PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor

Platelet-derived growth factors (PDGFs) are important in many types of mesenchymal cell. Here we identify a new PDGF, PDGF-C, which binds to and activates the PDGF alpha-receptor. PDGF-C is activated by proteolysis and induces proliferation of fibroblasts when overexpressed in transgenic mice. In situ hybridization analysis in the murine embryonic kidney shows preferential expression of PDGF-C messenger RNA in the metanephric mesenchyme during epithelial conversion. Analysis of kidneys lacking the PDGF alpha-receptor shows selective loss of mesenchymal cells adjacent to sites of expression of PDGF-C mRNA; this is not found in kidneys from animals lacking PDGF-A or both PDGF-A and PDGF-B, indicating that PDGF-C may have a unique function.     

Dominant inhibition of lens placode formation in mice

The lens in the vertebrate eye has been shown to be critical for proper differentiation of the surrounding ocular tissues including the cornea, iris and ciliary body. In mice, previous investigators have assayed the consequences of molecular ablation of the lens. However, in these studies, lens ablation was initiated (and completed) after the cornea, retina, iris and ciliary body had initiated their differentiation programs thereby precluding analysis of the early role of the lens in fate determination of these tissues. In the present study, we have ablated the lens precursor cells of the surface ectoderm by generation of transgenic mice that express an attenuated version of diphtheria toxin (Tox176) linked to a modified Pax6 promoter that is active in the lens ectodermal precursors. In these mice, lens precursor cells fail to express Sox2, Prox1 and αA-crystallin and die before the formation of a lens placode. The Tox176 mice also showed profound alterations in the corneal differentiation program. The corneal epithelium displayed histological features of the skin, and expressed markers of skin differentiation such as Keratin 1 and 10 instead of Keratin 12, a marker of corneal epithelial differentiation. In the Tox176 mice, in the absence of the lens, extensive folding of the retina was seen. However, differentiation of the major cell types in the retina including the ganglion, amacrine, bipolar and horizontal cells was not affected. Unexpectedly, ectopic placement of the retinal pigmented epithelium was seen between the folds of the retina. Initial specification of the presumptive ciliary body and iris at the anterior margins of the retina was not altered in the Tox176 mice but their subsequent differentiation was blocked. Lacrimal and Harderian glands, which are derived from the Pax6-expressing surface ectodermal precursors, also failed to differentiate. These results suggest that, in mice, specification of the retina, ciliary body and iris occurs at the very outset of eye development and independent of the lens. In addition, our results also suggest that the lens cells of the surface ectoderm may be critical for the proper differentiation of the corneal epithelium.     

Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis

The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.     

Platelet-derived growth factor receptor regulates salivary gland morphogenesis via fibroblast growth factor expression

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is beta-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor alpha is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.     

Analysis of lens cell fate and eye morphogenesis in transgenic mice ablated for cells of the lens lineage

Transgenic mice carrying the diphtheria toxin A gene driven by mouse gamma 2-crystallin promoter sequences manifest microphthalmia due to ablation of fiber cells in the ocular lens. Here we map ablation events in the lens by crossing animals hemizygous for the ablation construct with transgenic mice homozygous for the in situ lacZ reporter gene driven by identical gamma 2-crystallin promoter sequences. By comparing the spatial distribution of lacZ-expressing cells and the profile of gamma-crystallin gene expression in the lenses of normal and microphthalmic offspring, the contributions of specific cell types to lens development were examined. The results suggest that phenotypically and developmentally distinct populations of lens fiber cells are able to contribute to the lens nucleus during organogenesis. We also show that dosage of the transgene and its site of integration influence the extent of ablation. In those mice homozygous for the transgene and completely lacking cells of the lens lineage, we show that the sclera, cornea, and ciliary epithelium are reduced in size but, otherwise, reasonably well formed. In contrast, the anterior chamber, iris, and vitreous body are not discernible while the sensory retina is highly convoluted and extensively fills the vitreous chamber.     

Transforming growth factor-beta induces platelet-derived growth factor (PDGF) messenger RNA and PDGF secretion while inhibiting growth in normal human mammary epithelial cells

Platelet-derived growth factor (PDGF) is a potent mitogen in human serum which specifically stimulates the proliferation of mesenchymal cells. We have now examined normal human mammary epithelial cells (HMEC) derived from reduction mammaplasties and grown in a serum-free defined medium. Medium conditioned by HMEC contained a PDGF-like activity that competed with [125I]PDGF for binding to PDGF receptors in normal human fibroblasts. When conditioned media were incubated with antiserum specific for either PDGF-A or PDGF-B, only PDGF-A antiserum was capable of inhibiting binding of conditioned media to PDGF receptors. Using an RNase protection assay, mRNA from normal HMEC was probed for both the PDGF-A and PDGF-B chains. Little or no PDGF-B was found in HMEC strains, while a strong signal was seen with the PDGF-A probe. When HMEC were grown in the presence of transforming growth factor-beta (TGF beta) for 48 h, inhibition of growth was observed in association with a 20- to 40-fold stimulation of PDGF-B mRNA and a 2-fold stimulation of PDGF-A mRNA. This mRNA induction was extremely rapid (within 1 h), and secreted PDGF activity was induced 2- to 3-fold. Two other HMEC growth inhibitors and differentiating agents, sodium butyrate and phorbol ester 12-O-tetradecanoylphorbol-13-acetate, had no effect on PDGF mRNA regulation. The current study suggests that PDGF gene induction is an extremely rapid and specific indicator of TGF beta function regardless of whether TGF beta is acting in a growth stimulatory or inhibitory manner. Any role of PDGF-B in TGF beta modulation of differentiation of normal or malignant mammary gland remains to be determined.     

Angiogenesis during human extraembryonic development involves the spatiotemporal control of PDGF ligand and receptor gene expression.

We have examined the role of platelet-derived growth factor (PDGF) ligand and receptor genes in the angiogenic process of the developing human placenta. In situ hybridization analysis of first trimester placentae showed that most microcapillary endothelial cells coexpress the PDGF-B and PDGF beta-receptor genes. This observation indicates that PDGF-B may participate in placental angiogenesis by forming autostimulatory loops in capillary endothelial cells to promote cell proliferation. Endothelial cells of macro blood vessels maintained high PDGF-B expression, whereas PDGF beta-receptor mRNA was not detectable. In contrast, PDGF beta-receptor mRNA was readily detectable in fibroblast-like cells and smooth muscle cells in the surrounding intima of intermediate and macro blood vessels. Taken together, these data suggest that the PDGF-B signalling pathway appears to switch from an autocrine to a paracrine mechanism to stimulate growth of surrounding PDGF beta-receptor-positive mesenchymal stromal cells. Smooth muscle cells of the blood vessel intima also expressed the PDGF-A gene, the protein product of which is presumably targeted to the fibroblast-like cells of the mesenchymal stroma as these cells were the only ones expressing the PDGF alpha-receptor. PDGF-A expression was also detected in columnar cytotrophoblasts where it may have a potential role in stimulating mesenchymal cell growth at the base of the growing placental villi. We discuss the possibility that the regulation of the PDGF-B and beta-receptor gene expression might represent the potential targets for primary angiogenic factors.     

Immunoreactivity of the septins SEPT4, SEPT5, and SEPT8 in the human eye.

We aimed to examine the distribution of SEPT4, SEPT5, and SEPT8 in the human eye. For each septin, five to six normal human eyes were examined by immunohistochemical staining of paraffin sections using polyclonal antibodies against SEPT4, SEPT5, and SEPT8 and an avidin biotin complex immunodetection system. SEPT4 immunoreactivity (IR) was detected primarily in the epithelium of cornea, lens, and nonpigmented ciliary epithelium; in the endothelium of cornea and vessels of iris and retina; and in the retinal nerve fiber layer, the outer plexiform layer, the outer segments of the photoreceptor cells, the inner limiting membrane of the optic nerve head, and optic nerve axons. SEPT5-IR was present in corneal endothelial cells, iris tissue, nonpigmented ciliary epithelium, and epithelial cells of the lens. SEPT8-IR almost paralleled that of SEPT4, except for a lower SEPT8-IR of the outer photoreceptor segments and a positive staining of the meningothelial cell nests in the subarachnoidal space of the bulbar segment of the orbital optic nerve. In conclusion, SEPT4, SEPT5, and SEPT8 are expressed in various ocular tissues, each revealing a distinct expression pattern. Both physiological and potential pathophysiological role of septins in the human eye deserve further investigation.     

Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation

The ciliary body and iris are pigmented epithelial structures in the anterior eye segment that function to maintain correct intra‐ocular pressure and regulate exposure of the internal eye structures to light, respectively. The cellular and molecular factors that mediate the development of the ciliary body and iris from the ocular pigmented epithelium remain to be fully elucidated. Here, we have investigated the role of Notch signaling during the development of the anterior pigmented epithelium by using genetic loss‐ and gain‐of‐function approaches. Loss of canonical Notch signaling results in normal iris development but absence of the ciliary body. This causes progressive hypotony and over time leads to phthisis bulbi, a condition characterized by shrinkage of the eye and loss of structure/function. Conversely, Notch gain‐of‐function results in aniridia and profound ciliary body hyperplasia, which causes ocular hypertension and glaucoma‐like disease. Collectively, these data indicate that Notch signaling promotes ciliary body development at the expense of iris formation and reveals novel animal models of human ocular pathologies.     

Expression of PDGF and their receptors in human retinal pigment epithelial cells and fibroblasts: regulation by TGF-beta

Platelet derived growth factors (PDGF) are known to be associated with vitreoretinal disorders such as proliferative vitreoretinopathy (PVR). We have studied the expression of PDGF and their receptors in human retinal pigment epithelial cells (HRPE) and choroid fibroblasts (HCHF), and the regulation of PDGF and its receptors by various cytokines and growth factors. RT-PCR analyses showed enhanced expression of PDGF-A and PDGF-B mRNA in HRPE treated with TGF-beta, but not with other cytokines. A minimal increase was observed in PDGF-A mRNA in TGF-beta treated HCHF cells. PDGF-R alpha mRNA, which was expressed prominently in HCHF and at very low levels in HRPE, was not affected by any of the agents. PDGF-R beta was not detectable in either HRPE or HCHF. HRPE secreted PDGF-AA and AB constitutively, and this secretion was significantly enhanced by TGF-beta. In contrast, HCHF cultures did not secrete detectable levels of any of the three isoforms of PDGF (AA, AB, BB). All three human recombinant PDGF isoforms enhanced HCHF cell proliferation significantly, while only a minimal increase was observed in HRPE. PDGF isoforms also induced HCHF cell elongation and promoted migration of HCHF in an in vitro wound assay. The results presented in this study demonstrate that TGF-beta activated RPE cells produce PDGF that may act on fibroblasts and other mesenchyme derived cells which express PDGF receptors. These studies indicate that the promotion of the proliferation and migration of mesenchymal cells by RPE cell derived PDGF may facilitate the formation of fibrovascular tissues associated with PVR.     

Developmental analysis of ocular morphogenesis in alpha A-crystallin/diphtheria toxin transgenic mice undergoing ablation of the lens.

The role of the lens in early eye development was examined in transgenic mice carrying the cytotoxic diphtheria toxin A gene driven by hamster alpha A-crystallin promoter sequences. Mice hemizygous for this construct are microphthalmic and contain a vacuolated and highly disorganized lens, whereas adult homozygous mice are completely ablated of the lens and lack a pupil, aqueous and posterior chamber, vitreous humor, iris, and ciliary body and show extensive convolution of the sensory retina. Developmental analysis of animals homozygous for the transgene revealed that the optic cup and lens vesicle form normally and that ablation of the lens occurs as a gradual degenerative process beginning between Days 12 and 13 of gestation. Degeneration of the lens vesicle coincides with retarded growth and development of the neuroretina, sclera, and cornea. The anterior lip of the optic cup fails to differentiate into the normal epithelium of the iris and ciliary body and the vitreous body does not develop. Although the retinal layers apparently form normally, retinal folding becomes prominent following lens degeneration. These results suggest that development of a functional lens from Embryonic Day 12.5 onward is critical for formation of the ciliary epithelium, iris, and vitreous body, as well as for appropriate growth, development, and maintenance of morphology of the retina, cornea, sclera, and optic nerve. Our results also provide information on the time course of DT-A-mediated cell destruction in vivo and are discussed in context with previous lens ablation studies and the importance of developmental analysis for interpretation of the extent to which morphogenetic aberrations are concurrent with or secondary to genetic ablation of the target tissue.     

Spontaneous hepatic fibrosis in transgenic mice overexpressing PDGF-A

Platelet derived growth factor (PDGF) plays a central role in repair mechanisms after acute and chronic tissue damage. To further evaluate the role of PDGF-A in liver fibrogenesis in vivo, we generated transgenic mice with hepatocyte-specific overexpression of PDGF-A using the CRP-gene promoter. Transgenic but not wildtype mice showed expression of PDGF-A mRNA in the liver. Hepatic PDGF-A overexpression was accompanied by a significant increase in hepatic procollagen III mRNA expression as well as TGF-beta1 expression. Liver histology showed increased deposition of extracellular matrix in transgenic but not in wildtype mice. PDGF-A-transgenic mice showed positive sinusoidal staining for alpha-SMA indicating an activation of hepatic stellate cells. Since the profibrogenic effect of PDGF-A was accompanied by increased TGF-beta1 protein concentration in the liver of transgenic mice, it can be postulated that PDGF-A upregulates expression of TGF-beta1 which is a strong activator of hepatic stellate cells. Thus, these results point towards a fibrosis induction by PDGF-A via the TGF-beta1 signalling pathway. In conclusion, expression and functional analysis of PDGF-A in the liver of transgenic mice suggest a relevant profibrogenic role of PDGF-A via TGF-beta1 induction. Counteracting PDGF-A may therefore be one of the effects of tyrosine kinase inhibitors which showed protective effects in animal models of liver fibrosis.     

Platelet-Derived Growth Factor Is an Autocrine Stimulator for the Growth and Survival of Human Esophageal Carcinoma Cell Lines

Platelet-derived growth factors (PDGF) are important mitogens for mesenchyme-derived cells. Neither PDGF nor PDGF receptors (PDGFR) are expressed in epithelial cells under normal physiological conditions. However, we have found that PDGF-BB induces c-junexpression and promotes the growth of the human esophageal carcinoma cell line CE48T/VGH. Scatchard analysis revealed the presence of 6 × 10⁵binding sites for PDGF-BB per cell, with a Kd of 9.7 nM. Furthermore, our data indicate that CE48T/VGH expresses β type PDGFR (PDGFRβ) within vitroauto-kinase activity. We have also found that CE48T/VGH expresses the mRNA of the PDGF-A and PDGF-B chains and secretes PDGF molecules. Addition of anti-PDGF neutralizing antibody significantly decreased cell numbers of CE48T/VGH under serum-free conditions. The detached cells underwent apoptosis characterized by micronucleation. These results suggest that expression of the PDGF autocrine system may not only provide the growth advantage but also prevent the apoptosis for CE48T/VGH.     

Leydig cell loss and spermatogenic arrest in platelet-derived growth factor (PDGF)-A-deficient mice

Platelet-derived growth factor (PDGF)- A-deficient male mice were found to develop progressive reduction of testicular size, Leydig cells loss, and spermatogenic arrest. In normal mice, the PDGF-A and PDGF-Ralpha expression pattern showed positive cells in the seminiferous epithelium and in interstitial mesenchymal cells, respectively. The testicular defects seen in PDGF-A-/- mice, combined with the normal developmental expression of PDGF-A and PDGF-Ralpha, indicate that through an epithelial-mesenchymal signaling, the PDGF-A gene is essential for the development of the Leydig cell lineage. These findings suggest that PDGF-A may play a role in the cascade of genes involved in male gonad differentiation. The Leydig cell loss and the spermatogenic impairment in the mutant mice are reminiscent of cases of testicular failure in man.     

Glucosaminidase and Dedifferentiation of Newt Iris Epithelium

To try to understand the mechanism of the dedifferentiation process which occurs during metaplastic transformation of iris epithelial cells into lens cells in newt lens regeneration, the activity of N -acetylglucosaminidase in iris and iris epithelium was studied as a function of time after lentectomy. The activity was found to increase during the dedifferentiation phase of the iris epithelium. The dorsal iris, where definite dedifferentiation occurs side by side with incomplete dedifferentiation, shows significantly greater enhancement of the activity than the ventral iris, where only incomplete dedifferentiation takes place. When the cells complete dedifferentiation and engage in redifferentiation into lens cells, the level of activity drops, approaching that of the normal lens. Evidence is also presented for release of the enzyme into the ocular fluid during dedifferentiation. The possibility that the enzyme is involved in surface alterations of iris epithelial ceils engaged in dedifferentiation is discussed.     

Enlarged lateral ventricles and aberrant behavior in mice overexpressing PDGF-B in embryonic neural stem cells

Platelet-derived growth factor (PDGF) is important in central nervous system (CNS) development, and aberrant expression of PDGF and its receptors has been linked to developmental defects and brain tumorigenesis. We previously found that neural stem and progenitor cells in culture produce PDGF and respond to it by autocrine and/or paracrine signaling. We therefore aimed to examine CNS development after PDGF overexpression in neural stem cells in vivo.Transgenic mice were generated with PDGF-B under control of a minimal nestin enhancer element, which is specific for embryonic expression and will not drive adult expression in mice.The resulting mouse showed increased apoptosis in the developing striatum, which suggests a disturbed regulation of progenitor cells. Later in neurodevelopment, in early postnatal life, mice displayed enlarged lateral ventricles. This enlargement remained into adulthood and it was more pronounced in male mice than in transgenic female mice. Nevertheless, there was an overall normal composition of cell types and numbers in the brain and the transgenic mice were viable and fertile. Adult transgenic males, however, showed behavioral aberrations and locomotor dysfunction. Thus, a tightly regulated expression of PDGF during embryogenesis is required for normal brain development and function in mice.     

PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor

The term 'platelet-derived growth factor' (PDGF) refers to a family of disulphide-bonded dimeric isoforms that are important for growth, survival and function in several types of connective tissue cell. So far, three different PDGF chains have been identified - the classical PDGF-A and PDGF-B and the recently identified PDGF-C. PDGF isoforms (PDGF-AA, AB, BB and CC) exert their cellular effects by differential binding to two receptor tyrosine kinases. The PDGF alpha-receptor (PDGFR-alpha) binds to all three PDGF chains, whereas the beta-receptor (PDGFR-beta) binds only to PDGF-B. Gene-targeting studies using mice have shown that the genes for PDGF-A and PDGF-B, as well as the two PDGFR genes, are essential for normal development. Furthermore, overexpression of PDGFs is linked to different pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases. Here we have identify and characterize a fourth member of the PDGF family, PDGF-D. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulphide-linked homodimer, PDGF-DD. Upon limited proteolysis, PDGF-DD is activated and becomes a specific agonistic ligand for PDGFR-beta. PDGF-DD is the first known PDGFR-beta-specific ligand, and its unique receptor specificity indicates that it may be important for development and pathophysiology in several organs.     

In vivo chemotactic properties and spatial expression of PDGF in developing mesenteric microvascular networks

The recruitment of perivascular cells to developing microvessels is a key component of microvessel assembly. Whereas platelet-derived growth factor (PDGF) signaling is critical for this process during embryonic development, its role from the postnatal stages through adulthood remains unclear. We investigated the potential role of PDGF signaling during microvessel assembly by measuring in vivo the migration of labeled fibroblasts to PDGF in mesenteric connective tissue and by examining PDGF-B and PDGF receptor-beta (PGDFR-beta) expression in microvascular networks during normal maturation. PDGF-B homodimer (PDGF-BB; 30 ng/ml) application elicited a significant (P < 0.05) increase (7.8 +/- 4.1 cells) in labeled fibroblasts within 100 microm of the source micropipette after 2 h. PDGF-A homodimer (30 ng/ml) application and control solution did not elicit directed migration. PDGF-B was expressed in microvessel endothelium and smooth muscle, whereas PDGFR-beta was expressed in endothelium, smooth muscle, and interstitial fibroblasts. Given that PDGF-BB elicits fibroblast migration in the mesentery and that PDGF-B and PDGFR-beta are expressed in a pattern that indicates paracrine signaling from microvessels to the interstitium, the results are consistent with a role for PDGF-B in perivascular cell recruitment to microvessels.