Amy Willey

Virginia Commonwealth University Medical Center

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Martin F. Graham

Virginia Commonwealth University

Robert F. Diegelmann

Virginia Commonwealth University

Scott W. Walsh

Virginia Commonwealth University

Yanan Zhu

Xi'an Physical Education University

Alpha A. Fowler

Virginia Commonwealth University

Dorne Yager

Virginia Commonwealth University Medical Center

H J Sugerman

Virginia Commonwealth University

Steven Price

McGill University

Joëlla W. Adams

RTI Health Solutions

Gennady Rosenblat

Technion – Israel Institute of Technology

Cooperative Institutions

Virginia Commonwealth University Medical Center

Virginia Commonwealth University

Cairo University

Oita University

University of Richmond

Hunter Holmes McGuire VA Medical Center

Children's Hospital of Richmond at VCU

Harvard University

Technion – Israel Institute of Technology

Israel Institute

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Development of pseudointima and stenosis after transjugular intrahepatic portasystemic shunts: Characterization of cell phenotype and function

Hepatology (1998)

Arun J. Sanyal Melissa J. Contos Dorn Yager Yanan Zhu Amy Willey

The clinical utility of transjugular intrahepatic portasystemic shunts (TIPS) is frequently complicated by the ingrowth of tissue into the stent lumen, causing stent stenosis. These studies were undertaken to define the cellular and matrix components of the pseudointima, define the phenotype and function of the mesenchymal cells in the pseudointima and maintain them in culture, and to study the differences between stenotic and nonstenosed stents. A total of 35 stents were evaluated. TIPS pseudointima were examined histologically, by immunohistochemistry and in situ hybridization to determine the cellular and connective tissue constituents. Mesenchymal cells were grown from tissue within the TIPS and around it, and their phenotype was studied and compared with control smooth muscle cells and fibroblasts. Masson’s trichrome staining of histological sections demonstrated that TIPS tissue was composed of collagen and palisades of mesenchymal cells and was lined by an endothelium. Immunostaining demonstrated strong and uniform α-smooth muscle staining in TIPS mesenchymal cells and peri-TIPS cells. Type I procollagen mRNA expression was demonstrated in mesenchymal cells in and around the stent by in situ hybridization. TIPS mesenchymal cells secreted less radiolabeled fibronectin, and far more type III, relative to type I, collagen compared with peri-TIPS cells. TIPS cells also expressed high levels of type III procollagen mRNA compared with peri-TIPS cells. There was no difference between stenotic stents and nonstenosed stents with respect to clinical features, time from stenting, gross morphology, histology, presence of bile fistulae, and cell phenotype. However, smooth muscle cells (SMC) from stenotic stents demonstrated both greater cell proliferation and collagen I and III secretion compared with those from nonstenosed stents. These data demonstrate that TIPS stenosis results from an accumulation of collagen and proliferation of SMC within the stent lumen.

Masson's trichrome stain

Immunostaining

Type I collagen

10.1002/hep.510280105

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Citations (58)

Linoleic acid induces interleukin-8 production by Crohn's human intestinal smooth muscle cells via arachidonic acid metabolites

AJP Gastrointestinal and Liver Physiology (2004)

Mohammad Alzoghaibi Scott W. Walsh Amy Willey Dorne R. Yager Alpha A. Fowler

Previously we reported that linoleic acid (LA), but not oleic acid, caused a marked increase in the secretion of IL-8 by Crohn's human intestinal smooth muscle (HISM) cells. Antioxidants inhibited this response, implicating a role for oxidative stress and NF-kappaB, a transcription factor for IL-8 that is activated by oxidative stress. In this study, we examined two mechanisms whereby LA, the dietary precursor for arachidonic acid (AA), could increase the production of IL-8 via activation of AA pathways: 1) by generation of reactive oxygen species by the AA-pathway enzymes to activate NF-kappaB or 2) by AA metabolites. Normal and Crohn's HISM cells were exposed to LA, oxidizing solution (Ox), or oxidizing solution enriched with LA (OxLA). Exposure of cells to Ox or OxLA induced oxidative stress as determined by thiobarbituric acid reactive substances. In normal cells, Ox but not LA activated NF-kappaB as determined by transfection experiments and Western blot. In Crohn's cells, NF-kappaB was spontaneously activated and was not further activated by Ox or LA. In contrast, TNF-alpha markedly increased activation of NF-kappaB in both normal and Crohn's cells. These results indicated that LA did not increase IL-8 by activating NF-kappaB, so we evaluated the second mechanism of an effect of AA metabolites. In normal cells, OxLA, but not LA, markedly stimulated IL-8, whereas in Crohn's cells, both OxLA and LA stimulated IL-8. OxLA, also stimulated production of AA metabolites leukotriene B(4) (LTB(4)), PGE(2), and thromboxane B(2) (TXB(2)) by normal and Crohn's cells. To determine whether AA metabolites mediated the IL-8 response, cells were treated with OxLA plus indomethacin (Indo), a cyclooxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor. Both Indo and NDGA blocked the IL-8 response to OxLA. To determine more specifically a role for AA metabolites, AA was used. Similar to OxLA, OxAA stimulated production of IL-8 and AA metabolites. Pinane thromboxane, a selective thromboxane synthase inhibitor and receptor blocker, inhibited OxAA stimulation of TXB(2) and IL-8 in a dose-response manner. MK886, a selective 5-lipoxygenase inhibitor, inhibited OxAA stimulation of LTB(4) and IL-8 also in a dose-response manner. Analysis of specific gene products by RT-PCR demonstrated that HISM cells expressed receptors for both thromboxane and LTB(4). We conclude that AA metabolites mediated the IL-8 response to LA in HISM cells. Both cyclooxygenase and lipoxygenase pathways were involved. LA did not increase IL-8 by activating NF-kappaB, but NF-kappaB appeared to be involved, because LA increased IL-8 only in situations where NF-kappaB was activated, either spontaneously in Crohn's cells or by Ox in normal cells. We speculate that AA metabolites increased IL-8 production by enhancing NF-kappaB-dependent transcription of IL-8.

10.1152/ajpgi.00189.2003

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Citations (43)

Palmitoyl ascorbate: Selective augmentation of procollagen mRNA expression compared withL-ascorbate in human intestinal smooth muscle cells

Journal of Cellular Biochemistry (1999)

Gennady Rosenblat Amy Willey Yanan Zhu Adi Jonas Robert F. Diegelmann

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of l-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 μM. By contrast, l-ascorbate was required at 4–5 times the concentration for the same response. However, at 20 μM, both palmitoyl and l-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas l-ascorbate had no effect. Palmitoyl ascorbate and l-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or l-ascorbate on the activity of a procollagen α2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than l-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro. J. Cell. Biochem. 73:312–320, 1999. © 1999 Wiley-Liss, Inc.

Procollagen peptidase

Sodium ascorbate

10.1002/(sici)1097-4644(19990601)73:3<312::aid-jcb3>3.0.co;2-1

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Citations (10)

Corticosteroids increase procollagen gene expression, synthesis, and secretion by human intestinal smooth muscle cells

Gastroenterology (1995)

Martin F. Graham Amy Willey John W. Adams Robert F. Diegelmann

Procollagen peptidase

Northern blot

10.1016/0016-5085(95)90630-4

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Citations (39)

Palmitoyl ascorbate: Selective augmentation of procollagen mRNA expression compared with L‐ascorbate in human intestinal smooth muscle cells

Journal of Cellular Biochemistry (1999)

Gennady Rosenblat Amy Willey Yanan Zhu Adi Jonas Robert F. Diegelmann

Procollagen peptidase

Sodium ascorbate

10.1002/(sici)1097-4644(19990601)73:3<312::aid-jcb3>3.3.co;2-t

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Citations (1)

Glucocorticoid receptor characteristics, nuclear translocation & molecular effects in human interstinal smooth muscle (HISM) cells

Gastroenterology (1998)

M. Graham Amy Willey Yijun Zhu Jeremy Schreher Raphael J. Witorsch

10.1016/s0016-5085(98)84019-5

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Citations (0)

Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells

Journal of Cellular Physiology (1995)

Martin F. Graham Amy Willey John W. Adams Dorne Yager Robert F. Diegelmann

Abstract The role of ascorbate in the production and secretion of procollagen by human intestinal smooth muscle cells and the conditions in culture for optimal ascorbate bioefficacy were studied. Procollagen synthesis and secretion were determined by the incubation of cells with L‐[5‐ 3 H]proline, and the quantitation of radiolabelled procollagen bands in the cell layer and the culture medium by polycrylamide slab gel electrophoresis and densitometry. When cells were cultured without ascorbate in the culture medium, procollagen secretion into the medium was 75% less than in cells receiving fresh ascorbate daily. In the cell layer, in contrast, procollagen accumulation was fourfold greater in the scorbutic cells than in the ascorbate‐replete cells. These findings contrasted with those in a control line of scorbutic human dermal fibroblasts in which a 95% decrease in procollagen secretion was not associated with any procollagen accumulation in the cells. In the intestinal smooth muscle cells, the absence of ascorbate resulted in a 25 and 50% decrease in steady‐state levels of procollagen I and III mRNA, respectively, compared to a 40 and 75% decrease in fibroblasts. Heat inactivation of the serum in the culture medium augmented the promotion of procollagen secretion by ascorbate two‐ to fourfold. L‐ascorbate phosphate did not increase the activity of L‐ascorbate when replaced in medium either daily or every 4 days, and its efficacy was not augmented by serum heat inactivation. The changing of culture medium induced collagen secretion in the absence of ascorbate, but this process was markedly enhanced by ascorbate and induced a transient decrease in the steady‐state levels of both procollagen and nonprocollagen mRNAs. The predominant action of L‐ascorbate on HISM cells in vitro is to promote procollagen secretion and not procollagen synthesis. L‐ascorbate‐phosphate is not an adequate substitute for L‐ascorbate in this cell line. © 1995 Wiley‐Liss, Inc.

Procollagen peptidase

10.1002/jcp.1041620208

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Citations (27)

Redox Imbalance in Crohn's Disease Intestinal Smooth Muscle Cells Causes NF-κB-Mediated Spontaneous Interleukin-8 Secretion

Journal of Interferon & Cytokine Research (2001)

Ramesh Natarajan Shobha Ghosh Bernard Fisher Robert F. Diegelmann Amy Willey

Interleukin-8 (IL-8), a chemokine secreted by cells at injury sites, has recently been recognized as involved in the pathogenesis of Crohn's disease. However, the pathogenesis of enhanced spontaneous transcription of IL-8 by the bowel in patients with Crohn's disease is undefined. Although IL-8 is secreted primarily by neutrophils, macrophages, and endothelial and epithelial cells, we observed the involvement of mesenchymal cells in the inflammatory process. A smooth muscle cell line isolated from the ileum of a patient with Crohn's disease (CDISM) and maintained in culture exhibited spontaneous transcription and secretion of IL-8 when compared with intestinal smooth muscle cells obtained from a normal subject (NHISM). Furthermore, IL-8 transcription from CDISM cells was associated with remarkable spontaneous activation of the oxidant-sensitive transcription factor NF-κB, as assessed by transient transfection assays with an IL-8 promoter reporter construct, Western blot analysis, and electrophoretic mobility shift assays (EMSA). Finally, we report here that CDISM cells exhibit significantly altered redox balance. The antioxidant pyrrolidine dithiocarbamate (PDTC) restored the redox equilibrium by mechanisms that inhibit binding of NF-κB to its cognate site on the IL-8 promoter. These findings suggest that restoration of the redox balance could hold promise for therapeutic intervention in Crohn's disease.

Pyrrolidine dithiocarbamate

Pathogenesis

10.1089/107999001750277826

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Citations (14)

Isolation and Culture of Arterial Smooth Muscle Cells From Human Placenta

Hypertension (2004)

Courtney E. Leik Amy Willey Martin F. Graham Scott W. Walsh

A simple and economical technique was developed to isolate and culture human arterial smooth muscle cells from chorionic plate vessels. Placentas from healthy women were collected at the time of term delivery. Chorionic plate arteries were identified, excised, and cut into small pieces. An explant technique was used to grow cultures of placental arterial smooth muscle (PASM) cells. Small pieces of vessel with lumens down were placed in 100-mm culture plates and grown in Dulbecco modified eagle medium and 10% fetal bovine serum. Cells appeared from explants within 1 week and grew to confluence in approximately 4 weeks. At confluence, PASM cell cultures had a uniform cell morphology that was characterized by elongated cells in parallel rows, typical of smooth muscle cells. Smooth muscle cell phenotype was evaluated by morphology and by immunoblotting and immunofluorescence of smooth muscle myofilament proteins. All PASM cell cultures expressed α-smooth muscle actin, β-tropomyosin, and h-caldesmon. Expression was similar to that of human aortic smooth muscle cells, but not to endothelial cells or fibroblasts. PASM cells stained uniformly for α-smooth muscle actin and lacked staining for a fibroblast-specific antigen. PASM cells were evaluated for their response to inflammatory mediators, tumor necrosis factor-α, and interleukin-1β by measurement of interleukin-8 production. Cells cultured for 18 hours showed a progressive increase in interleukin-8 production with time. Treatment with inflammatory mediators increased interleukin-8 production by 3-fold as compared with media control. This technique provides a simple method to obtain normal human arterial smooth muscle cells for in vitro studies of physiology and pathophysiology.

Fetal bovine serum

10.1161/01.hyp.0000119191.33112.9c

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Citations (53)

IL-1β downregulates collagen and augments collagenase expression in human intestinal smooth muscle (HISM) cells

Gastroenterology (1995)

M.R. Graham Amy Willey Joëlla W. Adams Dorne Yager H J Sugerman

First-degree relatives

Concordance

Family aggregation

10.1016/0016-5085(95)27612-2

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Citations (53)