Octreotide prevents growth factor-induced proliferation of bovine retinal endothelial cells under hypoxia.

Ocular diseases such as proliferative diabetic retinopathy are the major cause of blindness in industrialized countries. The main pathologic features of these diseases are hypoxia and overproduction of growth factors resulting in pathologic proliferation of endothelial cells and new vessel formation. Particularly, hypoxia and growth factors, such as VEGF, IGF-1, bFGF and TGF beta(2), show a complex interaction in the onset and progression of the diseases. Therefore, to date, most therapeutic strategies for proliferative retinopathies have targeted proliferation of endothelial cells evoked by growth factors. Recently, a synthetic analog of somatostatin, octreotide, has been shown to inhibit the proliferation of various cells in vitro, including endothelial cells. In this study, we have investigated the proliferative response of bovine retinal endothelial cells (BREC) to growth factors under hypoxic conditions and the modulation by octreotide. We found a dose-dependent increase of cell proliferation with VEGF, IGF-1 and bFGF, and inhibition of hypoxia-induced cell proliferation with TGF beta(2). Moreover, growth factor-induced, but not hypoxia-induced, cell proliferation was attenuated in the presence of octreotide. In contrast, TGF beta(2) abolished hypoxia-induced BREC proliferation. Similar to octreotide BIM23027, a somatastatin receptor subtype 2 (SSTR2) receptor agonist was able to attenuate the growth factor-induced proliferation of BREC expressing mRNA and protein for SSTR2. Therefore, we postulate that octreotide exerts its effects mainly through binding to the SSTR2. Taken together, our findings point to octreotide as a promising candidate for the treatment of eye disorders involving growth factor-dependent proliferation of endothelial cells.

Octreotide inhibits growth factor-induced bovine choriocapillary endothelial cells in vitro.

BACKGROUND: Response to growth factors by the choriocapillaris is one of the main aspects in the development and progression of choroidal neovascularization in age-related macular degeneration (AMD). The aim of the study was to examine the effect of octreotide--an analogue of somatostatin with possible favorable effects in the treatment of age-related macular degeneration--on growth factor-activated bovine choriocapillary endothelial cells (BCEC). METHODS: Cultivated BCEC were stimulated with 20 ng/ml VEGF, 5 ng/ml bFGF, 50 ng/ml TGFbeta(2), or 50 ng/ml IGF-I for 24 h. Afterwards, the cells were incubated for a further 24 h with octreotide (10(-6) M). BCEC proliferation was determined by [(3)H]-thymidine incorporation. The experiments were performed in normal conditions and in O(2)-limited (5% O(2)) atmosphere. RESULTS: VEGF, bFGF, and IGF-I significantly stimulated BCEC proliferation under normoxic as well as under hypoxic conditions ( P<0.05); the value for TGFbeta(2)stimulation was not significant. Under normoxic conditions, after addition of octreotide to VEGF-, bFGF-, and IGF-I-stimulated cells there was no longer any significant [(3)H]-thymidine incorporation compared to the control medium. Under hypoxic conditions similar results were obtained after addition of octreotide to VEGF-, bFGF-, and IGF-I-stimulated cells. CONCLUSION: Octreotide has inhibitory properties after activation of BCEC with growth factors under normoxic and hypoxic conditions in vitro. Since activation of these cells is a crucial event in the development and progression of AMD, octreotide may be a potential drug for treatment of choroidal neovascularization in these patients.

Amphibian intestinal villin: isolation and expression during embryonic and larval development.

An actin-binding protein of M(r) 105,000 has been isolated from anuran amphibian intestinal mucosa. Polyclonal antibodies directed against chicken and pig intestinal villins and anti-porcine villin headpiece monoclonal antibody crossreact with the amphibian M(r) 105,000 protein. Furthermore, the latter possesses an NH2-terminal sequence that is very homologous to those of avian and mammalian villins. In addition, polyclonal antibodies directed against amphibian intestinal M(r) 105,000 protein crossreact with chicken and mouse intestinal epithelial cell villins. These data indicate that the amphibian intestinal M(r) 105,000 protein is immunologically and structurally related to villin, an actin-binding protein expressed in specific epithelial tissues in vertebrates. Morphological, immunocytochemical and immunoblotting techniques were then used to investigate the expression of villin during embryonic and larval intestinal development of Xenopus laevis. Villin is not found in the egg or the endoderm of the early embryo. It is first detected just before hatching in the apical domain of endodermal cells at a time when few surface microvilli are visible by transmission electron microscopy. In the newly hatched larva, villin accumulates as these cells differentiate. These results provide a detailed developmental profile of Xenopus intestinal villin expression and demonstrate that this protein is a useful marker for the presumptive intestinal endoderm.

Influence of triiodothyronine on the polypeptide composition of the intestinal brush border membrane during amphibian metamorphosis.

Brush border fragments (BBF) were isolated from homogenates of intestinal epithelium prepared from four groups of tadpoles: premetamorphic larvae, thyrostatic larvae, spontaneously metamorphosed larvae, and triiodothyronine (T3)-induced froglets. Isolation was accomplished by a combination of both Ca2+ precipitation and differential centrifugation methods. These preparations were routinely enriched seven- to-eleven-fold for the two amphibian brush border marker enzymes, gamma-glutamyltransferase and maltase. Comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining revealed the presence of a polypeptide of Mr 27,000 only after spontaneous and T3-induced metamorphosis. One-dimensional SDS-PAGE together with lectin staining showed six strongly concanavalin A reactive polypeptides (Mr 52,000, 57,000, 65,000, 80,000, 130,000 and 150,000) in both preparations examined. Immunoblot analyses allowed us to detect in both preparations the presence of villin (Mr 105,000), a cytoskeletal component of microvilli. Two-dimensional isoelectric focusing IEF/SDS-PAGE together with silver staining showed the polypeptides of Mr 41,500, 43,000, 60,500 and 101,000 to be specific components of the primary intestinal epithelium brush border. In contrast six polypeptides of Mr 27,000, 52,000, 58,000, 59,000 and 95,000 were only detected in intestinal BBF after spontaneous and T3-induced metamorphosis. Their presence is under the control of the thyroid hormone. The results provide new insight regarding the subcellular localization of polypeptides whose synthesis changes during spontaneous (Figiel et al., 1987) and T3-induced metamorphosis (Figiel et al., 1989).

Stage-specific polypeptide and villin expression during thyroid-hormone-induced substitution of the amphibian intestinal epithelium.

Treatment of anuran tadpoles with 5 nM 3,3',5-triiodo-L-thyronine (T3) results in the complete substitution of the intestinal epithelium. We have examined the developmental pattern of protein synthesis in Alytes obstetricans intestinal epithelium using two-dimensional gel electrophoresis. Four different types of changes have been observed. The group I polypeptides (Mr: 41,500; 44,500; 51,500; 55,000 and 101,000) are only synthesized during the first week of hormonal treatment. They are specific of the primary (larval) epithelium. On the other hand, polypeptides referred to as Group II (Mr: 47,000; 48,000; 58,000; 66,500, pl 5.2; 99,500 and 102,000) are not detected until day 8. They are characteristic of the secondary tissue. Polypeptides of Group III (Mr: 42,000, pl 5.15 and 5.25; 42,500, 47,500, pl 5.25 and 5.55) expressed between the 6th and 8th day of T3 treatment, are specific of growing stem cells. During this critical period, Group IV polypeptides (Mr: 63,500; 66,500, pl 6.35; 105,000, pl 5.5 and 5.55) are not synthesized. The protein of Mr 105,000 (pI 5.5 and 5.55) is immunologically related to villin, a core protein of intestinal microvilli. Expression of this protein has been analyzed by immunoreplica and immunocytochemical procedures during differentiation of basal stem cells into secondary absorptive epithelial cells. The results have been compared to that obtained during spontaneous metamorphosis.

Stage-specific polypeptides and villin expression during the intestinal epithelium substitution of the metamorphosing amphibian.

The amphibian intestinal epithelium provides an excellent aid to study the developmental pattern of protein synthesis during cell life. The metamorphosing tissue demonstrates a kaleidoscope of cell degeneration, proliferation and differentiation. These events occur at specific period in a synchronized cell population. Two-dimensional gel electrophoresis, together with histological studies, has been used to examine the changes in the patterns of protein synthesis during intestinal epithelium substitution in metamorphosing Alytes obstetricians larvae. Of the approximately 280 polypeptides detected by this method, 24 show major changes in their patterns of synthesis. Five polypeptides are only synthesized during the larval period and are characteristic of the primary epithelium. Six polypeptides are characteristic of the secondary intestinal epithelium, as they are only detected in the newly-metamorphosed juvenile. Four polypeptides of Mr 81,000, 78,000, 42,000 (pI, 5.1 and 6.2) are characteristic of the epithelium crisis, as they are only detected during climax. They may represent molecular markers of growing stem cells. On the other hand, two polypeptides, of Mr 66,500 and 63,500, are not synthesized during this critical period, but are synthesized before and after metamorphosis. Seven polypeptides show changes in the relative rate of their synthesis during metamorphosis of the intestinal epithelium. Among them, the protein of Mr 105,000 which presents two isoelectric variants (pI 5.5 and 5.55) is immunologically related to villin. Expression of this protein has been studied using immunoblotting of cell extracts onto nitrocellulose and immunodetection in tissue sections. The protein is localized in the brush border of primary and secondary epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)