Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints.

The synovium from patients with rheumatoid arthritis (RA) and LEW/N rats with streptococcal cell wall (SCW) arthritis, an experimental model resembling RA, is characterized by massive proliferation of synovial connective tissues and invasive destruction of periarticular bone and cartilage. Since heparin binding growth factor (HBGF)-1, the precursor of acidic fibroblast growth factor (FGF), is a potent angiogenic polypeptide and mitogen for mesenchymal cells, we sought evidence that it was involved in the synovial pathology of RA and SCW arthritis. HBGF-1 mRNA was detected in RA synovium using the polymerase chain reaction technique, and its product was immunolocalized intracellularly in both RA and osteoarthritis (OA) synovium. HBGF-1 staining was more extensive and intense in synovium of RA patients than OA and correlated with the extent and intensity of synovial mononuclear cell infiltration. HBGF-1 staining also correlated with c-Fos protein staining. In SCW arthritis, HBGF-1 immunostaining was noted in bone marrow, bone, cartilage, synovium, ligamentous and tendinous structures, as well as various dermal structures and developed early in both T-cell competent and incompetent rats. Persistent high level immunostaining of HBGF-1 was only noted in T-cell competent rats like the disease process in general. These observations implicate HBGF-1 in a multitude of biological functions in inflammatory joint diseases.

Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence.

Heparin-binding growth factor-1 (HBGF-1) is an angiogenic polypeptide mitogen for mesoderm- and neuroectoderm-derived cells in vitro and remains biologically active after truncation of the amino-terminal domain (HBGF-1 alpha) of the HBGF-1 beta precursor. Polymerase chain reaction mutagenesis and prokaryotic expression systems were used to prepare a mutant of HBGF-1 alpha lacking a putative nuclear translocation sequence (amino acid residues 21 to 27; HBGF-1U). Although HBGF-1U retains its ability to bind to heparin, HBGF-1U fails to induce DNA synthesis and cell proliferation at concentrations sufficient to induce intracellular receptor-mediated tyrosine phosphorylation and c-fos expression. Attachment of the nuclear translocation sequence from yeast histone 2B at the amino terminus of HBGF-1U yields a chimeric polypeptide (HBGF-1U2) with mitogenic activity in vitro and indicates that nuclear translocation is important for this biological response.

Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model.

Although matrix metalloproteinases (MMPs) are expressed in abundance in arterial aneurysms, their contribution to arterial wall degeneration, dilation, and rupture has not been determined. We investigated MMP function in a rat model of aneurysm associated with arterial dilation, elastin loss, medial invasion by mononuclear inflammatory cells, and MMP upregulation. Rupture was correlated with increased gelatinase B (MMP-9) and activated gelatinase A (MMP-2). Syngeneic rat smooth muscle cells retrovirally transfected with tissue inhibitor of matrix metalloproteinases (TIMP)-1 cDNA (LTSN) or with the vector alone as a control (LXSN) were seeded onto the luminal surface of the vessels. The seeding of LTSN cells resulted in TIMP-1 local overexpression. The seeding with LTSN cells, but not LXSN cells, decreased MMP-9, activated MMP-2 and 28-kD caseinase and elastase activity, preserved elastin in the media, and prevented aneurysmal degeneration and rupture. We conclude that MMP overexpression is responsible for aneurysmal degeneration and rupture in this rat model and that local pharmacological blockade might be a reasonable strategy for controlling the formation of aneurysms in humans.

Angiogenesis-directed implantation of genetically modified endothelial cells in mice.

By virtue of their location within blood vessels and their ability to express foreign genes, endothelial cells are attractive vehicles for the delivery of therapeutic molecules in vivo. We wished to determine whether i.v.-injected, genetically modified endothelial cells can become incorporated into sites of active angiogenesis in vivo. To do so, we studied the fate of i.v.-injected, lacZ-expressing human umbilical vein endothelial cells in athymic nude mice bearing lethally irradiated NIH 3T3 murine fibroblast cells transfected with a sp-hst/KS3:fibroblast growth factor-1 chimera that forces the secretion of the angiogenic protein, fibroblast growth factor-1. Following i.v. injection, lacZ-labeled human umbilical vein endothelial cells accumulated at sites of fibroblast growth factor-1-induced angiogenesis, persisting for at least 4 weeks. These results suggest that i.v.-administered, genetically modified endothelial cells can migrate into and survive within an angiogenic site. This strategy may be useful for delivery of therapeutic molecules to sites of pathological angiogenesis during tumor metastasis.

Differential expression in Escherichia coli of the alpha and beta forms of heparin-binding acidic fibroblast growth factor-1: potential role of RNA secondary structure.

Synthetic DNA fragments encoding the entire open-reading frame of human heparin-binding growth factor-1 (HBGF-1 beta) and its NH2-terminal truncated form (HBGF-1 alpha) were constructed. When both constructs were expressed in Escherichia coli under control of the trp-lac promoter, biologically active HBGF-1 alpha, but not HBGF-1 beta was produced in high yield. However, high level expression of HBGF-1 beta was obtained using the T7 polymerase expression vector. Computer analysis of HBGF-1 beta predicts the potential for the formation of exaggerated RNA secondary structure near the translation initiation codon and this could be implicated in contributing to the poor translation of HBGF-1 beta under the trp-lac promoter.

Cloning and characterization of a cDNA encoding the baboon tissue inhibitor of matrix metalloproteinase-1 (TIMP-1).

A baboon aortic smooth muscle cell (SMC) cDNA library was screened for the presence of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) by polymerase chain reaction (PCR); oligodeoxyribonucleotide primers corresponding to the coding frame of the known human TIMP-1 gene were used as primers. Sequencing of the PCR-amplified baboon cDNA demonstrated only eight single-nucleotide (nt) mismatches, when compared with the coding frame of human TIMP-1. The authenticity of the PCR-amplified TIMP-1 cDNA was further confirmed by clonal screening of the library with the PCR probe and sequencing of positive clones. On Northern blots from cultured baboon SMC, the baboon cDNA hybridized to a TIMP-1-specific mRNA of 800 bp. Phorbol ester (PMA) treatment of cultured baboon SMC produced a 2.5-fold increase in TIMP-1 transcript. TIMP-1 transcripts were also demonstrated in cultures of endothelial cells and fibroblasts obtained from baboon arteries. Immunohistochemical analysis demonstrated that TIMP-1 protein is localized to the adventitial layer of baboon artery. We conclude that TIMP-1 is a conserved molecule across species and localized to the tunica adventitia of baboon vessels.

Binding of tissue-specific factors to the enhancer sequence of hepatitis B virus.

We have identified tissue-specific factors, in human hepatoma cells, that bind specifically to the transcriptional enhancer sequence of the human hepatitis B virus (HBV). Two different types of protein factor were found in nuclear extracts of hepatoma cells by gel mobility shift assay. One factor was observed in human hepatoma cells but not in human kidney, lung, or vein cells, or in embryonic mouse cells. The other was discovered in both human hepatoma cells and human vein cells. DNase I footprint analysis, using the enhancer fragment (164 bp, AccI-SphI) from HBV, revealed that two specific sites are recognized by the nuclear factors. These sites contain consensus octamer sequences which have been found in many other enhancer elements. These results strongly suggest that the two nuclear factors found in hepatoma cells play key roles in the function of the HBV enhancer.

Generating antibodies against secreted proteins using vascular smooth muscle cells transduced with replication-defective retrovirus.

The traditional method of antibody (Ab) generation requires repeated injections of antigen (Ag). We have developed an alternative method that allows an investigator to generate a polyclonal antiserum with only a cDNA in hand. We cloned a cDNA encoding the coding frame for baboon tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Fischer rat arterial smooth muscle cells (SMC) transduced with the baboon TIMP-1 using a replication-defective retrovirus were propagated in culture. TIMP-1 overexpressing rat SMC were seeded into de-endothelialized rat carotid arteries. Three weeks after cell seeding in the rat, the presence of Ab to the baboon TIMP-1 was detected by dot blot and enzyme-linked immunosorbent assay in 5 of 6 of the animals. The major portion of the Ab generated against baboon TIMP-1 during the 12-month monitoring period after the cell seeding was identified as belonging to the IgG1 subtype. More interestingly, the titer of the Ab kept rising throughout an 8-month monitoring period. Among the salient features of this Ab are its capacity to block TIMP-1 activity and its utility for detecting TIMP-1 by immunohistochemistry. These results demonstrate that Ab against a secreted protein can be obtained in response to continuous expression of the cDNA by vascular SMC. Purified Ag is not required.

Long-term alcohol consumption increases matrix metalloproteinase-2 activity in rat aorta.

Altered degradation of extracellular matrix (ECM) underlies vascular remodeling, a hallmark in the pathogenesis of cardiovascular diseases including hypertension and aneurysmal dilatation. Although alcohol is recognized as a risk factor for certain cardiovascular disease states, its role in vascular remodeling has not been completely explored. We studied the effect of chronic alcohol consumption on upregulation of the enzymatic activity of matrix metalloproteinase-2 (MMP-2) as a possible pathway for large vessel remodeling. For this purpose, female rats were placed on one of three diets: a modified Lieber-DeCarli liquid diet containing 35% ethanol-derived calories, a pair-fed liquid diet with ethanol replaced by isocaloric maltose-dextrin, or a standard rat pellet. Weekly blood alcohol concentration averaged 117+/-7.9 mg/dl for the alcohol-fed rats. At 2, 4, and 72 weeks, aortas were removed and processed for measuring MMPs activity by gelatin zymography. Aortic extracts from rats on long-term (72 weeks), but not the short-term (2 and 4 weeks), alcohol diets showed increased MMP-2 activity. Furthermore, histochemical analysis of the aortas showed distinct disruption of the elastic fibers only in the 72 weeks alcohol-fed rats, compared to the control animals. These observations demonstrate that long-term alcohol consumption up-regulates MMP-2 activity, which is coincident with the alteration of aortic ECM composition through the degradation of vascular elastin components.

Elevated 80K-H protein in breast cancer: a role for FGF-1 stimulation of 80K-H.

An increase in fibroblast growth factor-1 (FGF-1) is established as part of the cause of several important cancers including breast cancer, but the mechanisms by which it induces malignant behavior are not known. We now report that the protein 80K-H, a substrate for PKC, appears to be part of this mechanism and that it is increased in breast cancer and localizes to the nucleus as part of the mechanism. Our conclusion is based on an examination of a total of 58 biopsy specimens from human breast cancer patients for the presence of relationships between the 80K-H protein and the following: fibroblast growth factor receptor-1 (FGFR-1), tumor grade, microvessel counts (MVC), estrogen receptor (ER) and progesterone receptor (PgR) status. Based on histological grading and immunohistochemical (IHC) assays, we found strong direct relationships between 80K-H and FGFR-1 (r = 0.49, p = 0.003) and tumor grade (r = 0.42, p = 0.006). A trend for a direct relationship was observed with PgR (r=0.27, p=0.087). Notably, 80K-H immunostaining was largely limited to the epithelial cells of the mammary ducts. Subsequently, we studied the effects of FGF-1 on 80K-H in cultured human mammary carcinoma epithelial cells in order to establish a more direct relationship between these two molecules. We observed that FGF-1 treatment of MCF-7 cells stimulated translocation of 80K-H protein to the cell nucleus, as demonstrated by subcellular fractionation studies. Maximal intranuclear 80K-H was observed approximately 30 minutes following FGF-1 treatment. In addition, FGF-1 treatment of MCF-7 cells increased growth and invasion of MCF-7 cells, as demonstrated by cell proliferation and a modified Boyden chamber assay, respectively. Further support for 80K-H nuclearization was provided by the immunostaining of human breast cancer specimens and computer-assisted identification of a putative nuclear localization signal (NLS) near the amino terminus of 80K-H protein structure. These data support the existence of a previously unrecognized FGF-1/80K-H nuclear pathway in progression of human breast cancer and suggest that 80K-H may be useful for the assessment of breast tumor progression.

Effects of temperature-sensitive variants of the Bacillus subtilis dnaB gene on the replication of a low-copy-number plasmid.

The dnaB gene of Bacillus subtilis is involved in the initiation of DNA replication and also in the binding of the chromosomal origin to the bacterial membrane. We studied the effect of temperature-sensitive dnaB mutants (dnaB1 and dnaB19) on the replication and on the DNA-membrane binding of the plasmid pKW1, which was derived from the low-copy-number plasmid pBS2. In the dnaB19 mutant, pKW1 was not able to replicate at the restrictive temperature. In the dnaB1 mutant, however, the dimeric form of pKW1 DNA was preferentially produced as the restrictive temperature, but the replication of the monomeric form was totally blocked. We also examined the effects of the dnaB(Ts) gene on the DNA-membrane binding of both the double-stranded and single-stranded DNA from pKW1. The single-stranded DNA from pKW1 was prepared from the DNA of the phage M13 mp19, which contained the origin of replication of pKW1. In the dnaB1 mutant, pKW1 DNA in both the double-stranded and single-stranded form was released from the membrane at the restrictive temperature. On the other hand, in the dnaB19 mutant, only double-stranded DNA, and not single-stranded DNA, was released from the membrane at the restrictive temperature. These results suggest that the product of the dnaB gene has at least two domains which influence the replication of DNA and the binding of DNA to the cell membrane in separate ways.

Identification of the product of dnaB gene in Bacillus subtilis.

In order to detect the product of dnaB gene in B. subtilis, a gene which is involved in the initiation of DNA replication and the formation of the DNA-membrane complex, we synthesized an origopeptide of 15 amino acids which corresponds to a region near the carboxyl-terminal of the gene product, and raised antibody against the synthetic peptide. We have also employed a filter binding assay to measure the predicted DNA binding activity of the product of the dnaB gene, using the plasmid pUB110. The binding activity was detected after fractionation of cell lysates of B. subtilis on sucrose-density gradients. When the active fraction was prepared from a mutant which was temperature-sensitive for the dnaB gene, the DNA binding activity in the fraction showed significant thermolability. Furthermore, the binding activity was inhibited by the purified antibody raised against the synthetic peptide. These results suggest that the product of the dnaB gene does indeed have DNA binding activity, and that the filter binding assay and the antibody can be used for the detection and characterization of the gene product.

Effects of dnats genes on the replication of plasmids in Bacillus subtilis.

An essential region (2.3 kb) for the replication of a low-copy-number plasmid, pBS-2, has been identified and cloned into plasmid pHV60 in Bacillus subtilis. The resultant plasmid, pKW1, and two other plasmids, pC194 (medium copy number) and pTP5 (high copy number), were examined by double radio-labelling and gel electrophoresis to determine which host functions are required for their replication in B. subtilis. Replication of pKW1 requires the functions of most dna genes, in particular dnaB, C, E, F, G and H; pC194 requires only dnaG and H; and pTP5 requires dnaE, F, G and H. Thus dnaG and dnaH are required for the replication of all three plasmids tested, even though each plasmid showed a different spectrum of dependency on other host functions. Because of its greater dependence on host functions and its low copy number, pKW1 should be a useful model with which to investigate the function of host genes in the replication of DNA in B. subtilis. pKW1 should also be a useful shuttle vector for cloning of genes in B. subtilis in cases when high gene dosage might be a problem.

Overexpression of a secretory form of FGF-1 promotes MMP-1-mediated endothelial cell migration.

A coordinated interaction between fibroblast growth factors (FGFs) and matrix metalloproteinases (MMPs) is implicated in migration of microvascular endothelial cells (ECs), an early stage of angiogenesis. Specifically, we investigated microvascular ECs migration in vitro, which can be initiated by the overexpression of a secretory form of the angiogenic fibroblast growth factor-1 (FGF-1) and mediated through the enzymatic activity of matrix metalloproteinase-1 (MMP-1). MMP-1 is a member of the MMP family with a propensity for degradation of interstitial type I collagen. We stably overexpressed a chimeric FGF-1 construct composed of the FGF-4 signal-peptide gene, linked in-frame to the FGF-1 coding frame gene (sp-FGF-1), in cultured postcapillary venular ECs. The presence of the biologically active form of FGF-1 was readily detected in the conditioned medium of ECs transfected with sp-FGF-1 construct as demonstrated by DNA synthesis assay. The sp-FGF-1-, but not the plasmid vector alone-transfected ECs, exhibited an altered morphology as demonstrated by their conversion from a classic cobblestone form to a fibroblastlike shape that featured prominent neuritelike extensions. Addition of the anti-FGF receptor 1 antibody (FGFR1 Ab) reverted the transformed phenotype of sp-FGF-1 transfectants. This suggests that the resulting phenotypic transformation in sp-FGF-1 transfectants requires an uninterrupted interaction between the FGF-1 ligand and its receptor. We studied migration of cells through matrices of either highly pure collagen I or reconstituted basement membrane (matrigel) and found that sp-FGF-1-transfected cells migrated two times and six times faster than the vector control transfectants in the respective matrices. We further demonstrated that the enhanced migration rate of sp-FGF-1-transfected EC coincided with the induction of their MMP-1 mRNA level and increased enzymatic activity. The enhanced migratory activity of sp-FGF-1 could be blocked with a selective inhibitor of MMP-1. These results suggest that the multipotent FGF-1 plays a key role in the early stages of angiogenesis, by mediating MMP-1 proteolytic activity.

Plasminogen activator inhibitor type 1 and tissue inhibitor of metalloproteinases-2 increase after arterial injury in rats.

Vascular injury induced by angioplasty causes smooth muscle cells to migrate, proliferate, and form a neointima. The neointima is further enlarged by the accumulation of matrix molecules synthesized by smooth muscle cells. Smooth muscle cell migration and matrix accumulation are associated with an increase in the expression of matrix-degrading enzymes and might be regulated by the balance of protease and anti-protease activity. We have studied the inhibitors of two major classes of matrix-degrading enzymes, the plasminogen activators and the matrix metalloproteinases (MMPs) to understand better the regulation of proteolytic activity following balloon catheter injury in the rat carotid artery. At various times after injury, protease inhibitor expression was analyzed by Northern blotting, reverse zymography, immunohistochemistry, and Western blotting. During the first month after injury, we found that the expression of two proteinase inhibitors (plasminogen activator inhibitor type 1 [PAI-1] and tissue inhibitor of metalloproteinases-2 [TIMP-2]) was modulated. PAI-1 mRNA expression reached a maximum 6 hours after injury before tapering off to baseline levels by 3 days. PAI-1 activity, as measured by reverse zymography, followed the same temporal profile. PAI-1, localized by immunohistochemistry, was expressed at low levels in the media of control arteries and was increased after injury primarily in the medial smooth muscle cells. TIMP-2 mRNA levels began to increase 24 hours after injury and reached a maximum at day 7. TIMP-2 activity, measured by reverse zymography, peaked at day 3 after injury. TIMP-2 protein was increased in the intima compared with the media and adventitia at day 7 after injury. The increase of PAI-1 and TIMP-2 after injury supports the hypothesis that changes in the proteolytic balance play an important role in smooth muscle cell migration after arterial injury.

Heat shock induces the release of fibroblast growth factor 1 from NIH 3T3 cells.

Fibroblast growth factor 1 (FGF-1) is a potent angiogenic and neurotrophic factor whose structure lacks a classical signal sequence for secretion. Although the initiation of these biological activities involves the interaction between FGF-1 and cell surface receptors, the mechanism responsible for the regulation of FGF-1 secretion is unknown. We report that murine NIH 3T3 cells transfected with a synthetic gene encoding FGF-1 secrete FGF-1 into their conditioned medium in response to heat shock. The form of FGF-1 released by NIH 3T3 cells in response to increased temperature (42 degrees C, 2 hr) in vitro is not biologically active and does not associate with either heparin or the extracellular NIH 3T3 monolayer matrix. However, it was possible to derive biologically active FGF-1 from the conditioned medium of heat-shocked NIH 3T3 cell transfectants by ammonium sulfate fractionation. The form of FGF-1 exposed by ammonium sulfate fractionation is similar in size to cytosolic FGF-1 and can bind and be eluted from immobilized heparin similarly to the recombinant human FGF-1 polypeptide. Further, the release of FGF-1 by NIH 3T3 cell transfectants in response to heat shock is reduced significantly by both actinomycin D and cycloheximide. These data indicate that increased temperature may upregulate the expression of a factor responsible for the secretion of FGF-1 as a biologically inactive complex that requires an activation step to exhibit the biological activity of the extracellular polypeptide mitogen.

Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in balloon-injured carotid artery.

Endothelial cells in normal blood vessels might prevent the unscheduled proliferation of smooth muscle cells (SMCs) by the expression of cell migration and growth inhibitors. NO, a potent vasodilator, generated by endothelium-specific constitutive NO synthase (ecNOS) might be such an inhibitor. To test this hypothesis, we overexpressed human ecNOS in syngeneic rat arterial SMCs using retrovirus-mediated gene transfer. Compared with SMCs transduced with vector alone (LXSN SMCs), DNA synthesis and cell proliferation were inhibited in the ecNOS-expressing SMCs (LCNSN SMCs). Basal and stimulated (by the calcium ionophore A23187) secretion of NO and intracellular cGMP were increased in LCNSN SMCs. Nomega-Nitro-L-arginine (L-NA), an inhibitor of NO synthesis, enhanced the proliferation of LCNSN SMCs but had no effect on LXSN SMCs. LCNSN SMCs seeded onto the luminal surface of balloon-injured rat carotid arteries inhibited neointimal formation by 37% and induced marked dilatation (3-fold increase in vessel diameter) at 2 weeks compared with LXSN SMC-seeded arteries. Orally administered L-NA blocked these changes. Phosphorylation of vasodilator-stimulated phosphoprotein, which is regulated in part by NO, was elevated in LCNSN SMCs and in LCNSN SMC-seeded arteries. This study demonstrates that NO generation by ecNOS inhibits SMC proliferation in vitro and modulates vascular tone locally in vivo.

Cytotoxic activity of chimeric proteins composed of acidic fibroblast growth factor and Pseudomonas exotoxin on a variety of cell types.

Chimeric proteins composed of acidic fibroblast growth factor (acidic FGF) and several forms of Pseudomonas exotoxin (PE) that cannot bind to the PE receptor have been produced in Escherichia coli by expressing chimeric genes in which DNA encoding acidic FGF is fused to various mutant forms of PE. These acidic FGF-PE fusion proteins were found to be cytotoxic to a variety of tumor cell lines including hepatocellular (PLC/PRF/5 and HEPG2), prostatic (LNCaP), colon (HT29), and breast (MCF-7) carcinomas at concentrations of 1-70 ng/ml. The cytotoxic effects of acidic FGF-PE were FGF-receptor specific as demonstrated by competition with excess acidic FGF and by showing that acidic FGF-PE bound to the FGF receptor with the same affinity as acidic FGF. Furthermore, the cell-killing activity of acidic FGF-PE was toxin-mediated, as an acidic FGF-PE mutant, which does not possess ADP-ribosylation activity, failed to kill cells. These findings demonstrate that acidic FGF-PE is a potent cytotoxic molecule that can be targeted to FGF receptor-bearing cells. Because acidic FGF is a potent angiogenic molecule, cytotoxic acidic FGF-PE chimeras may have utility as anti-angiogenic agents. These molecules could be helpful in determining the functional role of FGF receptors in cellular processes.