Fibroblast growth factor 2 control of vascular tone.

Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FGF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.

Reduced expression and function of bone morphogenetic protein-2 in bones of Fgf2 null mice.

Disruption of the fibroblast growth factor 2 (FGF-2) gene results in reduced bone mass in mice and impairs expression of bone morphogenic protein-2 (BMP-2) an important mediator of osteoblast and osteoclast differentiation. Since the relationship between FGF-2 and BMP-2 in bone remodeling has not been fully determined, in this study we examined whether endogenous FGF-2 was necessary for maximal effect of BMP-2 on periosteal bone formation in vivo and bone nodule formation and osteoclast formation in vitro in Fgf2-/- mice. We showed that BMP-2 significantly increased periosteal bone formation by 57% in Fgf2+/+ mice but the changes were not significant in Fgf2-/- littermates. In line with these results we found no significant increase in alkaline phosphatase positive (ALP) activity in calvarial osteoblasts or ALP mineralized colonies in stromal cultures from Fgf2-/- mice after BMP-2 treatment. Moreover, BMP-2 induced osteoclast formation was also impaired in marrow stromal cultures from Fgf2-/- mice. Interestingly, BMP-2 induced nuclear accumulation of the runt related transcription factor (Runx2) was markedly impaired in osteoblasts from Fgf2-/- mice. Examination of the effect of loss of FGF-2 on BMP-2 signaling pathway showed that BMP-2 caused a similar induction of phospho-Smad1/5/8 within 30 min in calvarial osteoblasts from both genotypes. In contrast BMP-2-induced p42/44 MAPK was reduced in Fgf2-/- mice. These findings strongly demonstrated that endogenous FGF-2 is important in the maximal responses of BMP-2 in bone and that this may be dependent on the p42/44 MAPK signaling pathway and downstream modulation of Runx2.

Changes in cerebral cortex size are governed by fibroblast growth factor during embryogenesis.

We show that fibroblast growth factor 2 (FGF2) and FGF receptors are transiently expressed by cells of the pseudostratified ventricular epithelium (PVE) during early neurogenesis. A single microinjection of FGF2 into cerebral ventricles of rat embryos at E15.5 increased the volume and total number of neurons in the adult cerebral cortex by 18% and 87%, respectively. Microinjection of FGF2 by the end of neurogenesis, at E20.5, selectively increased the number of glia. Mice lacking the FGF2 gene had fewer cortical neurons and glia at maturity. BrdU studies in FGF2-microinjected and FGF2-null animals suggested that FGF2 increases the proportion of dividing cells in the PVE without affecting the cell-cycle length. Thus, FGF2 increases the number of rounds of division of cortical progenitors.

Absence of the tight junctional protein AF-6 disrupts epithelial cell-cell junctions and cell polarity during mouse development.

BACKGROUND: The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS: Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS: AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.

Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation.

Basic fibroblast growth factor (FGF-2), an important modulator of cartilage and bone growth and differentiation, is expressed and regulated in osteoblastic cells. To investigate the role of FGF-2 in bone, we examined mice with a disruption of the Fgf2 gene. Measurement of trabecular bone architecture of the femoral metaphysis of Fgf2(+/+) and Fgf2(-/-) adult mice by micro-CT revealed that the platelike trabecular structures were markedly reduced and many of the connecting rods of trabecular bone were lost in the Fgf2(-/-) mice. Dynamic histomorphometry confirmed a significant decrease in trabecular bone volume, mineral apposition, and bone formation rates. In addition, there was a profound decreased mineralization of bone marrow stromal cultures from Fgf2(-/-) mice. This study provides strong evidence that FGF-2 helps determine bone mass as well as bone formation.

Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a pleiotropic growth factor detected in many different cells and tissues. Normally synthesized at low levels, FGF-2 is elevated in various pathologies, most notably in cancer and injury repair. To investigate the effects of elevated FGF-2, the human full-length cDNA was expressed in transgenic mice under control of a phosphoglycerate kinase promoter. Overexpression of FGF-2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly. Comparison by Western blot of FGF-2 transgenic mice to nontransgenic littermates showed expression of human FGF-2 protein in all major organs and tissues examined including brain, heart, lung, liver, kidney, spleen, and skeletal muscle; however, different molar ratios of FGF-2 protein isoforms were observed between different organs and tissues. Some tissues preferentially synthesize larger isoforms of FGF-2 while other tissues produce predominantly smaller 18-kDa FGF-2. Translation of the high molecular weight isoforms initiates from unconventional CUG codons and translation of the 18-kDa isoform initiates from an AUG codon in the FGF-2 mRNA. Thus the Western blot data from the FGF-2 transgenic mice suggest that tissue-specific expression of FGF-2 isoforms is regulated translationally.

Embryonic stem cell model systems for vascular morphogenesis and cardiac disorders.

To better understand the formation of the cardiovascular system and its disease states, models amenable to manipulation must be developed. In this article we present two models. One is a small animal model for an inflammatory disorder that can lead to heart failure. Production of this model is based on the ability of blastocyst-derived embryonic stem cells, which can be genetically altered in vitro by a technique called gene targeting, to reconstitute an entire animal when reintroduced into a blastocyst and allowed to colonize the germ line of the resulting chimeric embryo. The other model is based on the capacity of embryonic stem cells to differentiate in culture into embryo-like structures called embryoid bodies. Embryoid bodies contain angioblasts, or prevascular endothelial cells, which can be induced to undergo aspects of vascular development by manipulation of culture conditions.

Lack of the protein tyrosine phosphatase SHP-1 results in decreased numbers of glia within the motheaten (me/me) mouse brain.

Mice that are homozygous for the autosomal recessive motheaten allele (me/me) lack the protein tyrosine phosphatase SHP-1. Loss of SHP-1 leads to many hematopoietic abnormalities, as well as defects such as infertility and low body weight. However, little is known regarding the role SHP-1 plays in the development of the central nervous system (CNS). To define the role of SHP-1 in CNS development and differentiation, we examined the brains of me/me mice at various times after birth for neuronal and glial abnormalities. Although the brains of me/me mice are slightly smaller than age-matched wild-type littermates, both me/me and wild-type brains are similar in weight, possess an intact blood-brain barrier, and have largely normal neuronal architecture. Significantly, the current study reveals that me/me brain shows decreases in the number of glial fibriallary acidic protein (GFAP)+ astrocytes and F480+ microglia compared with wild-type mice. In addition, decreased immunostaining for the myelin-synthesizing enzyme CNPase was observed in me/me mice, confirming the loss of myelin in these animals, as reported (Massa et al. [2000] Glia 29:376-385). It is particularly significant that there is a decreased number of immunolabeled glia of all subtypes and that this deficit in glial number is not restricted to a particular class of glia. This suggests that SHP-1 is necessary for the normal differentiation and distribution of astrocytes, microglia, and oligendrocytes within the murine CNS.

FGF-2 dependent angiogenesis is a latent phenotype in basic fibroblast growth factor transgenic mice.

Overexpression of basic fibroblast growth factor (FGF-2) in transgenic (TgFGF2) mice results in a chondrodysplasia as the principle phenotype. Here we report a second phenotype in TgFGF2 mice that was previously undetected: A predisposition to angiogenic reactions with subsequent amplified angiogenesis that are both FGF-2 dependent. We used subcutaneous injection of extracellular matrix as an angiogenic assay. The matrix formed vascularized cysts in the TgFGF2 group after seven days, whereas the non-transgenic (NTg) group developed avascular cysts. Cysts from the TgFGF2 group contained 3-7X more hemoglobin (Hb), two-fold more vonWillebrand Factor (VWF), and 150X more FGF-2 than cysts from the NTg control group. Significant angiogenic reactions occurred only in the TgFGF2 group that express FGF-2 from the transgene. The TgFGF2 mice, therefore, constitute a unique experimental system to study FGF-2 dependent angiogenesis because they have no spontaneous or inherent vascular defects, but provision of an angiogenic substrate results in an amplified angiogenic response. In addition, we report development of an ELISA for VWF that provides a sensitive, quantitative assay for angiogenesis.

Activation of myocardial angiogenesis and upregulation of fibroblast growth factor-2 in transmyocardial-revascularization-treated mice.

OBJECTIVE: To evaluate the growth factor responses associated with myocardial angiogenesis. DESIGN: Mice were treated with transmyocardial revascularization (TMR) and evaluated for angiogenic and growth factor responses. METHODS: TMR was performed via thoractomy with a 27 g needle. At 2, 5, and 7 days post-treatment, hearts were removed from the TMR treated and control groups, then assayed for angiogenesis, fibroblast growth factor (FGF)-2 expression and vascular endothelial cell growth factor (VEGF) expression. RESULTS: TMR caused an angiogenic reaction in the myocardial blood vessels at 7 days post-TMR treatment. Elevated FGF-2 corresponded with increased TMR related angiogenesis. VEGF levels only increased in hearts that were prewounded then TMR treated. CONCLUSIONS: The data show that TMR stimulates myocardial angiogenesis. The angiogenic reaction is mediated by FGF-2 which increased in most experimental treatment groups. The VEGF response was more specific, requiring prewounding then TMR treatment for a VEGF increase.

Ontogenetic limb bone scaling in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a potent mitogen which is required for normal development, particularly the development of the skeletal system, where the inhibition of FGF binding to its receptor results in various skeletal malformations. The present study employed a newly engineered line of FGF-2 transgenic mice to determine the effects of overexpressing FGF-2 on limb bone ontogeny. We collected radiographic and weight data longitudinally and obtained the length, proximal, distal, and minimum diaphyseal widths of the humerus, femur, and tibia. Because growth is nonlinear with respect to time, we used the Gompertz mathematical model to obtain parameters describing rate and timing for each individual for each measurement. Differences in the parameters due to genotype and sex were subsequently tested with ANOVA. Transgenic animals exhibited consistently shorter limb bones which were generally wider at the epiphyses than those of controls. Parameters of early growth, including initial size and proportional rate of growth, appeared to be most directly responsible for significant differences in final size; however, exponential decay of growth was also a marginally significant factor. There were no differences between the genotypes in body weight, indicating that the shape anomalies observed in transgenic mice were a direct result of the action of FGF-2 rather than a general runting phenomenon.

Impaired bone anabolic response to parathyroid hormone in Fgf2-/- and Fgf2+/- mice.

Since parathyroid hormone (PTH) increased FGF2 mRNA and protein expression in osteoblasts, and serum FGF-2 was increased in osteoporotic patients treated with PTH, we assessed whether the anabolic effect of PTH was impaired in Fgf2-/- mice. Eight-week-old Fgf2+/+ and Fgf2-/- male mice were treated with rhPTH 1-34 (80mug/kg) for 4 weeks. Micro-CT and histomorphometry demonstrated that PTH significantly increased parameters of bone formation in femurs from Fgf2+/+ mice but the changes were smaller and not significant in Fgf2-/- mice. IGF-1 was significantly reduced in serum from PTH-treated Fgf2-/- mice. DEXA analysis of femurs from Fgf2+/+, Fgf2+/-, and Fgf2-/- mice treated with rhPTH (160mug/kg) for 10 days showed that PTH significantly increased femoral BMD in Fgf2+/+ by 18%; by only 3% in Fgf2+/- mice and reduced by 3% in Fgf2-/- mice. We conclude that endogenous Fgf2 is important for maximum bone anabolic effect of PTH in mice.

FGF and FGFR signaling in chondrodysplasias and craniosynostosis.

The first experimental mouse model for FGF2 in bone dysplasia was made serendipitously by overexpression of FGF from a constitutive promoter. The results were not widely accepted, rightfully drew skepticism, and were difficult to publish; because of over 2,000 studies published on FGF-2 at the time (1993), only a few reported a role of FGF-2 in bone growth and differentiation. However, mapping of human dwarfisms to mutations of the FGFRs shortly, thereafter, made the case that bone growth and remodeling was a major physiological function for FGF. Subsequent production of numerous transgenic and targeted null mice for several genes in the bone growth and remodeling pathways have marvelously elucidated the role of FGFs and their interactions with other genes. Indeed, studies of the FGF pathway present one of the best success stories for use of experimental genetics in functionally parsing morphogenetic regulatory pathways. What remains largely unresolved is the pleiotropic nature of FGF-2. How does it accelerate growth in one cell then stimulate apoptosis or retard growth for another cell in the same type of tissue? Some of the answers may come through distinguishing the FGF-2 protein isoforms, made from alternative translation start sites, these appear to have substantially different functions. Although we have made substantial progress, there is still much to be learned regarding FGF-2 as a most complex, enigmatic protein. Studies of genetic models in mice and human FGFR mutations have provided strong evidence that FGFRs are important modulators of osteoblast function during membranous bone formation. However, there is some controversy regarding the effects of FGFR signaling in human and murine genetic models. Although significant progress has been made in our understanding of FGFR signaling, several questions remain concerning the signaling pathways involved in osteoblast regulation by activated FGFR. Additionally, little is known about the specific role of FGFR target genes involved in cranial bone formation. These issues need to be addressed in future in in vitro and in vivo approaches to better understand the molecular mechanisms of action of FGFR signaling in osteoblasts that result in anabolic effects in bone formation.

Over-expression of fibroblast growth factor-2 causes defective bone mineralization and osteopenia in transgenic mice.

Over-expression of human FGF-2 cDNA linked to the phosphoglycerate kinase promoter in transgenic (TgFGF2) mice resulted in a dwarf mouse with premature closure of the growth plate and shortening of bone length. This study was designed to further characterize bone structure and remodeling in these mice. Bones of 1-6 month-old wild (NTg) and TgFGF2 mice were studied. FGF-2 protein levels were higher in bones of TgFGF2 mice. Bone mineral density was significantly decreased as early as 1 month in femurs from TgFGF2 mice compared with NTg mice. Micro-CT of trabecular bone of the distal femurs from 6-month-old TgFGF2 mice revealed significant reduction in trabecular bone volume, trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Osteoblast surface/bone surface, double-labeled surface, mineral apposition rate, and bone formation rates were all significantly reduced in TgFGF2 mice. There were fewer TRAP positive osteoclasts in calvaria from TgFGF2 mice. Quantitative histomorphometry showed that total bone area was similar in both genotypes, however percent osteoclast surface, and osteoclast number/bone surface were significantly reduced in TgFGF2 mice. Increased replication of TgFGF2 calvarial osteoblasts was observed and primary cultures of bone marrow stromal cells from TgFGF2 expressed markers of mature osteoblasts but formed fewer mineralized nodules. The data presented indicate that non-targeted over-expression of FGF-2 protein resulted in decreased endochondral and intramembranous bone formation. These results are consistent with FGF-2 functioning as a negative regulator of postnatal bone growth and remodeling in this animal model.

Developmental angiogenesis: quail embryonic vasculature.

We have examined the segregation and early morphogenesis of the embryonic vasculature by using a monoclonal antibody for immunofluorescence and by scanning electron microscopy. This antibody labels the presumptive endothelial cells (PECs) as they segregate from mesoderm. Similar embryos prepared for SEM revealed finer details of how these segregated cells interact to form the rudiments of the major blood vessels. Here we concentrate on the development of the dorsal aortae and the posterior cardinal veins. The dorsal aortae form from single PECs which segregate from the lateral mesoderm and aggregate into a loose cord ventral to the somites. These cells become more closely associated and a lumen forms. The posterior cardinal veins form from a loose plexus of cells segregated from the lateral mesoderm on its dorsal surface. These cells become intimately associated with the Wolffian ducts.

Embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos.

The development of the embryonic vasculature is examined here using a monoclonal antibody, QH-1, capable of labelling the presumptive endothelial cells of Japanese quail embryos. Antibody labelling is first seen within the embryo proper at the 1-somite stage. Scattered labelling of single cells appears ventral to the somites and at the lateral edges of the anterior intestinal portal. The dorsal aorta soon forms a continuous cord at the ventrolateral edge of the somites and continues into the head to fuse with the ventral aorta forming the first aortic arch by the 6-somite stage. The rudiments of the endocardium fuse at the midline above the anterior intestinal portal by the 3-somite stage and the ventral aorta extends craniad. Intersomitic arteries begin to sprout off of the dorsal aorta at the 7-somite stage. The posterior cardinal vein forms from single cells which segregate from somatic mesoderm at the 7-somite stage to form a loose plexus which moves mediad and wraps around the developing Wolffian duct in later stages. These studies suggest two modes of origin of embryonic blood vessels. The dorsal aortae and cardinal veins apparently arise in situ by the local segregation of presumptive endothelial cells from the mesoderm. The intersomitic arteries, vertebral arteries and cephalic vasculature arise by sprouts from these early vessel rudiments. There also seems to be some cell migration in the morphogenesis of endocardium, ventral aorta and aortic arches. The extent of presumptive endothelial migration in these cases, however, needs to be clarified by microsurgical intervention.

Vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern.

We are using a monoclonal antibody, QH-1, as a label for angioblasts in quail embryos to study vascular development. Our previous experiments showed that major embryonic blood vessels, such as the dorsal aortae and posterior cardinal veins, develop from angioblasts of mesodermal origin that appear in the body of the embryo proper (Coffin and Poole: Development, 102:735-748, '88). We theorized that there are two separate processes for blood vessel development that occur in quail embryos. One mechanism termed "vasculogenesis" forms blood vessels in place by the aggregation of angioblasts into a cord. The other mechanism, termed "angiogenesis," is the formation of new vessels by sprouting of capillaries from existing vessels. Here we report the results of microsurgical transplantation experiments designed to determine the extent of cell migration taking place during blood vessel formation. Comparison of the chimeras to normal embryos suggests that the vascular pattern develops, in part, from the normally restricted points of entry of angioblasts into the head from the ventral and dorsal aortae. Transplantations of quail mesoderm (1-15 somite stage) into the head of 5-15 somite chick hosts resulted in extensive sprouting and in migration of single and small groups of angioblasts away from the graft sites. Transplantations into the trunk resulted in incorporation of the graft into the normal vascular pattern of the host. Lateral plate mesoderm was incorporated into the dorsal aortae and individual sprouts grew between somites and along the neural tube to contribute to the intersomitic and vertebral arteries, respectively.

Endothelial cell origin and migration in embryonic heart and cranial blood vessel development.

Using the QH-1 monoclonal antibody as a marker for quail endothelium, blockage and transplant experiments were carried out to construct fate maps for the embryonic endocardium, to determine whether preendocardial angioblasts are migratory, and, if these cells are migratory, to outline the pathways that they use for directed migration in embryonic blood vessel development. Recent descriptive studies using QH-1 to make immunofluorescent whole mounts have described a sequence of events leading to the establishment of the embryonic heart tube. These reports suggest that the pattern for the endocardium and cranial vasculature is established by migrating angioblasts that form vascular cords which mature into blood vessels. Blockage experiments showed that the ventrolateral edge of the anterior intestinal portal serves as a substrate for the directed migration of pre-endocardial angioblasts and that the pattern of the cranioventral vasculature forms independent of the source of angioblasts. Transplant experiments showed that the origin for endocardial angioblasts lies in mesodermal tissue just anterior to Henson's node, that these cells undergo directed migration to the pericardial area, and that angioblasts are pluripotent with the ability to form different blood vessels. The transplant studies also showed that the embryonic mesoderm may contribute to extraembryonic blood vessels on the embryonic yolksac. These results support the hypothesis that embryonic blood vessels may develop by either the vasculogenesis or by the angiogenesis mechanism, and show that the endocardium of the primitive heart tube forms by vasculogenesis.

Angioblast differentiation and morphogenesis of the vascular endothelium in the mouse embryo.

Bandeiraea simplicifolia B4 isolectin (BSLB4) and polyclonal antisera against von Willebrand factor (VWF) were used to study the origin of endothelial cells and their organization into blood vessels in the postimplantation mouse embryo. Examination of BSLB4-stained whole mounted and sectioned embryos revealed intense staining of the endothelium, highlighting large vessels, capillaries, and many individual cells. Dorsal aorta formation was first obvious at E7 when many lectin-positive cells appeared in paraxial and lateral plate mesoderm. As development proceeded to E8, BSLB4-positive cells became organized into craniocaudal lines destined to become the aorta proper. At E9, BSLB4 stained all vessels of the embryo including the dorsal aorta, the intersomitic arteries, and the endocardium. VWF expression was not detected until E8 when BSLB4/VWF double-stained sections revealed the dorsal aortae as the first VWF-positive vessels, while other endothelium visible with BSLB4 remained negative for VWF immunostaining. By E12 many other vessels became VWF-positive, including the aortic arches, the intersomitic arteries, and the cardinal veins. However, many angioblasts and capillaries remained VWF-negative, reflecting the heterogeneous expression of VWF among endothelium that has been reported in adults of other species. The histochemical data reported here support the conclusions of earlier avian studies by showing distinct vascular patterns in the initial formation of vessels from isolated angioblasts (vasculogenesis), followed by the extension and organization of the initial vascular structures (angiogenesis). Moreover, our data suggest that the endothelium arises from distinct VWF-positive sources associated with the dorsal aorta, as well as VWF-negative sources associated with other vessels in the embryo.

Proline induced hemolytic anemia in fascioliasis.

A secondary anemia is characteristic of infections with the liver fluke, Fasciola hepatica, and previous studies had suggested that proline released by the worm might be involved in producing this condition. In the current study the effect of fascioliasis on erythropoiesis was compared to the effects of proline infusion. Both treatments produced effects characteristic of rapid erythrocyte turnover due to hemolysis. Moreover, infusion of a proline analog into animals with Fasciola or into animals infused with proline prevented the anemia. To our knowledge the production of hemolytic anemia by excessive proline has not been previously reported.