Quantitative Competitive Reverse Transcription-polymerase Chain Research Articles

Expression of GABA A receptor α 1 subunit mRNA and protein in rat neocortex following photothrombotic infarction

Photothrombotic infarcts of the neocortex result in structural and functional alterations of cortical networks, including decreased GABAergic inhibition, and can generate epileptic seizures within 1 month of lesioning. In our study, we assessed the involvement and potential changes of cortical GABA A receptor (GABA AR) α 1 subunits at 1, 3, 7, and 30 days after photothrombosis. Quantitative competitive reverse transcription-polymerase chain reaction (cRT-PCR) and semi-quantitative Western blot analysis were used to investigate GABA AR α 1 subunit mRNA and protein levels in proximal and distal regions of perilesional cortex and in homotopic areas of young adult Sprague-Dawley rats. GABA AR α 1 subunit mRNA levels were decreased ipsilateral and contralateral to the infarct at 7 days, but were increased bilaterally at 30 days. GABA AR α 1 subunit protein levels revealed no significant change in neocortical areas of both hemispheres of lesioned animals compared with protein levels of sham-operated controls at 1, 3, 7, and 30 days. At 30 days, GABA AR α 1 subunit protein expression was significantly increased in lesioned animals within proximal and distal regions of perilesional cortex compared with distal neocortical areas contralaterally (Student's t-test, p < 0.05). Short- and long-term alterations of mRNA and protein levels of the GABA AR α 1 subunit ipsilateral and contralateral to the lesion may influence alterations in cell surface receptor subtype expression and GABA AR function following ischemic infarction and may be associated with formative mechanisms of poststroke epileptogenesis.

Molecular characterization of M2 and M3 muscarinic receptor expression in bladder from women with refractory idiopathic detrusor overactivity

To examine the expression of muscarinic M2 and M3 receptors in human bladder detrusor and mucosa, from controls and patients with idiopathic detrusor overactivity (IDO), as antimuscarinic agents are the primary pharmacological treatment for IDO. Biopsies from the bladder body were collected at cystoscopy from 20 women with urodynamically confirmed refractory IDO (age range 25-86 years); biopsies were also collected from 30 asymptomatic female controls (age range 32-87 years). Samples were collected into RNA extraction medium and dissected into mucosa (urothelium plus lamina propria) and detrusor. RNA was extracted and the expression of M2 and M3 receptor mRNA determined by quantitative competitive reverse transcription-polymerase chain reaction. Results were normalized to beta-actin expression in the same sample. Expression of M3 receptor mRNA, in mucosa of IDO patients (median 0.057 pg M3/100 ng total RNA; interquartile range 0.03-0.13, 12 samples), was four times (P = 0.039, Mann-Whitney) lower than from the control (median 0.22 pg M3/100 ng total RNA; 0.13-0.51, 11 samples). The expression of muscarinic M3 receptor mRNA was higher (14-35 times) in detrusor (control median 3.17; 26 samples) than in mucosa and did not change in IDO (median 2.03; 14 samples). M2 expression was not significantly different with region or with IDO. These data show that M3 muscarinic receptor mRNA expression was significantly less in mucosa from IDO patients than from age-matched controls. The role of mucosal M3 receptors is unknown at present and elucidation of this role might provide a greater understanding of the aetiology of IDO.

Impaired nonrestricted cytolytic activity in systemic lupus erythematosus: involvement of a pathway independent of Fas, tumor necrosis factor, and extracellular ATP that is associated with little detectable perforin.

To determine the cytolytic effector pathway involved in impaired generation of nonrestricted cytolytic activity in systemic lupus erythematosus (SLE). Peripheral blood mononuclear cells (PBMC) from normal subjects and SLE patients were stimulated in vitro with anti-CD3 monoclonal antibody (MAb) and interleukin-2 to promote the generation of nonrestricted cytolytic activity. On day 13 of culture, the PBMC were assayed for cytolytic activity against Fas-Daudi cells and Fas+ Jurkat cells. The effects on cytotoxicity of calcium chelation, antagonist anti-Fas MAb, tumor necrosis factor (TNF) alpha and beta, and ATP were measured. Intracellular perforin expression was determined by intracellular staining, and perforin messenger RNA levels were determined by quantitative competitive reverse transcription-polymerase chain reaction. We demonstrated the existence of a cytolytic pathway that is independent of Fas, TNF alpha, TNF beta, and ATP, but is dependent upon extracellular calcium. Despite its calcium dependence, this pathway is associated with low-to-undetectable levels of perforin. Impaired generation of nonrestricted cytolytic activity in SLE is likely due to decreased activity of this Fas-, TNF alpha-, TNF beta-, ATP-independent pathway associated with very low levels of perforin.

Effects of interferon alpha on vascular endothelial growth factor gene transcription and tumor angiogenesis.

Interferon alpha (IFN-alpha) has antiangiogenic activity, although the underlying mechanism of action is unclear. Because human neuroendocrine (NE) tumors are highly vascularized and sensitive to IFN-alpha, we investigated whether the therapeutic effects of IFN-alpha result from an inhibition of angiogenesis mediated by a decrease in vascular endothelial growth factor (VEGF) gene expression. VEGF gene and protein expression was analyzed in NE tumors by immunohistochemistry and in NE tumor cell lines by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). VEGF promoter-reporter gene constructs containing various deletions or mutations and gel shift assays were used to identify minimal promoter requirements and potential transcription factors. A xenograft nude mouse model (five mice per group) was used to determine the effect of IFN-alpha on tumor growth (NE Bon cells and pancreatic Capan-1 cells) and microvessel density. Liver metastases from eight patients with NE tumors were analyzed for microvessel density, VEGF mRNA content, and VEGF plasma levels before and after initiation of IFN-alpha therapy. NE tumors and cell lines expressed VEGF mRNA and secreted VEGF protein. In vitro, IFN-alpha decreased transcription of VEGF gene expression through an Sp1- and/or Sp3-dependent inhibition of VEGF promoter activity. Compared with vehicle treatment in mice, IFN-alpha inhibited tumor growth by 36% and reduced microvessel density from 56 (95% confidence interval [CI] = 49 to 69) to 37 per x400 Field (95% CI = 32 to 41, P =.015). Patients with NE tumors had lower VEGF plasma levels and reduced VEGF mRNA levels and microvessel density in liver metastasis biopsy material after IFN-alpha treatment. IFN-alpha confers its antitumor activity, at least in part, by its antiangiogenic activity, which results from Sp1- and/or Sp3-mediated inhibition of VEGF gene transcription.

Matrix metalloproteinase inhibition modulates fibroblast-mediated matrix contraction and collagen production in vitro.

To investigate the effect of matrix metalloproteinase (MMP) inhibition on fibroblast-mediated matrix contraction and production. Free-floating fibroblast-populated type I collagen lattices were prepared with human Tenon's capsule fibroblasts. Lattice areas were photographed and digitally analyzed to indicate the degree of lattice contraction. Quantitative competitive reverse transcription-polymerase chain reaction (QCRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to quantify mRNA and protein respectively for MMP-1, -2, and -3 by fibroblasts during lattice contraction. Gelatin zymography demonstrated activity of MMPs released into the conditioned medium of contracting lattices. Concentrations of the broad-spectrum MMP inhibitors ilomastat, CellTech (Slough, UK), and BB-94 were added to the contracting fibroblast-populated collagen lattices. Secreted C-terminal propeptide of type I collagen was measured in conditioned medium of contracting lattices by ELISA. Fibroblast proliferation in the presence of concentrations of ilomastat was measured by using the reagent water-soluble tetrazolium-1 (WST-1). During contraction of type I collagen lattices, Tenon's capsule fibroblasts expressed MMP-1, -2, and -3 mRNA and protein. Zymography demonstrated the release of four gelatinolytic species into the conditioned medium of contracting lattices (57, 72, 91, and 100 kDa). Inclusion of MMP inhibitors in the zymogram-developing buffer reduced the proteolytic activity of the detected bands. MMP inhibition (1-100 microM) significantly reduced fibroblast-mediated collagen lattice contraction (P < 0.05), and this effect was found to be reversible. Ilomastat also significantly inhibited production of collagen in a dose-dependent manner (P < 0.05). No effect on fibroblast proliferation was found in the presence of ilomastat. MMPs are produced during Tenon's capsule fibroblast-mediated collagen lattice contraction. Broad-spectrum MMP inhibition significantly reduced matrix contraction and production without cell toxicity. Future clinical use of MMP inhibitors may be possible, because MMP inhibition significantly reduces fibroblast functions associated with contractile scarring.

Angiotensin II increases the expression of lectin-like oxidize low-density lipoprotein receptor- 1 in cultured human umbilical vein endothlial cells

OBJECTIVE: To observe whether angiotensin II (AngII) influences expression of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) in cultured human umbilical vein endothlial cells(HUVECs). METHODS: Cultured HUVECs were incubated with 10(-5)mol/L approximate, equals 10(-10) mol/L AngII for 24 h or with 10(-6) mol/L AngII for various time up to 48 h. Then HUVECs LOX-1 protein expression was measured by endothlial cell enzyme linked immunosorbent assay, and mRNA level of LOX-1 was detected by quantitative competitive reverse transcription-polymerase chain reaction. RESULTS: Incubation of HUVECs with AngII for 24 h significantly increased LOX-1 protein expression (P<0.001). The increase was dependent on AngII concentration (10(-5)mol/L approximate, equals 10(-9)mol/L). LOX-1mRNA expression was also induced by AngII, and after 24 h incubation of AngII(10(-5)mol/L approximate, equals 10(-8)mol/L), LOX-1mRNA expression increased 4.43, 4.25, 2.71, 1.84 times, respectively. After treatment with 10(-6) mol/L AngII for 3 h, LOX-1 expression (protein and mRNA) was elevated (P<0.001) in HUVECs, reaching its maxium at 24 approximate, equals 36 h. CONCLUSION: AngII upregulates LOX-1 expression concentration-dependently and time-dependently in HUVECs.

Localization and functional role of hepatocyte growth factor (HGF) and its receptor c-met in the rat developing cerebral cortex

Development of the cerebral cortex is a series of precisely timed proliferative, migratory, and maturational processes. Hepatocyte growth factor (HGF) is a pleiotrophic cytokine, which plays important roles in the organogenesis and regeneration of various tissues, both during development and in the adult, due to its mitogenic, motogenic and morphogenic activities. In the present study, we examined expression and functional roles of HGF and c-Met during development of the rat cerebral cortex. Quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) revealed that expression levels of c- met and HGF mRNAs were increased in the cerebral cortex during late embryonic development and peaked at E18. Immunohistochemical analyses revealed that c-Met-immunoreactivity (IR) was localized to the preplate (PP), with weaker-IR in neuroepithelial layer (NE) at embryonic day 14 (E14). At E16, c-Met-IR was present in the cortical plate (CP) and the intermediate zone (IZ), with a weak presence in the ventricular zone (VZ). On the other hand, HGF-IR was present in NE and VZ at E14 and E16, respectively. HGF-IR appeared in cortical plate tissue from E16 onward. Double labeling immunofluorescent cytochemical studies revealed that c-Met-IR was localized both in TuJ-1-IR- and non-TuJ-1-IR-cells, purified from E18 cerebral cortex in vitro, suggesting the presence of c-Met-IR in postmitotic neurons as well as in neuroepithelial cells. c-Met-IR was strong in cell bodies and neurites shortly after in vitro culture, while at 7DIV c-Met-IR decreased in neurites and was evident in growth cones. HGF dose dependently supported neuronal survival in vitro under serum-deprived conditions. In a transwell culture chamber, HGF increased neuronal migration, and co-incubation with functional blocking antibody against HGF abrogated this motogenic effect of HGF. These lines of evidence suggest that HGF is involved in the development and maintenance of cortical neurons during differentiation, motogenesis, neuritogenesis and neuronal survival.

Expression patterns and transcript concentrations of the autosomal DAZL gene in testes of azoospermic men.

The DAZ (Deleted in AZoospermia) gene cluster on the Y chromosome is a strong candidate for the azoospermia factor. The DAZ gene was derived from an autosomal homologue, DAZL (DAZ-Like). This study was designed to assess the functional role of DAZL in human spermatogenesis. The expression patterns and mRNA transcript levels of DAZL in the testes of 17 azoospermic men were therefore examined by immunohistochemical staining and quantitative competitive reverse transcription-polymerase chain reaction. DAZL protein was expressed in the cytoplasm of primary spermatocytes and weakly in spermatogonia. It was detected in the testicular tissues of all subjects with germ cells present. The copy number of the DAZL transcript in normal spermatogenesis (n = 4), hypospermatogenesis or maturation arrest (n = 6), and Sertoli cell-only syndrome (n = 7) ranged from 1.22 x 10(6) to 1.63 x 10(6) per ng of RNA, 1.19 x 10(5) to 2.82 x 10(5) per ng of RNA and 2.83 x 10(4) to 1.23 x 10(5) per ng of RNA respectively. DAZL transcripts were lower in men with spermatogenic failure, and a significant difference was found between the three groups (P < 0.0001). This study suggests that DAZL may play an important role in the human spermatogenic processes of both mitosis and meiosis.

Keratinocyte growth factor and coeliac disease

BACKGROUNDCoeliac disease is characterised by increased epithelial renewal associated with a mucosal T cell response to gliadin. Keratinocyte growth factor (KGF) is produced by cytokine activated gut stromal cells and...

Altered Glut4 mRNA levels in specific brain areas of hyperglycemic-hyperinsulinemic rats

The insulin sensitive glucose transporter Glut4 is expressed in neurons of the brain among which those of hypothalamic nuclei. It has been proposed that this transporter might be involved in the hypothalamic glucose-insulin sensing mechanism and thus in the nervous regulation of metabolism. In order to get further insights into its putative role, Glut4 expression was analyzed by quantitative competitive reverse transcription-polymerase chain reaction, in hypothalamic nuclei of hyperglycemic-hyperinsulinemic (HG-HI) rats, a model characterized by alteration of the autonomic nervous system activity. Glut4 mRNA content was decreased in the lateral hypothalamic area (33%) and arcuate nucleus (27%) but significantly only in the former. It was unchanged in other structures. These results are in favor of an alteration of Glut4 expression by short-term hyperglycemia and hyperinsulinemia that, in turn, could affect autonomic nervous system activity.

Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma

BACKGROUND/AIMSVascular endothelial growth factor (VEGF) plays a key role in regulation of tumour associated angiogenesis. In the current study we analysed expression of VEGF and its receptors in human hepatocellular...

Role of p38 mitogen-activated protein kinase in neointimal hyperplasia after vascular injury.

p38 mitogen-activated protein kinase (MAPK) is involved in intracellular signals that regulate a variety of cellular responses during inflammation. However, the role of p38 MAPK in atherosclerosis, a chronic inflammatory disorder, remains uncertain. The aim of the present study was to examine the role of p38 MAPK in the development of neointimal hyperplasia in balloon-injured rat carotid arteries. Immunohistochemical studies indicated that p38 MAPK was rapidly activated in the majority of medial cells in injured arterial walls. Rats treated with FR167653, a selective inhibitor of p38 MAPK, at a dosage of 10 mg x kg(-1) x d(-1), had a 29.4% lower intima-to-media ratio than the untreated controls at 14 days after balloon injury (P:<0.05). The percentage of proliferating nuclear antigen-positive cells in the media at 48 hours was significantly lower in the FR167653-treated group than in the control group. Quantitative competitive reverse transcription-polymerase chain reaction analysis revealed that interleukin-1beta mRNA expression in arteries was significantly inhibited by FR167653 (to 18.1% of control, P:<0.05) at 8 hours after balloon injury. Moreover, p38 MAPK activation and interleukin-1beta production by lipopolysaccharide-stimulated vascular smooth muscle cells were inhibited by FR167653 in a concentration-dependent manner in vitro. These results indicate that p38 MAPK is activated in vascular walls after injury and promotes neointimal formation and suggest that selective inhibition of p38 MAPK may be effective in the prevention of restenosis after percutaneous transluminal coronary angioplasty.

Extracellular collagen regulates expression of transforming growth factor-beta1 gene.

A complex interrelationship exists between the extracellular matrix and cytokine signaling in articular chondrocytes. We sought to determine whether the extracellular matrix serves as a regulatory component of transforming growth factor-beta1 expression. Bovine articular chondrocytes were isolated and resuspended in alginate, yielding final extracellular protein concentrations of 0 to 1.5% (wt/vol) for type-II or type-I collagen. Cultures were maintained for 7 days in the presence or absence of transforming growth factor-beta1-supplemented medium (10 ng/ml). The amount of transforming growth factor-beta1 mRNA was examined with quantitative competitive reverse transcription-polymerase chain reaction analysis. The results indicate that exogenous transforming growth factor-beta1 stimulates endogenous transforming growth factor-beta1 mRNA expression approximately 8-fold. This effect depended on the concentration of extracellular type-II collagen. As the concentration of extracellular type-II collagen is increased, the expression of transforming growth factor-beta1 mRNA decreases in both basal and transforming growth factor-beta1-stimulated cultures. Exogenous extracellular type-I collagen also served to negatively modulate transforming growth factor-beta1 gene expression but with a different concentration profile. The results demonstrate that transforming growth factor-beta1 mRNA expression was upregulated by exogenous transforming growth factor-beta1 and was downregulated by extracellular type-I and type-II collagens. The profoundly different effects on transforming growth factor-beta1 expression by the two collagens are consistent with those reported for mammary epithelial cells and likely serve as a negative feedback mechanism to preserve tissue homeostasis.

Cytokine and chemokine expression kinetics after corneal transplantation.

Allogeneic rejection is the most common cause of corneal graft failure. The aim of this work was to establish the kinetics of cytokine and chemokine mRNA expression before and after onset of corneal graft rejection. Intracorneal cytokine and chemokine mRNA levels were investigated in the Brown Norway-->Lewis inbred rat model in which rejection onset is observed at 8/9 days after grafting in all animals. Nongrafted corneas and syngeneic (Lewis-->Lewis) corneal transplants were used as controls. Donor and recipient cornea was examined by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) for hypoxyanthine phosphoribosyltransferase (HPRT), CD3, CD25, interleukin (IL)-1beta, IL-1RA, IL2, IL-6, IL-10, interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), transforming growth factor (TGF)-beta1, and macrophage inflammatory protein (MIP)-II and by nonquantitative RT-PCR for IL4, IL-5, IL-12 p40, IL-13, TGF-beta2, monocyte chemotactic protein-1 (MCP-1), MIP-1alpha, MIP-1beta, and RANTES (for regulated upon activation normal T cell expressed and secreted). A biphasic expression of cytokine and chemokine mRNA was found after transplantation. During the early phase (days 3-9), there was an elevation of the majority of the cytokines examined, including IL-1beta, IL-6, IL-10, IL-12 p40, and MIP-II. There was no difference in cytokine expression patterns between allogeneic or syngeneic recipients at this time. In syngeneic recipients, cytokine levels reduced to pretransplant levels by day 13, whereas levels of all cytokines rose after observed rejection onset in the allografts, including TGF-beta1, TGF-beta2, and IL-1RA. The T cell-derived cytokines IL-4, IL-13, and IFN-gamma were detected only during the rejection phase in allogeneic recipients. There is an early cytokine and chemokine response to the transplantation process, evident in syngeneic and allogeneic grafts, that probably drives angiogenesis, leukocyte recruitment, and affects leukocyte functions. After an immune response has been generated, allogeneic rejection results in the expression of Th1 cytokines (IL-2, IL-12 p40, IFN-gamma), Th2 cytokines (IL-4, IL-6, IL-10, and IL-13), and antiinflammatory/Th3 cytokines (TGF-beta1/2 and IL-1RA).

Deregulated expression of promyelocytic leukemia zinc finger protein in B-cell chronic lymphocytic leukemias does not affect cyclin A expression.

The promyelocytic leukemia zinc finger (PLZF) gene encodes a transcription factor expressed in myeloid, lymphoid and CD34(+) progenitor cells. Structurally related to BCL-6, which is involved in human lymphoma, PLZF may have a role in proliferation, differentiation and survival of hematopoietic cells, that could be mediated by transcriptional repression of the cyclin A gene. Quantitative competitive reverse transcription-polymerase chain reaction was used to measure the levels of expression of PLZF and cyclin A in normal leukocyte subsets (including CD19(+) lymphocytes, n=21) and malignant B lymphocytes (including B-chronic lymphocytic leukemias [B-CLL], n=63). Results obtained with this method were confirmed by Western and Northern blot analysis. Transactivation assays were performed using an expression construct for PLZF and two cyclin A promoter luciferase reporters in an Epstein-Barr virus (EBV)-transformed B-cell line. Cyclin A expression, cell growth kinetics, and cell cycle were analysed in stable clones of the Burkitt lymphoma (BL) B-cell line DG75 with inducible expression of PLZF, generated using the tetracycline-regulated expression system. Expression of PLZF was 100-fold downregulated in 90% B-CLL (56/63) compared to normal B lymphocytes (P<0.001). B-CLL patients with the highest levels of PLZF had a poorer survival (P<0.013). In transactivation assays, PLZF inhibited the activity of the cyclin A reporters by 50%, demonstrating that PLZF can repress cyclin A expression in non-malignant B lymphocytes. However, in B-CLL patients, the level of cyclin A expression was found to be within the normal range. Altered PLZF function in B lymphoid malignancies was further corroborated in the PLZF-regulatable DG75 clones, where induction of PLZF expression did not significantly alter the levels of cyclin A expression, the cell growth kinetics, or the cell cycle phase distribution. The lower survival of patients with the highest levels of PLZF suggests that this protein may be a marker of progression in B-CLL. The absence of co-ordinated regulation of PLZF and cyclin A genes in B-CLL and in a malignant B-cell line may indicate a loss of cyclin A control by PLZF in B-CLL and other B-cell disorders. Deregulation of PLZF could thus play a role in B-cell malignancy.

Quantitative analysis of leptin mRNA using competitive reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection.

Leptin, the protein hormone product of the obese (ob) gene, functions in the regulation of appetite, energy expenditure, and reproduction in animals and humans. Since changes in the level of circulating leptin can have marked physiological consequences, it is important to be able to accurately quantify leptin gene expression. Toward this goal, we have constructed a chicken leptin RNA competitor and successfully employed it as an internal standard in the development of a quantitative-competitive reverse transcription polymerase chain reaction (QC-RT-PCR) assay for leptin mRNA. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was utilized for the separation and analysis of chicken leptin target (261 bp) and competitor (234 bp) dsDNA products from QC-RT-PCR assay samples. Leptin amplicons were separated using a DB-1 coated capillary (27 cm x 100 microm ID) at a field strength of 300 V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethyl cellulose (HPMC) in 1 x TBE (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer with 0.5 microg/mL EnhanCE fluorescent intercalating dye. Samples were diluted 1:100 with deionized water and introduced into the capillary by electrokinetic injection. QC-RT-PCR/CE-LIF was used to quantify leptin mRNA in liver and adipose tissue from 8-week-old male and female broiler chickens. This study is the first report of quantitative analysis of leptin gene expression using QC-RT-PCR/CE-LIF.

Tumor necrosis factor-alpha upregulates angiotensin II type 1 receptors on cardiac fibroblasts.

Angiotensin II (Ang II) plays an important role in post-myocardial infarction (MI) remodeling. Most Ang II effects related to remodeling involve activation of the type 1 receptor (AT(1)). Although the AT(1) receptor is upregulated on cardiac fibroblasts post-MI, little is known about the mechanisms involved in the process. Consequently, we tested whether growth factors known to be present in the remodeling heart increased AT(1) mRNA levels. Using quantitative competitive reverse transcription-polymerase chain reaction, we found that norepinephrine, endothelin, atrial natriuretic peptide, and bradykinin had no significant effect on AT(1) mRNA levels. Ang II, transforming growth factor-beta(1), and basic fibroblast growth factor reduced AT(1) mRNA levels (P<0.02). Tumor necrosis factor-alpha (TNF-alpha), however, produced a marked increase in AT(1) mRNA. After 24 hours of TNF-alpha incubation, AT(1) mRNA increased by 5-fold above control levels (P<0.01). The EC(50) for the TNF-alpha effect was 4.6 ng/mL (0.2 nmol/L). Interleukin (IL)-1beta caused a 2.4-fold increase, whereas IL-2 and IL-6 had no significant effect. Studies of TNF-alpha enhancement of AT(1) mRNA levels demonstrate that the increase was not due to a change in transcript stability. TNF-alpha treatment for 48 hours also resulted in a 3-fold increase in AT(1) surface receptor and a 2-fold increase in Ang II-induced production of inositol phosphates. The present findings provide evidence for TNF-alpha regulation of AT(1) receptor density on cardiac fibroblasts. Because TNF-alpha concentration and AT(1) receptor density increase in the myocardium after MI, these results raise the possibility that TNF-alpha modulates post-MI remodeling by enhancing Ang II effects on cardiac fibroblasts.

Antisense Inhibition of Hyaluronan Synthase-2 in Human Articular Chondrocytes Inhibits Proteoglycan Retention and Matrix Assembly

In order to define the role of cell-associated hyaluronan in cartilage matrix retention, human articular chondrocytes as well as cartilage slices were treated with phosphorothioate oligonucleotides comprised of sequence antisense to the mRNA of human HA synthase-2 (HAS-2). As a prerequisite for these studies, it was necessary to determine which HA synthase (HAS), of three separate human genes capable of synthesizing HA, designated HAS-1, HAS-2, or HAS-3, is primarily responsible for HA synthesis in human articular chondrocytes. The copy number of each HAS mRNA expressed in cultured human articular chondrocytes was determined using quantitative (competitive) reverse transcription-polymerase chain reaction (RT-PCR). Only HAS-2 and HAS-3 mRNA expression was detected. The level of HAS-2 mRNA expression was 40-fold higher than that of HAS-3. Cultures of human articular chondrocytes and cartilage tissue slices were then transfected with HAS-2-specific antisense oligonucleotides. This treatment resulted in time-dependent inhibition of HAS-2 mRNA expression, as measured by quantitative RT-PCR, and a significant loss of cell-associated HA staining. Sense and reverse HAS-2 oligonucleotides showed no effect. The consequences of reduced HA levels (due to HAS-2 antisense inhibition) were a decrease in the diameter of the cell-associated matrix and a decreased capacity to retain newly synthesized proteoglycan. These results suggest that HA synthesized by HAS-2 plays a crucial role in matrix assembly and retention by human articular chondrocytes.

Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

Matrix metalloproteinase-2 production and its binding to the matrix are increased in abdominal aortic aneurysms.

Degradation of the elastic media is a hallmark of abdominal aortic aneurysms (AAAs). We examined the expression of 2 elastolytic matrix metalloproteinases (MMPs), MMP-2 and MMP-9, in AAA aortic tissues compared with those from atherosclerotic occlusive disease (AOD) and nondiseased control tissues. Quantitative competitive reverse transcription-polymerase chain reaction and gelatin zymography showed increased MMP-9 mRNA and protein in both AAA and AOD tissues compared with those in control tissue, but there was no significant difference between AAA and AOD. In contrast, MMP-2 mRNA and protein levels were significantly higher in AAA than in AOD or control tissues. Sequential extraction of the MMPs from the aortic tissue with a physiological salt solution, 2% dimethylsulfoxide (DMSO), and 10 mol/L urea showed that large amounts of MMP-2 and MMP-9 were bound to the matrix. The most conspicuous finding was that the levels of MMP-2 were significantly elevated in the DMSO fraction in AAA tissues compared with AOD and control tissues. In addition, a large portion of MMP-2 found in the DMSO and urea fractions was in the active 62-kDa form, indicating that the precursor of MMP-2 in AAA is largely activated locally and binds to the tissue matrix tightly. By immunolocalization, MMP-9 was found to be primarily produced by macrophages and MMP-2 by mesenchymal cells. The production of MMP-2 was prominent when mesenchymal cells were surrounded by inflammatory cells, suggesting paracrine modulation of MMP-2 expression in AAAs. These observations emphasize that MMP-2 participates in the progression of AAAs by degrading aortic tissue matrix components.