Targeting the long non-coding RNA MIAT for the treatment of fibroids in an animal model.

Our previous studies indicated that there is overexpression of MIAT in fibroids and MIAT is a sponge for the miR-29 family in these tumors. The objective of the present study was to determine if the knockdown of MIAT in fibroid xenografts will increase miR-29 levels and reduce the expression of genes targeted by this miRNA such as collagen and cell cycle regulatory proteins in a mouse model for fibroids. Ovariectomized CB-17 SCID/Beige mice bearing estrogen/progesterone pellets were implanted subcutaneously in the flank with equal weight of fibroid explants which had been transduced by lentivirus for either control (empty vector) or MIAT knockdown for four weeks (n=7). Knockdown of MIAT in fibroid xenografts resulted in a 30% reduction of tumor weight and a marked increase in miR-29a, -b, and -c levels in the xenografts. There was reduced cell proliferation and expression of cell cycle regulatory genes CCND1, CDK2, and E2F1 and no significant changes in apoptosis. The xenografts with MIAT knockdown expressed lower mRNA and protein levels of FN1, COL3A1, and TGF-ß3, and total collagen protein. Targeting MIAT, which sponges the pro-fibrotic miR-29 family, is an effective therapy for fibroids by reducing cell proliferation and thereby, tumor growth and accumulation of ECM, which is a hallmark of these benign gynecologic tumors.

The Effect of Race/Ethnicity and MED12 Mutation on the Expression of Long Non-Coding RNAs in Uterine Leiomyoma and Myometrium.

The objective of this study was to elucidate the expression of long non-coding RNA (lncRNA) in leiomyomas (Lyo) and paired myometrium (Myo) and explore the impact of race and MED12 mutation. Fold change analysis (Lyo/paired Myo) indicated the expression of 63 lncRNAs was significantly altered in the mutated group but not in the non-mutated Lyo. Additionally, 65 lncRNAs exhibited an over 1.5-fold change in the Black but not the White group. Fifteen differentially expressed lncRNAs identified with next-generation sequencing underwent qRT-PCR confirmation. Compared with Myo, the expression of TPTEP1, PART1, RPS10P7, MSC-AS1, SNHG12, CA3-AS1, LINC00337, LINC00536, LINC01436, LINC01449, LINC02433, and LINC02624 was significantly higher, while the expression of ZEB2-AS1, LINC00957, and LINC01186 was significantly lower. Comparison of normal Myo with diseased Myo showed significant differences in the expression of several lncRNAs. Analysis based on race and Lyo MED12 mutation status indicated a significantly higher expression of RPS10P7, SNHG12, LINC01449, LINC02433, and LINC02624 in Lyo from Black patients. The expression of TPTEP1, PART1, RPS10P7, MSC-AS1, LINC00337, LINC00536, LINC01436, LINC01449, LINC02433, and LINC02624 was higher, while LINC01186 was significantly lower in the MED12-mutated group. These results indicate that Lyo are characterized by aberrant lncRNA expression, which is further impacted by race and Lyo MED12 mutation status.

Therapeutic Effects of Long-Term Administration of Tranilast in an Animal Model for the Treatment of Fibroids.

Tranilast (N-3, 4-dimethoxycinnamoyl anthranilic acid) is an orally administered drug with antiallergic properties and approved in Japan and the Republic of Korea for the treatment of asthma and hypertrophic scars. Previous in vitro studies indicated that tranilast reduced fibroid growth through its inhibitory effects on cell proliferation and induction of apoptosis. The objective of this study was to determine the efficacy of tranilast for treatment of human-derived fibroids in a mouse model. SCID mice (ovariectomized, supplemented with estrogen and progesterone) were implanted with fibroid explants and treated for two months with tranilast (50 m/kg/daily) or the vehicle. After sacrifice, xenografts were excised and analyzed. Tranilast was well tolerated without adverse side effects. There was a 37% reduction in tumor weight along with a significant decrease in staining for Ki67, CCND1, and E2F1; a significant increase in nuclear staining for cleaved caspase 3; and reduced staining for TGF-ß3 and Masson's trichrome in the tranilast treated mice. There was a significant inhibition of mRNA and protein expression of fibronectin, COL3A1, CCND1, E2F1, and TGF-ß3 in the xenografts from the tranilast-treated mice. These promising therapeutic effects of tranilast warrant additional animal studies and human clinical trials to evaluate its efficacy for treatment of fibroids.

Differential Expression of MED12-Associated Coding RNA Transcripts in Uterine Leiomyomas.

Recent studies have demonstrated that somatic MED12 mutations in exon 2 occur at a frequency of up to 80% and have a functional role in leiomyoma pathogenesis. The objective of this study was to elucidate the expression profile of coding RNA transcripts in leiomyomas, with and without these mutations, and their paired myometrium. Next-generation RNA sequencing (NGS) was used to systematically profile the differentially expressed RNA transcripts from paired leiomyomas (n = 19). The differential analysis indicated there are 394 genes differentially and aberrantly expressed only in the mutated tumors. These genes were predominantly involved in the regulation of extracellular constituents. Of the differentially expressed genes that overlapped in the two comparison groups, the magnitude of change in gene expression was greater for many genes in tumors bearing MED12 mutations. Although the myometrium did not express MED12 mutations, there were marked differences in the transcriptome landscape of the myometrium from mutated and non-mutated specimens, with genes regulating the response to oxygen-containing compounds being most altered. In conclusion, MED12 mutations have profound effects on the expression of genes pivotal to leiomyoma pathogenesis in the tumor and the myometrium which could alter tumor characteristics and growth potential.

The Influence of Race/Ethnicity on the Transcriptomic Landscape of Uterine Fibroids.

The objective of this study was to determine if the aberrant expression of select genes could form the basis for the racial disparity in fibroid characteristics. The next-generation RNA sequencing results were analyzed as fold change [leiomyomas/paired myometrium, also known as differential expression (DF)], comparing specimens from White (n = 7) and Black (n = 12) patients. The analysis indicated that 95 genes were minimally changed in tumors from White (DF ≈ 1) but were significantly altered by more than 1.5-fold (up or down) in Black patients. Twenty-one novel genes were selected for confirmation in 69 paired fibroids by qRT-PCR. Among these 21, coding of transcripts for the differential expression of FRAT2, SOX4, TNFRSF19, ACP7, GRIP1, IRS4, PLEKHG4B, PGR, COL24A1, KRT17, MMP17, SLN, CCDC177, FUT2, MYO5B, MYOG, ZNF703, CDC25A, and CDCA7 was significantly higher, while the expression of DAB2 and CAV2 was significantly lower in tumors from Black or Hispanic patients compared with tumors from White patients. Western blot analysis revealed a greater differential expression of PGR-A and total progesterone (PGR-A and PGR-B) in tumors from Black compared with tumors from White patients. Collectively, we identified a set of genes uniquely expressed in a race/ethnicity-dependent manner, which could form the underlying mechanisms for the racial disparity in fibroids and their associated symptoms.

Mechanism underlying increased cardiac extracellular matrix deposition in perinatal nicotine-exposed offspring.

Although increased predisposition to cardiac fibrosis and cardiac dysfunction has been demonstrated in the perinatally nicotine-exposed heart, the underlying mechanisms remain unclear. With the use of a well-established rat model and cultured primary neonatal rat cardiac fibroblasts, the effect of perinatal nicotine exposure on offspring heart extracellular matrix deposition and the likely underlying mechanisms were investigated. Perinatal nicotine exposure resulted in increased collagen type I (COL1A1) and III (COL3A1) deposition along with a decrease in miR-29 family and an increase in long noncoding RNA myocardial infarction-associated transcript (MIAT) levels in offspring heart. Nicotine treatment of isolated primary neonatal rat cardiac fibroblasts suggested that these effects were mediated via nicotinic acetylcholine receptors including α7 and the induced collagens accumulation was reversed by a gain-of function of miR-29 family. Knockdown of MIAT resulted in increased miR-29 family and decreased COL1A1 and COL3A1 levels, suggesting nicotine-mediated MIAT induction as the underlying mechanism for nicotine-induced collagen deposition. Luciferase reporter assay and RNA immunoprecipitation studies showed an intense physical interaction between MIAT, miR-29 family, and argonaute 2, corroborating the mechanistic link between perinatal nicotine exposure and increased extracellular matrix deposition. Overall, perinatal nicotine exposure resulted in lower miR-29 family levels in offspring heart, while it elevated cardiac MIAT and collagen type I and III levels. These findings provide mechanistic basis for cardiac dysfunction in perinatal nicotine-exposed offspring and offer multiple novel potential therapeutic targets.NEW & NOTEWORTHY Using an established rat model and cultured primary neonatal cardiac fibroblasts, we show that nicotine mediated MIAT induction as the underlying mechanism for the excessive cardiac collagen deposition. These observations provide mechanistic basis for the increased predisposition to cardiac dysfunction following perinatal cigarette/nicotine exposure and offer novel potential therapeutic targets.

Role of miR-29 in mediating offspring lung phenotype in a rodent model of intrauterine growth restriction.

Considerable epidemiological and experimental evidence supports the concept that the adult chronic lung disease (CLD), is due, at least in part, to aberrations in early lung development in response to an abnormal intrauterine environment; however, the underlying molecular mechanisms remain unknown. We used a well-established rat model of maternal undernutrition (MUN) during pregnancy that results in offspring intrauterine growth restriction (IUGR) and adult CLD to test the hypothesis that in response to MUN, excess maternal glucocorticoids (GCs) program offspring lung development to a CLD phenotype by altering microRNA (miR)-29 expression, which is a key miR in regulating extracellular matrix (ECM) deposition during development and injury-repair. At postnatal day 21 and 5 mo, compared with the control offspring lung, MUN offspring lung miR-29 expression was significantly decreased in conjunction with an elevated expression of multiple downstream target ECM proteins [collagen (COL)1A1, COL3A1, COL4A5, and elastin], at both mRNA and protein levels. Importantly, MUN-induced changes in miR-29 and target gene expressions were at least partially blocked in the lungs of offspring of MUN dams treated with metyrapone, a selective GC synthesis inhibitor. Furthermore, dexamethasone treatment of cultured fetal rat lung fibroblasts significantly induced miR-29 expression along with the suppression of target ECM proteins. These data, along with the previously known role of miR-29 in regulating ECM deposition in vascular tissue in the MUN offspring, suggest miR-29 to be a common mechanistic denominator for the vascular and pulmonary phenotypes in the IUGR offspring, providing a novel potential therapeutic target.

miR-29c induction contributes to downregulation of vascular extracellular matrix proteins by glucocorticoids.

Maternal undernutrition increases maternal glucocorticoids (GCs) and alters microRNA expression in offspring. Given that the mechanisms of GC action on vascular development are not clear, this study examined the influence of GCs on microRNA 29c (miR-29c) and its predicted targets in primary rat aorta smooth muscle cells (RAOSMCs). Dexamethasone (Dex) and corticosterone (Cor) time-dependently increased miR-29c expression and reduced collagen type III (Col3A1), collagen type IV (Col4A5), elastin (ELN), and matrix metalloproteinase-2 (MMP2) protein in RAOSMCs. These effects were blocked by mifepristone. These genes were also targeted by miR-29c, as confirmed by a significant decrease in luciferase reporter activity of Col3A1 (34%), Col4A5 (45%), ELN (17%), and MMP2 (28%). In cells transfected with reporter plasmids, including the 3'-untranslated region of genes targeted by miR-29c, treatment with Dex or Cor also resulted in decreases in luciferase activity. Gain or loss of function of miR-29c significantly altered mRNA expression of Col3A1 (26% and 26%, respectively), Col4A5 (28% and 32%, respectively), and MMP2 (24% and 14%, respectively) but did not affect ELN. Gain or loss of function of miR-29c also significantly altered protein levels of Col3A1 (51% and 16%, respectively), Col4A5 (56% and 22%, respectively), ELN (53% and 71%, respectively), and MMP2 (28% and 53%, respectively). Coincubation of anti-miR-29c with Dex or Cor partially attenuated the effects of these steroids on protein expression of Col3A1 (25% and 24%, respectively), Col4A5 (26% and 44%, respectively), ELN (31% and 55%, respectively), and MMP2 (46% and 26%, respectively) in RAOSMCs compared with anti-miR negative controls. Our results demonstrate that GCs regulate the expression of Col3A1, Col4A5, ELN, and MMP2, at least in part, through induction of miR-29c.

Maternal food restriction modulates cerebrovascular structure and contractility in adult rat offspring: effects of metyrapone.

Although the effects of prenatal undernutrition on adult cardiovascular health have been well studied, its effects on the cerebrovascular structure and function remain unknown. We used a pair-fed rat model of 50% caloric restriction from day 11 of gestation to term, with ad libitum feeding after birth. We validated that maternal food restriction (MFR) stress is mediated by glucocorticoids by administering metyrapone, a corticosterone synthesis inhibitor, to MFR mothers at day 11 of gestation. At age 8 mo, offspring from Control, MFR, and MFR + Metyrapone groups were killed, and middle cerebral artery (MCA) segments were studied using vessel-bath myography and confocal microscopy. Colocalization of smooth muscle α-actin (SMαA) with nonmuscle (NM), SM1 and SM2 myosin heavy-chain (MHC) isoforms was used to assess smooth muscle phenotype. Our results indicate that artery stiffness and wall thickness were increased, pressure-evoked myogenic reactivity was depressed, and myofilament Ca(2+) sensitivity was decreased in offspring of MFR compared with Control rats. MCA from MFR offspring exhibited a significantly greater SMαA/NM colocalization, suggesting that the smooth muscle cells had been altered toward a noncontractile phenotype. MET significantly reversed the effects of MFR on stiffness but not myogenic reactivity, lowered SMαA/NM colocalization, and increased SMαA/SM2 colocalization. Together, our data suggest that MFR alters cerebrovascular contractility via both glucocorticoid-dependent and glucocorticoid-independent mechanisms.

The Immune Landscape of Uterine Fibroids as Determined by Mass Cytometry.

OBJECTIVE: To study the differences in immune cell profile in uterine fibroids (Fib) and matched myometrium (Myo). DESIGN: Observational Study SETTING: Laboratory Study PATIENTS: The study included tissue that was collected from ten pairs of fibroid and matched myometrium from women, not on hormonal medications, undergoing hysterectomy and myomectomy. INTERVENTIONS: None MAIN OUTCOME MEASURES: Differences in immune cell and cytokine composition between fibroid and matched myometrium RESULT(S): The mass cytometry analysis indicated that fibroids had a significantly higher number of NK cells, total macrophages, M2 macrophages, and conventional dendritic cells when compared to matched myometrium from the same patient. In contrast, fibroids had significantly fewer CD3 and CD4 T cells when compared to myometrium. The mass cytometry analysis did not show any significant difference in the number of resting mast cells. IFC and IHC imaging confirmed the CytoF results, showing a significantly higher (p<0.05) number of NK, tryptase-positive mast cells indicative of mast cell activation, total macrophage, and M2 cells in Fibroids and a significantly lower (p<0.05) number of CD3 and CD4 T cells. The cytokine assay revealed significantly increased (p<0.05) levels of IFNA2, Il-1α, and PDGF-AA and significantly lower levels of M-CSF and IL-1RA in Fib. CONCLUSION: Our results show significant differences in immune cell populations and cytokine levels between Fib and Myo. There was a significant increase in total number of macrophages, M2 macrophages, NK cells, and dendritic cells and a significant decrease in CD3 and CD4 T cells in Fib. IHC confirmed no differences in total resting mast cell count, but a significant increase in tryptase-positive mast cells in Fib. Fibs also expressed significantly higher levels of IFNA2, IL-1α, and PDGF-AA and significantly lower levels of IL-1RA and M-CSF as compared with matched myometrium. These findings provide a foundation for further studies exploring the role of immune cells in Fib development.

Therapeutic effects of in vivo administration of an inhibitor of tryptophan 2,3-dioxygenase (680c91) for the treatment of fibroids: a preclinical study.

OBJECTIVE: Fibroids are characterized by marked overexpression of tryptophan 2,3 dioxygenase (TDO2). The objective of this study was to determine the effectiveness of in vivo administration of an inhibitor of TDO2 (680C91) on fibroid size and gene expression. DESIGN: Animal and ex vivo human study. SETTING: Academic Research Institution. SUBJECTS: Severe combined immunodeficiency mice bearing human fibroid xenografts treated with vehicle and TDO2 inhibitor. INTERVENTION: Daily intraperitoneal administration of 680C91 or vehicle for 2 months and in vitro studies with fibroid explants. MAIN OUTCOME MEASURES: Tumor weight and gene expression profile of xenografts and in vitro mechanistic experiments using fibroid explants. RESULTS: Compound 680C91 was well-tolerated with no effects on blood chemistry and body weight. Treatment of mice with 680C91 resulted in 30% reduction in the weight of fibroid xenografts after 2 months of treatment and as expected lower levels of kynurenine, the byproduct of tryptophan degradation and an endogenous ligand of aryl hydrocarbon receptor (AhR) in the xenografts. The expression of cytochrome P450 family 1 subfamily B member 1 (CYP1B1), transforming growth factor ß3 (TGF-ß3), fibronectin (FN1), cyclin-dependent kinase 2 (CDK2), E2F transcription factor 1 (E2F1), interleukin 8 (IL-8) and secreted protein acidic and cysteine rich (SPARC) mRNA were lower in the xenografts of mice treated with 680C91 compared with vehicle controls. Similarly, the protein abundance of collagen, FN1, CYP1B1, and SPARC were lower in the xenografts of 680C9- treated mice compared with vehicle controls. Immunohistochemical analysis of xenografts indicated decreased expression of collagen, Ki67 and E2F1 but no significant changes in cleaved caspase 3 expression in mice treated with 680C91. The levels of kynurenine in the xenografts showed a direct correlation with the tumor weight and FN1 levels. In vitro studies with fibroid explants showed a significant induction of CYP1B1, TGF-ß3, FN1, CDK2, E2F1, IL8, and SPARC mRNA by tryptophan, which could be blocked by cotreatment with 680C91 and the AhR antagonist CH-223191. CONCLUSION: The results indicate that correction of aberrant tryptophan catabolism in fibroids could be an effective treatment through its effect to reduce cell proliferation and extracellular matrix accumulation.

Maturation and differentiation of the fetal vasculature.

Rapid postnatal growth and differentiation of fetal arterial smooth muscle is coordinated by a cacophony of growth factors, one of the most important of which is vascular endothelial growth factor (VEGF). In fetal arterial smooth muscle, VEGF influences both the expression and intracellular organization of contractile proteins and helps mediate hypoxic vascular remodeling. Numerous factors influence the expression of VEGF and its receptors, including chronic hypoxia, maternal food restriction, glucocorticoids, and miRNA. Continued study of the coupling between VEGF and transcription factors such as myocardin that govern smooth muscle differentiation, offers great promise for better clinical management of neonates at risk for cardiovascular dysregulation.

Chronic hypoxia and VEGF differentially modulate abundance and organization of myosin heavy chain isoforms in fetal and adult ovine arteries.

Chronic hypoxia increases vascular endothelial growth factor (VEGF) and thereby promotes angiogenesis. The present study explores the hypothesis that hypoxic increases in VEGF also remodel artery wall structure and contractility through phenotypic transformation of smooth muscle. Pregnant and nonpregnant ewes were maintained at sea level (normoxia) or 3,820 m (hypoxia) for the final 110 days of gestation. Common carotid arteries harvested from term fetal lambs and nonpregnant adults were denuded of endothelium and studied in vitro. Stretch-dependent contractile stresses were 32 and 77% of normoxic values in hypoxic fetal and adult arteries. Hypoxic hypocontractility was coupled with increased abundance of nonmuscle myosin heavy chain (NM-MHC) in fetal (+37%) and adult (+119%) arteries. Conversely, hypoxia decreased smooth muscle MHC (SM-MHC) abundance by 40% in fetal arteries but increased it 123% in adult arteries. Hypoxia decreased colocalization of NM-MHC with smooth muscle α-actin (SM-αA) in fetal arteries and decreased colocalization of SM-MHC with SM-αA in adult arteries. Organ culture with physiological concentrations (3 ng/ml) of VEGF-A(165) similarly depressed stretch-dependent stresses to 37 and 49% of control fetal and adult values. The VEGF receptor antagonist vatalanib ablated VEGF's effects in adult but not fetal arteries, suggesting age-dependent VEGF receptor signaling. VEGF replicated hypoxic decreases in colocalization of NM-MHC with SM-αA in fetal arteries and decreases in colocalization of SM-MHC with SM-αA in adult arteries. These results suggest that hypoxic increases in VEGF not only promote angiogenesis but may also help mediate hypoxic arterial remodeling through age-dependent changes in smooth muscle phenotype and contractility.

Altered endometrial expression of endothelial nitric oxide synthase in women with unexplained recurrent miscarriage and infertility.

Endothelial nitric oxide synthase (eNOS) has diverse roles in the female reproductive system including a role in blastocyst implantation. Aberrant expression of eNOS could therefore be significant in the pathogenesis of disorders of implantation. In this study, eNOS protein and mRNA levels in the endometrium of women with recurrent miscarriages, unexplained infertility and a control group were determined by compartmental quantitative immunohistochemistry and real-time reverse-transcription PCR. eNOS was found to be immunolocalized to all layers of the endometrium and vascular endothelium. eNOS protein was higher in glandular epithelium (P = 0.004) and luminal epithelium (P = 0.002), but not vascular endothelium and stroma, in women with recurrent miscarriage. Similarly, in women with unexplained infertility, eNOS was significantly higher (P < 0.03) in luminal epithelium but not in any other compartments compared with the control group. The levels of mRNA confirmed the protein data, demonstrating higher eNOS mRNA in the endometrium of women with recurrent miscarriage and unexplained infertility compared with controls. In conclusion, increased expression of eNOS in glandular and luminal epithelium of the endometrium in women with recurrent miscarriages and unexplained infertility suggests a detrimental effect of excess nitric oxide in endometrial receptivity and implantation.

Differential Expression of Super-Enhancer-Associated Long Non-coding RNAs in Uterine Leiomyomas.

Super-enhancer-associated long non-coding RNAs (SE-lncRNAs) are a specific set of lncRNAs transcribed from super-enhancer (SE) genomic regions. Recent studies have revealed that SE-lncRNAs play essential roles in tumorigenesis through the regulation of oncogenes. The objective of this study was to elucidate the expression profile of SE-lncRNAs with concurrent assessment of associated mRNAs in leiomyomas and paired myometrium. Arraystar SE-lncRNAs arrays were used to systematically profile the differentially expressed SE-lncRNAs along with the corresponding SE-regulated protein coding genes in eight leiomyomas and paired myometrium. The analysis indicated 7680 SE-lncRNAs were expressed, of which 721 SE-lncRNAs were overexpressed, while 247 SE-lncRNAs were underexpressed by 1.5-fold or greater in leiomyoma. Thirteen novel SE-lncRNAs and their corresponding protein coding genes were selected, and their expression was confirmed in eighty-one paired leiomyoma tissues by quantitative real-time PCR. The thirteen pairs of SE-lncRNAs and their corresponding protein coding genes included RP11-353N14.2/CBX4, SOCS2-AS1/SOCS2, RP1-170O19.14/HOXA11, CASC15/PRL, EGFLAM-AS1/EGFLAM, RP11-225H22/NEURL1, RP5-1086K13.1/CD58, AC092839.3/SPTBN1, RP11-69I8.3/CTGF, TM4SF1-AS1/TM4SF1, RP11-373D23/FOSL2, RP11-399K21.11/COMTD1, and CTB-113P19.1/SPARC. Among these SE-lncRNAs, the expression of SOCS2-AS1/SOCS2, RP11-353N14.2/CBX4, RP1-170O19.14/HOXA11, and RP11-225H22/NEURL1 was significantly higher in African Americans as compared with Caucasians. The expression of RP11-353N14.2/CBX4, SOCS2-AS1/SOCS2, CASC15/PRL, and CTB-113P19.1/SPARC was significantly higher in tumors with MED12-mutation-positive as compared with MED12-mutation-negative tumors. Collectively, our results indicate that the differential expression of SE in leiomyomas is another mechanism contributing to dysregulation of protein coding genes in leiomyomas and that race and MED12 mutation can influence the expression of a select group of SE.

Further characterization of tryptophan metabolism and its dysregulation in fibroids.

OBJECTIVE: To determine the expression of enzymes in tryptophan (Trp) catabolism in fibroids and matched myometrium and determine the effects of race and mediator complex subunit 12 gene (MED12) mutation on their expression. DESIGN: Experimental laboratory study. SETTING: Academic research laboratory. PATIENT(S): Women of reproductive age who underwent hysterectomy while on no hormonal medications before surgery. INTERVENTION(S): Fibroids and matched myometrium were obtained from patients who underwent hysterectomy from different race or ethnic groups. MAIN OUTCOME MEASURE(S): The expression of enzymes in the Trp catabolic pathway, tryptophan transporters, and the cytochrome P450 1B1 gene (CYP1B1) in the fibroids and matched myometrium of women from different race and ethnic groups and in tumors bearing the MED12 mutation and tumors without the mutation was determined using quantitative reverse-transcription polymerase chain reaction. The levels of serotonin, kynurenic acid (KYNA), and nicotinamide adenine dinucleotide (NAD) were determined using enzyme-linked immunosorbent assay. RESULT(S): In fibroids, the expression of tryptophan hydroxylase 1 (TPH1), kynurenine amino transferase (KAT)2, large neutral amino acid transporter small subunit 2 (SLC7A8), and large neutral amino acid transporter small subunit 1 (SLC7A5) messenger RNA (mRNA) was high and that of kynureninase (KYNU) and tryptophanyl-tRNA ligase (WARS1) mRNA was low, with no changes in the expression of WARS2, kynurenine formamidase (AFMID), kynurenine 3-monooxygenase (KMO), KAT1, KAT3, and KAT4 compared with that in the matched myometrium (n = 81). The expression of CYP1B1 mRNA, a marker of the activation of the aryl hydrocarbon receptor, was higher in fibroids. Tumors bearing the MED12 mutation expressed higher levels of CYP1B1 and lower levels of WARS1, KAT1, KAT3, and KAT4 mRNAs compared with tumors without the MED12 mutation. Race or ethnicity affected the expression of KYNU, with tumors from African American and Hispanic patients expressing lower levels of KYNU mRNA compared with those from Caucasian patients. We also quantified the levels of serotonin, KYNA, and NAD, which are the end products of Trp catabolism. There were no significant differences in the levels of serotonin and KYNA, whereas the levels of NAD were lower in fibroids than in the paired myometrium. This reduction in the levels of NAD was independent of race or ethnicity. CONCLUSION(S): In addition to the expression of tryptophan 2,3-dioxygenase or indoleamine-pyrrole 2,3-dioxygenase, there was marked dysregulation in the expression of other enzymes in the Trp metabolic pathway and Trp transporters in fibroids. Both MED12 mutation status and race or ethnicity had selective effects on the expression of the components of this pathway. Additional functional studies are necessary to establish the physiologic significance of the tryptophan degradation pathway in the pathogenesis of fibroids and its potential as a target for novel therapies.

Contributions of VEGF to age-dependent transmural gradients in contractile protein expression in ovine carotid arteries.

The present study explores the hypothesis that arterial smooth muscle cells are organized into layers with similar phenotypic characteristics that vary with the relative position between the lumen and the adventitia due to transmural gradients in vasotrophic factors. A corollary hypothesis is that vascular endothelial growth factor (VEGF) is a factor that helps establish transmural variations in smooth muscle phenotype. Organ culture of endothelium-denuded ovine carotid arteries with 3 ng/ml VEGF-A(165) for 24 h differentially and significantly influenced potassium-induced (55% increase) and stretch-induced (36% decrease) stress-strain relations in adult (n = 18) but not term fetal (n = 21) arteries, suggesting that smooth muscle reactivity to VEGF is acquired during postnatal maturation. Because inclusion of fetal bovine serum significantly inhibited all contractile effects of VEGF (adult: n = 11; fetus: n = 11), it was excluded in all cultures. When assessed in relation to the distance between the lumen and the adventitia in immunohistochemically stained coronal artery sections, expression of smooth muscle α-actin (SMαA), myosin light chain kinase (MLCK), and 20-kDa regulatory myosin light chain exhibited distinct protein-dependent and age-dependent gradients across the artery wall. VEGF depressed regional SMαA abundance up to 15% in adult (n = 6) but not in fetal (n = 6) arteries, increased regional MLCK abundance up to 140% in fetal (n = 8) but not in adult (n = 10) arteries, and increased regional MLC(20) abundance up to 28% in fetal arteries (n = 7) but decreased it by 17% in adult arteries (n = 9). Measurements of mRNA levels verified that VEGF receptor transcripts for both Flt-1 and kinase insert domain receptor (KDR) were expressed in both fetal and adult arteries. Overall, the present data support the unique hypothesis that smooth muscle cells are organized into lamina of similar phenotype with characteristics that depend on the relative position between the lumen and the adventitia and involve the direct effects of growth factors such as VEGF, which acts independently of the vascular endothelium in an age-dependent manner.

Functional role of the long noncoding RNA X-inactive specific transcript in leiomyoma pathogenesis.

OBJECTIVE: To determine the expression and functional roles of a long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) in leiomyoma. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for leiomyoma. INTERVENTION(S): Overexpression and underexpression of XIST; blockade of specific protein 1 (SP1). MAIN OUTCOME MEASURE(S): Expression of XIST in leiomyoma and its effects on microRNA 29c (miR-29c), miR-200c, and their targets. RESULT(S): Leiomyoma expressed statistically significantly more XIST as compared with matched myometrium, independent of race/ethnicity and menstrual cycle phase. By use of a three-dimensional spheroid culture system, we found reduced XIST levels in leiomyoma smooth muscle cells (LSMC) after treatment with 17ß-estradiol, progesterone, and their combination. The expression of XIST was down-regulated by treatment with the SP1-inhibitor mithramycin A and SP1 small interfering RNA. Knockdown of XIST resulted in inhibition of cell proliferation, up-regulation of miR-29c and miR-200c, and a concomitant inhibition of the target genes of these miRNAs, namely collagen type I (COL1A1), collagen type III (COL3A1), and fibronectin (FN1). By contrast, overexpression of XIST in myometrium smooth muscle cells repressed miR-29c and miR-200c, and induced COL1A1, COL3A1, and FN1 levels. By use of RNA immunoprecipitation analysis we confirmed XIST has sponge activity over miR-29c and miR-200c, which is more pronounced in leiomyoma as compared with myometrium. CONCLUSION(S): Our data demonstrate that increased expression of XIST in leiomyoma results in reduced expression of miR-29c and miR-200c with a consequent up-regulation of the genes targeted by these microRNAs including COL1A1, COL3A1, and FN1, which play key roles in extracellular matrix accumulation associated with fibroids.

Tryptophan catabolism is dysregulated in leiomyomas.

OBJECTIVE: To determine the expression and functional roles of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) in leiomyoma. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for leiomyoma. INTERVENTION(S): Blockade of IDO1 and TDO2. MAIN OUTCOME MEASURE(S): Expression of IDO1 and TDO2 in leiomyoma and the effects of their inhibitors on the extracellular matrix. RESULT(S): Leiomyoma expressed significantly higher levels of IDO1 and TDO2 messenger ribonucleic acid (mRNA; 60.3%, 35/58 pairs and 98.3%, 57/58 pairs, respectively) and protein (54%, 27/50 pairs and 92%, 46/50 pairs, respectively) as well as the enzyme activity marker kynurenine (78.3%, 36/46 pairs for IDO1/TDO2) compared with levels in matched myometrium. The expression of TDO2 but not IDO1 mRNA was significantly higher in fibroids from African American compared with that in Caucasian and Hispanic patients. The TDO2 but not the IDO1 protein and mRNA levels were more abundant in fibroids bearing the MED12 mutation compared with results in wild-type leiomyomas. Treatment of leiomyoma smooth muscle cell and myometrial smooth muscle cell spheroids with the TDO2 inhibitor 680C91 but not the IDO1 inhibitor epacadostat significantly repressed cell proliferation and the expression of collagen type I (COL1A1) and type III (COL3A1) in a dose-dependent manner; these effects were more pronounced in leiomyoma smooth muscle cells compared with myometrial smooth muscle cell spheroids. CONCLUSION(S): These results underscore the physiological significance of the tryptophan degradation pathway in the pathogenesis of leiomyomas and the potential utility of anti-TDO2 drugs for treatment of leiomyomas.

Long Noncoding RNA MIAT Modulates the Extracellular Matrix Deposition in Leiomyomas by Sponging MiR-29 Family.

The objective of this study was to determine the expression and functional role of a long noncoding RNA (lncRNA) MIAT (myocardial infarction-associated transcript) in leiomyoma pathogenesis. Leiomyoma compared with myometrium (n = 66) expressed significantly more MIAT that was independent of race/ethnicity and menstrual cycle phase but dependent on MED12 (mediator complex subunit 12) mutation status. Leiomyomas bearing the MED12 mutation expressed higher levels of MIAT and lower levels of microRNA 29 family (miR-29a, -b, and -c) compared with MED12 wild-type leiomyomas. Using luciferase reporter activity and RNA immunoprecipitation analysis, MIAT was shown to sponge the miR-29 family. In a 3-dimensional spheroid culture system, transient transfection of MIAT siRNA in leiomyoma smooth muscle cell (LSMC) spheroids resulted in upregulation of miR-29 family and downregulation of miR-29 targets, collagen type I (COL1A1), collagen type III (COL3A1), and TGF-ß3 (transforming growth factor ß-3). Treatment of LSMC spheroids with TGF-ß3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-ß3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma.