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W Casscells

Publications and source records attributed to W Casscells.

At least 37 records · Page 2Linked to original sources
PubMed1993

Localization of basic fibroblast growth factor in bovine endothelial cells: immunohistochemical and biochemical studies.

To gain insight into the mechanisms of synthesis, storage, and release of basic fibroblast growth factor (bFGF), we studied the immunohistochemical localization of bFGF in bovine coronary artery, coronary sinus, and adrenal capillary endothelial cells grown in culture. Light and electron microscopic immunohistochemical studies were performed using the ABC immunoperoxidase method on p-formaldehyde-fixed cells. Five different anti-bFGF antibodies gave similar results in all cell types. In subconfluent cells, immunoreactivity was noted in the nuclear chromatin, nucleoli, cytosol, cytoplasmic vesicles (some of which appeared to fuse with the plasma membrane), and extracellular matrix. No reaction was found in endoplasmic reticulum or Golgi zones. Confluent cells demonstrated less immunoreactivity in the nuclei and cytosol but more in the extracellular matrix. Some cells of senescent morphology showed only cytoplasmic staining; however, no cells were found with only nuclear staining. Biochemical studies showed that three forms of bFGF (18, 24, and 26 kDa) were present in endothelial cells and varied with different culture conditions. Protection analysis indicated that bFGF mRNA is less abundant in postconfluent cells than in subconfluent cells. These data suggest that subconfluent cells synthesize bFGF and transport it into the nucleus and exocytotic vesicles, while confluent cells synthesize little bFGF but store it in extracellular matrix, cytoplasmic vesicles, and nuclei.

Animals↗
PubMed1993

Smooth muscle cell abundance and fibroblast growth factors in coronary lesions of patients with nonfatal unstable angina. A clue to the mechanism of transformation from the stable to the unstable clinical state.

BACKGROUND: The mechanisms responsible for the transformation of stable angina to unstable angina, a major cause of morbidity and mortality, are commonly believed to be plaque rupture and thrombosis. We determined whether additional mechanisms are operative by analyzing the histopathology and immuno-histopathology of coronary plaques retrieved by directional atherectomy of patients with unstable angina in whom no intraluminal thrombus was demonstrated by angiography. METHODS AND RESULTS: The histological findings of atherectomy specimens from 34 patients with unstable angina were compared with those of 24 patients with postangioplasty restenosis, whose lesions are known to be composed of smooth muscle cells (SMCs), and 10 patients with stable angina, whose lesions contain relatively few SMCs. We also studied the expression of acidic and basic fibroblast growth factors (aFGF and bFGF), whose role in the vascular response to injury has been established. Specimens from unstable angina resembled those from postangioplasty restenosis in regard to SMC abundance (scale, 0 to 3; 1.4 +/- 0.9 versus 1.7 +/- 0.9; P = NS), and both differed from those of stable angina. Thrombus and/or hemorrhage occurred in only 34% of patients with unstable angina (compared with 8% of restenosis patients and in none of stable angina patients). Active lesions (defined as lesions (defined as lesions containing one or more of the following: thrombus, hemorrhage, abundant and disorganized SMCs in the presence of loose connective tissue, or inflammatory infiltrate) were observed in 56% of the unstable angina patients and in 50% of the restenosis patients but in none of the stable angina patients. The expression of aFGF and bFGF was detected in 80% to 100% of unstable angina (n = 11) and restenosis (n = 10) specimens but in only 1 of 5 stable angina specimens. CONCLUSIONS: Microscopic evidence of thrombosis and plaque rupture occurred in only one third of unstable angina patients, selected because they had no angiographic evidence of intracoronary thrombus. Moreover, their lesions resembled those of restenosis patients in regard to SMC abundance, lesion activity, and the expression of aFGF and bFGF. Our findings therefore suggest that an alternative mechanism to plaque rupture and thrombus formation may be operative in the precipitation of unstable angina; namely, in a subset of patients, SMC proliferation may lead to gradual plaque expansion and thereby to lumenal narrowing and unstable angina. Our data also suggest a role for aFGF and bFGF in this process.

Adult↗
PubMed1992

Elimination of smooth muscle cells in experimental restenosis: targeting of fibroblast growth factor receptors.

Factors in plasma and platelets do not fully account for the proliferation of smooth muscle cells in vascular injury, implying that additional factors are involved. Recently, we and others have observed that vascular injury regulates basic fibroblast growth factor, suggesting a further role for this pleiotropic factor. We report here that injury of rat arteries leads to an increase in fibroblast growth factor receptors in vascular smooth muscle cells. This up-regulation makes smooth muscle cells susceptible, in vitro and in vivo, to the lethal effects of a conjugate of basic fibroblast growth factor with the ribosome inactivator saporin. Saporin alone has no effect, whereas the conjugate kills proliferating, but not quiescent, smooth muscle cells in vitro. In vivo, one to three doses inhibit neointimal proliferation but have no apparent effect on the uninjured artery. Thus, the up-regulation of fibroblast growth factor receptors in vascular injury suggests new therapeutic possibilities for such refractory conditions as restenosis following balloon angioplasty.

Amino Acid Sequence↗
PubMed1992

Low level in vivo gene transfer into the arterial wall through a perforated balloon catheter.

BACKGROUND: Gene transfer into the arterial wall may provide a novel therapeutic strategy for the treatment of coronary artery restenosis. Previously described methods for gene transfer into the arterial wall require total vessel occlusion for 30 minutes. We sought to develop a protocol for gene transfer within a more clinically relevant time frame. METHODS AND RESULTS: We used a perforated balloon (Wolinsky) catheter to inject retroviral vector-containing virions into rabbit aortas in vivo. The virions were injected within 1 minute. Aortas were removed 5-14 days after injection and analyzed for evidence of gene transfer. In initial studies, nine rabbits were injected with a vector expressing the beta-galactosidase gene, and nine rabbits were injected with either non-beta-galactosidase-containing vectors or with a vehicle control. Histochemical staining of aortic tissues revealed blue (positive) cells in eight of nine experimental rabbits and six of nine controls. Because of the lack of specificity of the beta-galactosidase detection system, we adopted a polymerase chain reaction-based protocol in which oligonucleotide primers were used to amplify specific vector-related sequences from aortic tissue extracts. The polymerase chain reaction protocol, calibrated with standards containing known numbers of transduced cells, revealed low amounts of vector-related sequences in six of 12 vector-injected rabbits and in one of 13 controls (p less than 0.03). Comparison with standards indicated that fewer than 100 transduced cells were present in a 2-cm length of the injected aortic tissue. CONCLUSIONS: Although in vivo gene transfer through an infusion balloon catheter can be accomplished within 1 minute, the therapeutic use of this protocol is limited by the small number of cells that are transduced.

Animals↗
PubMed1992

Acidic and basic fibroblast growth factors in adult rat heart myocytes. Localization, regulation in culture, and effects on DNA synthesis.

Basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) are involved in the induction of embryonic mesoderm, angiogenesis, neuronal differentiation, and proliferation and survival of many cell types. In cardiac myocytes their roles are not well understood. Effects of fibroblast growth factors on reexpression of fetal actin genes have been reported. In freshly isolated adult rat cardiac myocytes, bFGF mRNA was not detectable by in situ hybridization, although the cells contained significant amounts of bFGF and aFGF as quantified by radioimmunoassays, mitogen assays with immunoneutralization, and Western blotting. After culturing, bFGF mRNA was detected (aFGF mRNA was not studied), and the cells contained 2.5-fold more bFGF and 60% more aFGF than freshly isolated cells. The FGFs were not found in conditioned medium. They were localized, especially in cultured cells, to the nucleus. Cultured myocytes bound fourfold more 125I-FGF than freshly isolated cells and expressed the fibroblast growth factor R-1 (flg) gene. The addition of bFGF or aFGF in serum-free medium to pure populations of myocytes (after 10 days in culture, at which time they are spread, beating, and multinucleated) led to increased thymidine incorporation. Expression of fibroblast growth factors and fibroblast growth factor receptors by adult cardiac myocytes that survive the shock and "dedifferentiation" of culturing may contribute to DNA synthesis and, by analogy, to other cell types, to regulation of ribosomal and actin genes, and to cell survival. These possibilities and their in vivo relevance will require further study.

Animals↗
PubMed1991

Culture-induced increase in acidic and basic fibroblast growth factor activities and their association with the nuclei of vascular endothelial and smooth muscle cells.

The activity of acidic and basic fibroblast growth factor-like mitogens (aFGF, bFGF) extracted from cultured bovine aortic endothelial (BAEC) and rat aortic smooth muscle cells (SMC) was compared with that of freshly isolated cells from the same tissues. Extracts of subendothelial extracellular matrix (ECM) and cell lysates of cultured BAEC contained 4-fold more bFGF-like activity than the extracts of fresh cells. ECM and cell lysates of SMC yielded 10-fold more bFGF-like activity than the fresh cell lysates. We consistently find aFGF-like activity in both cell types. In the case of BAEC, cultured cells and ECM contained 3-fold more aFGF-like activity when compared with freshly isolated cells, whereas in cultured SMC, aFGF-like activity in cell and ECM extracts was 8-fold higher than in fresh cell extracts. The mitogens extracted from cell lysates and from the ECM are closely related to aFGF or bFGF by the criteria that they bind to heparin-sepharose and elute at 1.1 M (aFGF) or 1.5 M (bFGF) NaCl, have molecular weights of about 18,000, and react with anti-aFGF (1.1 M), or anti-bFGF (1.5 M) antibodies when analyzed by Western blots and by radioimmunoassay specific for aFGF and bFGF. This mitogenic activity is inhibited by neutralizing antibodies to aFGF and bFGF. In addition, the column fractions are potent mitogens for Balb/c 3T3 fibroblasts. Acidic and basic FGF-like mitogenic activity could also be extracted from the cell nuclei. The subcellular localization of both FGFs was visualized in both nuclei and cytoplasm with immunoperoxidase. Compared with primary SMC, secondary SMC had an increased capacity to bind 125IaFGF to high affinity receptors, while binding to freshly isolated BAEC and SMC was negligible. We conclude that FGFs are present at low levels in freshly isolated cells and that propagation in cell culture provides a stimulus for production of these mitogens.

Animals↗
PubMed1991

Smooth muscle cell growth factors.

While the roles of the platelet-derived growth factors (PDGFs) in vascular smooth muscle cells (SMCs) continue to be elucidated, these cells, especially in their activated 'synthetic' state, have also been found to express, and proliferate in response to, many of the other families of polypeptide growth factors, such as the fibroblast growth factors. Other stimulators of DNA synthesis, and particularly of SMC hypertrophy, include the vasoconstrictor hormones such as angiotensin II, as well as physical forces, especially stretch or tension. For many of these ligands, multiple receptors have been identified and their means of signal transduction are being characterized rapidly. Regulatory regions of these genes are being identified as are transcription factors. Complex post-transcriptional regulation has also been shown by the findings that some growth factors are phosphorylated, or translocated to the nucleus or the extracellular matrix. Inhibitors have also been identified. These include some prostaglandins, calcium antagonists, agonists that activate guanylate and adenylate cyclases, inhibitors of angiotensin-converting enzyme, interferon gamma, and heparin. Future studies are likely to show that tyrosine phosphatases and recessive oncogenes also regulate growth. The existence of so many autocrine/paracrine mitogens--together with some experimental data--suggests some redundancy in the system as well as some additive effects. Redundancy may limit the efficacy of antibodies to a single growth factor to block cell proliferation. Their evolutionary conservation implies some unique roles for each growth factor but these have not been apparent from in vitro studies to date. Further insights are apt to come from the increasing recognition that growth factors have other effects--on cell attachment, migration, survival, production of extracellular matrix, thrombosis, vaso-constriction, regulation of cytokine synthesis, and inhibition of growth. Many of these effects may prove to be context-dependent, as with the case of growth inhibition by transforming growth factor-beta. Studies in monolayer cultures may not obtain the same results as studies using cocultures of endothelial and smooth muscle cells, or 3-dimensional matrix cultures, organ cultures, or in the intact animal. In vivo descriptive studies of growth factors expressed in vascular embryogenesis, hypertension, atherosclerosis, acute balloon injury and thrombosis are being supplemented by interventions such as infusions with growth factors, antibodies, and toxin conjugates. These studies, and studies using transgenic mice and homologous recombination, should yield information as to mechanisms and may also suggest new therapies.

Animals↗
PubMed1991

Acidic fibroblast growth factor in the developing rat embryo.

Compared to basic fibroblast growth factor (bFGF), a widely distributed, broad spectrum mitogen and mesoderm inducer, acidic fibroblast growth factor (aFGF) is reported to have an essentially neural distribution and to be undetectable in the early embryo. In the present investigation, we used immunoblotting and immunochemistry to assess the cellular and tissue distributions of aFGF and bFGF in 11-20-d rat embryos. Immunoblotting of crude and heparin-bound embryo extracts revealed faint bands at the expected 17-18-kD and predominant bands at an apparent molecular mass of 26 to 28-kD (despite reducing conditions) using multiple specific antibodies for aFGF and bFGF. Pretreatment with 8 M urea yielded 18-20-kD aFGF and bFGF and some 24-26-kD bFGF. Immunoreactivity for both aFGF and bFGF was positive and similar in the cytoplasm, nuclei, and extracellular matrix of cells of neuroectodermal and mesodermal origin, while it was negative in endoderm-derived cells. The distribution of immunoreactive aFGF and bFGF also showed changes during development that were associated with the process of cellular and tissue differentiation. For example, intensity and extent of immunoreactivity for both peptides progressively increased in the middle layer of the spinal cord with increasing differentiation of the neural cells. The immunostaining patterns were very similar for aFGF and bFGF for each organ and at each stage. In conclusion, high molecular mass forms of immunoreactive aFGF and bFGF are present in the rat embryo. Acidic FGF and bFGF are both widely distributed in tissues of neuroectodermal and mesodermal origin, and their distribution was very similar.

Animals↗
PubMed1991

A second messenger RNA species of transforming growth factor beta 1 in infarcted rat heart.

Transforming growth factor-beta 1 (TGF-beta 1) is encoded predominantly by a 2.4-kb mRNA in most tissues. However, an additional transcript of 1.9 kb can be detected in rat heart after experimental myocardial infarction caused by ligation of the left coronary artery. This transcript level is significantly higher in infarcted heart tissue than in normal heart tissue, suggesting an important role for this mRNA species in response to injury. Structural characterization of the 1.9-kb mRNA showed that it included the entire coding sequence present in the 2.4-kb TGF-beta 1 mRNA, but also contained an additional nonhomologous 3'-untranslated region (UTR). The junction between the shared and unique 3' sequence in the 1.9-kb mRNA occurred only two nucleotides before the proposed polyadenylation site of the rat TGF-beta 1 2.4-kb mRNA. The unique 3'-UTR and the deduced shortened 5'-UTR in the novel 1.9-kb TGF-beta 1 mRNA suggest different transcriptional and translational regulatory mechanisms under conditions of tissue injury.

Amino Acid Sequence↗
PubMed1991

Immunohistochemical localization of basic and acidic fibroblast growth factors in the developing rat heart.

BACKGROUND: We used biochemical and immunohistochemical techniques to investigate the expression and distribution of immunoreactive basic and acidic fibroblast growth factors (bFGF and aFGF, respectively) in the hearts of rat embryos (11-20 days of gestation) and of postnatal rats (1-35 days after birth). Our purpose was to assess the relation between the cellular distribution of these growth factors and histogenetic and morphogenetic events in the developing heart. METHODS AND RESULTS: Western-blot analysis of heparin-bound material from neonatal heart extracts identified a single band with a molecular weight of approximately 18 kD for both bFGF and aFGF. Five antibodies for bFGF and three for aFGF showed superimposable distribution of immunoreactive bFGF and aFGF in the heart at each stage examined. At the cellular level, these peptides were localized in the cytoplasm and extracellular matrix. In the myocytes, immunostaining was positive throughout the embryonic and neonatal periods. In the majority of the mesenchymal cells of the cushions and endothelial cells of endocardium and vessels, staining was also positive. In the smooth muscle cells of the aorta, other large arteries, and coronary arteries, immunostaining was intensely positive at early stages of development but became faint or negative with increasing cell differentiation. CONCLUSIONS: The wide distribution of immunoreactive bFGF and aFGF that we identified in the developing rat heart suggests that these growth factors play an important role in heart cytodifferentiation and morphogenesis. Their superimposable distribution may reflect functional interaction. The progressive changes in their distribution suggest a changing role for these peptides during organogenesis.

Animals↗
PubMed1991

Effects of acidic fibroblast growth factor on normal and ischemic myocardium.

We sought to determine the effects of acidic fibroblast growth factor (FGF) on ischemic and normal myocardium and to determine whether direct application of acidic FGF to the heart could promote angiogenesis. Eighteen dogs underwent placement of an ameroid constrictor on the left anterior descending coronary artery (LAD). Three weeks later, a left internal mammary artery (IMA) pedicle was positioned over the LAD territory, with a sponge saturated with acidic FGF (n = 12) or saline (n = 4) interposed between the pedicle and the heart. Polytetrafluoroethylene fiber or collagen I sponges were used to deliver the acidic FGF. Weekly angiography of the IMA was performed in all dogs, but significant IMA to coronary collaterals were not demonstrable in any dog. Eight dogs had histological evidence of subendocardial infarction in the LAD territory (five acidic FGF, three control, p = NS). Striking smooth muscle cell hyperplasia was present in arterioles and small arteries exclusively in areas of subendocardial infarction in all of the acidic FGF-treated dogs but in none of the control dogs (p less than 0.05). Noninfarcted myocardium appeared normal in all dogs. In two additional dogs, ameroid constrictors were not placed on the LAD, such that acidic FGF-treated sponges were placed on normally perfused myocardium of the LAD territory. Histological evaluation of those hearts revealed normal myocardium, without evidence of myocardial infarction or smooth muscle cell hyperplasia. Thus, when acidic FGF is delivered to the myocardium via an epicardial sponge in dogs whose coronary flow is compromised, acidic FGF does not cause an angiogenic response in viable myocardium but causes vascular smooth muscle cell hyperplasia in areas subjected to ischemic injury.

Animals↗
PubMed1990

Influence of extracellular magnesium on capillary endothelial cell proliferation and migration.

We investigated the role of extracellular magnesium on capillary endothelial cell migration and proliferation, components of endothelial cell function that play an important role in angiogenesis and wound healing. Cell migration and proliferation were tested in six different MgSO4 concentrations and in various culture conditions. The Boyden chamber procedure was used to evaluate migration of bovine adrenal cortex capillary endothelial cells. We found that low magnesium concentration inhibited cell migration, but a dose-dependent increase in migration was observed when magnesium level was increased beyond the normal serum concentration (up to 2.4 mM magnesium; p less than 0.0001). Cell proliferation was also inhibited by very low magnesium concentration, an effect observed under all conditions studied. When cell proliferation was stimulated by acidic or basic fibroblast growth factors, it appeared that a ceiling was reached, an increasing magnesium concentration had no additional stimulatory effect. However, a dose-dependent increase in proliferation (p less than 0.005) was observed when magnesium concentration was increased above the normal serum level (0.8 mM) in culture conditions that did not cause marked cell proliferation. Thus, magnesium has an important role in endothelial cell migration and proliferation: very low extracellular magnesium concentrations inhibit and supranormal levels enhance both migration and proliferation. These results suggest that magnesium deficiency might adversely influence the healing and reendothelialization of vascular injuries and the healing of myocardial infarction and might also result in delayed or inadequate angiogenesis, effects potentially leading to infarct expansion and inadequate collateral development.

Animals↗
PubMed1990

Isolation, characterization, and localization of heparin-binding growth factors in the heart.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are angiogenic polypeptide mitogens for cells of mesodermal and neuroectodermal origin. In this report we describe the purification from several normal human hearts (including a very fresh, nonischemic sample) of heparin-binding, acid-, heat- and trypsin-sensitive 14-18-kD peptides that crossreact with antisera against aFGF and bFGF. Further evidence includes (a) prevention of mitogenicity by protamine and by anti-bFGF, (b) displacement of 125I-bFGF from cell membranes, and (c) stimulation of capillary endothelial cell migration. Specific immunohistochemistry localized bFGF to endothelial cells and, surprisingly, to cardiac myocytes, with almost no immunoreactivity in smooth muscle cells. These peptides may function in cardiac embryogenesis, hypertrophy, atherogenesis, angiogenesis, and wound healing, and may also have endocrine, neurotropic, or vasomotor functions.

Aged↗