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Biomedical subjects

J Baffi

Publications and source records attributed to J Baffi.

15 recordsLinked to original sources
PubMed2003

CXCR4 expression in vitreoretinal membranes.

BACKGROUND/AIM: Proliferative vitreoretinopathy (PVR) and macular pucker (MP) vitreoretinal membranes are caused by abnormal cell migration. By their role in chemotactism, chemokine receptors represent good candidates to sustain this process. The authors thus investigated the expression of one of them, CXCR4, in these pathologies. METHODS: Three PVR and four MP membranes were surgically removed and processed for immunochemical studies with antibodies for CXCR4, cytokeratins or smooth muscle actin. RESULTS: CXCR4 expression was found in all membranes. There was no relation between severity of PVR or MP and presence of CXCR4. In addition, there was no difference in CXCR4 expression between MP and PVR. CONCLUSION: CXCR4 is expressed in PVR and MP. Further experiments are needed to test if CXCR4 and other chemokine receptors are implicated in vitreoretinal membrane formation.

Antibodies↗
PubMed2002

Safety and pharmacokinetics of intravitreal 2-methoxyestradiol implants in normal rabbit and pharmacodynamics in a rat model of choroidal neovascularization.

Choroidal neovascularization (CNV) is the leading cause of severe vision loss associated with age-related macular degeneration. As the pathogenesis of CNV formation is better understood, mechanism-based therapies, including the use of antiangiogenesis inhibitors, have been investigated. 2-methoxyestradiol (2ME2), an endogenous metabolite of estradiol, has been shown in the chick allantoic membrane model and the corneal micropocket assay to have antiangiogenic properties. The authors sought to determine the safety and pharmacokinetics of sustained-release intravitreal 2ME2 implants in normal rabbit and their efficacy in a rat model of CNV. 2ME2 implants were constructed using two designs: implant A, a silicone-based reservoir implant for the rabnbit eye, and implant B, a microimplant matrix design for the rat eye. In vitro release rates of both implants were determined. New Zealand white (NZW) rabbits had implant A placed in the vitreous cavity of one eye and the ocular toxicity was evaluated by clinical examination, serial electroretinography (ERG), and histopathology over a 28 week period. The steady state clearance of 2ME2 in the rabbit eye was calculated from in vivo release rates divided by steady state vitreous concentrations. A CNV model in the Brown-Norway rat was performed by injecting an adenoviral vector encoding human vascular endothelial growth factor in the subretinal space. Following the injection, a 2ME2 or sham (no drug) microimplant was placed in the vitreous cavity. Animals were killed over a 3 week period and the eyes examined for CNV by histopathology. Results showed that following a short burst, the release rate of implant A followed zero-order kinetics, typical of reservoir devices, and the cumulative release of implant B was proportional to the square root of time, as expected for a matrix delivery device. The safety studies in normal rabbit showed no ocular toxicities by clinical examination, ERG, and histopathology. Pharmacokinetic evaluation in the rabbit showed mean 2ME2 vitreous levels within the therapeutic range for the inhibition of endothelial cell proliferation. The experimental rat model showed a significant reduction in CNV in eyes treated with the 2ME2 implant. In conclusion, sustained-release 2ME2 intravitreal implants, which can be designed to deliver potentially therapeutic vitreous levels of 2ME2 for an extended period of time, appeared to be safe in normal rabbit and effective in a rat model of CNV. Sustained-release 2ME2 intravitreal implants may hold promise in the treatment of recurrent CNV refractory to standard therapy.

2-Methoxyestradiol↗
PubMed2001

Aqueous humor and plasma vascular endothelial growth factor in uveitis-associated cystoid macular edema.

PURPOSE: To determine the association between cystoid macular edema and vascular endothelial growth factor concentration in the aqueous humor and plasma of uveitis patients. METHODS: Cross-sectional study. Vascular endothelial growth factor concentrations were measured by enzyme-linked immunosorbent assay in the aqueous humor of 20 uveitis patients (9 with and 11 without cystoid macular edema), and in the plasma of 40 uveitis patients (20 with and 20 without cystoid macular edema) and 20 healthy volunteers. RESULTS: Mean aqueous humor vascular endothelial growth factor concentrations for uveitis patients with and without cystoid macular edema were 152.3 and 109.5 pg/ml, respectively, P =.044. Mean plasma vascular endothelial growth factor concentrations in uveitis patients with and without cystoid macular edema and in healthy volunteers were 32.2, 29.6, and 55.0 pg/ml, respectively. Uveitis patients had lower plasma vascular endothelial growth factor levels than did healthy volunteers, P =.0002. CONCLUSION: In uveitis patients, vascular endothelial growth factor concentration is increased in the aqueous humor of eyes with cystoid macular edema. It may be useful to investigate vascular endothelial growth factor antagonists as a treatment for uveitis-associated cystoid macular edema.

Adolescent↗
PubMed2001

Transcriptional regulation of vascular endothelial growth factor expression in epithelial and stromal cells during mouse mammary gland development.

Accompanying changes in the development and function of the mammary gland is the establishment of a vascular network of critical importance for lactogenesis and tumorigenesis. A potent angiogenic and permeability factor that regulates vascular development in association with epithelial-stromal interactions is vascular endothelial growth factor (VEGF). Analysis of VEGF transcription by RT-PCR revealed mRNA for all three VEGF isoforms (VEGF120, 164, 188) within the mammary gland of nulliparous females. During pregnancy the level of VEGF188 declined and became undetectable during lactation in association with the increased abundance of VEGF120 and VEGF164 mRNAS: All three isoforms were expressed at consistent levels within the cleared mammary fat pad throughout development. Furthermore, the presence of VEGF188 mRNA in omental adipose tissue at various stages established that VEGF188 is expressed specifically in adipose tissue within the mammary gland. Using 3T3-L1 preadipocytes it was demonstrated that VEGF188 mRNA transcription occurs as a late event during lipogenesis distinct from earlier induction of VEGF120 and VEGF164 mRNA during differentiation. In contrast, HC11 mammary epithelial cells only expressed mRNA for VEGF120 and VEGF164. Localization of VEGF mRNA and protein revealed that VEGF is expressed in stromal cells of the mammary gland in nulliparous females and then undergoes a transition to epithelial expression during lactation. By contrast, mRNA for the VEGF receptors, Flk-1 and Flt-1, localized to stromal cells within the mammary fat pad during virgin and gestational development and was expressed in the interstitial tissue basal to epithelial cells during lactation. Taken together, these results support the conclusion that VEGF is differentially transcribed by specific cell types within the mammary gland, and that under hormonal regulation it functions in an autocrine/paracrine manner.

Adipocytes↗
PubMed2001

Regulated heat shock protein 27 expression in human retinal pigment epithelium.

PURPOSE: To examine the expression and regulation of an injury-related protein, heat shock protein (Hsp) 27, in retinal pigment epithelium (RPE), since RPE injury may be an important feature of age-related macular degeneration (ARMD). METHODS: Retinal cross sections from eyes of Lewis rats were examined for Hsp27 in vivo by immunohistochemistry, and in vitro expression of Hsp27 in human ARPE-19 cells was determined by Northern and Western blot analysis. Oxidant-mediated injury was performed by exposing ARPE-19 cells to myeloperoxidase and hydrogen peroxide. Cell lines stably expressing green fluorescent protein (GFP) targeted to the cell membrane were used to study injury-induced membrane blebbing, and XTT conversion was used to detect cell viability. RESULTS: High level of Hsp27 expression was detected in vivo in ganglion cells, RPE, and photoreceptor outer segments of rat retina. ARPE-19 cells also expressed high levels of Hsp27 in vitro. Oxidative injury in ARPE-19 cells resulted in transcriptional and translational activation of Hsp27 and induced extensive membrane blebbing. A high level of Hsp 27 protein was detected within membrane blebs. Increased expression of Hsp27 was also observed in differentiated ARPE-19 cells when compared with dividing cells. Higher Hsp27 levels in differentiated RPE cells correlated with increased viability and phenotypically different blebbing after exposure to the injury stimulus. In addition, sublethal injury doses caused a moderate amount of membrane blebbing, which was well tolerated by differentiated ARPE-19 cells. CONCLUSIONS: These results indicate that Hsp27 may be an important component of the RPE injury response and may contribute to injury-induced membrane blebbing in differentiated RPE cells. It is hypothesized that Hsp27 levels may play a role in disease states in the retina, such as ARMD.

Animals↗
PubMed2000

Saturation of, and competition for entry into, the apical secretory pathway.

To investigate mechanisms of apical sorting in the secretory pathway of epithelial cells, we expressed varying amounts of the 165 amino acid isoform of vascular endothelial growth factor (VEGF(165)) and transforming growth factor beta1 (TGF-beta1) via replication defective adenoviruses. Apical sorting of both proteins was efficient at low expression levels but saturated or was reversed at high expression levels. High expression levels of TGF-beta1 were effective at competing VEGF(165) out of the apical pathway; however, VEGF(165) did not compete out TGF-beta1. Tunicamycin inhibition experiments showed that the apical polarity of VEGF(165) was independent of N-glycosylation. We conclude that the apical sorting of these two molecules is a saturable, signal-mediated process, involving competition for apical sorting receptors. The sorting of the two proteins does not appear to involve N-glycans as sorting signals, or lectin sorters. The observations are particularly relevant to gene therapy because they demonstrate that overexpression of a transgene can result in undesirable missorting of the encoded protein.

Adenoviridae↗
PubMed2000

Choroidal neovascularization in the rat induced by adenovirus mediated expression of vascular endothelial growth factor.

PURPOSE: To determine the effects of an adenovirus vector encoding vascular endothelial growth factor(165) (Ad.VEGF) delivered to the subretinal space in the rat. METHODS: An E1-deleted adenoviral vector encoding VEGF was injected into the subretinal space of Long-Evans rats. Immunohistochemistry identified VEGF expression. Histopathologic changes in the retina were determined by light and electron microscopy, immunohistochemistry, fluorescein angiography, and examination of wholemounts of choroid and retina. RESULTS: Increased expression of VEGF only in the retinal pigment epithelium (RPE) was detected after Ad.VEGF injection. Histopathology of these eyes revealed minimal subretinal exudation at 1 week followed by the appearance of vascular structures in the subretinal space by week 2, which persisted up to 4 weeks. Shortening of photoreceptor outer segments and reduction of the outer nuclear layer were present overlying areas of neovascularization. Fluorescein angiography of animals injected with fluorescein-dextran revealed a deep complex of new vessels. Choroidal flatmounts showed new vessel formation, verified by detection of endothelial cells via immunohistochemistry, arising from the choroid with absence of change in the overlying retinal vasculature. Electron microscopy confirmed the presence of sub-RPE endothelial cells and pericytes and the loss of integrity of Bruch's membrane, and serial sectioning demonstrated choroidal vascular growth through Bruch's membrane. CONCLUSIONS: These results support the hypothesis that overexpression of VEGF from RPE cells is capable of inducing choroidal neovascularization in the rat and provide a framework for further examining angiogenic processes in the RPE-choroid complex.

Adenoviruses, Human↗
PubMed1999

Cloning and characterization of a novel orphan G-protein-coupled receptor localized to human chromosome 2p16.

We report the identification and characterisation of a novel human orphan G-protein-coupled receptor (GPR) which maps to chromosome 2p16. We have determined the full-length coding sequence and genomic structure of a gene corresponding to the anonymous expressed sequenced tag, WI-31133. This gene encodes a novel protein that is 540 amino acids in length. Protein sequence analysis predicts the presence of seven transmembrane domains, a characteristic feature of GPRs. In situ hybridisation to human retina and Northern blot analysis of human retinal pigment epithelium (RPE) showed localisation of this transcript to the RPE and cells surrounding retinal arterioles. In contrast, the transcript was localised to the photoreceptor inner segments and the outer plexiform layer in mouse sections. Northern blot analysis demonstrated a 7 kb transcript highly expressed in the brain. No mutations were identified during a screen of patients suffering from Doyne's honeycomb retinal dystrophy (DHRD), an inherited retinal degeneration which maps to chromosome 2p16.

Amino Acid Sequence↗
PubMed1998

Hypoalgesia in mice with a targeted deletion of the tachykinin 1 gene.

The tachykinin neuropeptides, substance P and substance K, are produced in nociceptive primary sensory neurons and in many brain regions involved in pain signaling. However, the precise role and importance of these neuropeptides in pain responses has been debated. We now show that mice that cannot produce these peptides display no significant pain responses following formalin injection and have an increased pain threshold in the hotplate test. On the other hand, the mutant mice react normally in the tail flick assay and acetic acid-induced writhing tests. These results demonstrate that substance P and/or substance K have essential functions in specific responses to pain.

Acetic Acid↗
PubMed1995

Stress-induced changes in messenger RNA levels of N-methyl-D-aspartate and AMPA receptor subunits in selected regions of the rat hippocampus and hypothalamus.

The postsynaptic AMPA/kainate and N-methyl-D-aspartate-selective glutamate receptors are formed by several different subunits and the overall subunit composition of the receptor appears to determine its physiological and pharmacological properties. Although glutamatergic mechanisms have been implicated in various forms of hippocampal stress responses, the impact of stress on glutamate receptor subunit composition has not yet been elucidated. We have used cell-by-cell quantitative in situ hybridization to assess stress-induced changes in transcript levels of N-methyl-D-aspartate and AMPA receptor subunit genes in subdivisions of the rat hippocampus and hypothalamus that are implicated in the stress response. We found that 24 h after a single immobilization stress there was a significant increase in the cellular level of NR1 subunit messenger RNA (about 35-45% above control values) in hippocampal CA3 and CA1 pyramidal cells as well as in neurons of the hypothalamic supraoptic and paraventricular nuclei. Moreover, in the CA3 area we have detected a concomitant increase (50% above controls) in the level of NR2B subunit messenger RNA, while the expression of NR2A subunit gene did not change after stress. Stress induced a selective decrease in the level of AMPA receptor subunit glutamate receptor A messenger RNA in neurons of both the CA3 and CA1 areas (18 and 24%, respectively, below control values). These results suggest that the regulation of specific subunit messenger RNAs of the N-methyl-D-aspartate and AMPA receptors may be involved in altered hippocampal and hypothalamic responsiveness to glutamate and thus could play a critical role in stress-induced changes in their function.

Animals↗
PubMed1994

Stressor-specific increase of vasopressin mRNA in paraventricular hypophysiotrophic neurons.

Cellular levels of vasopressin (VP) and corticotropin-releasing factor (CRF) RNA transcripts were determined in hypophysiotrophic neurons after open-field and immobilization stress using quantitative in situ hybridization. We found that 8 min open-field stress is sufficient to produce a significant up-regulation of CRF mRNA, without any concomitant changes in the level of VP mRNA. In contrast, 8 min immobilization stress resulted in an increased labeling density of both CRF and VP mRNAs. These results suggest that the level of CRF and VP transcripts in parvicellular hypophysiotrophic neurons is differentially regulated in a stressor-specific manner.

Animals↗
PubMed1993

Up-regulation of vasopressin mRNA in paraventricular hypophysiotrophic neurons after acute immobilization stress.

Cellular levels of vasopressin (VP) and corticotropin-releasing factor (CRF) mRNAs were determined in the rat hypothalamic paraventricular nucleus after acute immobilization stress. Messenger RNA levels were measured by in situ hybridization histochemistry using 35S-labeled synthetic DNA oligonucleotide probes and quantitative autoradiography. Analysis of histograms related to density values revealed that two hours after immobilization the average cellular VP and CRF mRNA levels were significantly increased (145 +/- 30.3 and 68 +/- 21.3%, respectively, above control values). Moreover, the number of VP-expressing parvicellular neurons was doubled in stressed rats relative to controls. These results indicate that single acute immobilization stress is sufficient to up-regulate VP as well as CRF mRNA production in the hypothalamic CRF-neurosecretory system.

Animals↗
PubMed1992

Neuropeptides in the human superior cervical ganglion.

Superior cervical ganglia from 7 human cadavers (3-7 h post mortem) were immunostained for tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and 14 different neuropeptides. The results show that ganglionic cells contain TH, DBH, neuropeptide Y (NPY), somatostatin, vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP). These substances were present predominantly within large ganglionic cells. Inside the ganglion, the number and topographical distribution of various types of immunoreactive cells differed from one another. NPY and CGRP immunoreactivities were found in some TH-positive cells, but that co-localization never exceeded the 30% of the TH cells. Leu-enkephalin showed a weak immunoreactivity, which was restricted to fibers or varicosities. Neuropeptides like substance P, dynorphin A and B, cholecystokinin, galanin, corticotropin-releasing factor, thyrotropin-releasing hormone, angiotensin II and neurotensin showed no immunoreactivity in the human superior cervical ganglion.

Calcitonin Gene-Related Peptide↗