Amy Willey

Isolation and Culture of Arterial Smooth Muscle Cells From Human Placenta

Abstract—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 &agr;-smooth muscle actin, &bgr;-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 &agr;-smooth muscle actin and lacked staining for a fibroblast-specific antigen. PASM cells were evaluated for their response to inflammatory mediators, tumor necrosis factor-&agr;, and interleukin-1&bgr; 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.

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

BACKGROUND & AIMS Collagen synthesis by smooth muscle cells plays an important role in intestinal fibrosis. Corticosteroids inhibit collagen synthesis in fibroblasts. The aim of this study was to examine the effect of corticosteroids on the expression of collagen by human intestinal smooth muscle (HISM) cells in vitro. METHODS Collagen synthesis was determined by the sensitivity of radiolabeled protein to collagenase. Secretion was determined by polyacrylamide gel electrophoresis of radiolabeled procollagen in the medium. Procollagen messenger RNA was determined by Northern blot analysis. RESULTS Collagen synthesis by confluent HISM cells was not affected by corticosteroids at 10(-10) to 10(-5) mol/L but, in subconfluent cultures, was nonspecifically increased 50% at 10(-5) mol/L. Procollagen secretion was nonspecifically increased 60% at 10(-6) mol/L dexamethasone without any effect on the type III/I ratio. Procollagen I and III messenger RNA levels responded in a biphasic manner: a 45%-65% increase at 10(-10) mol/L and a 15% and 30% decrease at 10(-8) and 10(-6) mol/L. In fibroblasts, collagen synthesis was inhibited 85% by dexamethasone, procollagen secretion was decreased 70%, the type III/I ratio decreased from 70:1 to 18:1, and procollagen messenger RNA was inhibited 25% and 60% (types I and III). CONCLUSIONS Collagen expression by HISM cells is refractory to corticosteroids and, at certain concentrations, is augmented.

Corticosteroids repress the interleukin 1 beta-induced secretion of collagenase in human intestinal smooth muscle cells

BACKGROUND & AIMS The cytokine interleukin (IL)-1 beta induces collagenase expression and inhibits collagen expression in human intestinal smooth muscle (HISM) cells. Corticosteroids cause transrepression of certain genes, including the collagenase gene. The aim of this study was to determine if corticosteroids repress the induction of collagenase expression and the inhibition of collagen secretion by IL-1 beta in HISM cells. METHODS HISM cells were exposed to IL-1 beta (1-100 pmol/L) for 24 hours in the presence or absence of dexamethasone (10(-6) mol/L). Collagenase messenger RNA (mRNA) levels were determined by ribonuclease protection assay. Collagenase and tissue inhibitor of metalloproteinase protein secretion were determined by enzyme-linked immunosorbent assay of culture medium. Procollagen and collagen secretion were determined by polyacrylamide slab gel analysis of radiolabeled proteins in culture medium. RESULTS A 30-fold induction of collagenase mRNA and collagenase protein secretion by IL-1 beta was completely abrogated by dexamethasone. Dexamethasone at 10(-6) mol/L also reduced basal levels of collagenase mRNA by 50% and blocked the IL-1 beta-induced inhibition of collagen secretion. CONCLUSIONS Corticosteroids repress the collagenolytic action of IL-1 beta on HISM cells in vitro and therefore should promote healing in the inflamed intestine.

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

Interleukin-8 (IL-8), a chemokine secreted by cells at injury sites, has recently been recognized as involved in the pathogenesis of Crohns disease. However, the pathogenesis of enhanced spontaneous transcription of IL-8 by the bowel in patients with Crohns 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 Crohns 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-kappaB, 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-kappaB 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 Crohns disease.

Palmitoyl Ascorbate: Selective Augmentation of Procollagen mRNA Expression Compared With L-Ascorbate in Human Intestinal Smooth Muscle Cells

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.

Effects of antibodies against odorant binding proteins on electrophysiological responses to odorants

Monoclonal antibodies against two olfactory mucosal proteins, one with affinity for anisole-like and the other for benzaldehyde-like compounds, were applied to mouse olfactory epithelium. Responses to three odorants (anisole, benzaldehyde and amyl acetate) were measured. Of 26 antibodies, three (12%) inhibited responses only to the odorant with affinity for the antigen, nine (35%) inhibited responses to all three odorants, and 14 (54%) were without effect. None reduced responses by as much as 50%. The data support the hypothesis that there is a class of related proteins in olfactory neuronal cell membranes that function as receptor molecules and that other mechanisms also mediate odorant stimulation.

Benzaldehyde Binding Protein from Dog Olfactory Epitheliuma

A protein with a high affinity for o-methyl phenols, of which the odorant anisole is the simplest compound, has been extracted from dog olfactory epithelium.’ Called anisole binding protein, it was hypothesized to be a receptor for odorants of the anisole type. Further studies on it and isolation of a benzaldehyde binding protein are reported here. “Anisole” and “benzaldehyde” columns were prepared by covalently coupling panisic acid and p-carboxybenzaldehyde, respectively, to o-aminobutyl agarose via the carboxyl groups. Olfactory epithelium from dog nasal septum was extracted with 0.1 M Tris buffer, pH 7.2, and the residue was reextracted with the same buffer to which 0.1% sodium dodecyl sulfate had been added. The resulting extract was subjected to affinity chromatography on the two columns consecutively, and the anisole and benzaldehyde binding proteins were displaced from the columns by addition of 1 mMpanisic acid or p-carboxybenzaldehyde, respectively. The eluates from the columns were subjected to polyacrylamide gel slab electrophoresis? Protein on the slabs was visualized by silver ~taining.~ Rabbits were immunized with anisole or benzaldehyde binding protein, and sera from them were tested for their effects on electroolfactograms (EOGs) of sagitally bisected heads of male ICR mice? Except for the method of EOG recording and the use of slab rather than disc electrophoresis, the methods used in this study were essentially the same as those described previously.’ The electrophoretic patterns obtained from elution of the columns are shown in FIGURE 1. Nearly all the protein passes through the columns freely, eluting with Tris buffer (tracks B and E). After the buffer-elutable protein has cleared the column (tracks C and F), p-anisic acid and p-carboxybenzaldehyde displace additional protein from the anisole and benzaldehyde columns, respectively (tracks D and G). These are the anisole and benzaldehyde binding proteins. From their mobilities relative to those of the standards, their molecular masses are estimated to be about 61,000 daltons. Effects of antisera and control sera (sera from the same rabbits, taken before immunization) on EOG responses to anisole, benzaldehyde and isoamyl acetate are shown in TABLE 1. The control sera have no significant effects. The serum from the rabbit immunized with anisole binding protein abolished the response to anisole and reduced the responses to the other odorants to about one-third. Serum from the rabbit immunized with benzaldehyde binding protein reduced responses to all three odorants to about one-third.