Gary D. Shipley

A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.

Cultured human retinal pigment epithelial cells express basic fibroblast growth factor and its receptor

Basic fibroblast growth factor (bFGF) has been implicated in the maintenance of neuronal differentiation, the induction of neovascularization and intravitreal proliferative diseases. We have found that human retinal pigment epithelial (RPE) cells grown in vitro transcribe the bFGF gene and synthesize a peptide that crossreacts with anti-bFGF antibodies. In culture, these cells appear to release activity with biological and biochemical properties similar to bFGF. RPE cells have specific bFGF receptors and proliferate in response to bFGF. Thus, it is possible that the RPE cell is an important source of retinal bFGF and may respond to bFGF in an autocrine manner.

Cardiac Allograft Vasculopathy Association With Cell-Mediated but Not Humoral Alloimmunity to Donor-Specific Vascular Endothelium

BACKGROUND Cardiac allograft vasculopathy (CAV) is an accelerated form of coronary artery disease responsible for the majority of late deaths after cardiac transplantation. Although most consider this complication a manifestation of chronic allograft rejection, it has not been established whether this disease is a consequence of humoral or cell-mediated alloreactivity. METHODS AND RESULTS Human aortic endothelial cells (HAECs) were isolated from donor aortas obtained at the time of organ acquisition for 52 cardiac allograft recipients. Serum and peripheral blood mononuclear cells were obtained from these 52 allograft recipients at several time points during the first year after transplantation. Lymphocyte proliferation (LP) in response to donor-specific HAECs and alloantibody binding to interferon-gamma-treated donor-specific HAECs were performed and correlated with clinical parameters, including HLA matching, acute cellular rejection, and coronary artery disease on surveillance angiography. Ten of the 52 patients studied had angiographic or autopsy evidence of coronary artery disease in the first posttransplantation year (CAV+ group). The CAV+ group had higher LP responses to their donor HAECs at 1 week, 3 months, and 6 months after transplantation compared with the CAV- group (1 week: 1439 +/- 222 versus 824 +/- 141 counts per minute [cpm], P = .026; 3 months: 1282 +/- 388 versus 884 +/- 94 cpm, P = .07; 6 months: 2504 +/- 635 versus 1540 +/- 209 cpm, P = .036; CAV+ versus CAV-, respectively). Only 8 of the 52 patients had donor-specific alloantibodies, and there was no relation between antibody presence and CAV. Other clinical parameters that correlated with CAV included the level of HLA-DR mismatch and the presence of late acute rejection. CONCLUSIONS CAV is associated with donor-specific cell-mediated alloreactivity to vascular endothelium. Humoral immunity does not appear to have a major role in this disease.

Cardiac allograft vasculopathy : Preferential regulation of endothelial cell-derived mesenchymal growth factors in response to a donor-specific cell-mediated allogeneic response

We have previously reported that cell-mediated immunity to vascular endothelium is associated with the development of cardiac allograft vasculopathy (CAV). The mechanism by which a cell-mediated immune response to the coronary vascular is translated into the development of CAV is, however unknown. Peripheral blood mononuclear cells (PBMCs) obtained serially following cardiac transplantation were cocultured with donor-specific human aortic endothelial cells (HAECs) in 47 allograft recipients, 9 of whom had CAV (CAV+) at 1 year by angiography. At 20 hr following coculture, HAEC poly (A+) RNA was isolated, reverse-transcribed, and the cDNA-amplified (PCR) for a panel of growth factors (GFs) known to alter smooth muscle cell proliferation or migration. Relative quantitation of PCR product was performed using high-pressure liquid chromatography (HPLC). Three patterns of GF regulation were observed depending on the GF, the time posttransplant, and whether the patient had CAV: (1) no regulation (TGF-beta, PDGF-A early post-tx); (2) upregulation irrespective of CAV (bFGF, PDGF-B, TGF-alpha early post-tx); and (3) preferential or exclusive upregulation by CAV+ patients (PDGF-A and TGF-alpha late post-tx, HB-EGF early and late post-tx). For example, using PBMCs as stimulators, obtained 6 months posttransplant from CAV+ patients, increases in HAEC-derived PDGF-A chain (31 +/- 7 to 69 +/- 11), TGF-alpha (97 +/- 27 to 201 +/- 23), and HB-EGF (78 +/- 16 to 173 +/- 27) mRNA were demonstrated (all P<0.05 or greater using HPLC peak area as units). These data demonstrate that cell-mediated activation of vascular endothelial cells in patients with CAV results in preferential upregulation of certain endothelial-derived mesenchymal growth factors capable of stimulating smooth muscle cell proliferation and migration.

HUMAN DERMAL FIBROBLASTS EXPRESS MULTIPLE bFGF AND aFGF PROTEINS

SummaryWe investigated the regulation of expression of bFGF and aFGF in cultures of normal human dermal fibroblasts grown in a defined, serum-free medium which did not contain FGF. Under these conditions we detected three molecular weight forms of bFGF protein [18.0, 23.0, and 26.6 kiloDaltons (kD)] and three molecular weight forms of aFGF protein (18.4, 19.2, and 28.6 kD) in these cells using western blot analysis. The addition of fetal bovine serum (FBS) to these cultures caused an accumulation of all three molecular weight forms of bFGF protein with a more dramatic accumulation of the 23.0 and 26.6 kD forms. In contrast, the addition of FBS to the cultures had no effect on the level of aFGF proteins. Analysis of mRNA isolated from cells grown in serum-free medium revealed multiple species of both bFGF and aFGF RNA with molecular weights that correlated with our previous observations. The abundance of all bFGF mRNA species increased dramatically after serum treatment while the abundance of aFGF mRNA species increased only slightly. Our observations demonstrate that factor(s) present in FBS elevate the levels of bFGF mRNA and protein beyond the levels already present in the cultures growing in serum-free medium. Moreover, both bFGF and aFGF protein are present in these cells as multiple molecular weight species. Some of these forms are higher in apparent molecular weight than would be predicted from ATG-initiated primary translation products of these genes. We also show that the cells used for this study proliferate in response to bFGF and aFGF, thus, it is possible that the growth of these cells could be subject to autocrine/paracrine control in certain conditions.

INHIBITION OF AUTONOMOUS HUMAN KERATINOCYTE PROLIFERATION AND AMPHIREGULIN MITOGENIC ACTIVITY BY SULFATED POLYSACCHARIDES

SummaryWe previously demonstrated that human keratinocyte cultures proliferate in the absence of polypeptide growth factors (autonomous growth) and that this autonomous growth is blocked by interaction of heparin with a human keratinocyte-derived autocrine factor (KAF) which we identified as amphiregulin (AR). In the present study, we demonstrate that sulfated polysaccharides other than heparin (low and high molecular weight dextran sulfates) also inhibit the AR-mediated autonomous proliferation of human keratinocytes. Furthermore, sulfated polysaccharides such as high and low molecular weight dextran sulfates, heparan sulfate and, to a lesser extent, chondroitin sulfates B and C were also shown to be inhibitors of human keratinocyte-derived AR (k-d AR)-stimulated DNA synthesis in quiescent murine AKR-2B cell cultures. Our results demonstrate that sulfation of polysaccharides is required for AR inhibitory activity, and that several sulfated polysaccharides (other than heparin) can act as inhibitors of AR-mediated autonomous proliferation in human epidermal keratinocytes and as inhibitors of k-d AR-mediated mitogenic activity in AKR-2B cells.

The pattern of cytokine messenger RNA expression in human aortic endothelial cells is different from that of human umbilical vein endothelial cells.

Endothelial cells most readily available and most frequently used in investigations of alloimmunity and cytokine expression and function are derived from human umbilical veins. It is unclear whether cells derived from fetal venous tissue are relevant to phenomena related to the adult allograft, especially in areas such as cardiac allograft vasculopathy, a chronic rejection process directed against the coronary arteries. Human aortic endothelial cells (HAECs) were compared to human umbilical vein endothelial cells (HUVECs) for their constitutive expression of poly (A)+ RNA coding for a group of cytokines known to stimulate smooth muscle cell proliferation, including acidic fibroblast growth factor, basic fibroblast growth factor, transforming growth factor alpha, transforming growth factor beta, platelet-derived growth factor A-chain, platelet-derived growth factor B-chain and amphiregulin. Poly (A)+ RNA coding for basic fibroblast growth factor and transforming factor-beta was consistently expressed by all nine isolates of HAECs, but platelet-derived growth factor A- and B-chain were expressed in only six of the nine isolates. In most cases this was related to the presence of transforming growth factor alpha expression. In contrast, HUVECs consistently expressed basic fibroblast growth factor, transforming growth factor-beta, and both platelet-derived growth factor chains. Transforming growth factor alpha expression was never seen in the HUVEC isolates. No endothelial cell isolate expressed mRNA coding for acidic fibroblast growth factor or amphiregulin. There appear to be differences between cytokine gene expression patterns by endothelial cells from different vascular beds.

A serum-free [3H]thymidine incorporation assay for the detection of transforming growth factors

A rapid mitogenic assay suitable for the detection of transforming growth factors in the extracts of tissues or cells or in the medium conditioned by tumor cells in vitro is described. The method utilizes a nontumorigenic mouse embryo cell line (AKR-2B cells) maintained in serum-free conditions. Three classes of growth factors can be distinguished using this assay.

Stimulation of arachidonic acid release and eicosanoid biosynthesis in an interleukin 2-dependent T cell line

Previous studies have provided pharmacologic evidence that T lymphocyte function may be regulated in part by the intracellular production of various arachidonic acid (AA) metabolites in response to cellular stimulation. However, the specific AA metabolic capabilities of homogeneous T cell populations have not been clearly defined. In the present studies, we have employed an accessory cell-free T cell line, HT-2, as a model system for the examination of stimulus-induced eicosanoid biosynthesis in T lymphocytes. HT-2 cells were biosynthetically labeled with [3H]-AA and challenged briefly with various agents that stimulate the hydrolytic release of AA from cellular phospholipids. The bee venom peptide melittin stimulated a profound AA release response in the cells and the concomitant synthesis of both cyclooxygenase (PGF2 alpha, PGE2 and PGD2) and lipoxygenase (5-,12-,15-HETE and possibly 5-,12-diHETE) metabolites of AA. The formation of PGs was blocked by 5 microM indomethacin, demonstrating that this cell line contains cyclooxygenase activity functionally similar to that described in macrophages and other cell types. The high activity of melittin in this system was shown to result largely from a synergy between the peptide itself and a persistent bee venom phospholipase A2 contaminant. However, experiments with melittin freed of detectable phospholipase A2 activity by heating, and with synthetic homopolymers of (L)-lysine and (L)-arginine demonstrated that HT-2 cells contain sufficient endogenous, stimulus-responsive phospholipase A2 to provide both the cyclooxygenase and lipoxygenase pathways of AA metabolism ith substrate. In contrast, Ca++ ionophores, which are known to stimulate AA release and metabolism in certain cell types, stimulated only AA release but no detectable eicosanoid biosynthesis in HT-2 cells. Experiments with exogenous bacterial phospholipase C suggested that this cell line can also generate free AA for eicosanoid biosynthesis from membrane-derived 1,2-diacylglycerol. These results indicate that multiple intracellular pathways of AA metabolism are present HT-2 cells, and that the stimulus-induced release of AA and the production of eicosanoid second messengers may result from activation of either phospholipase A2 or phospholipase C.

3 – Transforming Growth Factors

The term transforming growth factor (TGF) has been applied to peptides that have the ability to confer the transformed phenotype on untransformed fibroblastic indicator cells in vitro. Peptides representing two distinct classes of TGFs have been purified to homogeneity. Type alpha and type beta TGFs are distinguished both chemically by their unique amino acid sequences and biologically by their different activities on cells. Type alpha TGFs are single chain peptides of 50-53 amino acids cross-linked by three disulphide bonds. They have strong homology to epidermal growth factor with which they compete for receptor binding. Type beta TGFs have a homodimeric structure comprised of two chains of 112 amino acids, each containing nine cysteine residues; TGF beta binds to a unique cell surface receptor. Type alpha TGFs are usually mitogenic for fibroblasts, whereas type beta TGFs have bifunctional effects on cell growth and can either stimulate or inhibit growth of the same cells, depending on conditions. The interactions of type alpha and beta TGFs can be either synergistic or antagonistic. Though the development of peptide antagonists to type alpha TGFs may have therapeutic potential for the treatment of malignancy, type beta TGFs may inhibit tumorigenesis.