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P A Campochiaro

Publications and source records attributed to P A Campochiaro.

At least 19 recordsLinked to original sources
PubMed2006

Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1.

In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mug of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mug of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential.

Animals↗
PubMed2006

Potential applications for RNAi to probe pathogenesis and develop new treatments for ocular disorders.

The eye is a relatively isolated tissue compartment, which provides advantages for utilization of small interfering RNA (siRNA). Feasibility of using siRNA for treatment of choroidal neovascularization has been demonstrated using siRNA directed against vascular endothelial growth factor (VEGF) or VEGF receptor 1 (VEGFR1), and both of these approaches are being tested in clinical trials. The results with VEGFR1 siRNA show that VEGFR1 is pro-angiogenic in the eye and is not a decoy receptor as it is in developmental angiogenesis. Topical delivery of siRNAs directed against VEGF or its receptors has also been shown to suppress corneal neovascularization. Signaling through transforming growth factor-beta receptor 2 (TGFbetaR2) has been implicated in excessive ocular scarring and TGFbetaR2 siRNA has shown benefit in a model relevant to excessive scarring after glaucoma filtration surgery. RNAi has been used to identify genes that promote apoptosis or oxidative damage in retinal cells and could be the basis of new treatments for glaucoma or photoreceptor degenerations. In cultured cells derived from ocular tissues, siRNA has become a valuable tool to explore the potential role of various genes in ocular disease processes. Based upon this early experience in vivo and in vitro, it appears that siRNAs may be valuable to help define the pathogenesis and develop new treatments for several ocular diseases.

Animals↗
PubMed2006

Sustained expression after nonviral ocular gene transfer using mammalian promoters.

The CMV promoter drives high transgene expression and is one of the most commonly used promoters for gene transfer. Tissue-specific mammalian promoters provide an alternative, and it would be useful to have a system to directly compare them to viral promoters free from potential confounding vector-related effects. In this study, we describe how electroporation after subretinal injection of plasmid DNA can be used to perform comparative quantitative analysis of promoter activities. Luciferase assay of eyecup homogenates was carried out after coinjection/electroporation of pGL2, a plasmid containing the promoter fragment of interest coupled to the firefly luciferase gene, and pRL-CMV, a plasmid containing the CMV promoter coupled to the Renilla luciferase gene for normalization. This technique was used to compare activity of different fragments of the 5'-upstream region of the vitelliform macular dystrophy 2 (VMD2) gene, which is selectively expressed in the retinal pigmented epithelial (RPE) cells, and results indicated positive regulatory elements between -104 and -154 bp and between -424 and -585 bp. Addition of a fragment from intron 1 reduced the activity of the -585/+38 bp fragment by 75%. Deletion analysis implicated a 342 bp region near the 5'-end of intron 1 in the repression. Results of transient transfections in two cell lines that constitutively express VMD2 were similar, and results in transgenic mice were consistent, providing validation for promoter analysis by in vivo electroporation. We then explored the time course of expression of the -585/+38 VMD2 promoter fragment and found that compared to cassettes driven by CMV or SV40 promoters, which showed peak luciferase activity on day 2 followed by a rapid decrease in activity, the VMD2 promoter fragment showed lower activity initially, but the activity was sustained for up to 56 days (longest time point measured). A promoter fragment from another RPE-specific gene, Rpe65, showed a similar pattern of sustained expression for at least 112 days. These data indicate that nonviral gene transfer can be used to quantitatively evaluate the activity of promoter fragments independent of influence from viral vectors. A potentially important finding using this new technique is the demonstration that relatively sustained passenger gene expression can be achieved with nonviral gene transfer using mammalian rather than viral promoters.

Animals↗
PubMed2005

Nonviral ocular gene transfer.

In this study, we explored the use of electroporation or media that promote lipoplex formation for nonviral gene transfer in the eye. There was no detectable staining for LacZ after subretinal, intravitreous, or periocular injection of a plasmid containing a CMV promoter expression cassette for LacZ, but when plasmid injection in each of the three sites was combined with electroporation, there was efficient transduction. Specific staining for LacZ was seen in retinal pigmented epithelial (RPE) cells after subretinal injection of a plasmid containing a vitelliform macular dystrophy 2 (VMD2) promoter expression cassette, demonstrating that this approach can be used to evaluate purported tissue-specific promoters in vivo. Electroporation with 10 V/mm resulted in strong LacZ staining, but was damaging to photoreceptors; substantial transduction with no evidence of retinal damage was seen using 3.4 V/mm. Staining for LacZ was also seen after subretinal or periocular, but not intravitreous, injection of plasmid DNA in medium containing 40% Lipofectamine2000 (Lf). Injection of 40% Lf into the subretinal space caused damage to photoreceptors, but subretinal injection of plasmid DNA in medium containing 10% NeuroPorter resulted in transduction of RPE cells with no adverse effects on retinal morphology or function as assessed by electroretinograms (ERGs). After either electroporation or lipofection, LacZ staining was detectable for at least 14 days, and could be reinduced by a second procedure. These data suggest that electroporation or lipofection can be used as experimental tools for ocular gene transfer to evaluate tissue-specific promoter fragments or to evaluate the effects of transgene expression in the retina. Also, with additional optimization, nonviral gene transfer may prove to be a valuable approach for the treatment of retinal and choroidal diseases.

Animals↗
PubMed2004

Angiopoietin 1 inhibits ocular neovascularization and breakdown of the blood-retinal barrier.

Several retinal and choroidal diseases are potentially treatable by intraocular delivery of genes whose products may counter or neutralize abnormal gene expression that occurs as part of the diseases. However, prior to considering a transgene, it is necessary to thoroughly investigate the effects of its expression in normal and diseased eyes. An efficient way to do this is to combine tissue-specific promoters with inducible promoter systems in transgenic mice. In this study, we used this approach to evaluate the effects of ectopic expression of angiopoietin-1 (Ang1) in normal eyes and those with ocular neovascularization. Adult mice with induced expression of Ang1 ubiquitously, or specifically in the retina, appeared normal and had no identifiable changes in retinal or choroidal blood vessels or in retinal function as assessed by electroretinography. Increased expression of Ang1 in eyes with severe retinal ischemia or in eyes with rupture of Bruch's membrane significantly suppressed the development of retinal or choroidal neovascularization, respectively. This inhibition of ocular neovascularization is particularly interesting and noteworthy, because overexpression of Ang1 in skin stimulates neovascularization. Ang1 also significantly reduced VEGF-induced retinal vascular permeability. These data suggest that intraocular delivery of ang1 has potential for treatment of ocular neovascularization and macular edema.

Angiopoietin-1↗
PubMed2003

Periocular injection of an adenoviral vector encoding pigment epithelium-derived factor inhibits choroidal neovascularization.

Gene transfer provides an exciting new approach for the treatment of retinal and choroidal diseases. Two areas of concern are the potential for vector-related toxicity and uncertainties associated with prolonged transgene expression. One way to address these concerns for transfer of genes encoding secreted proteins is to transduce cells on the outside of the eye, provided the gene product can gain access to the eye and have the desired effect. In this study, we investigated the feasibility of this approach. Periocular injection of an adenoviral vector encoding beta-galactosidase (AdLacZ.10) resulted in LacZ-stained cells throughout the orbit and around the eye. Compared to periocular injection of 5 x 10(9) particles of control vector, periocular injection of 5 x 10(9) or 1 x 10(9) particles of an adenoviral vector expressing pigment epithelium-derived factor (PEDF) regulated by a CMV promoter (AdPEDF.11) resulted in significantly elevated intraocular levels of PEDF and suppression of choroidal neovascularization. Periocularly injected recombinant PEDF was also found to diffuse through the sclera into the eye. Although similar experiments are needed in an animal with a human-sized eye, these data suggest that periocular gene transfer deserves consideration for the treatment of choroidal diseases.

Adenoviridae↗
PubMed2001

Expression and permeation properties of the K(+) channel Kir7.1 in the retinal pigment epithelium.

Bovine Kir7.1 clones were obtained from a retinal pigment epithelium (RPE)-subtracted cDNA library. Human RPE cDNA library screening resulted in clones encoding full-length human Kir7.1. Northern blot analysis indicated that bovine Kir7.1 is highly expressed in the RPE. Human Kir7.1 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The macroscopic Kir7.1 conductance exhibited mild inward rectification and an inverse dependence on extracellular K+ concentration ([K+]o). The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.013) > Na+ (0.003) approximately Li+ (0.001) and the sequence based on conductance ratios was Rb+ (9.5) >> K+ (1.0) > Na+ (0.458) > Cs+ (0.331) > Li+ (0.139). Non-stationary noise analysis of Rb+ currents in cell-attached patches yielded a unitary conductance for Kir7.1 of approximately 2 pS. In whole-cell recordings from freshly isolated bovine RPE cells, the predominant current was a mild inwardly rectifying K+ current that exhibited an inverse dependence of conductance on [K+]o. The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.021) > Na+ (0.003) approximately Li+ (0.002) and the sequence based on conductance ratios was Rb+ (8.9) >> K+ (1.0) > Na+ (0.59) > Cs+ (0.23) > Li+ (0.08). In cell-attached recordings with Rb+ in the pipette, inwardly rectifying currents were observed in nine of 12 patches of RPE apical membrane but in only one of 13 basolateral membrane patches. Non-stationary noise analysis of Rb+ currents in cell-attached apical membrane patches yielded a unitary conductance for RPE Kir of approximately 2 pS. On the basis of this molecular and electrophysiological evidence, we conclude that Kir7.1 channel subunits comprise the K+ conductance of the RPE apical membrane.

Animals↗
PubMed2001

Pigment epithelium-derived factor inhibits retinal and choroidal neovascularization.

In this study, we investigated whether overexpression of pigment epithelium-derived factor (PEDF) by gene transfer can inhibit neovascularization by testing its effect in three different models of ocular neovascularization. Intravitreous injection of an adenoviral vector encoding PEDF resulted in expression of PEDF mRNA in the eye measured by RT-PCR and increased immunohistochemical staining for PEDF protein throughout the retina. In mice with laser-induced rupture of Bruch's membrane, choroidal neovascularization was significantly reduced after intravitreous injection of PEDF vector compared to injection of null vector or no injection. Subretinal injection of the PEDF vector resulted in prominent staining for PEDF in retinal pigmented epithelial cells and strong inhibition of choroidal neovascularization. In two models of retinal neovascularization (transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors and mice with oxygen-induced ischemic retinopathy), intravitreous injection of null vector resulted in decreased neovascularization compared to no injection, but intravitreous injection of PEDF vector resulted in further inhibition of neovascularization that was statistically significant. These data suggest that sustained increased intraocular expression of PEDF by gene therapy might provide a promising approach for treatment of ocular neovascularization.

Adenoviridae↗
PubMed2001

Neurotrophic signaling in normal and degenerating rodent retinas.

Several types of insult cause up-regulation of neurotrophic factors and their receptors in the retina resulting in decreased photoreceptor cell death from subsequent injury. This phenomenon is more prominent in rats than in mice and neurotrophic factors are more efficacious in rats than mice. If up-regulation of neurotrophic factor receptors on photoreceptor cells early in the course of degenerations contributes to neurotrophic factor survival-promoting activity, it may also increase the ability to detect neurotrophic factor-induced signaling in photoreceptors, particularly in rats. In this study, these hypotheses were investigated by performing immunohistochemical staining for the phosphorylated form of extracellular receptor kinase (pERK) or c-fos after intravitreous injection of neurotrophic factors in wild type rats or mice, or those with inherited retinal degenerations. In both rats and mice either early or late in the course of degeneration, or in wild type animals, intravitreous injection of brain-derived neurotrophic factor, ciliary neurotrophic factor, or fibroblast growth factor-2 caused immunostaining for pERK and c-fos in cells of the inner retina, particularly Müller cells, but not in photoreceptors. These data add to the mounting evidence suggesting that neurotrophic factors act indirectly through Müller cells to promote photoreceptor survival.

Animals↗
PubMed2001

Inhibition of choroidal neovascularization by intravenous injection of adenoviral vectors expressing secretable endostatin.

Endostatin is a cleavage product of collagen XVIII that inhibits tumor angiogenesis and growth. Interferon alpha2a blocks tumor angiogenesis and causes regression of hemangiomas, but has no effect on choroidal neovascularization (CNV). Therefore, inhibitors of tumor angiogenesis do not necessarily inhibit ocular neovascularization. In this study, we used an intravenous injection of adenoviral vectors containing a sig-mEndo transgene consisting of murine immunoglobulin kappa-chain leader sequence coupled to sequence coding for murine endostatin to investigate the effect of high serum levels of endostatin on CNV in mice. Mice injected with a construct in which sig-mEndo expression was driven by the Rous sarcoma virus promoter had moderately high serum levels of endostatin and significantly smaller CNV lesions at sites of laser-induced rupture of Bruch's membrane than mice injected with null vector. Mice injected with a construct in which sig-mEndo was driven by the simian cytomegalovirus promoter had approximately 10-fold higher endostatin serum levels and had nearly complete prevention of CNV. There was a strong inverse correlation between endostatin serum level and area of CNV. This study provides proof of principle that gene therapy to increase levels of endostatin can prevent the development of CNV and may provide a new treatment for the leading cause of severe loss of vision in patients with age-related macular degeneration.

Adenoviridae↗
PubMed2001

Fibroblast growth factor-2 decreases hyperoxia-induced photoreceptor cell death in mice.

Fibroblast growth factor-2 (FGF2) has neurotrophic effects in vitro and in vivo. It has been demonstrated to decrease photoreceptor cell death in rats exposed to constant light and in rats with an inherited defect in retinal pigmented epithelium (RPE) phagocytosis, but the effects of intravitreous injections of FGF2 in mice are equivocal. In this study, we used transgenic mice with increased expression of FGF2 in photoreceptors (rhodopsin promoter/FGF2 transgenics) to investigate the effects of sustained increased expression of FGF2 in mice with various types of photoreceptor degeneration, including rd mice that are homozygous for mutated phosphodiesterase beta subunit, Q344ter mice that undergo photoreceptor degeneration because of expression of mutated rhodopsin, and mice exposed to 75% oxygen for 1 or 2 weeks. At P21, the outer nuclear layer was markedly reduced in rd mice or Q344ter mice regardless of whether they inherited the rhodopsin promoter/FGF2 transgene. However, after 2 weeks of exposure to 75% oxygen, outer nuclear layer thickness was significantly reduced in littermate control mice compared to FGF2 transgenic mice (P = 0.0001). These data indicate that increased expression of FGF2 in photoreceptors protects them from hyperoxia-induced damage, but does not decrease cell death related to expression of mutated proteins involved in the phototransduction pathway. This suggests that FGF2 protects photoreceptors from oxidative damage, which may play a role in complex genetic diseases such as age-related macular degeneration.

Aging↗
PubMed2001

Eye pain after vitreoretinal surgery: a prospective study of 185 patients.

PURPOSE: To assess subjective levels of eye pain, nausea, and sedation following vitreoretinal surgery performed with intravenous sedation and retrobulbar anesthesia. METHODS: One hundred eighty-five consecutive patients who underwent vitreoretinal surgery and had access to a standard postoperative analgesic regimen prospectively quantified levels of eye pain, nausea, and sedation 2 hours and 5 hours after surgery by using a standard visual analog scale. Analgesic requests were recorded. Responses were statistically analyzed. RESULTS: Fifty-six percent of patients had some eye pain after vitreoretinal surgery; 48% of patients requested an analgesic within 5 hours after surgery. Twenty-seven percent of patients required narcotic analgesia. There was a significant relationship between the presence of eye pain and surgery duration of >2 hours (P < 0.02). Sixteen percent of patients had postoperative nausea, which more likely occurred in those who received a narcotic analgesic (P < 0.02). Eighty percent of patients had postoperative sedation, which more likely occurred in those who received a narcotic analgesic (P < 0.02). CONCLUSION: One half of individuals undergoing vitreoretinal surgery, especially those who have lengthy procedures (>2 hours), will request pain medication within 5 hours after surgery; one half of these patients will need narcotic analgesia for pain control. Narcotics may result in nausea and sedation.

Aged↗
PubMed2001

TIMP-1 promotes VEGF-induced neovascularization in the retina.

Proteolysis of vascular basement membranes and surrounding extracellular matrix is a critical early step in neovascularization. It requires alteration of the balance between matrix metalloproteinases (MMPs) and proteins that bind to and inactivate MMPs, tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 has been demonstrated to inhibit neovascularization in chick chorioallantoic membranes. However, TIMP-1 has also been shown to either promote or inhibit cell proliferation and migration in different settings. To determine whether genetic alteration of the MMP/TIMP-1 ratio would alter retinal neovascularization, we crossed mice that express vascular endothelial growth factor (VEGF) in photoreceptors with TIMP-1-deficient mice or mice that overexpress TIMP-1. Compared to VEGF transgene-positive/TIMP-1-sufficient mice, VEGF transgene-positive/TIMP-1-deficient mice showed smaller neovascular lesions. There was also no difference between the two groups of mice in the appearance of the neovascularization by light or electron microscopy. Compound VEGF/TIMP-1 transgenic mice had increased expression of both VEGF and TIMP-1 in the retina, and had more neovascularization than mice that had increased expression of VEGF alone. These gain- and loss-of-function data suggest that alteration of the TIMP-1/MMP ratio modulates retinal neovascularization in a complex manner and not simply by altering the proteolytic activity and thereby invasiveness of endothelial cells.

Actins↗
PubMed2000

Angiopoietin 2 expression in the retina: upregulation during physiologic and pathologic neovascularization.

Vascular development in the embryo requires coordinated signaling through several endothelial cell-specific receptors; however, it is not known whether this is also required later during retinal vascular development or as part of retinal neovascularization in adults. The Tie2 receptor has been implicated in stabilization and maturation of vessels through action of an agonist ligand, angiopoietin 1 (Ang1) and an antagonistic ligand, Ang2. In this study, we have demonstrated that ang2 mRNA levels are increased in the retina during development of the deep retinal capillaries by angiogenesis and during pathologic angiogenesis in a model of ischemic retinopathy. Mice with hemizygous disruption of the ang2 gene by insertion of a promoterless beta-galactosidase (beta gal) gene behind the ang2 promoter, show constitutive beta gal staining primarily in cells along the outer border of the inner nuclear layer identified as horizontal cells by colocalization of calbindin. During development of the deep capillary bed or retinal neovascularization, other cells in the inner nuclear layer and ganglion cell layer, in regions of neovascularization, stain for beta gal. Thus, there is temporal and spatial correlation of Ang2 expression with developmental and pathologic angiogenesis in the retina, suggesting that it may play a role.

Angiopoietin-1↗
PubMed2000

Retinal and choroidal neovascularization.

The unique vascular supply of the retina, the ability to visualize the vasculature in vivo, and the ability to selectively express genes in the retina make the retina an ideal model system to study molecular mechanisms of angiogenesis. In addition, this area of investigation has great clinical significance, because retinal and choroidal neovascularization are the most common causes of severe visual loss in developed countries and new treatments are needed. As a result, interest in ocular neovascularization is rapidly growing and there has been considerable recent progress. Use of genetically engineered mice in recently developed murine models provides a means to investigate the role of individual gene products in neovascularization in two distinct vascular beds, the retinal vasculature and the choroidal vasculature. It appears that angiogenesis in different vascular beds has common themes, but also has tissue-specific aspects. This review summarizes recent progress in the field of ocular neovascularization and the prospects that it provides for the development of new treatments.

Adult↗
PubMed2000

Cell injury unmasks a latent proangiogenic phenotype in mice with increased expression of FGF2 in the retina.

Fibroblast growth factor-2 (FGF2) is a potent mitogen for vascular endothelial cells and exogenous administration of FGF2 stimulates angiogenesis. However, increased expression of FGF2 in the retina does not cause angiogenesis. One possible explanation is that FGF2 may not be capable of initiating angiogenesis unless it is administered in pharmacologic levels or there is coexpression of another angiogenic factor. Alternatively, there may be control mechanisms that sequester FGF2 in vivo, preventing it from manifesting its in vitro angiogenic activity. We tested the first hypothesis by crossing mice that express FGF2 in the retina with mice that express vascular endothelial growth factor (VEGF) in the retina. Surprisingly, despite comparable levels of VEGF expression, mice that expressed both FGF2 and VEGF had significantly less neovascularization than mice that expressed VEGF alone. The second hypothesis was tested by treating Rho/FGF2 transgenic mice with low-intensity laser photocoagulation that disrupts photoreceptors, but does not rupture Bruch's membrane, or intense laser that ruptures Bruch's membrane. In Rho/FGF2 transgenics, but not wild type mice, choroidal neovascularization developed in areas of low-intensity laser. Both wild type and transgenic mice developed choroidal neovascularization in areas of intense laser that ruptured Bruch's membrane, but the area of neovascularization was significantly greater in transgenics. These data suggest that increased retinal expression of FGF2 is angiogenic only when it is accompanied by cell injury that overcomes sequestration control mechanisms.

Animals↗
PubMed2000

Sensitivity of different vascular beds in the eye to neovascularization and blood-retinal barrier breakdown in VEGF transgenic mice.

Neovascularization (NV) causes visual deficits in ocular disorders such as diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. An understanding of the angiogenic factors promoting this abnormal vascular growth is necessary to devise a therapeutic approach to inhibit NV. One factor known to promote NV is vascular endothelial growth factor (VEGF), which can also induce a breakdown of the blood-retinal barrier (BRB) leading to macular edema, another major cause of visual loss in a variety of ocular disorders. To investigate the role of VEGF on ocular NV, transgenic mice have been produced that over-express VEGF in the photoreceptors under control of the rhodopsin promoter. Eyes from these mice and from littermates not expressing the transgene were examined using immunohistochemistry, griffonia simplicifolia isolectin-B4 (GSA) staining to clearly visualize vessels, and electron microscopy. Levels of transgene expression were determined by the polymerase chain reaction. In normal mice, retinal vessels are organized into a superficial and a deep capillary bed with some vessels forming a shunt between both beds. In a transgenic line of mice that over-expresses VEGF (V-6), NV originates from the deep capillary bed at about postnatal day 10 (P10) and extends through the photoreceptor layer to form vascular complexes in the subretinal space with BRB breakdown occurring only in the area of NV. The superficial capillary bed and the choroidal vasculature are unaffected. In another line of transgenic mice with a higher expression rate of VEGF (V-24), photoreceptor degeneration begins at P7-8, soon after the onset of transgene expression, without widespread NV, as was observed in V-6 mice. In conclusion, overexpression of VEGF in transgenic mice is sufficient to cause retinal NV, but only the deep capillary bed is responsive. Increasing the expression of VEGF does not necessarily increase the amount of NV. A better understanding of the specific factors and conditions that result in a particular pattern of ocular NV may provide clues regarding the pathogenesis of ocular neovascular disease.

Animals↗