Nasser Chegini

Expression Profile of MicroRNAs and mRNAs in Human Placentas From Pregnancies Complicated by Preeclampsia and Preterm Labor

MicroRNAs (miRNAs) have emerged as key regulators of gene expression stability implicated in cell proliferation, apoptosis, and development, whereas their altered expression has been associated with various pathological disorders. The objective of this study was to assess the expression profile of miRNAs and their predicted target genes in placentas from patients with preeclampsia (PC) and preterm (PT) labor as compared to normal term (NT) pregnancies. Using microarray profiling of 820 miRNAs and 18,630 mRNA transcripts, the analysis indicated that 283 of these miRNAs and 9119 mRNAs were expressed in all placentas, of which the relative expression of 20 miRNAs (P < .05 and ≥1.5-fold) and 120 mRNAs (P < .05, and 2-fold cutoff) was differentially expressed in PT and PC as compared to NT. The expression of miR-15b, miR-181a, miR-200C, miR-210, miR-296–3p, miR-377, miR-483–5p, and miR-493 and a few of their predicted target genes: matrix metalloproteinases (MMP-1, MMP-9), a disintegrin and metalloproteinase domains (ADAM-17, ADAM-30), tissue inhibitor of metalloproteinase 3 (TIMP-3); suppressor of cytokine signaling 1 (SOCS1); Stanniocalcin (STC2); corticotropin-releasing hormone (CRH), CRH-binding protein (CRHBP); and endothelin-2 (EDN2) were validated in these cohorts using real-time polymerase chain reaction (PCR), some displaying an inverse correlation with the expression of their predicted target genes. Functional analysis indicated that the products of these genes regulate cellular activities considered critical in normal placental functions and those affected by PC and PT labor. In conclusion, the results provide further evidence that placentas affected by PC and PT labor display an altered expression of a number of miRNAs with potential regulatory functions on the expression of specific target genes whose altered expression and function have been associated with these pregnancy complications.

Effect of transforming growth factor β on postoperative adhesion formation and intact peritoneum

Transforming growth factor beta (TGF beta) is an extremely potent chemoattractant for macrophages, mononuclear leukocytes, and fibroblasts. It also acts as a potent stimulant for collagen and fibronectin synthesis and inhibits epithelial cell growth. TGF beta plays an important role in healing many types of wounds, but its role in peritoneal adhesion formation is not known. These studies were performed to determine if TGF beta could affect postoperative wound healing in a rat model. In the first experiment, 20 rats were divided into two groups and received either 2 micrograms TGF beta or control diluent IP daily for 5 days after surgical injury to the uterine horns. The severity of the adhesions were graded 2 weeks postoperatively using a score of 0-3. The TGF beta group showed a higher adhesion score at 2 weeks compared to control, 2.9 +/- 0.34 and 1.6 +/- 0.61, respectively (P less than 0.001). On H&E stained sections of the adhesions, there was an increase in the number of both inflammatory cells and fibroblasts in the TGF beta-treated animals. A comparison trial of bone-derived TGF beta (a gift from Collagen Corporation, Palo Alto, CA) versus recombinant TGF beta (a gift from Oncogen, Seattle, WA) versus control using the same protocol as above showed that both sources of TGF beta were more effective in promoting postoperative adhesions when compared to controls, and there was no difference between TGF beta groups, 3.0 +/- 0 for both TGF beta groups, and 2.2 +/- 0.91 for control (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Retracted: differential expression of microRNAs in myometrium and leiomyomas and regulation by ovarian steroids.

Given the emerging roles of microRNAs (miRNAs) as key regulator of mRNA stability we assessed their expression profile in paired myometrium and leiomyoma, their isolated smooth muscle cells (MSMC and LSMC), a spontaneously transformed leiomyoma smooth muscle cells (T‐LSMC) and SK‐LMS‐1, a leiomyosarcoma cell line using microarray and real time PCR.Based on global normalization of expression values of 385 miRNAs and statistical analysis (anova), 91 miRNAs were expressed above the threshold levels in myometrium, with a progressive decline in numbers in leiomyomas, MSMC, LSMC, T‐LSMC and SK‐LMS‐1 (P<0.05).We selected and validated the expression of miR‐20a, miR‐21, miR‐26a, miR‐18a, miR‐206, miR‐181a and miR‐142–5p and found their differential expression in tissue and cell‐specific manners (P<0.05).Treatments of MSMC and LSMC with 17β estradiol and medroxyprogesterone acetate (10−8M), or ICI‐182780 and RU‐486 (10−6M) resulted in differential regulation of these miRNAs (P<0.05).In conclusion, the expression of a number of miRNAs in myometrium and leiomyoma with their progressive aberrant from normal MSMC into LSMC, transformed and cancerous stage, suggests that miRNAs and their regulation by ovarian steroids play a key role in pathogenesis of leiomyoma through gene expression stability.

Growth factors and ocular wound healing

Protein growth factors regulate many of the processes in vitro that are essential for the process of normal ocular wound healing, including migration, mitosis and differentiation of cells. This has led to the hypothesis that peptide growth factors play key roles in regulating normal ocular wound healing in vivo. A corollary to this concept is that insufficient action of growth factors causes impaired healing, and prolonged action of growth factors produces excessive scarring. If both of these concepts are correct, then the addition of exogenous protein growth factors should enhance healing of chronic ocular wounds and reducing prolonged actions of growth factors should limit excessive scarring. Although much remains to be understood about the role of growth factors in ocular development and wound healing, results of a substantial number of laboratory and clinical experiments indicate that these hypotheses are generally correct. This article reviews the results of pre-clinical experiments and clinical trials investigating the roles of protein growth factors in ocular development and wound healing.

MicroRNA Signature and Regulatory Functions in the Endometrium during Normal and Disease States

During the menstrual cycle, human endometrium undergoes extensive cyclic morphologic and biochemical modifications in preparation for embryo implantation. These processes are highly regulated by ovarian steroids and various locally expressed gene products and involve inflammatory reaction, apoptosis, cell proliferation, angiogenesis, differentiation (tissue formation), and tissue remodeling. MicroRNAs (miRNAs) have emerged as key regulators of gene expression, and their altered and/or aberrant expression has been associated with establishment and progression of various disorders, including tumorigenesis. This review highlights the endometrial expression of miRNAs and their potential regulatory functions under normal and pathologic conditions such as endometriosis, dysfunctional uterine bleeding, and endometrial cancer. Given the key regulatory function of miRNAs on gene expression stability, understanding the underlying mechanisms of how endometrial miRNAs are regulated and identifying their specific target genes and their functions might lead to the development of preventive and therapeutic strategies by regulating specific target genes associated with such reproductive disorders.

The Expression and Ovarian Steroid Regulation of Endometrial Micro-RNAs

Toloubeydokhti T, Pan Q, Luo X, Bukulmez O, Chegini N. The expression and ovarian steroid regulation of endometrial micro-RNAs. Reprod Sci. 2008;15:993-1001. Following an investigation by the University of Florida providing evidence of the senior (last) author’s use of falsified or fabricated data in Figures 1, 2 and 3, the above-mentioned article has been retracted. None of the other authors, Tannaz Toloubeydokhti, Qun Pan, Xiaoping Luo, and Orhan Bukulmez, were the subject of the investigation MicroRNAs (miRNAs) which regulate gene expression stability displayed an aberrant expression profile in ectopic endometrium (ECE) as compared to eutopic (EUE) and normal endometrium (NE). We assessed the expression of miR-17-5p, miR-23a, miR-23b and miR-542-3p, their predicted target genes, steroidogenic acute regulatory protein, aromatase and cyclooxygenase-2, and influence of ovarian steroids on their expression in endometrial stromal (ESC) and glandular epithelial cells (GEC). The results indicated a lower expression of miR-23b and miR-542-3p and higher level of miR-17-5p in paired ECE and EUE as compared with NE. These levels were elevated and inversely correlated with the level of expression of their respective target genes in ECE. The expression of these miRNAs and genes was differentially regulated by 17β- estradiol, medroxyprogesterone acetate, ICI-182780 and RU-486, or their respective combinations in ESC and GEC. We concluded that altered expression of specific miRNAs in ECE, affecting the stability of their target genes expression, has direct implications in pathogenesis of endometriosis.

Proinflammatory and Profibrotic Mediators: Principal Effectors of Leiomyoma Development as a Fibrotic Disorder

Leiomyomas are believed to derive from the transformation of myometrial smooth muscle cells/connective tissue fibroblasts. Although the identity of the molecule(s) that initiate such cellular transformation and orchestrate subsequent growth is still unknown, conventional evidence indicates that ovarian steroids are essential for leiomyoma growth. Ovarian steroid action in their target cell/tissue is mediated in part through local expression of various growth factors, cytokines, and chemokines. These autocrine/paracrine molecules with proinflammatory and profibrotic activities serve as major contributing factors in regulating cellular transformation, cell growth and apoptosis, angiogenesis, cellular hypertrophy, and excess tissue turnover, events central to leiomyoma growth. This review addresses the key regulatory functions of proinflammatory and profibrotic mediators and their molecular mechanisms, downstream signaling that regulates cellular events that result in transformation, and commitments of specific cells into forming a cellular environment with a possible role in development and subsequent growth of leiomyomas.

Expression of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases in mesothelial cells and their regulation by transforming growth factor-beta1.

Tissue injury and pelvic inflammation often results in peritoneal scar tissue formation. The objective of this study was to determine whether mesothelial cells which line the peritoneal cavity express matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), and if their expression is regulated by transforming growth factor‐β1, a key regulator of tissue fibrosis. For this purpose we used Met‐5A cells, a cell line derived from human normal mesothelial cells, and for comparative analysis we used U‐937 cells, a human monocytic/macrophage cell line. The cells were treated with transforming growth factor‐β1 (1 ng/ml) for various time periods and the levels of MMP and TIMP mRNA and protein expression were determined using quantitative reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. The results indicate that the mesothelial cells and macrophages express MMP‐1 (collagenase‐1), MMP‐3 (stromelysin‐1), TIMP‐1 and TIMP‐2 mRNA and protein at various levels, with significantly higher TIMPs than MMPs, and higher MMP‐1 than MMP‐3 (p < 0.001). The mesothelial cells express significantly less MMP‐1, higher MMP‐ 3 and similar levels of TIMP mRNA compared to macrophages. In a time‐dependent manner, treatment of the mesothelial cells with transforming growth factor‐β1 resulted in a significant decrease in the expression of MMP‐1, while increasing the expression of TIMP‐1 mRNA (p = 0.05). In contrast, MMP‐3 and TIMP‐2 expression was unaffected in mesothelial cells and in macrophages, compared to untreated controls. There was a significant increase in secreted MMP‐1 and TIMP‐2 by mesothelial cells following transforming growth factor‐β1 treatments in a time‐dependent manner (p = 0.05 and p = 0.01), without affecting the secretion of these proteins by macrophages. A major portion of MMP‐1 in the culture conditioned media of both cell types was found in complex with TIMP‐1. The ratios of MMP‐1/TIMPs production were significantly higher than MMP‐3/TIMPs in mesothelial cells and macrophages, and progressively decreased following transforming growth factor‐β1 treatments (p < 0.05). In conclusion, these results indicate that mesothelial cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by transforming growth factor‐β1, a mechanism that in part may influence the outcome of peritoneal tissue repair and adhesion formation.

Expression of matrix metalloproteinase-26 and tissue inhibitors of metalloproteinases TIMP-3 and -4 in benign endometrium and endometrial cancer☆

OBJECTIVE Matrix metalloproteinases (MMPs) and their physiological inhibitors, the tissue inhibitors of MMPs (TIMPs), play a key role in tumor cell invasion, angiogenesis, and growth. The aim of this study was to determine the expression and cellular distribution of MMP-26, TIMP-3, and TIMP-4 in endometrial cancers and benign endometrium throughout the menstrual cycle and the correlation with tumor histological subtype, stage, and grade. METHODS Immunohistochemical analysis using polyclonal antibodies generated against pro- and active MMP-26, and mono- and polyclonal antibodies specific to TIMP-3 and TIMP-4, respectively, was performed. RESULTS MMP-26, TIMP-3, and TIMP-4 are expressed in endometrial carcinomas (N = 86) and benign endometrium (N = 50) from various stages of the menstrual cycle. Semi-quantitative analysis of staining intensity indicated that endometrial carcinomas expressed more MMP-26, TIMP-3, and TIMP-4 compared to benign endometrium from the postmenopausal period, but not from the secretory phase of the menstrual cycle. The highest staining intensity was associated with endometrial epithelial cells, followed by vascular endothelial cells, myometrial smooth muscle cells, and endometrial stromal cells. Increased staining intensity of MMP-26 and TIMP-3 correlated with grade III tumors and MMP-26 and TIMP-4 with the depth of myometrial invasion in tumors histologically characterized as endometrioid adenocarcinoma, clear-cell, and papillary serous carcinoma staged/graded based on FIGO criteria. CONCLUSION MMP-26 and TIMP-4 are expressed in endometrium and endometrial carcinoma and their elevated expression and correlation with myometrial invasion suggests that MMP-26 and TIMP-4 may play a key role in endometrial tumor progression.

Modulation by benzo[a]pyrene of epidermal growth factor receptors, cell proliferation, and secretion of human chorionic gonadotropin in human placental cell lines

Clinical observations indicate that maternal cigarette smoking has significant detrimental effects on fetoplacental development. The present study used human trophoblastic choriocarcinoma cell lines of placental origin to investigate the effects of benz[a]pyrene (BaP) on epidermal growth factor (EGF) receptors, cell proliferation and human chorionic gonadotropin (hCG) secretion. BaP decreased 125I-EGF binding and EGF receptor protein in a concentration-related manner in both BeWo and JEG-3 cell lines. The steady-state level of EGF receptor mRNA, however, was not changed significantly by BaP in either cell line. Cell proliferation was unchanged or slightly increased following exposure to 10 and 50 microM BaP in the presence of serum, whereas proliferation progressively decreased in cells exposed under serum-free conditions. The mitogenic effect of EGF was inhibited by cotreatment with BaP in both cell lines. Further study of trophoblast endocrine function showed that both basal and EGF-stimulated secretion of hCG was reduced significantly by BaP exposure in BeWo cells, whereas no adverse effect was seen in JEG-3 cells. Finally, cytochrome P450 1A1 (CYP1A1) was induced in a concentration-dependent manner by BaP in both cell lines. Thus, data indicate that the BaP-mediated loss of EGF receptors alters trophoblast proliferation and endocrine function, and that different mechanisms may be involved in the regulation of hCG secretion in BeWo and JEG-3 cells. In addition, this study supports the feasibility of using the BeWo and JEG-3 trophoblastic choriocarcinoma cell lines to investigate biomarkers and mechanisms of placental toxicity.