Biomedical subjects

C E Hart

Publications and source records attributed to C E Hart.

At least 37 records · Page 2Linked to original sources

PubMed1997

Rapid activation of PDGF-A and -B expression at sites of lung injury in asbestos-exposed rats.

The development of interstitial pulmonary fibrosis is associated with a variety of inflammatory mediators, including peptide growth factors and cytokines. In the work presented here, we have asked whether or not platelet-derived growth factor (PDGF)-A and -B genes and proteins are expressed in anatomic and temporal patterns consistent with this factor playing a role in the disease process. Using an established rat model of asbestos-induced fibroproliferative lung disease, we demonstrate elevated levels of PDGF-A and -B mRNAs in total lung RNA immediately after a single 5-h exposure to approximately 1,000 fibers/ml of chrysotile asbestos. In situ hybridization revealed the PDGF-A and -B in RNAs primarily in macrophages and bronchiolar-alveolar epithelial cells at sites of initial fiber deposition and lung injury. There was clear evidence of PDGF-A and -B mRNAs in interstitial cells as well. The pattern of in situ hybridization was entirely consistent with the appearance (established by immunohistochemistry) of PDGF-A and -B proteins by 24 h post-exposure in the same cell types. Both mRNAs and proteins remained detectable at the fiber deposition sites for almost 2 wk post-exposures. These findings are consistent with our previous studies showing increased mesenchymal cell proliferation and fibroproliferative lesions that progress at the sites where PDGF-A and -B are expressed. Although it is clear that multiple growth factors are produced simultaneously at sites of initial injury, we suggest that the PDGF isoforms could be playing a central role in the disease process based upon their potent mitogenic effects upon mesenchymal cells.

Animals↗

PubMed1997

Characterization of cementum derived growth factor as an insulin-like growth factor-I like molecule.

Cementum is the thin calcified outer layer through which tooth-root surfaces are anchored to soft periodontal connective tissues. A variety of growth factors and adhesion molecules are sequestered in the extracellular matrix of cementum, and we have purified and characterized one of the growth factors. This growth factor, the cementum derived growth factor (CGF), was purified from bovine cementum by acetic acid extraction followed by heparin affinity chromatography and HPLC using cation exchange, molecular sieve, and reverse-phase columns. NaDodSO4-polyacrylamide gel electrophoresis of purified CGF preparation revealed the presence of two major protein bands migrating with Mr 18,000-22,000 and 14,000-16,000. The latter was associated with the major part of the mitogenic activity. The activity of CGF was inhibited by antibodies to insulin-like growth factor-I (IGF-I) and IGF-I receptor. Both CGF and IGF-I were mitogenic to human gingival fibroblasts and alveolar bone cells, but the bone cells responded better to CGF than to IGF-I. The IGF-I did not bind to heparin-sepharose, while CGF bound to it and was eluted with 0.6M NaCl from heparin-sepharose columns. Heparin-sepharose 0.2M NaCl fractions of cementum extracts contained IGF-I migrating with Mr 7,500, but its mobility was not affected by N-glycosidase treatment. Western analysis using anti-IGF-I antibodies showed that CGF preparations contained cross-reacting species migrating with Mr 18,000-22,000, 14,000-16,000 and 11,000-12,000, however after treatment with N-glycosidase the Mr 18,000-22,000 component was absent. Internal amino acid sequences of six tryptic peptides of CGF were determined by microsequencing. The sequence of one 15-amino acid long peptide was the same as the receptor binding domain of IGF-I, and another 9-amino acid peptide had 78 % homology to a sequence derived from an untranslated region of sheep IGF-I exon 1. Four other peptides had no apparent homology with IGF-I. From these results we conclude that the CGF is an IGF-I like molecule.

Amino Acid Sequence↗

PubMed1997

Human papillomavirus type in anal epithelial lesions is influenced by human immunodeficiency virus.

BACKGROUND AND OBJECTIVE: Infection with human immunodeficiency virus (HIV) increases the risk for human papillomavirus (HPV)-associated genital neoplasia. Human immunodeficiency virus-infected patients also have higher rates of treatment failure and more rapid neoplastic progression. Impaired immune function does not entirely explain these clinical observations. This pilot project was designed to investigate the hypothesis that HIV infection is associated with changes in HPV type and integration within anogenital lesions that could explain the increased risk of neoplastic progression. METHODS: Anal neoplastic lesions from patients with and without HIV infection were analyzed for the presence, type, and integration status of HPV by colorimetric in situ hybridization. Tissue localization of HIV was evaluated by p24 immunohistochemistry and HIV-1 DNA polymerase chain reaction. Results for matched histology were compared for the two patient groups. RESULTS: For all lesions, the presence of high-risk HPV types and multiple HPV types was strongly associated with HIV infection (P = .003 and .0003, respectively). For lesions with matched histology there was no association of HPV integration with HIV status. Tissue localization of HIV did not significantly influence HPV type or integration. CONCLUSIONS: The presence of high-risk HPV types and multiple types within low-grade lesions may explain the increased risk of neoplastic progression in HIV patients. Colocalization of HIV and HPV does not appear to be required for this effect. There is no evidence that HPV integration is influenced by HIV infection.

Adolescent↗

PubMed1996

Analysis of human immunodeficiency virus type 1 mRNA splicing patterns during disease progression in peripheral blood mononuclear cells from infected individuals.

HIV-1 produces more than 20 mRNAs encoding the viral proteins. We have used a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) approach to determine HIV-1 transcriptional patterns during the course of viral infection in unstimulated peripheral blood mononuclear cells (PBMCs) from different patients. Several sets of PCR primers, used in parallel reactions, allowed the amplification and specific detection of almost all individual HIV-1 transcripts. We investigated the transcriptional profile in two individuals during primary acute and early chronic infection. In these individuals, HIV-1 mRNA expression was elevated at the first time points examined and declined over time. In addition, we performed a detailed study of HIV-1 expression in several individuals over a minimum of 7 years following seroconversion. We found that long-term asymptomatic individuals had undetectable or low levels of the three classes of HIV-1 transcripts (unspliced, singly spliced, and multiply spliced). Individuals who demonstrated disease progression showed either a general increase in the amount of expression of all transcripts or elevated levels of unspliced transcripts in late-stage disease. The splicing pattern in each patient was conserved over the years and differed among the different individuals. No evidence of major changes in the splicing pattern was found during disease progression within the same individual. Thus, HIV-1 transcriptional patterns are viral strain specific rather than disease stage specific. These results indicate that high-level expression of any class of HIV-1 transcripts is associated with clinical progression. Our analysis also demonstrates the importance of using more than one set of primers to evaluate HIV-1 RNA expression, since virus in patient PBMCs showed sequence heterogeneity in conserved regions.

CD4 Lymphocyte Count↗

PubMed1996

PDGF-A is required for normal murine cardiovascular development.

Several lines of evidence suggest that platelet-derived growth factor A chain (PDGF-A) is required for normal embryonic cardiovascular development. To test this directly, we introduced anti-PDGF-A neutralizing antibodies into mouse deciduas in utero at Embryonic Days (E) 8.5, 9.5, and 10.5. This resulted in the selective disruption of PDGF-A ligand-receptor interactions in vivo for a period of 18-24 hr and allowed us to assess both if PDGF-A is required for cardiovascular development and when it is required. Embryos collected 48 hr after antibody treatment displayed severe cardiovascular abnormalities. These included both atrial and ventricular myocardial hypertrophy, epicardial and endocardial abnormalities, and aortic dilation, among others. Although heart abnormalities were observed in embryos treated at all three ages, they were more common in embryos treated at E8.5. In contrast, only embryos treated at E10.5 exhibited significant aortic dilation. This work (1) demonstrates directly for the first time that PDGF-A is required for normal cardiovascular development, (2) identifies several processes that require PDGF-A, and (3) defines discreet developmental periods during which these PDGF-A-dependent processes require the factor.

Animals↗

PubMed1996

Selective inhibition of factor Xa is more efficient than factor VIIa-tissue factor complex blockade at facilitating coronary thrombolysis in the canine model.

OBJECTIVES: We determined the effect of adjunctive inhibition of the extrinsic coagulation pathway by factor VIIa-tissue factor complex inhibitors, DEGR VIIa and tissue factor pathway inhibitor (TFPI), and the selective factor Xa inhibitor, tick anticoagulant peptide (TAP), after thrombolytic therapy with tissue-type plasminogen activator (t-PA) in a canine model of electrically induced coronary thrombosis. BACKGROUND: Ongoing thrombin generation is considered an important component of the heightened thrombin activity associated with thrombolytic therapy and may be responsible for reperfusion failure and reocclusion. METHODS: Forty-two dogs with electrically induced coronary thrombus undergoing thrombolysis with t-PA (1 mg/kg over 20 min) were randomly assigned to one of the following adjunctive regimens: TAP (30 micrograms/kg body weight per min for 90 min, n = 10); TFPI (100 to 150 micrograms/kg per min for 90 min, n = 10); DEGR VIIa (1- to 2-mg/kg bolus, n = 10) and saline control (n = 12). The dogs were observed for 120 min after thrombolysis for reocclusion. RESULTS: All three active study agents accelerated the time to reperfusion by an average of 12 min (all p < 0.05). Duration of reflow was greatest with TAP (117 +/- 8 min, p < 0.05 compared with saline control), whereas DEGR VIIa and TFPI did not prolong the duration of reflow. Reocclusion rates were similar among control, DEGR VIIa and TFPI groups (70%, 78% and 67%, respectively). Tick anticoagulant peptide reduced the occurrence of reocclusion (0%, p < 0.05 compared with saline control). CONCLUSIONS: In this experimental model, during systematic blockade of various extrinsic coagulation pathway proteins, we demonstrated that whereas acceleration of thrombolysis occurs with factor VIIa-tissue factor complex inhibition, optimal enhancement of thrombolysis was achieved through specific factor Xa blockade.

Animals↗

PubMed1996

Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

Amino Acid Sequence↗

PubMed1995

Influence of blockade at specific levels of the coagulation cascade on restenosis in a rabbit atherosclerotic femoral artery injury model.

BACKGROUND: The relation among the coagulation cascade, its individual proteins, and the response to vascular injury is largely undefined. We have evaluated the effect of four probes that block specific levels of coagulation cascade on neointimal hyperplasia in the atherosclerotic rabbit arterial injury model. METHODS AND RESULTS: Focal femoral atherosclerosis was induced by air-desiccation injury and hypercholesterolemic diet in 48 New Zealand White rabbits, followed by balloon angioplasty. Active-site inactivated factor VIIa (DEGR-VIIa), which blocks the binding of factor VIIa to tissue factor, was administered (n = 12 arteries) by intravenous bolus (1 mg/kg) at the time of balloon angioplasty and followed by infusion of 50 micrograms.kg-1.h-1 for 3 days; for the control (n = 13 arteries), 150 U heparin was injected as bolus and followed by infusion of saline at 50 microL.kg-1.min-1. Recombinant tissue factor pathway inhibitor (TFPI), which binds factor Xa and inhibits the tissue factor-factor VIIa complex and factor Xa, was given as a 1 mg/kg bolus followed by 15 micrograms.kg-1.min-1 infusion for 3 days (n = 17 arteries). Recombinant tick anticoagulant peptide (TAP; n = 15 arteries) and hirudin (n = 14 arteries), which block factor Xa and thrombin, respectively, were administered as a 1 mg/kg bolus followed by 5 micrograms.kg-1.min-1 infusion for 3 days. These three groups had their own controls (n = 14 arteries). There were no differences among treatment groups in preangioplasty and postangioplasty minimal luminal diameter (MLD) by angiography. The mean MLD 21 days after balloon angioplasty was significantly different between control and DEGR-VIIa-treated groups (0.74 +/- 0.25 and 1.24 +/- 0.27 mm, respectively; P = .0001) and between the TFPI-treated group and others (0.88 +/- 0.21 mm for control, 0.97 +/- 0.22 mm for hirudin-treated, 0.98 +/- 0.14 mm for TAP-treated, and 1.32 +/- 0.21 mm for TFPI-treated arteries; P = .0001 by ANOVA). By quantitative histological analysis, the ratio of neointimal cross-sectional area compared with the area of internal elastic lamina in the DEGR-VIIa-treated group was significantly less than control (0.48 +/- 0.12 versus 0.67 +/- 0.12, P = .0001), and the ratio of neointimal cross-sectional area to the area demarcated by the internal elastic lamina of the TFPI-treated group was significantly reduced compared with the other groups (0.46 +/- 0.20 for TFPI-treated, 0.67 +/- 0.15 for hirudin-treated, 0.61 +/- 0.15 for TAP-treated, and 0.64 +/- 0.13 for control groups; P = .003). CONCLUSIONS: Treatment with DEGR-VIIa or TFPI for 3 days in this rabbit atherosclerotic injury model reduced angiographic restenosis and decreased neointimal hyperplasia compared with controls. These findings highlight the importance of early initiators of the extrinsic coagulation pathway, especially factor VII and tissue factor, in the response to arterial injury.

Angioplasty, Balloon↗

PubMed1995

Time course of growth factor staining in a rabbit model of traumatic tractional retinal detachment.

BACKGROUND: This study examined the relationship between growth factor expression and cellular proliferation during the evolution of traumatic tractional retinal detachment (TRD) in a rabbit model. METHODS: TRD was induced in 15 pigmented rabbits by treating the inferior retina with cryopexy and making a scleral incision superiorly. Sections from varied time points were stained in the same assay with mouse monoclonal antibodies (MAb) specific for basic fibroblastic growth factor (bFGF) and platelet-derived growth factor (PDGF-BB/AB). RESULTS: Initially, the eyes exhibited intense vitritis; discrete membranes were present at 7 days and progressed to tractional retinal detachment at 17 and 28 days, after which there was no clinical change. At 6 and 24 h, bFGF, PDGF, and proliferating cell nuclear antigen (PCNA) were not detectable in membranes or wound sites (except for PDGF-positive inflammatory cells). On days 7, 17, 28, and 52, bFGF and PDGF were readily detectable in most membranes. Cellular proliferation as detected by PCNA staining was also present on days 7, 17, and 28, but was absent by day 52 despite growth factor staining. At all times, PCNA staining, which was most intense at the wound site, showed only limited correlation with staining for either growth factor for individual cells. Müller cells stained positively for PDGF-BB/AB in 13 of the 15 TRD eyes, but in none of the normal eyes. CONCLUSIONS: Since cellular proliferation correlated incompletely with the staining for bFGF and PDGF, these growth factors may not account exclusively for cellular proliferation within the membrane. Their distribution, however, including PDGF staining of Müller cells and bFGF staining at the vitreous-membrane interface, suggests that they may have roles in the pathogenesis of TRD.

Animals↗

PubMed1995

A human chromosome 12-associated 83-kilodalton cellular protein specifically binds to the loop region of human immunodeficiency virus type 1 trans-activation response element RNA.

trans activation of human immunodeficiency virus type 1 (HIV-1) involves the viral trans-activator protein (Tat) and a cellular factor(s) encoded on human chromosome 12 (HuChr12) that targets the trans-activation response element (TAR) in the viral long terminal repeat. Because nascent TAR RNA is predicted to form a secondary structure that specifically binds cellular proteins, we investigated the composition of the TAR RNA-protein complex for HuChr12-specific proteins. UV cross-linking of TAR RNA-nuclear protein complexes formed in vitro identified an 83-kDa protein in human cells and in a human-hamster hybrid cell containing only HuChr12. The 83-kDa TAR RNA-binding protein was absent in the parental hamster cells. TAR RNA mutations that inhibited binding of the 83-kDa protein in vitro also inhibited HuChr12-dependent Tat trans activation. These TAR mutations changed the native sequence or secondary structure of the TAR loop. The TAR RNA binding activity of the 83-kDa protein also correlated with a HuChr12-dependent increase in steady-state HIV-1 RNA expression during Tat trans activation. Our results suggest that either a species-specific 83-kDa TAR RNA loop-binding protein is directly encoded on HuChr12 or a HuChr12 protein(s) induces the expression of an 83-kDa TAR-binding protein in nonprimate cells.

Animals↗

PubMed1995

Expression of basic fibroblast growth factor in synovial tissue from patients with rheumatoid arthritis and degenerative joint disease.

BACKGROUND: Recent studies have implicated polypeptide growth factors in the development of rheumatoid arthritis (RA), which is characterized by synoviocyte hyperplasia and neovascularization. One such polypeptide, basic fibroblast growth factor (bFGF), is of particular interest because of its potent mitogenic and angiogenic activities. We have previously reported that cultured human synoviocytes synthesize and bind bFGF and also proliferate in response to it (1). Recently, we found a close association between increased bFGF expression and destructive changes in arthritic joints from rats (2). Now we extend our study by detecting in vivo expression of bFGF in human synovial tissues obtained from patients with RA. EXPERIMENTAL DESIGN: Human synovial tissues from patients with RA, degenerative joint disease (DJD), and trauma were collected during joint surgery. The expression of bFGF protein and mRNA by the synovia was examined by immunolocalization, Western blot, Northern blot, and RNase protection assays. Synovium from patients with DJD and trauma was used to compare with rheumatoid synovium. Double immunostaining with cell type-specific antibodies was carried out to identify cellular sources of bFGF. RESULTS: Both polypeptide and mRNA for bFGF were detected in the synovial samples examined. Increased bFGF staining was found in synovium-cartilage interface where joint destruction occurred and in hyperplastic synoviocytes of a subset of rheumatoid synovium. Strong cytoplasmic bFGF staining was localized in the majority of mast cells and vascular cells. CONCLUSIONS: Synovial tissue from patients with RA, DJD, and trauma express bFGF. Increased bFGF staining in the hyperplastic lining synoviocytes and at the pannus-cartilage interface suggests that bFGF may play a role in synovial hyperplasia and joint destruction. Strong cytoplasmic bFGF staining found in mast cells and vascular cells indicates that these cells are the major sources of tissue bFGF.

Adult↗

PubMed1995

Characterization of human aortic smooth muscle cells expressing HPV16 E6 and E7 open reading frames.

A comparative study of human papillomavirus type 16 E6E7-transfected and normal human aortic smooth muscle cells by morphological, electron microscopic, immunofluorescent, and biochemical analyses demonstrated that the E6E7-expressing cells retained much of the phenotype of normal aortic smooth muscle cells, including expression of smooth muscle markers and appropriate growth responses to PDGF and heparin. These cells differed from normal vascular smooth muscle cells in that they had slightly altered morphology and a higher growth rate that was not due to an autocrine response to secreted PDGF, and they contained more polyribosomes than normal smooth muscle cells.

Aorta↗

PubMed1994

Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro.

Osteopontin is an Arg-Gly-Asp-containing acidic phosphoprotein recently shown to be upregulated in vascular smooth muscle during rat arterial neointima formation and in human atherosclerotic plaques. Functional studies showed that osteopontin promoted adhesion of both cultured aortic endothelial cells and aortic smooth muscle cells. Adhesion of vascular cells to osteopontin was dose dependent and half maximal when solutions containing 7 and 30 nmol/L osteopontin were used to coat wells for endothelial and smooth muscle cells, respectively. Smooth muscle cells adherent to osteopontin were spread after 60 minutes, whereas endothelial cells remained round, although flattened, at this time point but were spread at 90 minutes. Cell spreading on osteopontin was accompanied by the formation of focal adhesion plaques. A newly developed anti-osteopontin antibody completely inhibited adhesion of both cell types to osteopontin but not to fibronectin or vitronectin. In addition, the peptide GRGDSP blocked adhesion to osteopontin, suggesting that integrins mediate Arg-Gly-Asp-dependent adhesion. Indeed, an antibody against the alpha v beta 3 integrin neutralized adhesion of both endothelium and smooth muscle cells to osteopontin by approximately 50%, demonstrating that alpha v beta 3 is one osteopontin receptor on vascular cells. Osteopontin also promoted the migration of smooth muscle cells in a Boyden-type chamber, with half-maximal effects observed at 77 nmol/L osteopontin. Checkerboard analysis demonstrated that this stimulus was chemotactic in nature. Our findings suggest that osteopontin may be functionally important as an adhesive and chemotactic molecule for vascular cells, particularly when levels of osteopontin are dramatically increased, as is the case after arterial angioplasty and in atherosclerotic plaques.

Animals↗

PubMed1994

Different functions of the platelet-derived growth factor-alpha and -beta receptors for the migration and proliferation of cultured baboon smooth muscle cells.

Migration of medial smooth muscle cells (SMCs) and their proliferation in the intima contribute to thickening of injured and atherosclerotic vessels. These events have been proposed to be regulated in part by platelet-derived growth factor (PDGF). Two separate PDGF receptors have been identified, PDGF-R alpha and PDGF-R beta. To study the functions of PDGF-R alpha and PDGF-R beta in vascular SMCs, neutralizing monoclonal antibodies (mAbs) specific for each of the two receptors were used. These antibodies allowed us to evaluate the role of each receptor for PDGF-induced proliferation and migration of cultured baboon SMCs. Both PDGF-AA and PDGF-BB stimulated SMC growth, with PDGF-BB being more potent than PDGF-AA. Studies with anti-PDGF-R alpha and anti-PDGF-R beta mAbs revealed that both PDGF receptors promoted the stimulatory signals for proliferation. In contrast, PDGF-BB stimulated SMC migration, whereas PDGF-AA had no stimulatory activity on its own. Additionally, PDGF-AA was able to suppress migration induced by PDGF-BB or fibronectin in modified Boyden's chamber assay. When PDGF-BB-induced migration was separated into chemotactic and chemokinetic activities, only the chemotactic component was inhibited by PDGF-AA. The suppression of SMC migration by PDGF-AA was eliminated by anti-PDGF-R alpha mAb. In addition, PDGF-BB, in the presence of anti-PDGF-R beta, bound only to PDGF-R alpha and caused suppression of SMC migration induced by fibronectin. These results suggest that when activated by ligand binding, both PDGF-R alpha and PDGF-R beta stimulate proliferation. In contrast, only activation of PDGF-R beta stimulates migration, whereas ligand binding to PDGF-R alpha leads to inhibition of cell migration. These observations provide support for the conclusion that PDGF-R alpha and PDGF-R beta may play different roles in SMC function and may be involved in different regulatory mechanisms during vascular remodeling.

Animals↗

PubMed1994

Immunolocalization of basic fibroblast growth factor and platelet-derived growth factor-A during adjuvant arthritis in the Lewis rat.

A prerequisite in defining the role of a growth factor in a disease is knowledge of its expression kinetics during the natural course of the disease. We, therefore, used immunohistochemical and immunoblot analyses to examine tissue distribution of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF-A) during the development of destructive arthropathy in the rat adjuvant arthritis model. In normal joints, bFGF was primarily localized in endothelial cells. In inflamed joints, increased staining for bFGF was found in the invading panni, hyperplastic synovium, and thickened periosteum where bFGF was also co-localized with two cell proliferation markers. Staining for bFGF began to increase at the onset of arthritis (days 11 to 13), reached peak level on days 17 to 24, and gradually declined afterward. In contrast, PDGF-A staining did not change until day 17 and the increased staining was restricted to areas of newly formed bone. The district temporal and spatial distribution pattern of these two growth factors during the destructive arthropathy strongly suggests that they play different roles during arthritis. Although PDGF-A seems to be exclusively related to osteogenesis, bFGF may have a more extensive impact on synovial proliferation and bone destruction as well as bone formation.

Animals↗

PubMed1994

Platelet-derived growth factor ligands and receptors immunolocalized in proliferative retinal diseases.

PURPOSE: Platelet-derived growth factor (PDGF) and its receptors could contribute to the development of proliferative retinal membranes, because PDGF is angiogenic and is both mitogenic and chemotactic for retinal pigment epithelial (RPE) and glial cells, components of membranes. The authors sought to determine whether PDGF ligands and their receptors were present in proliferative retinal membranes. METHODS: To localize PDGF ligands and receptors, the authors examined normal postmortem control retinas, intact eyes with proliferative vitreoretinopathy (PVR) or proliferative diabetic retinopathy (PDR), and membranes removed by vitrectomy from patients with PVR, epimacular proliferation, PDR, or PVR with PDR of previous onset. Sections were stained with antibodies specific for each PDGF ligand and receptor, using an avidin-biotin-complex immunohistochemical technique. To correlate PDGF receptor beta (PDGFR beta) and ligand immunostaining, sections were doubled labelled with antibodies specific for either PDGF-A or PDGF-B. RESULTS: Ligands. In the normal retina and choroid, staining for the A-chain was limited to vascular cells. Only the nerve fiber layer and vessels were positive for the B-chain. In diseased tissue, PDGF-A immunoreactivity was detected as intense staining ( ) of all but one of the proliferative retinal membranes; some RPE cells were positive for PDGF-A, especially in the eye with PDR. PDGF-B was also present in many proliferative retinal membranes but not in RPE cells. Receptors. In the normal retina and choroid, both PDGFR alpha and PDGFR beta were detected only in vessels. In proliferative retinal membranes, both receptors were detected in vessels. Long strands of RPE-like cells at the edges of PVR membranes were strongly positive for PDGFR beta but negative or +/-, respectively, for PDGFR alpha. Double-label assays showed that PDGFR beta was often colocalized with each PDGF ligand, especially in pigmented cells. CONCLUSIONS: PDGF ligands and receptors are widespread in proliferative retinal membranes of different origin. Because PDGFR beta and PDGF-B were colocalized in many of the same cells, the potential for autocrine and paracrine stimulation of cell migration and growth exists. These results are consistent with a role for PDGF ligands and receptors in the pathogenesis of different proliferative retinopathies.

Adolescent↗

PubMed1993

Identification of a soluble receptor for platelet-derived growth factor in cell-conditioned medium and human plasma.

We have discovered a soluble form of the platelet-derived growth factor (PDGF) alpha receptor, designated sPDGF-R alpha, that is produced by and secreted into the conditioned medium of the human osteosarcoma cell line, MG-63. Additionally, sPDGF-R alpha activity has been detected in normal human blood plasma and serum. We have achieved partial purification of this protein by column chromatography using three different affinity matrices: anti-PDGF-R alpha monoclonal antibody (mAb) 292.15-Sepharose, PDGF-BB-Sepharose, and wheat germ agglutinin-agarose. All three matrices have been shown to purify a 90-kDa protein that is recognized by mAbs specific for the PDGF-R alpha extracellular domain. sPDGF-R alpha is capable of binding PDGF ligand in solution and can compete with cell-associated PDGF receptors for ligand binding. We provide three pieces of data suggesting that the sPDGF-R alpha is generated by proteolytic clipping of the full-length PDGF-R alpha protein. First, the conditioned medium of an expression cell line transfected with a cDNA construct designed to produce only full-length PDGF-R alpha exhibits sPDGF-R alpha activity. Second, a truncated intracellular fragment of the PDGF-R alpha, presumably representing the intracellular counterpart of the clipped sPDGF-R alpha, can be immunoprecipitated from the MG-63 osteosarcoma cell extracts using antiserum raised against an intracellular portion of PDGF-R alpha. Finally, we have been unable to detect alternative splicing in the PDGF-R alpha transcript using reverse transcription-polymerase chain reaction.

Antibodies↗

PubMed1993

Distribution of FGF-2 suggests it has a role in chick limb bud growth.

We developed and characterized antibodies specific for FGF-2 and used them to locate FGF-2 during chick embryo development. A series of micrographs demonstrated the progression of FGF-2 staining during development of the different tissues and organs. FGF-2 was present in the ectoderm covering the entire embryo, muscle cells, nervous system, neural crest cells, and mesonephros. FGF-2 was also present in the limb from initiation of budding through differentiation. The limb ectoderm and subjacent mesoderm showed the strongest immunostaining, with lower levels in the center of the bud. However, the distribution of FGF-2 positive cells in the mesoderm was not homogeneous. This heterogeneity was not due to cell cycle specific distribution of FGF-2 protein, as flow cytometric analysis showed that FGF-2-positive cells were distributed throughout the cell cycle. However, the amount of anti-FGF-2 fluorescence varied most during G1, consistent with the possibility that FGF-2 is low after M phase and increases during G1. A bioassay was used to demonstrate FGF-2 levels in the wing ectoderm were approximately 2.7-fold greater than in the mesoderm. We propose that the location of FGF-2 in the embryo is consistent with a role in epithelial-mesenchymal interactions; in the limb bud it may prevent differentiation and permit limb outgrowth and subsequent expression of patterning events.

Amino Acid Sequence↗