PubMed Health⌕ Search

Biomedical subjects

Frank J Lovicu

Publications and source records attributed to Frank J Lovicu.

14 recordsLinked to original sources
PubMed2007

Crim1KST264/KST264 mice display a disruption of the Crim1 gene resulting in perinatal lethality with defects in multiple organ systems.

Crim1 is a transmembrane protein, containing six vWF-C type cysteine-rich repeats, that tethers growth factors to the cell surface. A mouse line, KST264, generated in a LacZ insertion mutagenesis gene-trap screen, was examined to elucidate Crim1 function in development. We showed that Crim1(KST264/KST264) mice were not null for Crim1 due to the production of a shortened protein isoform. These mice are likely to represent an effective hypomorph or a dominant-negative for Crim1. Transgene expression recapitulated known Crim1 expression in lens, brain, and limb, but also revealed expression in the smooth muscle cells of the developing heart and renal vasculature, developing cartilage, mature ovary and detrusor of the bladder. Transgene expression was also observed in glomerular epithelial cells, podocytes, mesangial cells, and urothelium in the kidney. Crim1(KST264/KST264) mice displayed perinatal lethality, syndactyly, eye, and kidney abnormalities. The severe and complex phenotype observed in Crim1(KST264/KST264) mice highlights the importance of Crim1 in numerous aspects of organogenesis.

Abnormalities, Multiple↗
PubMed2006

Pax7 and superior collicular polarity: insights from Pax6 (Sey) mutant mice.

Pax genes are important modulators of CNS development. Pax7 and Pax6 polarise the neural tube and regionalise the brain. Pax7 is pivotal in specifying the superior colliculus/tectum, an important centre for integration of visuomotor responses and a target for Pax6+ retinal ganglion cell axons during retinocollicular mapping. Whilst initial Pax7-specification of the mesencephalon is well-established, a role in regulating polarity within the maturing mouse superior colliculus is yet to be defined, although already detailed for the chick tectum. We therefore quantified Pax7 cellular distribution and expression levels at three functionally distinct stages of superior collicular development, and analysed Pax7 expression in response to aberrant axonal input and altered forebrain/midbrain boundary placement in Pax6 mutant mice. Comparative expression profiles of ephrin-A2 and its co-localisation with Pax7 were determined in wildtype and Pax6 mutant mice. Results indicate that graded Pax7 expression in wildtype mice is perturbed in Pax6 mutant mice; changes manifest as a shift in polarity, loss of graded expression and dramatically reduced protein levels during RGC synaptogenesis. Ephrin-A2 expression is similarly altered. These results implicate Pax7 as an important determinant of polarity within the mouse superior colliculus, and suggest a role in retinotopic mapping.

Animals↗
PubMed2006

Sef and Sprouty expression in the developing ocular lens: implications for regulating lens cell proliferation and differentiation.

In many developmental systems, growth factor signalling must be temporally and spatially regulated, and this is commonly achieved by growth factor antagonists. Here, we describe the expression patterns of newly identified growth factor inhibitors, Sprouty and Sef, in the developing ocular lens. Sprouty and Sef are both expressed in the lens throughout embryogenesis, and become restricted to the lens epithelium, indicating that lens cell proliferation and fibre differentiation may be tightly regulated by such antagonists. Future studies will be aimed at understanding how these negative regulatory molecules modulate growth factor-induced signalling pathways and cellular processes in the lens.

Animals↗
PubMed2006

Renin enhancer is critical for control of renin gene expression and cardiovascular function.

The important cardiovascular regulator renin contains a strong in vitro enhancer 2.7 kb upstream of its gene. Here we tested the in vivo role of the mouse Ren-1c enhancer. In renin-expressing As4.1 cells stably transfected with Ren-1c promoter with or without enhancer, expression of linked beta-geo reporter, stable expression, and colony formation were dependent on the presence of the enhancer. We then generated mice carrying a targeted deletion of the enhancer (REKO mice) and found marked depletion of renin in renal juxtaglomerular and submandibular ductal cells, as well as hyperplasia of macula densa cells. Plasma creatinine was increased, but electrolytes were normal. Male REKO mice implanted with telemetry devices had 9 +/- 1 mm Hg lower mean arterial pressure (p < 0.001), which was partly normalized by a high NaCl diet. Locomotor activity was lower, and baroreflex sensitivity was normal. Markedly reduced mean arterial pressure variability in the midfrequency band indicated a contribution of reduced sympathetic vasomotor tone to the hypotension. In conclusion, the renin enhancer is critical for renin gene expression and physiological sequelae, including response to alteration in salt intake. The REKO mouse may be useful as a low renin expression model.

Animals↗
PubMed2006

Differing effects of dexamethasone and diclofenac on posterior capsule opacification-like changes in a rat lens explant model.

Posterior capsular opacification (PCO) arises from lens cells that remain associated with the lens capsule after cataract surgery and subsequently become abnormal, proliferate and migrate into the visual pathway. In this study, a rat lens explant model was used to assess the effects of the prototype steroidal and non-steroidal anti-inflammatory drugs, dexamethasone (DEX) and diclofenac (DIC), on epithelial cells undergoing PCO-like changes. Such drugs are widely used at the time of cataract surgery. TGFbeta2 and FGF-2 were added sequentially and explants were cultured for up to 30 days, with or without addition of DEX or DIC at a clinically relevant concentration. Without DEX or DIC, explants became multilayered and cells tended to retract into PCO-like plaques. Inclusion of DEX, but not DIC, resulted in transient formation of needle-like cells, enhanced cell coverage, and the retention a monolayer of migratory cells surrounding PCO-like plaques. With or without drug addition, most cells became aberrant, as indicated by loss of Pax6 expression and the presence of PCO markers alpha-smooth muscle actin and type I collagen; however, DEX and DIC both strongly enhanced type I collagen accumulation. Furthermore, DEX enhanced cell coverage in explants treated with TGFbeta alone. Thus the behaviour of lens cells was significantly and differentially affected by the presence of DEX and DIC, highlighting the possibility that drugs used to control inflammation after cataract surgery, and the clinician's choice of drugs, may influence PCO development.

Actins↗
PubMed2006

Aqueous humour- and growth factor-induced lens cell proliferation is dependent on MAPK/ERK1/2 and Akt/PI3-K signalling.

The aqueous humour of the eye is a rich source of growth factors, many of which have been shown to be lens cell mitogens; however, the identity of the endogenous mitogen(s) for lens cells is still unknown. As a first approach to identify the mechanisms by which these aqueous humour-derived growth factors induce lens cell proliferation, the present study set out to examine MAPK/ERK1/2 and PI3-K/Akt signalling associated with lens cell proliferation. Using a lens explant system, we examined the effects of different lens mitogens (aqueous humour, FGF, PDGF, IGF and EGF) using 5'-2'-bromo-deoxyuridine incorporation. In addition, we adopted immunolabelling techniques to compare the roles that the ERK1/2 and PI3-K signalling pathways play in regulating lens cell proliferation. We showed that the aqueous humour, and all the other growth factors examined, could activate ERK1/2 and PI3-K/Akt signalling. By targeting these pathways using specific pharmacological inhibitors, we were able to show that both ERK1/2 and PI3-K signalling are required for growth factor-induced lens cell proliferation, and that there was a strong correlation between the spatial distribution of proliferating cells in lens explants with ERK1/2 labelling. Furthermore, our blocking studies confirmed that PI3-K/Akt signalling can act upstream of ERK1/2, potentiating ERK1/2 phosphorylation in growth factor-induced lens cell proliferation. A better understanding of the signalling pathways required for aqueous humour-induced lens cell proliferation may ultimately allow us to identify the mitogen(s) that are important for regulating lens cell proliferation in situ.

Animals↗
PubMed2005

Posterior capsule opacification-like changes in rat lens explants cultured with TGFbeta and FGF: effects of cell coverage and regional differences.

Following cataract surgery, many patients suffer secondary loss of vision because of posterior capsule opacification (PCO), which arises when residual lens epithelial cells become aberrant and migrate into the light path. Transforming growth factor-beta (TGFbeta)-induced transdifferentiation of lens cells appears to play a key role in this process. Fibroblast growth factor (FGF) may also play a role by promoting the survival of TGFbeta-affected cells and influencing their subsequent behaviour. In the present study, the effects of two different TGFbeta and FGF treatment regimes were compared in rat lens epithelial explants with either low or high initial cell coverage. Explants treated with 50 pg ml(-1) TGFbeta2 and 20 ng ml(-1) FGF-2 sequentially (day 0, day 1) or simultaneously (day 0), then cultured for up to 30 days with FGF, were assessed by light microscopy and immunolocalisation of markers for transdifferentiation (alpha-smooth muscle actin (alphaSMA) and type I collagen) or lens epithelial phenotype (Pax6) and fibre differentiation (beta-crystallin). By day 4, most cells had lost Pax6 reactivity, alphaSMA reactivity was evident, and there were differences between growth factor treatment groups, low and high initial cell coverage explants, and peripheral and central regions of explants. On day 30 of culture, all explants were well populated with cells, irrespective of treatment and initial cell coverage, and exhibited diverse PCO-like morphological changes, with expression of transdifferentiation markers and beta-crystallin in virtually all cells. Such overall resilience to variations in conditions may contribute to the insidious nature of PCO, while factors related to observed early differences between groups may contribute to PCO pleiomorphism.

Actins↗
PubMed2004

Aberrant lens fiber differentiation in anterior subcapsular cataract formation: a process dependent on reduced levels of Pax6.

PURPOSE: TGFbeta can induce development in lenses of opaque subcapsular fibrotic plaques that have many features of human subcapsular cataracts. To understand further the events associated with the onset and progression of TGFbeta-induced cataract, several different models for anterior subcapsular cataract (ASC) were used and characterized. METHODS: Anterior subcapsular plaques were induced in rat lenses cultured with TGFbeta and in transgenic mice overexpressing TGFbeta in the lens. ASC was also examined in lenses of mice haploinsufficient for Pax6, as well as in human biopsy specimens. Immunofluorescence and in situ hybridization labeling were used to examine changes in patterns of gene expression associated with cataract formation in these models. RESULTS: Examination of TGFbeta-induced cataract in transgenic mice established that the subcapsular plaques are composed of a heterogenous cell population: a population of myofibroblastic cells as well as a population of lens-fiber-like cells. Further support for phenotypic change comes from the observation that the cells in these plaques no longer expressed lens epithelial markers, such as Pax6 and Connexin43. Subsequent examination of human biopsy specimens of ASC, as well as lenses from Pax6-deficient mice, showed that the anterior subcapsular plaques in both cases were also composed of a heterogenous population of cells. In contrast, anterior subcapsular plaques that developed in vitro in response to TGFbeta did not have this same cellular heterogeneity, as no fiber-like cells were present. CONCLUSIONS: These findings suggest that in vivo, during TGFbeta-induced cataract formation, some lens epithelial cells transform into myofibroblastic cells, whereas others differentiate into fiber cells. As this pathologic change is accompanied by altered expression of genes characteristic of the normal lens epithelial cell phenotype and as lenses from Pax6-deficient mice exhibit development of anterior subcapsular plaques closely resembling those induced by TGFbeta in transgenic mice, the authors propose that a reduction in Pax6 levels may be essential for this pathologic process to progress. Furthermore, it is clear from these in vitro studies that TGFbeta alone cannot reproduce the same morphologic and molecular changes associated with ASC formation in vivo, indicating that additional molecule(s) in the eye are important in this process.

Actins↗
PubMed2004

Expression of Frizzleds and secreted frizzled-related proteins (Sfrps) during mammalian lens development.

Recent studies indicate a role for Wnt signaling in regulating lens cell differentiation (Stump et al., 2003). Here we investigated expression patterns of Wnt receptors, the Frizzleds (Fzs) and the Wnt signaling regulators, the secreted frizzled-related proteins (Sfrps), during rodent lens development. RT-PCR showed that Fz receptors, Fz1-Fz8 are expressed in lens. In situ hybridization showed that all the Fz genes examined have similar expression patterns. Fzs are expressed throughout the early lens primordium. At embryonic day 14.5 (E14.5), Fz gene expression is predominantly localized to the epithelium and elongating cells at the lens equator. Fz expression is absent from lens fibers. This pattern of Fz gene expression continues throughout early postnatal development. Immunolocalization studies showed that Fz protein distribution closely follows that of the mRNAs. In addition, epithelial cells in FGF-treated explants show strongest Fz reactivity in cellular protrusions as they migrate and elongate. Sfrp1- Sfrp5 are expressed and all, except Sfrp2, have similar patterns of expression to each other and to the Fzs during lens development. Sfrp2 is strongly expressed in all lens pit cells but becomes restricted to the presumptive epithelial cells of the lens vesicle. By E14.5, Sfrp2 is only present in a few cells above the lens equator. Sfrp2 is not detected in the lens at E18.5 or at later stages. This study shows that multiple Fz and Sfrp genes are expressed during lens morphogenesis and differentiation. This is consistent with a role for Wnt-Fz signaling during both embryonic and postnatal lens development.

Animals↗
PubMed2003

A role for Wnt/beta-catenin signaling in lens epithelial differentiation.

The differentiation of epithelial cells and fiber cells from the anterior and posterior compartments of the lens vesicle, respectively, give the mammalian lens its distinctive polarity. While much progress has been made in understanding the molecular basis of fiber differentiation, little is known about factors that govern the differentiation of the epithelium. Members of the Wnt growth factor family appear to be key regulators of epithelial differentiation in various organ systems. Wnts are ligands for Frizzled receptors and can activate several signaling pathways, of which the best understood is the Wnt/beta-catenin pathway. The presence of LDL-related protein coreceptors (LRPs) 5 or 6 has been shown to be a requirement for Wnt signaling through the beta-catenin pathway. To access the role of this signaling pathway in the lens, we analyzed mice with a null mutation of lrp6. These mice had small eyes and aberrant lenses, characterized by an incompletely formed anterior epithelium resulting in extrusion of the lens fibers into the overlying corneal stroma. We also showed that multiple Wnts, including 5a, 5b, 7a, 7b, 8a, 8b, and Frizzled receptors 1, 2, 3, 4, and 6, were detected in the lens. Expression of these molecules was generally present throughout the lens epithelium and extended into the transitional zone, where early fiber elongation occurs. In addition to both LRP5 and LRP6, we also showed the expression of other molecules involved in Wnt signaling and its regulation, including Dishevelleds, Dickkopfs, and secreted Frizzled-related proteins. Taken together, these results indicate a role for Wnt signaling in regulating the differentiation and behavior of lens cells.

Animals↗
PubMed2002

Influence of platelet-derived growth factor on lens epithelial cell proliferation and differentiation.

The expression pattern of platelet-derived growth factor (PDGF) and its receptor suggest a role in lens cell proliferation. PDGF is strongly expressed in the iris and ciliary body, situated opposite the proliferative cells of the lens epithelium which express the PDGF-alpha receptor. In this study, using lens epithelial explant cultures, we report that PDGF can induce a dose and time dependent increase in lens cell DNA synthesis. Culturing lens explants with both PDGF and FGF (a mitogen and differentiation factor for lens cells) resulted in responses greater than those induced by either growth factor alone. PDGF did not induce any changes typical of fibre differentiation; however, in combination with FGF it potentiated the fibre differentiating activity of FGF. Results obtained in this study support previous indications that PDGF has an important role in regulating lens cell proliferation. In addition, PDGF may have a role in potentiating FGF-induced lens fibre differentiation in vivo.

Animals↗
PubMed2002

Apoptosis is a feature of TGF beta-induced cataract.

BACKGROUND: Studies in our laboratory have shown that transforming growth factor beta (TGF beta) induces rodent lens epithelial cells to undergo aberrant growth and differentiation that reproduces morphological and molecular features of human anterior subcapsular cataract and posterior capsule opacification. In addition, features of apoptosis have been described in some forms of human cataract. In the present study we investigated apoptotic changes induced by TGF beta in our rodent models. METHODS: Lens epithelial explants and whole lenses from rats were cultured with TGF beta. Morphological analysis and TUNEL were used to detect apoptotic changes. Transgenic mice expressing a self-activating form of human TGF beta 1 in the lens were included in the analysis. RESULTS: TGF beta-induced cell loss in epithelial explants coincided with increased numbers of pyknotic nuclei. Some of these nuclei were TUNEL-positive. Studies on lenses cultured with TGF beta and lenses from transgenic mice showed that the subcapsular plaques that developed contained pyknotic nuclei and that many of these were TUNEL-positive. DISCUSSION: This study shows that cells develop morphological and molecular features of apoptosis in TGF beta-induced cataract models. This confirms that apoptosis can be included as another TGF beta-induced cellular change that mimics events in human cataract.

Animals↗
PubMed2002

TGFbeta induces morphological and molecular changes similar to human anterior subcapsular cataract.

BACKGROUND: Transforming growth factor beta (TGFbeta) has been shown to induce subcapsular plaques in cultured rat lenses as well as in lenses of transgenic mice. In the present study the authors have extended their analysis of these cataract models to determine how closely they mimic human cataract. In particular, they studied the maturation of cataract in the transgenic model to determine if it develops similar features as previously described for anterior subcapsular cataract (ASC) in humans. Furthermore, they investigated whether both of these animal models express the range of molecular markers that have now been shown to be present in human ASC. METHODS: Histology and periodic acid Schiff staining were used to study the development and maturation of subcapsular plaques in transgenic mice overexpressing TGFbeta1 in the lens. Immunolabelling methods were used to identify the molecular markers for ASC in both the transgenic mouse model and in rat lenses cultured with TGFbeta2. RESULTS: Histological analysis showed that the subcapsular plaques that develop in adult transgenic mouse lenses bear a striking similarity to mature human ASC, including the formation of a new epithelial-like layer extending between the subcapsular plaque and the underlying fibre mass. All known molecular markers for human ASC were induced in both rodent models, including collagen types I and III, tenascin, and fibronectin. They also identified the presence of desmin in these plaques, a putative novel marker for human cataract. CONCLUSIONS: In both transgenic mouse and rat lens culture models TGFbeta induces markers similar to those found in human ASC. Atypical expression of these cataract markers is also characteristic of posterior capsular opacification (PCO). The molecular markers expressed are typical of a myofibroblastic/fibroblastic phenotype and suggest that a common feature of ASC and PCO may be induction of an epithelial-mesenchymal transition by TGFbeta.

Animals↗