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Biomedical subjects

Paul Hiscott

Publications and source records attributed to Paul Hiscott.

At least 19 recordsLinked to original sources
PubMed2006

Trafficking of osteonectin by retinal pigment epithelial cells: evidence for basolateral secretion.

Osteonectin is a glycoprotein that modulates several aspects of cellular behaviour including proliferation and adhesion. The retinal pigment epithelium forms a continuous monolayer of polarised cells immediately bellow the neuroretina, and is integral to the homeostasis of photoreceptor cells. While osteonectin is expressed by normal retinal pigment epithelium in situ, its expression is significantly increased in retinal pigment epithelial cells associated with several common retinal diseases. This pattern of expression implies an important role for osteonectin in the biology of retinal pigment epithelial cells. However, the trafficking, processing, and eventual fate of osteonectin in these cells is not clear at present. Although the theoretical report of a leader sequence within the osteonectin open reading frame and its extracellular presence in some tissues indirectly support secretion of the protein, there is no direct experimental demonstration of the secretion route to date. As a first step towards understanding the role of osteonectin in retinal pigment epithelium, we studied the intracellular distribution and trafficking of the protein in living cells. Here, we present experimental evidence that a precursor osteonectin fusion protein is targeted to the endoplasmic reticulum/Golgi pathway, with a likely basal secretion in retinal pigment epithelial cells. In addition, we show that the precursor osteonectin protein having the leader sequence masked fails to undergo secretion leading to cell death, a phenotype which may be of relevance not only for retinal pathology, but also for other diseases such as the bone disorder known as pseudoachondroplasia that is associated with a lack of osteonectin secretion.

Achondroplasia↗
PubMed2006

Expression of p53-induced apoptosis effector PERP in primary uveal melanomas: downregulation is associated with aggressive type.

Expression of PERP (p53 apoptosis effector related to PMP-22) was investigated in primary uveal melanomas and its variation was analyzed in relation to clinico-pathological and cytogenetical characteristics of these tumors. The transcriptional level of PERP gene was measured by quantitative real-time RT-PCR in 26 uveal melanomas with known chromosomes 3 and 8 status. PERP protein levels were assessed by Western blot analysis of 22 fresh-frozen tumors and by immunohistochemical analysis of 16 paraffin-embedded tumor specimens. Differential expression of PERP was identified in primary choroidal melanoma specimens, both at transcriptional and protein level. Reduced PERP mRNA level was significantly associated with monosomy 3 (two-way ANOVA and t-test, p=0.004) but not with gains in chromosome 8. Transcriptional downregulation of PERP did not present a statistically significant association with ciliary body involvement, size, PAS-positive loops or cell type. Immunoblotting and immunohistochemistry further demonstrated significantly reduced PERP protein level in monosomy 3 melanomas, as compared with disomy 3 tumors. The altered expression of PERP highlighted this apoptosis-specific target of p53 as a possible contributor to apoptosis in uveal melanoma with PERP downregulation being particularly relevant to the aggressive (monosomy 3) type of uveal melanoma. As PERP is a novel type of p53 effector that is likely to stimulate apoptosis through a mechanism distinct from that of Bcl-2-related mitochondrial effectors, further elucidation of its role in uveal melanoma pathogenesis will assist in the design of novel therapeutic approaches aimed at increasing the rate of apoptosis in this tumor.

Adult↗
PubMed2006

A dual Golgi- and mitochondria-localised Ala25Ser precursor cystatin C: an additional tool for characterising intracellular mis-localisation leading to increased AMD susceptibility.

An artificial mutant Ala25Ser precursor cystatin C was created to help elucidate the cause of intracellular mis-localisation of the biochemically related variant B (Ala25Thr) precursor cystatin C to the mitochondria. Homozygotes of variant B precursor cystatin C were reported to carry an increased susceptibility to developing the exudative form of AMD. Ala25Ser precursor cystatin C shows a dual distribution to the Golgi apparatus and to the mitochondria. This localisation is thus intermediary between that of wild-type cystatin C (targeted to ER/Golgi compartment) and that of variant B precursor cystatin C. Furthermore, the level of secretion of Ala25Ser cystatin C by RPE cells is intermediary between wild type and variant B cystatin C. Ala25Ser precursor cystatin C thus represents a biochemical intermediate between the wild type and the AMD-associated cystatin C and as such, is a novel tool for the investigation of the mechanism of intracellular mis-localisation of variant B cystatin C. Our findings further support the hypothesis that substitution of the alanine residue in the penultimate position of precursor cystatin C signal sequence with a less hydrophobic amino acid residue, such as threonine (as in variant B cystatin C) or serine is sufficient to impair the intracellular trafficking and processing of the protein.

Cystatin C↗
PubMed2006

Choroidal neovascular membrane in a series of cases of malignant melanoma of the choroid.

PURPOSE: To examine a series of choroidal melanoma specimens to determine the frequency of overlying choroidal neovascularization (CNV) and to ascertain whether CNV over choroidal malignant melanoma is associated with any particular histological tumour characteristics. METHODS: We carried out a retrospective histological analysis of globes containing choroidal melanomas for evidence of choroidal neovascular membranes. RESULTS: Microscopic evidence of choroidal neovascular membranes was evident in 6% of cases. Choroidal neovascularization was not associated with any particular histological tumour characteristic. CONCLUSION: Choroidal neovascularization over choroidal malignant melanoma is not an infrequent occurrence and possibly appears as frequently as CNV over choroidal naevi. The presence of a choroidal neovascular membrane over a pigmented fundal lesion should not be taken as reassurance that the lesion is benign.

Aged↗
PubMed2005

Thrombospondin 1, thrombospondin 2 and the eye.

Thrombospondin 1 and thrombospondin 2 (TSP1 and TSP2), which comprise the subgroup A thrombospondins, are matricellular proteins. As matricellular proteins, they modulate interactions between cells and the cellular environment, regulate cell adhesion and typically are expressed during tissue formative processes. In general, TSP1 and TSP2 counter angiogenesis (including tumour angiogenesis) and play important but contrasting roles during cutaneous repair. The two proteins are involved in development, including that of the eye, although evidence suggests that they have their greatest impact during tissue production in the adult. In the normal adult eye, they tend to be found at sites of ongoing matrix synthesis or cell-matrix interactions. At these sites, the two proteins possibly influence cellular differentiation and/or basement membrane deposition. TSP1 is also present in the intraocular fluids and drainage pathway, where it may function in maintaining the anti-angiogenic environment and in intraocular pressure control, respectively. TSP1 could also be involved in ocular immune privilege. Unlike in skin wounds, where TSP1 is derived from the blood and is present only in the early phases of repair, ocular tissue damage appears to lead to protacted TSP1 synthesis by local cells. This response might help suppress angiogenesis in the transparent tissues of the eye and so lessen visual axis opacification following injury. However, TSP2, which is also produced by damaged ophthalmic tissue and may be especially important in matrix organisation, seems to augment contraction in anomalous intraocular fibrosis. Elucidating the roles of TSP1 and TSP2 in ocular physiology and pathobiology may lead to improved therapies for neovascular, neoplastic, reparative and other ophthalmic diseases.

Animals↗
PubMed2005

Suppression of thrombospondin 1 and 2 production by herpes simplex virus 1 infection in cultured keratocytes.

PURPOSE: Stromal vascularization is a frequent occurrence in herpes simplex keratitis (HSK) and carries a poor prognosis for penetrating keratoplasty. The pathogenesis may involve disruption of the normal equilibrium between angiogenic and anti-angiogenic factors in and around the cornea. Thrombospondin (TSP) 1 and 2 are multifunctional matricellular glycoproteins with potent anti-angiogenic properties and are expressed by human keratocytes in a stromal wound repair model. We hypothesize that the synthesis of these anti-angiogenic proteins by keratocytes is inhibited by HSV1 and that such a mechanism may contribute to stromal vascularization in HSK. METHODS: Nonconfluent monolayers of human keratocytes were infected with HSV1 at a multiplicity of infection of 5 virus particles/keratocyte. Expression of TSP1 and TSP2 was determined by immunohistochemistry and SDS-polyacrylamide gel electrophoresis at 0, 2, 4, 6, 8, 24, 48, and 72 h after infection (ai). Expression of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) served as a control. Expression of immediate early and late viral proteins was also determined. Protein expression was quantified by densitometric analysis of the immunoblot bands. RESULTS: Human keratocytes supported the growth of HSV1 at all times ai. TSP1 and TSP2 were downregulated as early as 4 h ai to a 50% reduction by 8 h (p<0.002), and were absent from 24 h ai (p<0.001). There was no change in the level of expression of GAPDH throughout the duration of the experiment. Immediate early viral proteins (HSV1:ICP27) could be detected from 6 h ai reaching maximum intensity 24 h ai and late proteins (HSV:1gD) were expressed from 24 h. CONCLUSIONS: The synthesis of TSP1 and TSP2 is selectively downregulated by HSV1 infection in human keratocytes. Addition of these proteins or their angio-active peptides in early stage HSK therapy may be an important adjuvant in controlling HSV1 induced corneal vascularization.

Blotting, Western↗
PubMed2005

Evidence for persistence of adenovirus in the tear film a decade following conjunctivitis.

Chronic papillary conjunctivitis has been described following adenoviral conjunctivitis. It is unknown however, how long adenovirus is able to persist in the tear film and conjunctiva. To determine if adenovirus persists in the ocular surface following adenoviral conjunctivitis, 304 patients with a history of adenovirus conjunctivitis from whom an adenovirus had been isolated 10 years previously were sent a questionnaire regarding persistent or recurrent symptoms and were invited to attend. Patients were examined and samples of tears and conjunctival cells were collected from both eyes using tear film washes, filter paper, and swabs, the latter for virus isolation. Extracted DNA from the ocular samples was amplified using primers for herpes simplex virus (thymidine kinase) and adenovirus (hexon) genes. Adenovirus amplicons were sequenced and compared to original serotype. Thirty patients attended, 19 of whom had persistent papillary conjunctivitis. Evidence of adenovirus DNA was detected in 17 of 30 patients, 15 of whom also had evidence of a chronic papillary conjunctivitis. Adenovirus DNA was significantly associated with papillary conjunctivitis (P = 0.03). Adenovirus amplicons were successfully sequenced from six patients. Four patients harbored type 3 adenovirus, the same serotype with which they were infected originally 10 years previously. Two patients were infected originally with adenovirus serotype 3 but the current serotype was type 4. Infection of the ocular surface with adenovirus may predispose to the development of a persistent or recurrent conjunctivitis, the presence of which, appears to be associated with evidence of long term persistence of adenovirus DNA.

Adenovirus Infections, Human↗
PubMed2005

Detection of the slipped extraocular muscle after strabismus surgery.

PURPOSE: To estimate the frequency of slipped extraocular muscle (SM) in cases of repeat strabismus surgery and to compare the results of a number of putative preoperative tests for the detection of SM, with direct intraoperative inspection and histologic confirmation. DESIGN: Retrospective analysis of audit records followed by prospective interventional case series. PARTICIPANTS: Case records of 715 adults presenting for repeat surgical correction of horizontal strabismus. Five patients suspected of having horizontal SM were consecutively recruited for the prospective case series. METHODS: Slipped extraocular muscle frequency was determined retrospectively from surgical outcome data in 715 adult horizontal strabismus surgeries in 1 center. A separate prospective study examined 5 participants with putative SM in detail. Gaze-dependent clinical tests (squint magnitude, eye movement range, palpebral fissure widening, naked-eye saccadic velocity estimation, intraocular pressure change) and saccade main sequence parameters (infrared eye-tracking) were recorded before and 2 weeks after corrective surgery. Intraoperative diagnosis was made by direct muscle examination and confirmed by histologic examination. Length of slippage and forced duction tests were intraoperative outcome measures. RESULTS: Slipped extraocular muscle frequency was 10.6%. The muscles of 3 participants in the prospective series were slipped. Direct intraoperative inspection accurately identified all SM cases. All other clinical tests produced false-positive and false-negative results, although SM was suggested preoperatively by limited eye movement range. Distorted saccadic velocity profiles were significantly (P<0.0001) associated with prior strabismus surgery. Saccade main sequence parameters were not diagnostic for SM. CONCLUSIONS: Direct intraoperative inspection can accurately diagnose SM. Preoperative diagnosis of long-standing SM was not possible. In contradiction to a previous report, SM was not reliably associated with reduced peak saccadic velocity. The association between distorted saccadic velocity profiles and previous strabismus surgery is, to our knowledge, reported here for the first time.

Adult↗
PubMed2005

Differential expression of angioregulatory matricellular proteins in posterior uveal melanoma.

Metastases from uveal melanoma, the most common primary malignant eye tumour in adults, develop solely via their vascular bed due to the absence of intraocular lymphatics. The present study investigated the expression in this tumour of three matricellular proteins--Secreted Protein Acidic and Rich in Cysteine (SPARC), thrombospondin 1 (TSP1) and thrombospondin 2 (TSP2)--with putative contrasting roles in the regulation of angiogenesis. Immunohistochemical analysis of the three proteins was carried out in paraffin-embedded specimens from 27 posterior uveal melanomas and was corroborated with Western blot analysis of fresh-frozen samples from seven of the tumours. SPARC immunoreactivity was detected in all specimens and defined two categories of tumour: SPARC-rich (21 of 27 specimens) and SPARC-patchy (six of 27 specimens) uveal melanomas. SPARC-rich tumours had a significantly higher proportion of specimen area occupied by blood vessels (P=0.04) and showed a positive association with the presence of epithelioid-type tumoral cells (P=0.101). TSP1 was not detected by either of the methods in any of the tumours analysed. Some immunopositivity for TSP2 was detected in tumour cells in approximately 40% of specimens, but was not associated with survival, tumour vascularity or any other histopathological indices of survival. The pattern of expression of these matricellular proteins in uveal melanoma is consistent with a cooperative mechanism for establishing an enhanced environment favourable to angiogenesis. Interventions inducing TSP1 expression and/or inhibiting SPARC expression may be candidates for therapies directed towards the inhibition of angiogenesis in posterior uveal melanoma.

Adult↗
PubMed2005

Bullous keratopathy and corneal decompensation secondary to iridoschisis: a clinicopathological report.

PURPOSE: To report a case of localized bullous keratopathy and total corneal decompensation secondary to iridocorneal touch in iridoschisis. DESIGN: Single interventional case report with pathologic correlation. METHODS: An 80-year-old man with bilateral iridoschisis presented with loss of vision in the left eye (OS). Examination of the OS showed shallow anterior chamber (AC), cortical cataract, and free-floating iris fibers causing iridocorneal touch, leading to localized bullous keratopathy and total corneal decompensation. Ultrasound biomicroscopy (UBM) confirmed the area of iridocorneal contact. RESULTS: Penetrating keratoplasty combined with cataract extraction and posterior chamber lens implantation was performed in the OS. Histopathology of the corneal button showed features of corneal edema and endothelial cell loss with evidence of iridocorneal contact. CONCLUSIONS: Iridoschisis is an uncommon cause of bullous keratopathy and corneal decompensation. Patients with iridoschisis may need specular microscopy to determine the status of the corneal endothelium.

Aged, 80 and over↗
PubMed2004

Distal arthrogryposis type IIB: unreported ophthalmic findings.

We describe four members spanning three generations of a Caucasian family affected with distal arthrogryposis (DA). Based on Hall's original classification, we have placed our family under type IIB and present previously unreported ophthalmic features. All the members had different degrees of ophthalmoplegia, ptosis, astigmatism, and strabismus. Other findings in affected family members included keratoconus in the index patient, which was associated with abnormal electron microscopy of the affected cornea and increased thickness of the central cornea, small axial length of the globe and choroidal folds in the others.

Adult↗
PubMed2004

Unexpected intracellular localization of the AMD-associated cystatin C variant.

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.

Aging↗
PubMed2003

Expression of ADAMTS metalloproteinases in the retinal pigment epithelium derived cell line ARPE-19: transcriptional regulation by TNFalpha.

ADAMTS (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type I motifs) are multidomain proteins with demonstrated metalloproteinase functionality and have potential roles in embryonic development, angiogenesis and cartilage degradation. We present here investigations of ADAMTS expression in an ocular cell type, ARPE-19, with a view to implicating them in retinal matrix turnover. Expression analysis was undertaken using a combination of reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting experiments, which together detected the expression of mRNAs for several ADAMTS proteins, all of which have active site motifs characteristic of matrix metalloproteases (MMPs). These included ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS5, ADAMTS6, ADAMTS7 and ADAMTS9. The expression of mRNA isoforms for ADAMTS7 and ADAMTS9 were also detected. Following stimulation with TNFalpha, ADAMTS1, ADAMTS6 and both ADAMTS9 transcripts expressed in ARPE-19 cells showed a potent upregulation. The expression of ADAMTS genes in ARPE-19 cells and the transcriptional stimulation of some family members by TNFalpha may implicate them in inflammatory eye disease and the compromise of retinal matrix structure, which is evident in age-related macular degeneration (ARMD) and other retinal pathologies.

ADAM Proteins↗
PubMed2003

Effects of the matricellular protein SPARC on human retinal pigment epithelial cell behavior.

PURPOSE: To determine the effects of the matricellular protein SPARC (Secreted Protein, Acidic and Rich in Cysteine) on human retinal pigment epithelial (HRPE) cell behavior in vitro. METHODS: Proliferation and migration assays were performed on HRPE cells exposed to various concentrations of SPARC. Additionally, HRPE cells were seeded on top of collagen matrices (a 2D model of the retinal scarring disorder known as proliferative vitreoretinopathy or PVR) and were exposed to SPARC over a 7 day period. Changes in matrix contraction were recorded. RESULTS: HRPE cell proliferation was significantly inhibited at 1 and 10 microg/ml SPARC (p<0.01). SPARC protein did not stimulate HRPE cell migration at any of the concentrations used. SPARC did not significantly affect fibronectin-induced HRPE cell migration at SPARC concentrations up to 10 microg/ml. HRPE cell-seeded collagen matrices demonstrated a significant inhibition of matrix contraction by 1 and 10 microg/ml SPARC (t-test; p<0.02 and 0.001, respectively) compared to controls. CONCLUSIONS: SPARC protein has anti-proliferative effects on HRPE cells in vitro. In addition, SPARC appears to have an inhibitory effect on HRPE-mediated contraction of 2D collagen matrices. These results are consistent with an important role for SPARC in modulating cell behavior in vitro and may indicate a role for SPARC in modifying HRPE cell activities during the development of PVR and other proliferative retinal diseases.

Adult↗
PubMed2003

Keratocyte matrix interactions and thrombospondin 2.

PURPOSE: To determine whether human keratocytes synthesize thrombospondins 2 and 3 (TSP-2, TSP-3) in a collagen matrix and the effect of addition of antibodies to TSP-2 and TSP-3 on keratocyte-populated collagen matrix contraction. METHODS: Keratocyte-populated collagen matrices were evaluated for TSP-2 and TSP-3 mRNA. Sections of matrices were stained by immunohistochemistry for TSP-2 and TSP-3. Keratocyte populated collagen matrices were treated with antibodies specific for TSP-2 and TSP-3 and these preparations were evaluated for contraction and keratocyte morphology. RESULTS: Keratocyte derived fibroblasts in collagen matrices contained TSP-2 and TSP-3 mRNA, and the cells were immunoreactive for both proteins. Compared to controls, an antibody specific to the N-terminal domain of TSP-2 significantly inhibited matrix contraction for up to 10 days at a concentration of 20 microg/ml antibody. At 2 microg/ml TSP-2 antibody concentration significant inhibition occurred up to 3 days. Removal of the antibody from the media reversed the inhibitory effect. Cultured keratocytes in TSP-2 treated collagen matrices appeared more rounded than keratocytes in control matrices. An antibody specific to TSP-3 had no effect on matrix contraction or keratocyte morphology. CONCLUSIONS: Keratocyte derived fibroblasts synthesize TSP-2 and TSP-3 when seeded in collagen matrices. Antibody specific to TSP-2 reversibly inhibits matrix contraction. TSP-2 may play a key role in keratocyte/collagen matrix interactions, as may occur during corneal stromal repair.

Cell Adhesion Molecules↗