Corpus luteum angiogenic factor is related to fibroblast growth factor

An angiogenic growth factor present in bovine corpus luteum (CL) has been purified to apparent homogeneity by a combination of differential salt precipitation, ion exchange chromatography, and heparin-Sepharose chromatography. It is a single chain polypeptide with an apparent mol wt of 15,000 and an amino acid composition similar to that previously reported for pituitary and brain fibroblast growth factor (FGF). Sequence analysis of the first 17 residues of the CL-derived growth factor identified the sequence; His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-X-Lys-Asn-Gly-Gly-X-Phe-Leu. This sequence is identical to residues 16-33 of bovine pituitary and brain FGF, indicating that the CL-derived growth factor is an amino-terminally truncated form of FGF and is otherwise similar, if not identical, to FGF. The biological activity of CL FGF is indistinguishable from that of pituitary or brain FGF. It is highly active in triggering the proliferation of cultured bovine vascular endothelial cells derived either from large vessels (aortic arch) or from corpus luteum and adrenal cortex capillaries (half-maximal stimulation at 20-40 pg/ml and saturation at 400-600 pg/ml). In vivo implants containing 50 ng to 1 microgram CL-derived growth factor stimulate neovascularization in the chorioallantoic membrane of the chick embryo. In addition to being mitogenic for vascular endothelial cells, CL FGF also stimulates the proliferation of a wide variety of mesoderm- and neuroectoderm-derived cells, including vascular smooth muscle cells, granulosa and adrenal cortex cells, rabbit costal chondrocytes, and corneal endothelial cells.     

Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells.

A growth factor with specificity for vascular endothelial cells has been identified in conditioned medium of pituitary-derived folliculo stellate cells. This factor, named folliculo stellate-derived growth factor (FSdGF), was purified to homogeneity by a combination of heparin-Sepharose affinity chromatography, Bio-Gel P-60 exclusion chromatography, Mono S ion-exchange chromatography, and hydrophobic chromatography on a C4 reverse-phase HPLC column. FSdGF was characterized as a homodimer composed of two subunits with a molecular mass of 23 kDa. FSdGF was a potent mitogen for vascular endothelial cells with activity detectable at 25 pg/ml and saturation at 500 pg/ml. It did not stimulate the proliferation of other cell types such as bovine vascular smooth muscle cells, corneal endothelial cells, adrenal cortex cells, granulosa cells, BALB/MK cells, or BHK-21 cells. Microsequencing revealed an N-terminal sequence having no significant homology to any known protein. The release of FSdGF by pituitary cells and its target cell specificity raise the possibility that FSdGF may play a role in angiogenesis.     

Isolation and partial molecular characterization of pituitary fibroblast growth factor.

Fibroblast growth factor (FGF) has been purified to homogeneity from bovine pituitaries by two methods. Starting material for both methods was an FGF preparation partially purified as described by Gospodarowicz [Gospodarowicz, D. (1975) J. Biol. Chem. 250, 2515-2520]. Purification procedure I involved cation-exchange and reversed-phase HPLC, while procedure II employed gel filtration and ion-exchange chromatography. Isolation was monitored by testing column fractions for their capacity to stimulate the proliferation of vascular endothelial cells in vitro. The growth factor has an approximate molecular weight of 16,000. Its amino-terminal sequence was determined as Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe-Pro-Pro-Gly. Sequence and amino acid composition indicate that the structure of pituitary FGF is different from that of other known growth factors. Pituitary FGF, as isolated under nonacidic conditions (procedure II), has high potency and intrinsic activity to stimulate adult bovine aortic endothelial cells (half-maximal proliferation at 2 pM). Acidic conditions as in procedure I, however, lead to about 90% loss of potency while the intrinsic activity remains intact (identical maximal stimulation values). By all other criteria (molecular weight, amino acid composition, amino-terminal sequence), the two preparations are indistinguishable. Antibodies were raised in rabbits against a synthetic peptide representing the first nine residues of the amino-terminal sequence of the pituitary FGF. The polyclonal antibodies recognize the synthetic peptide and the purified growth factor on an equimolar basis and are capable of inhibiting mitogenic activity in vitro. This report describes a partial chemical characterization of a pituitary FGF and demonstrates rigorously that the characterized protein possesses the mitogenic activity commonly referred to as "basic pituitary FGF."     

Basic fibroblast growth factor: production and growth stimulation in cultured adrenal cortex cells

Cultured bovine adrenal cortex cells express the basic fibroblast growth factor (bFGF) gene and contain, but under normal conditions apparently do not release, bFGF. However, once released, bFGF can stimulate proliferation of the cells, indicating that it could act as a self-stimulating growth factor for adrenal cortex cells. It is conceivable that the intracellular bFGF is released upon injury of the adrenal cortex and that it may be involved in the subsequent tissue repair mechanisms by stimulating the proliferation of adrenal cortical and vascular endothelial cells.     

Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF.

The two major mitogenic polypeptides for endothelial cells have been purified to homogeneity. The complete primary structure of bovine pituitary basic fibroblast growth factor (FGF) and the amino-terminal amino acid sequence of bovine brain acidic FGF have been established by gas-phase sequence analyses. Homogeneous preparations of these polypeptides are potent mitogens (basic FGF, ED50 approximately equal to 60 pg/ml; acidic FGF ED50 approximately equal to 6000 pg/ml) for many diverse cell types including capillary endothelial cells, vascular smooth muscle cells, and adrenocortical and granulosa cells; in vivo, basic FGF is a powerful angiogenic agent in the chick chorioallantoic membrane assay. The available protein sequence data demonstrate the existence of significant structural homology between the two polypeptides.     

Pituitary follicular cells secrete an inhibitor of aortic endothelial cell growth: identification as leukemia inhibitory factor.

Medium conditioned by bovine pituitary follicular cells paradoxically inhibits the growth of adult bovine aortic endothelial (ABAE) cells at dilutions that are instead mitogenic to adrenal cortex capillary endothelial (ACCE) cells, suggesting that follicular cells secrete a growth inhibitor with a selectivity for ABAE cells. The ABAE cell inhibitory activity was purified to apparent homogeneity by a combination of size-exclusion chromatography, ion-exchange chromatography, and two reversed-phase steps on a C4 column. Microsequencing of the purified material revealed a single NH2-terminal amino acid sequence, identical to that of leukemia inhibitory factor (LIF), a glycoprotein originally identified by its ability to inhibit the growth of MT1 mouse leukemia cells and subsequently found to have numerous effects. Recombinant human LIF inhibited the growth of ABAE cells as effectively as transforming growth factor beta (TGF beta 1). However, it failed to inhibit markedly the growth of ACCE cells, whereas TGF beta 1 dramatically inhibited their growth. Recombinant human LIF also failed to induce a significant angiogenic response in the chicken chorioallantoic membrane, indicating that, unlike TGF beta, LIF probably does not induce the release of direct-acting angiogenic factors from inflammatory cells. The presence of LIF in follicular cells may relate to the peculiar vascular organization of the pituitary gland, where no arteries reach the pars distalis and all of the blood supply to this area is by capillaries.     

Differential Effects of Cartilage-Derived Growth Factor Stimulation of Collagen Secretion by Bovine Aortic and Microvascular Endothelial Cells

Cartilage-derived growth factor (CDGF), a protein closely related to basic fibroblast growth factor, is known to have both mitogenic and chemokinetic properties in microvascular endothelial cells (MVEC). Becaue of the angiogenic properties of CDGF and its rate in accelerating wound repair, the capacity of this factor to stimulate both proliferation and matrix synthesis was compared in distinct populations of vascular endothelial cells: MVEC from bovine adrenal cortex and macrovascular endothelial cells from the bovine aorta (BAEC). No significant differences in the responses to mitogenic stimulation using CDGF (5-100 units/ml) were observed using MVEC and BAEC. Only rapidly dividing MVEC, however, showed significant increases in collagen secretion in the presence of CDGF. The differential responsiveness of these two cell populations to a defined growth factor underscores the phenotypic diversity of endothelium.     

Control of proliferation of human fetal adrenal cells in vitro

The influence of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on the proliferation of cultured human fetal adrenal cells has been examined. Separated human definitive zone and fetal zone adrenal cells plated at low density in the presence of 10% serum and maintained on plastic culture dishes proliferated slowly. If the cultures were exposed to either FGF or EGF, the growth rate of the cells from each zone increased significantly. Half-maximal stimulation of cell proliferation for both zones occurred at a concentration of 3 X 10(-11) M for EGF and 8 X 10(-9) M for FGF. In addition, 125I-labeled EGF binding to both definitive and fetal zone cells demonstrated high affinity (Kd = 10(-9) M). To investigate the influence of an extracellular matrix (ECM) on cell proliferation, separated fetal adrenal cells maintained on plastic culture dishes were compared with cells maintained on a recently described ECM prepared from bovine corneal endothelial cells. Fetal adrenal cells maintained on the ECM had a significantly higher growth rate than cells maintained on plastic alone. These results demonstrate 1) the mitogenic role of EGF and FGF for human fetal adrenal cells, and 2) that the type of substrate upon which fetal adrenal cells are maintained has a profound influence on their proliferation.     

Heparin affinity of anionic and cationic capillary endothelial cell growth factors: analysis of hypothalamus-derived growth factors and fibroblast growth factors.

Bovine hypothalamus-derived growth factors (HDGF), pituitary fibroblast growth factor (FGF), and brain FGF were analyzed by chromatography on immobilized heparin and tested for the ability to stimulate the proliferation of capillary endothelial (CE) cells. Two distinct CE cell growth factors were found in hypothalamus, one anionic (aHDGF; pI of about 5) and one cationic (cHDGF; pI of about 8). Both aHDGF and cHDGF adhered tightly to immobilized heparin. They were eluted with 0.9-1.1 M NaCl and 1.3-1.5 M NaCl, respectively. Pituitary and brain FGF were also found to bind to immobilized heparin and to stimulate CE cell proliferation. Pituitary FGF was eluted at 1.4-1.6 M NaCl. The elution profile of brain FGF showed that two peaks of CE cell growth factor activity were eluted from the heparin column, one at 1.0 M NaCl and a second at 1.4-1.6 M NaCl. The tight binding of all of these growth factors to heparin (particularly aHDGF, whose binding is unexpected because of its negative charge) is presented as evidence that CE cell growth factors all share an affinity for heparin.     

Isolation and characterization of ovine luteal pericytes and effects of nitric oxide on pericyte expression of angiogenic factors

We have demonstrated that vascular endothelial growth factor (VEGF) is expressed in capillary pericytes of the developing corpus luteum (CL) and others have shown that basic fibroblast growth factor (FGF2) and angiopoietins (ANGPT) are present in the CL. VEGF and FGF2 target endothelial cells to initiate angiogenesis and stimulate nitric oxide (NO) production. Conversely, NO may increase VEGF expression by vascular smooth muscle cells and pericytes. To investigate the relationship between these angiogenic factors and NO in the CL, microvascular pericytes and endothelial cells were isolated from CL collected from superovulated ewes (n = 5) on d 9 of the estrous cycle. Pericytes were identified by their morphology in culture and by immunofluorescent staining for smooth muscle cell actin. Pericytes were incubated with or without varying doses of the NO-donor DETA-NO for 8 h. Then, total cellular RNA was extracted from the cells and evaluated for expression of mRNA for VEGF, FGF2, ANGPT1, ANGPT2, and NO receptor, guanylate cyclase 1, soluble beta3 (GUCY1B3), using real-time quantitative RT-PCR. NO caused a dose-dependent increase in VEGF (p < 0.001), FGF2 (p < 0.001), ANGPT2 (p < 0.06), and GUCY1B3 (p < 0.03) mRNA expression. Expression of mRNA for ANGPT1 in luteal pericytes was not affected by the NO treatment. These data provide further evidence of the role of the luteal pericyte and NO in angiogenic factor expression, and of the potential interactions of pericytes with endothelial cells via NO production.     

Mitogenic functions of endocrine gland-derived vascular endothelial growth factor and Bombina variegata 8 on steroidogenic adrenocortical cells

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and its homolog Bombina variegata (Bv8), also termed prokineticin-1 and -2 (PK1 and PK2) respectively, are newly identified peptides with specific mitogenic activity on endocrine gland-derived endothelial cells. In the present study, we analyzed the sites of expression of EG-VEGF/PK1, Bv8/PK2, and their receptors (PKR1 and PKR2) in the adrenal cortex and checked for new biological functions of these factors on the endocrine cell compartment. RT-PCR and immunostaining analyses revealed that glomerulosa and fasciculata cells express both factors and both receptors. EG-VEGF/PK1 had no effect on the steroidogenic activity of both bovine glomerulosa and fasciculata cells but appeared to be mitogenic for both cell types. Binding of EG-VEGF/PK1 to fasciculata cells stimulated the phosphorylation of ERK1/2. Pretreatment with pertussis toxin suppressed this effect, indicating that it was Gi mediated. EG-VEGF/PK1 also increased the phosphorylation of Akt in endocrine cells of the adrenal cortex. EG-VEGF/PK1 and Bv8/PK2 thus represent new regulatory peptides acting as autocrine mitogens for endocrine cells.     

An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization.

Extracts of bovine hypothalamus were found to contain a significant level of mitogenic activity when tested in a Swiss 3T3 cell [3H]dThd incorporation assay and in a human umbilical vein endothelial cell growth assay. The mitogenic activity responsible for 3T3 cell activity was purified and characterized as a fibroblast growth factor (FGF)-like mitogen. Neither the biologically active FGF-like mitogen purified from the hypothalamus extracts nor FGF purified from bovine pituitary glands was mitogenic when added to human endothelial cells in vitro, suggesting the presence of more than one mitogen in the hypothalamic extracts. The 3T3 and endothelial cell biological activities of hypothalamic extracts were both found to be inactivated by trypsin, subtilisin, and heat treatment, but were stable to dialysis. The endothelial cell growth factor activity could be efficiently separated from the FGF activity by gel exclusion chromatography. The endothelial cell mitogen possessed a molecular weight of approximately 75,000, whereas that of FGF was approximately 15,000. The endothelial cell growth factor activity was found to be inactivated with reducing agents whereas the 3T3 cell mitogenic activity was stable after incubation with 2-mercaptoethanol. Significant levels of endothelial cell mitogenic activity were also found in extracts of bovine brain and pituitary glands.     

Dual hormonal regulation of endocrine tissue mass and vasculature by adrenocorticotropin in the adrenal cortex

The mass of healthy adult tissues is stable and their vasculature is quiescent, but this equilibrium is disrupted under certain physiological or pathological situations. There is an emerging concept indicating that these trophic changes may be initiated by modifications of the vasculature. In the current study, we documented over a period of 14 d the serial alterations occurring in both endocrine and endothelial compartments during adrenal atrophy induced by ACTH suppression in mice. After dexamethasone perfusion, a rapid fall of plasmatic ACTH and corticosterone concentrations was observed within the first 24 h. During the first 4 d of treatment, adrenal weight and adrenal cortex cellularity decreased rapidly. This was correlated with an inhibition of cell proliferation and a massive induction of endocrine cell apoptosis. Between d 4 and d 14, a slower but sustained decay of adrenal cortex size and cellularity was observed. This second phase was associated with progressive loss of vascular endothelial growth factor protein expression in the endocrine cells and regression of the vascular network. These data support the concept that ACTH controls adrenal cortex trophicity through a dual mechanism involving its antiapoptotic effect on endocrine cells and its indirect vascular endothelial growth factor-mediated action on endothelial cells.     

N-terminal proopiomelanocortin acts as a mitogen in adrenocortical tumor cells and decreases adrenal steroidogenesis

There is evidence that proopiomelanocortin (POMC)-derived peptides other than ACTH are involved in pituitary-dependent adrenal growth. We have synthesized the human N-terminal POMC fragment 1-28-POMC with the disulfide bridges in the correct position between cysteine residues 2-24 and 8-20 and studied the activity of these peptides in adrenocortical tumor cells in vitro. 1-28-POMC stimulated cell proliferation in human NCI-h295 and mouse Y-1 adrenal cancer cell lines and also in primary cultures of bovine adrenocortical cells in a concentration-dependent manner. 1-28-POMC led to rapid activation of the MAPKs extracellular signal-regulated kinases-1 and -2, but not c-Jun N-terminal kinase and p38, pathways. Steroid hormone production (cortisol, 17-hydroxyprogesterone, and dehydroepiandrosterone sulfate) in NCI-h295 cells was decreased by 1-28-POMC in a concentration-dependent fashion. However, protein levels of important regulators of steroidogenesis [steroidogenic factor-1, DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome 1), steroidogenic acute regulatory protein, and cytochrome P450 side-chain cleavage enzyme] remained unaffected by 1-28-POMC treatment. Our results provide evidence that synthetic 1-28-POMC induces adrenal tumor cell proliferation, inhibits adrenal steroidogenesis, and mediates its action by signaling via the extracellular signal-regulated kinase pathway. The distinct roles of 1-28-POMC and ACTH in the regulation of adrenal growth and steroidogenesis suggest that the adrenal cortex is under the dual opposing control of fragments from the same mother peptide POMC.     

Basic fibroblast growth factor induces angiogenesis in vitro.

Fibroblast growth factors (FGFs) are potent mitogens for vascular and capillary endothelial cells in vitro and can stimulate the formation of blood capillaries (angiogenesis) in vivo. A crucial event in this process is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. Using a recently developed in vitro model of angiogenesis, we show here that highly purified basic pituitary FGF can induce capillary endothelial cells to invade a three-dimensional collagen matrix and to organize themselves to form characteristic tubules that resemble blood capillaries. We also show that basic FGF concomitantly stimulates endothelial cells to produce a urokinase-type plasminogen activator, a protease that has been implicated in the neovascular response. The results demonstrate that basic FGF can stimulate processes that are characteristic of angiogenesis in vivo, including endothelial cell migration, invasion, and production of plasminogen activator.     

Effects of tetrandrine on growth factor-induced DNA synthesis and proliferative response of rat pulmonary artery smooth muscle cells

The objective of the present study was to investigate the effect of tetrandrine (a plant alkaloid isolated from Stephenia tetrandra) on growth factor-induced DNA synthesis and proliferative responses of rat pulmonary artery smooth muscle cells. Male rat and bovine pulmonary artery smooth muscle cells (PASMC) were cultured in Medium 199 containing FBS (10%). DNA synthesis was monitored from [(3)H]-thymidine uptake and cell proliferation by direct cell counting. In the present study FBS (1% v/v) caused a small increase in DNA synthesis above basal levels in rat and bovine PASMC (6% and 11% respectively). Platelet-derived growth factor (PDGF, 50 ng/ml), fibroblast growth factor (FGF, 50 ng/ml) or interleukin-1alpha (IL-1alpha, 100 pg/ml) alone increased rat PASMC proliferation (69-85%) and DNA synthesis above basal levels (76-92%). The addition of these growth factors in combination with FBS (1%) resulted in higher increases in DNA synthesis above basal levels (rat PASMC:PDGF, 465%; FGF, 421%; IL-1alpha, 406%; bovine PASMC:PDGF, 279%). Tetrandrine (10(-5) M) inhibited FBS (10%)-induced rat PASMC proliferation (90.5%) and DNA synthesis (89.0%). Tetrandrine significantly inhibited cell proliferation (86.5-98.5%) and DNA synthesis (79.9-89.0%) induced by FBS (1%) in combination with one of the following mitogens; PDGF (50 ng/ml), FGF (50 ng/ml), IL-1alpha (100 pg/ml). The inhibitory effects of tetrandrine were observed between 10(-6) and 10(-5)M and PASMC viability was not affected by tetrandrine below 3x10(-5) m. In summary, these results suggest that tetrandrine can exert anti-proliferative effects against a range of mitogenic stimuli for vascular smooth muscle cells in vitro. Such effects may contribute to the inhibitory effect of tetrandrine on pulmonary vascular remodelling associated with pulmonary hypertension.     

Pituitary fibroblast growth factor as a stimulator of growth in cultured rabbit articular chondrocytes.

Growth promoting activity for rabbit chondrocytes has been described as a contaminant of partially pruified TSH and LH prepared from bovine and ovine pituitaries. We have investigated fibroblast frowth factor (FGF), a small growth promoting peptide isolated from bovine pituitary tissue, in a rabbit chondrocyte system. The results suggest to us that FGF is the factor or one of the factors responsible for chondrocyte growth stimulating activity previously described in the pituitary hormone preparations. DNA synthesis in these cells is stimulated by FGF at final medium concentrations of 10(-9) g/ml. Bovine NIH-LH is not stimulatory below concentrations of 10(-7) g/ml. FGF also stimulates cell growth in the presence of 10% fetal bovine serum. Dexamethasone, at concentrations of 10(-5) to 10(-9) g/ml exerts a synergistic effect with FGF on both DNA synthesis and cell growth. Over a concentration range of 10(-6) to 10(-9) g/ml, FGF does not stimulate synthesis of sulfated mucopolysaccharides.     

Identification of a receptor for N-POMC peptides

The adrenal cortex is a dynamic organ in which the cells of the outer cortex continually divide. It is well known that this cellular proliferation is dependent on constant stimulation from peptides derived from the ACTH precursor proopiomelanocortin (POMC) since disruption of pituitary corticotroph function results in rapid atrophy of the gland. Although ACTH has often been assumed to be the adrenal mitogen, results from our laboratory suggest that the true mitogen is a fragment derived from the N-terminal of POMC that does not contain the gamma-MSH sequence. Since these peptides are not generated during the processing of POMC in the pituitary it has been proposed that the mitogen is generated from circulating pro-gamma-MSH by an adrenal protease. We have recently substantiated this hypothesis by characterizing a serine protease expressed by the adrenal necessary for growth and lead us to propose that N-POMC (1-52) is the adrenal mitogen. Using N-POMC (1-28) linked to a solid support we have extended these studies in an attempt to identify the receptor through which this peptide elicits its actions. Using this approach we have isolated a 80 KDa candidate protein from membranes prepared from the adrenal cortical Y1 cell line.