Ujjwal K. Rout

Expression pattern and regulation of genes differ between fibroblasts of adhesion and normal human peritoneum

BackgroundInjury to the peritoneum during surgery is followed by a healing process that frequently results in the attachment of adjacent organs by a fibrous mass, referred commonly as adhesions. Because injuries to the peritoneum during surgery are inevitable, it is imperative that we understand the mechanisms of adhesion formation to prevent its occurrence. This requires thorough understanding of the molecular sequence that results in the attachment of injured peritoneum and the development of fibrous tissue. Recent data show that fibroblasts from the injured peritoneum may play a critical role in the formation of adhesion tissues. Therefore, identifying changes in gene expression pattern in the peritoneal fibroblasts during the process may provide clues to the mechanisms by which adhesion develop.MethodsIn this study, we compared expression patterns of larger number of genes in the fibroblasts isolated from adhesion and normal human peritoneum using gene filters. Contributions of TGF-beta1 and hypoxia in the altered expression of specific genes were also examined using a semiquantitative RT-PCR technique.ResultsResults show that several genes are differentially expressed between fibroblasts of normal and adhesion peritoneum and that the peritoneal fibroblast may acquire a different phenotype during adhesion formation. Genes that are differentially expressed between normal and adhesion fibroblasts encode molecules involved in cell adhesion, proliferation, differentiation, migration and factors regulating cytokines, transcription, translation and protein/vesicle trafficking.ConclusionsOur data substantiate that adhesion formation is a multigenic phenomenon and not all changes in gene expression pattern between normal and adhesion fibroblasts are the function of TGF-beta1 and hypoxia that are known to influence adhesion formation. Analysis of the gene expression data in the perspective of known functions of genes connote to additional targets that may be manipulated to inhibit adhesion development.

Transforming growth factor-β1 modulates expression of adhesion and cytoskeletal proteins in human peritoneal fibroblasts

OBJECTIVE To determine the effects of TGF-beta1 on the expression of the alpha1, alpha2, alpha5, alpha(v), and alpha6 integrin subunits and on vinculin, and F-actin in human peritoneal fibroblasts. DESIGN Descriptive study using cell culture, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescent and confocal microscopy. SETTING Academic medical center. PATIENT(S) Gynecological surgery patients. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Effects of TGF-beta1 on the steady state levels of alpha5, alpha(v), and alpha6 integrin transcripts were examined in the normal peritoneal fibroblasts using RT-PCR. Expression levels of the alpha1, alpha2, alpha5, alpha(v), and alpha6 integrin subunits and of F-actin were measured by immunofluorescent microscopy. The distribution pattern of the integrin subunits, vinculin, and F-actin were examined using confocal microscopy. RESULT(S) TGF-beta1 significantly up-regulated the expression levels of the alpha5, alpha(v), and alpha6 integrin subunits and modulated their expression pattern, resulting in relatively higher levels of these subunits in the focal contacts of peritoneal fibroblasts. It allocated vinculin expression primarily to the focal contacts of cells and caused distortion of F-actin structure. The transcript levels of the alpha5, alpha(v), and alpha6 integrin subunits were not altered by TGF-beta1 treatment. CONCLUSION(S) TGF-beta1 may promote postoperative adhesion formation by inducing the migration of peritoneal fibroblasts by altering the expression levels and patterns of specific integrin subunits, vinculin, and F-actin.

Altered expressions of VEGF mRNA splice variants during progression of uterine-peritoneal adhesions in the rat.

PROBLEM: Postoperative pelvic adhesions contribute to infertility, pelvic pain, bowel obstruction, and difficult reoperative procedures.
 METHOD OF STUDY: In the present study, a rat uterine‐peritoneal adhesion model was developed to study the progression of adhesion formation during a course of 7 days following pelvic surgery. The distal 1 cm of each uterine horn and its adjacent peritoneum was abraded by six scratches with a scalpel blade, producing punctate bleeding. The scratched portion of uterine horn and the peritoneum was then held with Vicryl 3‐0 to promote adhesion. The uterine tissue and the portion of peritoneum, held with suture, were then excised from a group of four rats, each at 6, 12, 24, 48, 72 hr and 5 and 7 days following surgery. Total RNA was isolated from these tissues and the expression pattern of different splice variants of vascular endothelial growth factors (VEGF) was examined using relative abundance reverse transcriptase polymerase chain reaction (RA‐RT‐PCR) method.
 RESULTS: Three known splice variants of VEGF mRNA (VEGF120, VEGF164 and VEGF188), as well as an additional band (∼510 bp), were amplified from these tissues. The relative abundance of known VEGF isoforms demonstrated altered expression during adhesion progression. When compared with noninjured uterine tissues, VEGF120 and VEGF188 demonstrated up‐regulation during early stages of adhesion formation, whereas VEGF164 rather demonstrated down‐regulation 24 and 48 hr following surgery.
 CONCLUSIONS: The up‐regulation of VEGF isoforms during the progression of uterine‐peritoneal adhesion may be a compensatory mechanism regulating angiogenesis in order to provide nutrients and oxygen to the injured tissues.

Ethanolminduced intracellular calcium mobilization rapidly alters gene expression in the mouse blastocyst

The induction of intracellular Ca2+ release in pre-implantation mouse embryos accelerates their subsequent rate of development in vitro through a calmodulin-dependent mechanism [Stachecki J.J., Armant D.R. Transient release of calcium from inositol 1,4,5-trisphosphate-specific stores regulates mouse pre-implantation development. Development 1996; 122: 2485-2496]. To examine the hypothesis that intracellular Ca2+ signaling alters embryonic gene expression, individual transcript levels were compared by mRNA differential display before and 1 h after intracellular Ca2+ mobilization with ethanol in mouse blastocysts. Ten up-regulated and four down-regulated genes were observed, representing 3.5% of approximately 400 transcripts that were resolved. After sequencing, most of the DNA fragments appeared to be novel; however, two amplicons that increased after Ca2+ mobilization were identified as arginase and ubiquitin conjugating enzyme (E2). The up-regulation of arginase mRNA (3.5-fold after 2 h) was confirmed by reverse transcription and the polymerase chain reaction using specific oligonucleotide primers derived from the deduced mouse embryo sequence. A corresponding 2.5-fold increase in arginase enzymatic activity peaked 9 h after ethanol exposure. Increased expression of arginase and other genes may mediate the onset of rapid cell proliferation and differentiation that is induced by Ca2+ signaling during pre-implantation development.

Role of plasminogen activators during healing after uterine serosal lesioning in the rat.

OBJECTIVE Study the expression pattern of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activators during uterine serosal healing after lesioning in a rat model. DESIGN Descriptive study with an adhesion model using reverse transcriptase-polymerase chain reaction (PCR) and immuncytochemical techniques. SETTING Academic medical center. PATIENT(S) None. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Expression profiles of t-PA and u-PA were examined in the uteroperitoneal adhesion tissues of rat from the time of injury until day 7 after surgery using relative abundance reverse transcriptase-PCR and immunocytochemical techniques. RESULT(S) The t-PA transcript levels dropped in uteroperitoneal tissues by 6 hours after surgery and remained low until day 5. Thereafter, a rapid up-regulation of this transcript was observed at day 7. In contrast, the u-PA transcript levels demonstrated a biphasic profile during the progression of adhesion development with peaks at 2 and 5 days after surgery. Immunocytochemical staining demonstrated expression of these plasminogen activators in the newly formed blood vessels. CONCLUSION(S) Altered levels of t-PA and u-PA transcripts and its expression in newly formed blood vessels during healing and adhesion development indicate involvement of these plasminogen activators in serosal healing and adhesion development.

Expression of genes for alcohol and aldehyde metabolizing enzymes in mouse oocytes and preimplantation embryos.

Alcohols and aldehydes are metabolized primarily by alcohol (ADH) and aldehyde (ALDH) dehydrogenase isozymes. Although significant progress has been made towards understanding the involvement of these isozymes in the oxidation of alcohol and aldehydes in the body, it is not known how these compounds are handled during fertilization and preimplantation embryogenesis. In this study, reverse transcription and the polymerase chain reaction (RT-PCR) was used to determine which ADH and ALDH isozymes are expressed at the oocyte, zygote, morula, and blastocyst stages of preimplantation development in the mouse. Transcripts of beta-actin and vimentin, assayed as controls, were detected at all stages, as well as Class III ADH (Adh-2) and Class 3 ALDH (Ahd-4), involved in the detoxification of formaldehyde and aromatic aldehydes, respectively. In contrast, transcripts for the major ethanol oxidizing isozyme, Class I ADH (Adh-1) was not detected during preimplantation development. Cytosolic retinol dehydrogenase (Adh-3) transcripts were marginally detected in oocytes and zygotes. The mRNA for cytosolic retinal dehydrogenase (Ahd-2), microsomal short-chain retinol dehydrogenases (RoDH Type I), and the mitochondrial low-Km acetaldehyde dehydrogenase (Ahd-5) only appeared as maternal transcripts. Microsomal ALDH (Ahd-3), which is induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was not expressed until the blastocyst stage. ADH and ALDH enzyme systems may guard mouse preimplantation embryos against the toxic effects of industrial pollutants, such as formaldehyde and TCDD, as well as peroxidatic aldehydes generated during lipid peroxidation. The absence of enzymes to convert ethanol to acetaldehyde, coupled with oocyte expression of the acetaldehyde-degrading enzyme, Ahd-5, may be protective for the early embryo.

Valproate, thalidomide and ethyl alcohol alter the migration of HTR-8/SVneo cells

BackgroundValproate, thalidomide and alcohol (ethanol) exposure during the first trimester of pregnancy is known to cause several developmental disorders. All these teratogens are known to pass the placental barrier and interfere directly with the normal development of the fetus. However, these teratogens also alter the formation and function of the placenta itself which may in turn affect the proper nourishment and development of the fetus. Optimum development of the placenta requires adequate invasion of trophoblast into the maternal uterine tissues. Changes in the migratory behavior of trophoblast by maternal exposure to these teratogens during placentogenesis may therefore alter the structure and function of the placenta.MethodsIn the present study, the effects of sodium valproate, thalidomide and alcohol on the migration of human first trimester trophoblast cell line (HTR-8/SVneo) were examined in vitro. Cells were cultured in the wells of 48-well culture plates as mono or multilayers. Circular patches of cells were removed from the center of the wells by suction, and the migration of cells into the wound was studied using microscopy. Effects of low and high concentrations of valproate, thalidomide and alcohol were examined on the healing of wounds and on the migration rate of cells by determining the wound areas at 0, 3, 6, 12, 24 and 48 h. Effects of drugs and alcohol on the proliferation and the expression levels of integrin subunits beta1 and alpha5 in cells were examined.ResultsThe migration rates of trophoblast differed between wounds created in mono and multilayers of cells. Exposure to teratogens altered the migration of trophoblast into mono and multilayer wounds. The effects of valproate, thalidomide and alcohol on the proliferation of cells during the rapid migratory phase were mild. Drug exposure caused significant changes in the expression levels of beta1 and alpha5 integrin subunits.ConclusionResults suggest that exposure to valproate, thalidomide or alcohol during the first trimester of pregnancy may change the ultrastructure of the placenta by altering the migration of trophoblast cells and this effect may be mediated by drug- or alcohol-induced changes in the expression levels of beta1 and alpha5 integrin subunits.

Alcohol dehydrogenases and aldehyde dehydrogenases among inbred strains of mice: multiplicity, development, genetic studies and metabolic roles

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the major enzymes responsible for the metabolism of alcohols and aldehydes in the body. Both exist as a family of isozymes in mammals, and have been extensively studied in animal models, particularly among inbred strains of mice. Mouse ADH exists as at least three major classes, which are predominantly localized in liver (classes I and III), and in stomach/cornea (class IV). Mouse ALDH exhibits extensive multiplicity, several forms of which have been characterized, including ALDH1 (liver cytoplasmic/class 1 isozyme); ALDH2 (liver mitochondrial/class 2.); ALDH3 (stomach cytosolic/class 3); ALDH4 (liver microsomal/class 3); and ALDH5 (testis cytosolic/class 3). Biochemical, genetic and molecular genetic analyses have been performed on several of these enzymes, including studies on variant forms of ADH and ALDH. Distinct metabolic roles are proposed, based upon their tissue and subcellular distribution characteristics and the biochemical properties for these enzymes.