent Hmoxl inducer, decreased sFlt-<NUM> level at both <NUM> and <NUM> (<FIG>). Furthermore another Hmoxl inducer, hemin, decreased sFlt-<NUM> release as expected at <NUM> in endothelial cells and this reduction cannot be recovered by stimulation of VEGF-E, implying VEGF-E and hemin are individual regulators of sFlt-<NUM> (<FIG>).

To identify the microRNAs that respond to hemin stimulation, a qPCR-based microarray was performed using RNA samples isolated from hemin treated HUVEC cells. Briefly, HUVEC cells were treated with <NUM> Hemin for <NUM> hours and the total RNA was isolated for Human miFinder RT2 miRNA PCR Array (Qiagen, Cat. The results were analyzed using the programme provided by manufacture and the differently expressed microRNAs upon Hemin treatment were determined. In summary, there are <NUM> up-regulated and <NUM> down-regulated microRNAs have been identified. To further select the microRNAs directly target sFlt-<NUM>, the up-regulated microRNAs were analyzed for the possibility of directly targeting sFlt-<NUM> mRNA using online bioinformatic tools, including microRNA. org, PicTar and Target Scan. Finally, <NUM> microRNAs, miR-<NUM>, miR-<NUM> and miR-374b, were predicted to be the ones directly target sFlt-<NUM> and used for further study.

Since miR-<NUM>, miR-<NUM> and miR-374b are predicted to be direct regulator of sFlt-<NUM> and VEGF or Hmoxl inducers regulate sFlt-<NUM> release, we tested whether these microRNAs can be regulated by VEGF or Hmoxl inducers. HUVECs were treated with hemin (<NUM>), VEGF-A (20ng/ml) or VEGF-E (20ng/ml) and the expression of these microRNAs was determined by qPCR. Treatment with hemin increased miR-<NUM> and miR-374b expression by <NUM>-fold and <NUM>-fold respectively (<FIG>), but had no significant effect on miR-<NUM> expression (data not shown). Moreover, both <NUM> and <NUM> pravastatin stimulation significantly induced miR-<NUM> and mir-374b expression by <NUM>-<NUM>-fold and <NUM>-fold respectively (<FIG>). Inversely, decreased miR-<NUM> expression was decreased by <NUM>% and <NUM>% in VEGF-A and VEGF-E stimulation (<FIG>, <FIG>). The expression of miR-374b was reduced by <NUM>% and <NUM>% respectively upon VEGF-A and VEGF-E treatment (<FIG>, <FIG>).

As Hmoxl inducers modulate miR-<NUM> and miR-374b expression, we investigated whether these microRNAs can be regulated directly by Hmoxl. HUVEC cells were infected with adenovirus overexpressing Hmoxl and the expression of miR-<NUM> and miR-374b in these cells were quantified by qPCR. As expected, overexpression of Hmoxl increased miR-<NUM> and miR-<NUM> expression by <NUM>-fold and <NUM>-fold respectively (<FIG>), but the miR-<NUM> expression was not changed. Conversely, HUVEC cells transfected with siRNA against Hmox1 showed decreased expression of miR-<NUM> and miR-374b at <NUM>% and <NUM>% respectively (<FIG>).

The previous bioinformatic analysis predicted that miR-<NUM> and miR-374b can directly bind to the <NUM>'-untranslated region (UTR) of sFlt-<NUM> mRNA (<FIG>). To further confirm that sFlt-<NUM> mRNA is directly targeted by these microRNAs, the luciferase assay using <NUM>'-UTR of sFlt-<NUM> and mimic of miR-<NUM> (mi-<NUM>) or miR-374b (mi-374b) was performed. The plasmids containing <NUM>'-UTR of sFlt-<NUM> either with intact sequence or mutated sequence which the miR-<NUM> and miR-374b binding sites have been modified were fused to the firefly luciferase and transfected into HEK293 cells. These cells were also co-transfected with mimic control, mi-<NUM> and mi-374b alone or in combination. The luciferase activity assay revealed that mi-<NUM> and mi-374b transfection significantly suppressed luciferase activity compared to mimic control transfected cells, while this suppression was not observed in the mutant plasmids containing modified microRNA binding sites (<FIG>). Interestingly, the combination of mi-<NUM> and mi-374b did not reduce the luciferase activity further than the individual transfection of mi-<NUM> or mi-374b (<FIG>). The targeting of sFlt-<NUM> by miR-<NUM> and mir-374b was confirmed additionally by ELISA measuring sFlt-<NUM> in the culture medium of HUVECs transfected with mi-<NUM> and mi-374b alone or in combination. The sFlt-<NUM> level in the medium of HUVECs transfected with mi-<NUM> or mi-374b was reduced around <NUM>% compared to mimic control in the condition of both vehicle and VEGF-E stimulation (<FIG>). Reversely, HUVECs transfected with antagomir of miR-<NUM> (anti-<NUM>) and miR-374b (anti-374b) released significantly more sFlt-<NUM> into the medium especially in the condition of statin stimulation (<FIG>). More importantly, these microRNAs act in the downstream of Hmoxl to target sFlt-<NUM> expression. HUVECs were transfected with siHO-<NUM> in combination with mi-<NUM> or mi-374b and the sFlt-<NUM> release was measured by ELISA. As expected, siHO-<NUM> transfection significantly increased sFlt-<NUM> expression, while co-transfection with mi-<NUM> or mi-374b completely abolished this increase mediated by siHO-<NUM> (<FIG>).

Since the angiogenic imbalance is the main cause of preeclampsia and sFlt-<NUM> antagonists VEGF signalling pathway (Levine et al. , <NUM>; Venkatesha et al. , <NUM>; Ramma and Ahmed, <NUM>), we measured the expression of miR-<NUM> and miR-374b by qPCR in the placenta of preeclamptic patients and RuPP preeclampsia mouse model. The expression of miR-<NUM> and miR-374b in the preeclamptic placenta was decreased by around <NUM>% and <NUM>% respectively compared to age matched controls (<FIG>). Furthermore, their expression in the placenta of RuPP mice was decreased by about <NUM>% and <NUM>% respectively compared to sham control (<FIG>). More importantly, the expression of miR-<NUM> and miR-374b was negatively correlated with the blood pressure in the RuPP mice (<FIG>, N=<NUM>, R=-<NUM> and R=-<NUM> respectively) and ad-sFlt-<NUM> virus injected mouse model (<FIG>, N=<NUM>, R=-<NUM> and R=-<NUM> respectively). Moreover, this correlation has been further confirmed in the severe preeclampsia patient. The expression of miR-<NUM> and miR-374b in the placenta of severe preeclampsia patient was negatively correlated with their systolic and diastolic blood pressure (SBP and DBP) (<FIG>, N=<NUM>-<NUM>).

Recombinant growth factors, vascular endothelial growth factor A (VEGF-A), VEGF-E, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and interleukin <NUM> beta (IL-1β) were purchased from RELIATech (Brauschweig, Germany). Rabbit antibody against ITGA5, Caspase <NUM>, Flt-<NUM>, CAV2 and NRP1 were obtained from Cell Signaling Technology (Danvers, MA <NUM>, United States). Mouse anti-β-Actin antibody and rabbit anti-GAPDH were obtained from Sigma-Aldrich (A5441, St. Louis, MO, United States) and Santa Cruze Biotech respectively. Quantitative PCR primer against miR-<NUM>, qScript™ microRNA cDNA and Synthesis PerfeCTa® SYBR® Green SuperMix Kit were purchased from Quanta Biosciences. Growth factor reduced Matrigel purchased from Becton Dickinson (Oxford, UK). M199 medium was purchased from Invitrogen (Paisley, UK). Calcein AM Fluorescent Dye was purchased from BD Bioscience (P. Box <NUM> Sparks, MD, USA <NUM>). DyLight <NUM> labelled Lycopersicon Esculentum (Tomato) Lectin (LEL, TL) was purchased from Vector Laboratories (Burlingame, CA <NUM>). Alexa Fluor® <NUM> Conjugated Isolectin GS-IB4 was purchased from Life Technologies (Paisley, UK). OCT compound was purchased from VWR International Ltd. (Leicestershire, LE17 4XN, England).

Human umbilical vein endothelial cells (HUVECs) were isolated and cultured in M199 medium as described previously (Bussolati et al, <NUM>). Experiments were performed on third or fourth passage HUVEC. First trimester placental tissues (<NUM>-<NUM> weeks gestational age) were retrieved from normal pregnancies that had undergone elective termination. Placental villus tissue explants were prepared as described previously (Ahmad and Ahmed, <NUM>). Briefly, human placental villus explants were incubated under stimulation of test substances or hypoxia condition and collected for quantitative PCR of miR-<NUM>. To create hypoxic condition, <NUM>-<NUM>% confluent HUEVCs or placental explants were cultured in an incubator with <NUM>% O<NUM> and <NUM>% CO<NUM> at <NUM>.

Chemically synthesized double-stranded microRNA mimic and single-stranded inhibitor against miR-<NUM> (mimic-<NUM> and anti-<NUM>) were purchased from Qiagen.

HUVECs were trypsinized, and <NUM>× <NUM><NUM> cells were electroporated with ≈<NUM> ug mimic-<NUM>, ≈<NUM> ug anti-<NUM> or control molecules using electroporation (Amaxa GmbH, Cologne, Germany) as described previously (Cudmore et al.

Adenovirus, ad-sFlt-<NUM>, was a gift from Prof. Richard Mulligan (Harvard Medical School, Boston, USA). The ad-sFlt-<NUM> and ad-CMV control virus were amplified and titered and <NUM><NUM> PFU adenoviruses were injected into C57BL/<NUM> mice at E <NUM> via tail vein to over-express sFlt-<NUM>.

Adeno-associated virus (serotype <NUM>) over-expressing miR-<NUM> (AAV-<NUM>) was generated and titered by Vector Biolabs (Philadelphia, USA). The GFP was fused with miR-<NUM> cDNA as reporter gene. In the hind limb muscle injection, <NUM> × <NUM><NUM> GC of AAV1-con or AAV1-<NUM> AAVs were injected locally into left side adductor muscle of C57BL/<NUM> mice and the same volume saline solution was injected into the right side as control. The virus infection was confirmed by co-expressed GFP reporter protein and over-expression of miR-<NUM> was determined in the muscle <NUM> weeks after injection by quantitative PCR (<FIG>).

Total protein from HUVECs or animal tissue was lysed in RIPA buffer and assayed as previously described (Ahmad and Ahmed, <NUM>).

Enzyme-linked immunosorbent assay (ELISA) kits for human sFlt-<NUM>, sEng and P1GF were obtained from R&D Systems and performed according to the manufacturer's specifications.

HUVECs were electroporated with mimic-<NUM> or control mimic-con. After <NUM>, <NUM>×<NUM><NUM> mimic-<NUM> or mimic-con transfected cells were plated in the <NUM>% gelatin coated <NUM>-well plate and treated with VEGF-A (20ng/ml) or VEGF-E (20ng/ml) for <NUM> mins. Thereafter, cells were washed three times with PBS, stained with Calcein AM Fluorescent Dye and proceeded to fluorescent microscopy. The number of the adhesive cells per field under the <NUM> × magnification was counted.

Formation of capillary-like structures of mimic-<NUM>, anti-<NUM> or mimic-<NUM> and ad-ITGA5 treated HUEVCs on growth factor reduced Matrigel was determined as previously described (Bussolati et al, <NUM>).

HUVEC cells were electroporated with anti-<NUM> or mimic-<NUM>. After overnight recovery, transfected cells were trypsinized and plated into <NUM>-well plate with <NUM>×<NUM><NUM> per well under the vehicle or VEGF-A (20ng/ml) treatment. After <NUM>, these cells were proceeded to MTT assay using cell growth determination kit (CDG1, Sigma-Aldrich). For cell proliferation assay, transfected cells were plated in <NUM>-well plate with 4X <NUM><NUM> per well and stimulated with VEGF-A (20ng/ml) or VGEF-E (20ng/ml). After <NUM> treatment, cells were trypsinized and the cell number per well was counted under microscope using a hemocytometer.

HUEVC cells were electroporated with anti-<NUM> or mimic-<NUM>. After overnight recovery, the scratch was made in the centre of the transfected confluent cells in a <NUM>- or <NUM>-well plate. The width of the scratches was measured immediately at <NUM> and <NUM>-<NUM> after the scratches were generated. The cell migration distance was calculated by subtracting the width of scratches at <NUM> and <NUM>-<NUM>.

Animal study protocols were approved by Aston University Ethical Review Committee and conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, <NUM>. Male C57Bl/<NUM> mice (<NUM>-<NUM> month old) were injected intraterially with ad-<NUM> or ad-CMV then subjected to HLI by surgical excising left femoral artery as previously described (Murdoch et al. The left femoral artery was isolated. A temporary proximal ligation of both vein and artery was placed on before canulation and injection of ad-<NUM> or ad-CMV into the left femoral artery. After <NUM> minutes two further ligations were added just to the artery at a proximal and distal location before excising approximately <NUM>. The temporary ligation to the vein was removed. Blood flow perfusion was measured by LASER Doppler (Moor Instruments UK) on plantar aspects of the feet of anesthetized mice ( ketamine (<NUM>/kg) xylazine (<NUM>/kg); i. ) as previously described (Murdoch et al. Blood flow recovery was calculated as a ratio of blood flow observed in left ischemic foot compared to right non-ischemic foot. Capillary density was quantified in non-ischemic and ischemic gastrocnemius muscle by histological assessment, by Isolectin B4 staining.

Three- to four-month-old C57BL/<NUM> mice were mated. The first day of pregnancy (E0. <NUM>) was defined by the presence of a vaginal plug the following morning. Pregnant mice were randomly assigned into two groups at day E11. <NUM> and injected intraperitoneally with ad-<NUM> or ad-CMV with a dose of <NUM> × <NUM><NUM> PFU per mouse. After <NUM> days, the pregnant mice were mice were anesthetized using a Ketamine/Xylazine cocktail and subsequently sacrificed for sampling. The live foetuses and placentas were counted and weighed. Their blood was taken and kidneys, livers, spleens and placentas were collected. The expression of miR-<NUM> was measured by qPCR in blood and placentas. The placental tissue was later stained with Isolectin B4 for analyzing of the placental vasculature.

The Homo sapiens cDNA containing untranslated region (UTR) of ITGA5 was purchased from Origene (Rockville, MD <NUM>, USA) and the UTR region was subcloned into pMirTarget to generate ITGA5-UTR-Luciferase (firefly) expression plasmid, pmiR-ITGA5. The successful transfection of pmiR-ITGA5 in endothelial cells was validated by the expression of red fluorescent protein (RFP) (<FIG>). The plasmid harbouring mutant on miR-<NUM> and ITGA5-UTR binding site was generated using site-directed mutagenesis kit (Stratagene) with PCR primers containing the mutant site and the resulted plasmid was designated as pmiR-ITGA5M. The primers used for PCR amplification were mITGA5-F, TCCCTCCCCCCCATGCTGTGG, and mITGA5-R, TGTAAACAAGGGTCCACAGCA. For microRNA target assay, HEK293 cells were transfected with pmiR-ITGA5 or pmiR-ITGA5M together with mimic-con or mimic-<NUM>. The plasmid containing renilla luciferase gene was also transfected as the internal control. After overnight incubation, the relative firefly luciferase activity was measured and normalized to the renilla activity according to the manufacture's protocol of Dual-Luciferase® Reporter Assay System (E1910, Promega).

The direct targets of miR-<NUM> were predicted using online programs, microRNA. org (http://www. org/microma/home. do), PicTar (http://pictar. mdc-berlin. de/) and Target Scan (http://www. targetscan.

HUVEC cells were transfected with mimic-<NUM> and the total RNA sample was collected for whole genome gene array analysis using Affymetrix genechip Human Gene <NUM> ST. The up-regulated and down-regulated genes upon mimic-<NUM> transfection are selected using a ±<NUM>-fold cut off.

Although Zhu and colleagues demonstrated the elevated expression of miR-<NUM> in preeclampsia (Zhu et al. , <NUM>), the placental samples used in their study were collected from preeclampsia women with an average gestational age of <NUM> weeks. To investigate the miR-<NUM> expression in more precisely defined severe preeclampsia patient, the placental samples from preeclampsia women with an average gestational age of <NUM> weeks were collected and examined for miR-<NUM> expression by quantitative PCR. Quantitative PCR revealed that miR-<NUM> in the preeclamptic placenta (N=<NUM>) was significantly increased compared to gestation age matched control normotensive placenta (N=<NUM>), but not changed in IUGR placenta (N=<NUM>), implying miR-<NUM> is up-regulated specifically in preeclampsia (<FIG>.

To further confirm the up-regulation of miR-<NUM> in preeclampsia, the expression of miR-<NUM> was examined in two animal models of preeclampsia. The pregnant mice receiving ad-sFlt-<NUM> adenovirus injection exhibited significant increased placental miR-<NUM> level (<FIG> as well as elevated sFlt-<NUM> expression and mean artery pressure compared to ad-CMV control virus injected mice (<FIG>). Moreover, the miR-<NUM> expression in placenta of pregnant Sprague Dawley rats that undergone reduced uterine perfusion pressure surgery (N=<NUM>) was also significantly increased compared to normal pregnant rat (N=<NUM>, <FIG>, suggesting that miR-<NUM> elevation is associated with pathogenesis of preeclampsia. Severe preeclampsia patients are normally associated with fetal growth restriction (IUGR), thus we examined the miR-<NUM> level in IUGR patient placentas. The expression of miR-<NUM> in the IUGR patients (N=<NUM>) was increased by <NUM>% compared to the gestational age-matched controls (N=<NUM>, <FIG>).

Since obesity pregnant women pose a high risk to preeclampsia, we examined the miR-<NUM> expression in the adipose tissues of obesity pregnant women. As expected, miR-<NUM> expression in the visceral (N=<NUM>) and subcutaneous fat (N=<NUM>) of obesity pregnant women was increased significantly compared to lean pregnant women (N=<NUM>-<NUM>) (<FIG>). Moreover, we tested the miR-<NUM> level in liver and mesenteric fat of the diabetic db/db mouse. The results confirmed the significant elevation of miR-<NUM> expression in db/db mice compared to controls (N=<NUM>, <FIG>).

Preeclamptic placenta is associated with hypoxia, inflammation condition as well as the elevation of VEGF and FGF-<NUM> growth factors (Hunter et al. , <NUM>; Ozkan et al. , <NUM>), thus the miR-<NUM> expression in the placental explants and endothelial cells under hypoxia, inflammation and VEGF stimulation was determined by qPCR. Stimulation of mixed inflammatory cytokines in placental explants increased miR-<NUM> by approximate <NUM>% (<FIG>). Furthermore, hypoxia treatment increased miR-<NUM> expression by <NUM>% and <NUM>% in placental explants (<FIG>) and endothelial cells (Fig <NUM> D) respectively. The combination of hypoxia and inflammatory cytokine mixture stimulation increased miR-<NUM> expression by <NUM>% in placental explants (<FIG>). Moreover, VEGF and FGF-<NUM> stimulation elevated miR-<NUM> expression by approximately <NUM>% and <NUM>% respectively (Fig <NUM> E and F). These data confirmed that miR-<NUM> is regulated by the pathological conditions in preeclamptic placenta.

The release of anti-angiogenic factors, sFlt-<NUM> and sEng, and the inhibition of placental growth factor expression are the main cause of preeclampsia (Powe et al. , <NUM>; Ahmad and Ahmed, <NUM>). Moreover, VEGF and inflammatory cytokines stimulation increases sFlt-<NUM> and sEng release (Cudmore et al. , <NUM>), therefore we examined whether miR-<NUM> modulates sFlt-<NUM>, sEng or PlGF expression in endothelial cells. HUVECs were transfected with mimic-<NUM> to over-express miR-<NUM> (<FIG>) and the sFlt-<NUM>, sEng and P1GF levels were examined by ELISA. Overexpression of miR-<NUM> in HUVECs decreased P1GF expression under both vehicle and VEGF-E stimulation (Fig <NUM> I). Although the expression of sFlt-<NUM> (Fig <NUM>) and sEng (Fig <NUM>) was unaltered, the P1GF level was significantly reduced, thus the ratio of sFlt-<NUM>/PlGF was increased. These data further support the concept that miR-<NUM> upregulation may contribute to the pathogenesis of preeclampsia.

Since endothelium dysfunction is the main cause of preeclampsia, we tested whether miR-<NUM> modulates endothelial cell functions. HUVEC cells were transfected with either synthetic inhibitor (anti-<NUM>) (I) or the mimic of miR-<NUM> (mimic-<NUM>) (II) and the MTT (<FIG> or cell proliferation assay (<FIG> was performed. Although anti-<NUM> significantly increased cell viability in HUVECs, the mimic-<NUM> did not change the cell viability under vehicle or VEGF-A stimulation (<FIG>. Moreover, cell proliferation assay confirmed that either anti- or mimic-<NUM> had no effect on HUEVC cell proliferation (<FIG>. Furthermore, western blot analysis revealed that the expression of cleaved Caspase-<NUM> in HUVECs transfected with anti- or mimic-<NUM> was not changed (<FIG>.

The effects of miR-<NUM> in endothelial cell adhesion, migration and angiogenesis were further tested. HUVEC cell adhesive ability was dramatically decreased in the mimic-<NUM> transfected cells regardless the stimulation of VEGF-A and VEGF-E. Cell counting confirmed an <NUM>% reduction of the numbers of adhesive cells in the mimic-<NUM> transfected HUVECs under vehicle condition, while approximate <NUM>% reduction under VEGF-A and VEGF-E stimulation compared to mimic-con transfected HUVECs (<FIG>.

Since miR-<NUM> inhibits angiogenesis in tumor cells, we speculated that miR-<NUM> also inhibits angiogenesis in placental endothelial cells to contribute to the pathogenesis of preeclampsia. HUVEC cells transfected with mimic-<NUM> exhibited decreased tube formation on Matrigel under vehicle, VEGF-A or VEGF-E stimulation (<FIG>. Quantitative analysis revealed a <NUM>% reduction in the tube formation of mimic-<NUM> transfected cells under vehicle condition and <NUM>% to <NUM>% reduction under VEGF-A and VEGF-E stimulation. Conversely, anti-<NUM> transfection increased tube formation in the HUVEC cells by approximate <NUM>% under VEGF-A stimulation (<FIG>. These data confirmed that miR-<NUM> negatively modulates placental endothelial cell in vitro angiogenic ability.

Furthermore, the scratch assay revealed that overexpression of miR-<NUM> increased (<FIG> while inhibition of miR-<NUM> significantly decreased (<FIG> HUVEC cell migration ability under vehicle or growth factor stimulation.

To further test the anti-angiogenic property of miR-<NUM> in vivo, mouse hind limb ischemia model was utilized. Adenovirus overexpressing miR-<NUM> was injected into the femoral artery which was later been removed. The expression of miR-<NUM> in the Gastrocnemius muscle was increased approximate <NUM>-fold on day <NUM> and <NUM>% on day <NUM> compared to non-ischemic muscle (<FIG>. Hind limb ischemic blood flow recovery was compared in ad-<NUM> injected mice to ad-CMV injected controls. Blood flow was assessed serially after HLI surgery (days <NUM>, <NUM>, <NUM>, and <NUM>) by LASER Doppler in the plantar aspect of the paws (<FIG>. The blood flow recovery was evidenced in ad-CMV injected mice on day <NUM> and the ad-<NUM> injected mice exhibited consistent lower blood flow at day <NUM> and day <NUM> (<FIG>. At day <NUM>, the blood flow in ad-<NUM> injected mice showed a maximum reduction of approximate <NUM>% compared to controls (<FIG>. Since blood flow recovery relates to the increased tissue capillary density, we examined the capillary density in gastrocnemius muscle (<FIG>. As expected, capillary density was increased in the ischemic gastrocnemius muscle of both ad-<NUM> and ad-CMV injected mice; however in the ischemic ad-<NUM> injected muscle, there was a marked reduction of the capillary increase (<FIG>. Moreover, in agreement with the in vitro observation, the expression of miR-<NUM> in the ischemic muscle of control mice was significantly increased compared to control non-ischemic muscle (Fig <NUM> F).

To investigate the effect of miR-<NUM> overexpression in pregnancy, the adenovirus overexpressing miR-<NUM> was injected intraperitoneally into pregnant mice at E11. After six days of injection, the blood from ad-CMV and ad-<NUM> injected mice was taken and the expression of miR-<NUM> was measured by qPCR. The ad-<NUM> injection significantly increased miR-<NUM> expression compared to ad-CMV control (<FIG>. In line with these data, the fetal growth in the ad-<NUM> injected pregnant mice was significantly restricted (<FIG>. This observation was further confirmed by the measurement of average fetal weight in these mice. The foetuses from ad-<NUM> injected mice exhibited around <NUM>% reduction in body weight compared to the ad-CMV injected mice (<FIG>.

More importantly, the fetal growth restriction could be caused by the impaired neovasculature in the placenta as evidenced by the avasculated placenta in the ad-<NUM> injected mice (<FIG>. Histological analysis of placental labyrinth zones further confirmed this observation. The labyrinth zone consists of cells of trophoblast and fetal endothelial cells, forming a large surface area for nutrient and gas exchange between the mother and foetus. Using isolectin B4 to highlight the fetal endothelial cells, the anatomical features of labyrinth zone in ad-<NUM> and ad-CMV injected mice were analyzed. The vasculature in the labyrinth zone of control mice was well organized with high density of fetal vascular branching. However, the vascular structure in the ad-<NUM> injected labyrinth was observed as irregular branching with much lower density (Fig <NUM> E).

To identify the direct targets of miR-<NUM>, gene expression microarray was performed using RNA samples from mimic-<NUM> and mimic-con transfected HUVEC cells. The <NUM> up-regulated and <NUM> down-regulated genes upon miR-<NUM> over-expression were selected with ±<NUM>-fold cut off. These expression altered genes have broad functions on vascular remodelling, cell adhesion, angiogenesis, cell mobility and survival. Moreover, the down-regulated genes were further analyzed using online prediction tools, microRNA. org, PicTar and Target Scan, to confirm the possible direct targets of miR-<NUM> by sequence alignment. Finally, <NUM> out of <NUM> down-regulated genes were selected for candidates of miR-<NUM> direct target and the expression of some of these candidate targets were further tested by western blot in HUVEC cells (<FIG>.

Among the targets tested, ITGA5 was the one most regulated by miR-<NUM>. ITGA <NUM> was predicted to be miR-<NUM> direct target by the online prediction tools (<FIG> and also confirmed by western blot that miR-<NUM> overexpression decreased while inhibition of miR-<NUM> increased ITGA5 expression in HUVECs (<FIG>. MicroRNA target assay further proved that transfection of mimic-<NUM> significantly inhibited luciferase activity in the pmiR-ITGA5 transfected HEK293 cells, but not in the mutant pmiR-ITGA5M transfected cells (<FIG> C; <FIG>). To validate that miR-<NUM> target ITGA5 to fulfil its role on endothelial dysfunction, we tested whether overexpression of ITGA5 could rescue the mimic-<NUM> mediated inhibition of angiogenesis. As expected, overexpression of ITGA5 increased spontaneously tube formation and significantly recovered mimic-<NUM> inhibited tube-like structure in HUVEC cells (<FIG>.

More importantly, the regulation of ITGA5 by miR-<NUM> was also confirmed in animal models used in this study. AAV-<NUM> virus injection mediated overexpression of miR-<NUM> (<FIG>) in the muscle of mouse limb significantly decreased ITGA5 expression compared to control AAV virus injected limb muscle (<FIG>. Furthermore, in hind limb ischemia condition, the ITGA5 expression in the ischemic muscle of mice injected with ad-<NUM> was decreased compared to ad-CMV injected ischemic muscle (<FIG>. Moreover, the ITGA5 expression in the placenta of ad-<NUM> injected pregnant mice decreased dramatically compared to ad-CMV injected placenta (Fig <NUM> C).

Since miR-<NUM> is up-regulated in preeclampsia patients and ITGA5 is identified to be the direct target of miR-<NUM>, we speculate that the expression of miR-<NUM> and ITGA5 in the preeclampsia patients was negatively correlated. The expression of miR-<NUM> and ITGA5 was examined from <NUM> preeclampsia and <NUM> gestational age matched control patients using qPCR and western blot respectively (Fig <NUM> D). The negative correlation of miR-<NUM> and ITGA5 expression was found in the preeclamptic placentas (R=-<NUM>, Fig <NUM> E), but not in the control normotensive placentas, suggesting miR-<NUM> targets ITGA5 specifically in the pathological condition of preeclampsia.

Although microarray profiling and next generation sequencing techniques revealed that microRNAs are differently expressed in the placenta of preeclamptic women (Yang et al. , <NUM>; Wu et al. , <NUM>; Hromadnikova et al. , <NUM>; Pan et al. , <NUM>), only very limited number of microRNAs have been characterized for their roles in preeclampsia pathogenesis, especially in the dysfunction of endothelium. Mir-<NUM> expression has been shown to be up-regulated in the placenta of preeclampsia patients by microarray profiling (Zhu et al. In line with this, our data confirmed the elevated expression of miR-<NUM> in a more precisely defined population of severe preeclampsia patients with an average gestational age of <NUM> weeks. Moreover, we proved the up-regulated miR-<NUM> expression in the mouse and rat models of preeclampsia, further strengthened the possibility that miR-<NUM> is associated with preeclamptic pathogenesis.

The preeclamptic placenta is associated with hypoxia and inflammatory conditions (Lockwood et al. , <NUM>; Soleymanlou et al. Our data demonstrated that hypoxia and inflammatory cytokines increased miR-<NUM> expression in placental explants suggesting miR-<NUM> could be the molecular clue to understand the pathogenesis of preeclampsia. In addition, the imbalance of angiogenesis has been highlighted as the primary culprit in preeclampsia (Ramma and Ahmed, <NUM>; Ramma et al. In our study, miR-<NUM> was identified to be a negative regulator of PlGF, an important growth factor for placental function, in endothelial cells. Although sFlt-<NUM> and sEng levels were not changed by overexpression of miR-<NUM>, the sFlt-<NUM>/PlGF ratio was increased due to the decreased level of PlGF. Since the elevated sFlt-<NUM>/PlGF ratio is an important indicator of angiogenic imbalance and a reliable biomarker in the assessment of preeclampsia (De Vivo et al. , <NUM>; Verlohren et al. , <NUM>), the elevated miR-<NUM> expression in the preeclamptic placenta may contribute to the angiogenic dysfunction in preeclampsia pathogenesis.

Mir-<NUM> has been shown to inhibit cell proliferation and adhesion (Zhou et al. , <NUM>; Mancini et al. , <NUM>) as well as tumor cell angiogenesis (Zheng et al. , <NUM>; Xu et al. , <NUM>), but not in the endothelial cells. Our data demonstrated that miR-<NUM> decreased endothelial cell adhesive and angiogenic abilities, and increased cell migration, but had no effect on cell proliferation. Endothelial dysfunction plays a central role in the pathogenesis of preeclampsia and causes most clinical symptoms of preeclampsia (Poston <NUM>; Baumwell and Karumanchi, <NUM>), thus miR-<NUM> may contribute to the pathogenesis of preeclampsia by modulation of endothelial functions.

ITGA5 belongs to the integrin alpha chain family which promotes cell adhesion, invasion and migration in cancer cells (Hood and Cheresh, <NUM>; Wang et al. , <NUM>; Qin et al. Moreover, it has been shown to promote vasculogenesis and angiogenesis in endothelial cells and mouse embryo (Francis et al. , <NUM>; Bonauer et al. In our study, ITGA5 was found to be the direct target of miR-<NUM> and also the mediator of miR-<NUM> induced endothelial dysfunction. By directly targeting ITGA5, miR-<NUM> inhibited endothelial cell adhesion and the reduced cell adhesion subsequently led to the promoted cell migration (Moh and Shen, <NUM>; Grzesiak et al. More importantly, overexpression of ITGA5 rescued the mimic-<NUM> inhibited angiogenesis in endothelial cells suggesting that ITGA5 is the downstream effector of miR-<NUM> mediated endothelial dysfunction at least on the disruption of angiogenesis. Notably, our microarray data revealed more possible targets of miR-<NUM> which regulate vascular remodelling, cell adhesion, angiogenesis, cell mobility and survival processes. These potential targets of miR-<NUM> may further contribute to the different clinical manifestation of preeclampsia.

Since effective treatment to preeclampsia is not currently available, the early prediction of high-risk women is important for the prevention and primary care of this medical condition (Leslie et al. Moreover, a recent meta-analysis suggests that low-dose aspirin started before <NUM> weeks' gestation could prevent up to <NUM>% of PE, severe PE, and intrauterine growth restriction (IUGR) in high-risk women (Bujold et al. Thus a biomarker that accurately predicts preeclampsia is of great clinical value. Recently, miR-<NUM> has been implicated in the serum of pregnant women and suggested for the potential use as biomarker (Williams et al. Based on our current study that miR-<NUM> elevated in severe preeclampsia, miR-<NUM> expression in the serum of early pregnancy of preeclampsia high-risk women should be examined to validate its biomarker potential.

Preeclampsia is a multifactorial disease which causes maternal and fetal morbidity and mortality worldwide (Pennington et al. , <NUM>), thus there is a pressing need for novel approaches to tackle this complex medical condition. Our data revealed that miR-<NUM> is elevated in the preeclampsia and subsequently leads to the endothelial dysfunction via targeting ITGA5 suggesting that miR-<NUM> and ITGA5 may serve as potential therapeutic targets in preeclampsia. More importantly, the miR-<NUM> and ITGA5 expression in the placenta of preeclamptic women was negatively correlated, further confirming the therapeutic potential of miR-<NUM> and ITGA5 in preeclampsia.

Recombinant growth factors, vascular endothelial growth factor A (VEGF-A), VEGF-E, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and interleukin <NUM> beta (IL-1β) were purchased from RELIATech (Brauschweig, Germany). Rabbit antibody against total eNOS and phosphor-eNOS were obtained from Cell Signaling Technology (Danvers, MA <NUM>, United States). Mouse anti-β-Actin antibody was obtained from Sigma-Aldrich (A5441, St. Louis, MO, United States). Quantitative PCR primer against miR-<NUM>, qScript™ microRNA cDNA and Synthesis PerfeCTa® SYBR® Green SuperMix Kit were purchased from Quanta Biosciences. Growth factor reduced Matrigel purchased from Becton Dickinson (Oxford, UK). M199 medium was purchased from Invitrogen (Paisley, UK). Calcein AM Fluorescent Dye was purchased from BD Bioscience (P. Box <NUM> Sparks, MD, USA <NUM>).

Institutional Ethics Committee approved the placental tissue collection and written informed consent was obtained. All women were followed prospectively from enrolment until delivery. Different gestational placenta tissues from pregnant women of 1st trimester (N=<NUM>), 2nd trimester (N=<NUM>) and 3rd trimester (N=<NUM>) was collected and measured for relative miR-<NUM> expression by qPCR. Human adipose tissues were collected from pregnant women with BMI over <NUM> (N=<NUM> and <NUM> respectively) or with normal BMI (<NUM>-<NUM>) (N=<NUM> for visceral and <NUM> for subcutaneous fat) and miR-<NUM> expression was determined by qPCR. Moreover, human placental tissues were collected from women with pregnancy complicated by preeclampsia (N=<NUM>) and Intrauterine growth restriction (IUGR, N=<NUM>), and from normotensive pregnant women (N=<NUM>). The placental tissues collected were further used for quantitative PCR. Preeclampsia was defined as blood pressure > <NUM>/<NUM> Hg on at least two consecutive measurements and maternal proteinuria of at least <NUM>/<NUM> and IUGR was defined as a foetus with estimated weight below the 10th percentile for its gestational age and abdominal circumference below the <NUM>. 5th percentile.

Human umbilical vein endothelial cells (HUVECs) were isolated and cultured in M199 medium as described previously (Bussolati et al, <NUM>). Experiments were performed on third or fourth passage HUVEC. First trimester placental tissues (<NUM>-<NUM> weeks gestational age) were retrieved from normal pregnancies that had undergone elective termination. Placental villus tissue explants were prepared as described previously (Ahmad and Ahmed, <NUM>). Briefly, human placental villus explants were incubated under stimulation of test substances or hypoxia condition and collected for quantitative PCR of miR-<NUM>. To create hypoxic condition, <NUM>-<NUM>% confluent HUEVCs or placental explants were cultured in an incubator with <NUM>% O<NUM> and <NUM>% CO<NUM> at <NUM>. To mimic inflammatory condition, HUVECs or placental explants were stimulated in cyto-mix cocktail containing TNF-α (20ng/ml), IFN-γ (20ng/ml) and IL-1β (2ng/ml).

Chemically synthesized double-stranded microRNA mimic and single-stranded inhibitor against miR-<NUM> (mi-<NUM> and anti-<NUM>) were purchased from Qiagen. The efficiency of these molecules was tested by qPCR using primer against miR-<NUM> (<FIG>.

HUVECs were trypsinized, and <NUM>×<NUM><NUM> cells were electroporated with ≈<NUM> ug mi-<NUM>, ≈<NUM> ug anti-<NUM> or equivalent control molecules using electroporation (Amaxa GmbH, Cologne, Germany) as described previously (Cudmore et al.

Adenovirus overexpressing eNOS constitutively activated form eNOSS1177D, ad-eNOSS1177D, was a gift from Prof. Ingrid Fleming (Johann Wolfgang Goethe University, Germany). The adenovirus overexpressing miR-<NUM>, ad-<NUM>, were purchased from Vector Biolabs (Philadelphia, PA, USA) and the efficiency was tested using Qpcr (<FIG>.

Adenovirus, ad-sFlt-<NUM>, was a gift from Prof. Richard Mulligan (Harvard Medical School, Boston, USA). The pregnant C57BL/<NUM> mice were injected with <NUM><NUM> PFU adenovirus, ad-CMV or ad-sFlt-<NUM>, at E <NUM> via tail vein injection. The sFlt-<NUM> expression level in the circulation was measured using ELISA against Flt-<NUM> and the mean artery pressure (MAP) was evaluated in the carotid artery at E <NUM> (<FIG>). Placenta tissue from ad-CMV control virus (N=<NUM>) and ad-sFlt-<NUM> virus (N=<NUM>) injected mice was collected and assayed for miR-<NUM> expression using qPCR.

Sample preparation and real-time quantitative PCR was performed as described previously (Cudmore et al. Reverse transcription kit and primers against miR-<NUM> were purchased from Quanta BioSciences.

Enzyme-linked immunosorbent assay (ELISA) kit for human P1GF was obtained from R&D Systems and performed according to the manufacturer's specifications.

HUVEC cells were electroporated with anti-<NUM> or mi-<NUM>. After overnight recovery, transfected cells were trypsinized and plated into <NUM>-well plate with <NUM>×<NUM><NUM> per well under the vehicle or VEGF-A (20ng/ml) treatment. After <NUM>, these cells were proceeded to MTT assay using cell growth determination kit (Cat No. CDG1, Sigma-Aldrich). For cell proliferation assay, transfected cells were plated in <NUM>-well plate with 4X <NUM><NUM> per well and stimulated with VEGF-A (20ng/ml) or VGEF-E (20ng/ml). After <NUM> treatment, cells were trypsinized and the cell number per well was counted under microscope using a hemocytometer.

HUVECs were electroporated with mi-<NUM> or control mi-con. After <NUM>, <NUM>×<NUM><NUM> mi-<NUM> or mi-con transfected cells were plated in the <NUM>% gelatin coated <NUM>-well plate and treated with VEGF-A (20ng/ml) or VEGF-E (20ng/ml) for <NUM> mins. Thereafter, cells were washed three times with PBS, stained with Calcein AM Fluorescent Dye and proceeded to fluorescent microscopy. The number of the adhesive cells per field under the <NUM> × magnification was counted.

Formation of capillary-like structures of HUVECs transfected with mi-<NUM> or anti-<NUM> or mi-<NUM> co-infected with ad-eNOSs1177D was determined on growth factor reduced Matrigel as previously described (Bussolati et al, <NUM>).

HUEVC cells were electroporated with anti-<NUM> or mi-<NUM>. After overnight recovery, the scratch was made in the centre of the transfected confluent cells in a <NUM>- or <NUM>-well plate. The width of the scratches was measured immediately at <NUM> and <NUM>-<NUM> after the scratches were generated. The cell migration distance was calculated by subtracting the width of scratches at <NUM> and <NUM>-<NUM>.

Total NO in conditioned media was assayed as nitrite, the stable breakdown product of NO, using a Sievers NO chemiluminescence analyzer (Analytix, Sunderland, UK) as described previously (Ahmad et al. Briefly, HUVECs were infected with ad-con or ad-<NUM> with MOI=<NUM> and the condition medium were collected for NO measurement.

The Homo sapiens cDNA containing untranslated region (UTR) of eNOS was purchased from Origene (Rockville, MD <NUM>, USA) and the UTR region was subcloned into pMirTarget to generate eNOS-UTR-Luciferase (firefly) expression plasmid, pmiR-eNOS. The successful transfection of pmiR-eNOS in endothelial cells was validated by the expression of red fluorescent protein (RFP) (Fig <NUM>). The plasmid harbouring eNOS UTR mutant form of miR-<NUM> binding site was generated using site-directed mutagenesis kit (Stratagene) with PCR primers containing the mutant site and the resulted plasmid was designated as pmiR-eNOSM. The primers used for PCR amplification were mNOS3-F (CTCTCAGGAGTAGAGTACCTGTAAAGGAGAATCTCTAAATCAAGT) and mNOS3-R (ACTTGATTTAGAGATTCTCCTTTACAGGTACTCTACTCCTGAGAG).

For microRNA target assay, HEK293 cells were transfected with pmiR-eNOS or pmiR-eNOSM together with mi-con or mi-<NUM>. The plasmid containing renilla luciferase gene was also transfected as the internal control. After overnight incubation, the relative firefly luciferase activity was measured and normalized to the renilla activity according to the manufacture's protocol of Dual-Luciferase® Reporter Assay System (E1910, Promega).

The direct targets of miR-<NUM> were predicted using online programs, microRNA. org (http://www. org/microma/home. do), PicTar (http://pictar. mdc-berlin. de/), Target Scan (http://www. targetscan. org/), RNAhybrid <NUM> and RNA22 microRNA target prediction.

HUVEC cells were transfected with mi-<NUM> and the total RNA sample was collected for whole genome gene array analysis using Affymetrix genechip Human Gene <NUM> ST. The up-regulated and down-regulated genes upon mi-<NUM> transfection are selected using a ±<NUM>-fold cut off.

Various microarray studies revealed that miR-<NUM> expression was dysregulated in the placentas of preeclamptic women (Xu et al. , <NUM>; Zhu et al. , <NUM>; Hu et al. Thus we examined the expression pattern of miR-<NUM> in different human tissues that are related to the pathogenesis of preeclampsia. First, the miR-<NUM> expression in the placentas of pregnant women with different gestational age was determined by qPCR. The miR-<NUM> expression in the <NUM>rd trimester placenta (N=<NUM>) was up-regulated by <NUM>-fold and <NUM>-fold respectively compared to <NUM>st (N=<NUM>) and <NUM>nd (N=<NUM>) trimester placenta (<FIG>). Furthermore, as obesity pregnant women pose a high risk to preeclampsia, we examined the miR-<NUM> expression in the adipose tissues of obesity pregnant women. The miR-<NUM> expression in the visceral (N=<NUM>) and subcutaneous fat (N=<NUM>) of obesity pregnant women was increased by <NUM>-fold and <NUM>-fold respectively compared to lean pregnant women (N=<NUM>-<NUM>) (<FIG>). Although various microarray studies demonstrated the dysregulated expression of miR-<NUM> in preeclampsia, the results from these studies are contradictive. More importantly, the placental samples used in these studies were collected from late gestational age of preeclampsia women range from <NUM> to <NUM> weeks. To investigate the miR-<NUM> expression in more precisely defined early stage severe preeclampsia patients, the placental samples from preeclampsia women with an average gestational age of <NUM> weeks were collected and examined for miR-<NUM> expression by qPCR. Quantitative PCR revealed that miR-<NUM> expression in the preeclamptic placenta (N=<NUM>) and IUGR placenta (N=<NUM>) was increased by <NUM>-fold and <NUM>-fold respectively compared to gestation age matched control normotensive placenta (N=<NUM>) (<FIG>).

To further confirm the up-regulation of miR-<NUM> in preeclampsia, the expression of miR-<NUM> was examined in two animal models of preeclampsia. The pregnant mice receiving ad-sFlt-<NUM> adenovirus injection (N=<NUM>) exhibited significant increased placental miR-<NUM> level (<FIG>) as well as elevated sFlt-<NUM> expression and mean artery pressure compared to ad-CMV control virus injected mice (N=<NUM>) (<FIG>). Moreover, the miR-<NUM> expression in placenta of pregnant Sprague Dawley rats that undergone reduced uterine perfusion pressure surgery (N=<NUM>) was also increased by <NUM>-fold compared to normal pregnant rat (N=<NUM>, Fig 22F). Taken together, these data suggest that miR-<NUM> elevation is associated with pathogenesis of preeclampsia.

Preeclamptic placenta is associated with hypoxia, inflammation condition as well as the elevation of VEGF and FGF-<NUM> growth factors (Hunter et al. , <NUM>; Ozkan et al. , <NUM>), thus the miR-<NUM> expression in the endothelial cells and placental explants under hypoxia and inflammation stimulation was determined by qPCR. Stimulation of mixed inflammatory cytokines in HUVEC cells increased miR-<NUM> by approximate <NUM>% (Fig <NUM>). Furthermore, the combination of hypoxia and inflammatory cytokine mixture stimulation increased miR-<NUM> expression by <NUM>% and <NUM>% in HUVECs and placental explants respectively (Fig <NUM> and I). These data confirmed that miR-<NUM> expression is regulated by the pathological conditions that associated with preeclamptic placenta.

The release of anti-angiogenic factors, sFlt-<NUM> and sEng, and the inhibition of placental growth factor (PlGF) expression are the main cause of preeclampsia (Powe et al. , <NUM>; Ahmad and Ahmed, <NUM>), therefore we examined whether miR-<NUM> modulates sFlt-<NUM>, sEng or P1GF expression in endothelial cells. HUVECs were transfected with mi-<NUM> to over-express miR-<NUM> (<FIG>) and the sFlt-<NUM>, sEng and P1GF release was examined by ELISA. Although overexpression of miR-<NUM> in HUVECs did not change sFlt-<NUM> and sEng expression, the P1GF level was significantly reduced under both vehicle and VEGF stimulation (<FIG>). As a result, the ratio of sFlt-<NUM>/PlGF was increased. These data further support the concept that miR-<NUM> upregulation may contribute to the pathogenesis of preeclampsia.

Since the impaired endothelium is the main cause of preeclampsia, we tested whether miR-<NUM> modulates endothelial cell functions. HUVECs were transfected with synthetic mimic (mi-<NUM>) and the cell proliferation assay was performed. Overexpression of miR-<NUM> decreased cell number by <NUM>% to <NUM>% compared to control under vehicle, VEGF-A or VEGF-E stimulation (<FIG>). Moreover, MTT assay in the HUVECs transfected with mi-<NUM> further confirmed that miR-<NUM> overexpression decreased cell viability by <NUM>% or <NUM>% under vehicle or VEGF stimulation respectively (Fig 23C).

The effect of miR-<NUM> overexpression in endothelial cell adhesion was also tested. The adhesive ability was dramatically decreased in the mi-<NUM> transfected cells regardless the stimulation of VEGF-A or VEGF-E. Cell counting confirmed an approximate <NUM>% reduction of adhesive cells in the mi-<NUM> transfected HUVECs under vehicle, VEGF-A or VEGF-E stimulation compared to mi-con transfected cells (Fig 23D).

Since miR-<NUM> inhibits angiogenesis in tumor cells, we speculated that miR-<NUM> also inhibits angiogenesis in placental endothelial cells to contribute to the pathogenesis of preeclampsia. HUVECs transfected with mi-<NUM> exhibited decreased tube formation on Matrigel under vehicle, VEGF-A or VEGF-E stimulation (Fig 23E). Quantitative analysis revealed a <NUM>% reduction in the tube formation of mi-<NUM> transfected cells under vehicle condition and <NUM>% to <NUM>% reduction under VEGF-A or VEGF-E stimulation.

Moreover, the role of miR-<NUM> in endothelial cell migration was determined by scratch assay. HUVECs transfected with anti-<NUM> showed <NUM>% to <NUM>% decrease of migration ability compared to anti-con transfected control cells under vehicle or VGEF stimulation (<FIG>). Conversely, Overexpression of miR-<NUM> in HUVECs significantly increased cell migration ability by approximate <NUM>-fold under vehicle or VEGF-A stimulation (<FIG>).

To identify the direct targets of miR-<NUM>, gene expression microarray was performed using RNA samples from mi-<NUM> and mi-con transfected HUVEC cells. The <NUM> up-regulated and <NUM> down-regulated genes upon miR-<NUM> over-expression were selected with ±<NUM>-fold cut off. These expression altered genes have broad functions on vascular remodeling, cell adhesion, mobility and angiogenesis. Moreover, the down-regulated genes were further analyzed using online prediction tools to confirm the possible direct targets of miR-<NUM> by sequence alignment. Finally, four down-regulated genes were selected for candidates of miR-<NUM> direct target.

Claim 1:
An miRNA modulator or a combination thereof for use in the treatment of preeclampsia and/or fetal growth restriction, wherein
the modulator is an miRNA inhibitor, and wherein the miRNA inhibitor is a nucleic acid molecule substantially complementary to the miRNA to allow a duplex to form and is selected from an inhibitor of miR-<NUM> or miR-<NUM>, or a combination thereof.