William J. Brunken - Academia.edu (original) (raw)

Papers by William J. Brunken

Investigative Ophthalmology & Visual Science, 2008

Investigative Ophthalmology & Visual Science, 2013

Investigative Ophthalmology & Visual Science, 2010

Investigative Ophthalmology & Visual Science, 2014

Program Number: 3014 Presentation Time: 11:00 AM–11:15 AM Changes in retinal vessel caliber with ... more Program Number: 3014 Presentation Time: 11:00 AM–11:15 AM Changes in retinal vessel caliber with flicker light stimulation in eyes with diabetic retinopathy Laurence S. Lim1, 2, Peng Guan Ong1, E SHyong Tai1, 2, Gemmy C. Cheung1, Wallace S. Foulds1, Tien Y. Wong1, 2. 1Ophthalmology, Singapore National Eye Center, Singapore, Singapore; 2Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. Purpose: Changes in retinal vessel calibre in response to flickering light are believed to be mediated by nitric oxide release from the retinal microvascular endothelium. This study investigated the responses of retinal vessels to flickering light in diabetic patients with various grades of diabetic retinopathy(DR). Methods: This cross-sectional observational study evaluated adult subjects with diabetes mellitus. The Dynamic Vessel Analyser (DVA) was used to measure retinal vascular responses to diffuse illuminance flicker. DR was graded from retinal photography. Each eye was assigned a retinopathy severity score according to the modified Airlie House classification system, and categorized as minimal nonproliferative diabetic retinopathy (NPDR), mild NPDR, moderate NPDR, severe NPDR, or proliferative retinopathy. Eyes were also classified as having any DR (minimal NPDR or worse), moderate DR (moderate NPDR or worse), or vision-threatening DR (severe NPDR or worse, or clinically significant macular edema) according to the Eye Diseases Prevalence Research Group definitions. Results: There were 279 subjects in total, with a mean age of 59.9±9.2 years. The majority were male (73%) and the mean HbA1c level and mean duration of diabetes were 7.7±1.4% and 13.9±10.4 years respectively. After adjustments for age, sex, smoking, duration of diabetes, HbA1c, hypertension and hyperlipidemia, retinal arteriolar and venular dilation responses to flicker stimulation decreased continuously with increasing severity of diabetic retinopathy.(p = 0.008 and <0.001 respectively). Subjects with reduced arteriolar dilation responses were more likely to have any DR [odds ratio (OR) 1.20 (95% confidence interval 1.01 – 1.45) per standard deviation (SD) decrease, p=0.045]. Subjects with reduced venular dilation responses were more likely to have any DR [OR 1.27(1.04 – 1.53) per SD decrease, p=0.02], moderate DR [OR 1.27 (1.06 – 1.49) per SD decrease, p = 0.007] and vision-threatening DR [OR 1.51(1.14 – 1.50) per SD decrease, p = 0.002]. Conclusions: Retinal arteriolar and venular dilation responses to flickering light are diminished in subjects with DR, and decrease progressively with more severe stages of DR. Our findings suggest that the severity of DR is correlated with measurable differences in retinal microvascular endothelial function, supporting a role for the latter in the pathogenesis of DR. Commercial Relationships: Laurence S. Lim, None; Peng Guan Ong, None; E SHyong Tai, None; Gemmy C. Cheung, None; Wallace S. Foulds, None; Tien Y. Wong, None Support: NMRC grant number R710/60/2009 Program Number: 3015 Presentation Time: 11:15 AM–11:30 AM Amacrine cell-derived VEGF is required for development and maintenance of the retinal vasculature in mice Yoshihiko Usui, Toshihide Kurihara, Peter D. Westenskow, Edith Aguilar, Liliana P. Paris, Stacey K. Moreno, Carli M. Wittgrove, Daniel Feitelberg, Martin Friedlander. Cell Biology, The Scripps Research Institute, San Diego, CA. Purpose: The retinal vasculature of many organisms including humans and mice consists of three distinct plexus layers. While it is clear that the inner retinal vascular layer develops over a pre-existing astrocytic network and that development of vascular and neuronal networks are co-dependent, it is unclear how the outer retinal vascular networks form. As retinal neurons populate the retina and mature, oxygen demands change and activation of the oxygen sensing VHL/HIF-α/VEGF pathway in maturing neurons may be a strong driving force for development and maintenance of the outer plexus layers. In this study, we examined the contribution of amacrine and horizontal cells due to their close proximity to the intermediate and outer retinal vascular layers. Methods: Transgenic mice expressing Cre recombinase specifically in amacrine and horizontal cells (Ptf1a-Cre mice) were mated with floxed VHL, HIF-1α, HIF-2α and/or VEGF mice to generate conditional knockouts. Amacrine and horizontal cells were genetically ablated using Ptf1a-Cre and forced expression of diphtheria toxin (DT) receptors. Results: We show that amacrine and horizontal cell processes tightly associate with intermediate and outer plexus retinal capillaries. Pseudo-hypoxia in Ptf1a-Cre; VHL mutants induces formation of a dense intermediate plexus compared to controls, while a dramatically attenuated intermediate plexus is observed in Ptf1a-Cre; VEGF and Ptf1a-Cre; HIF-1α mutants. Co-deletion of HIF-1α, but not HIF-2α, rescued the vascular phenotypes of Ptf1a-Cre; VHL KO…

Investigative Ophthalmology & Visual Science, 2006

Cell reports, Jan 31, 2017

Synapses in the developing brain are structurally dynamic but become stable by early adulthood. W... more Synapses in the developing brain are structurally dynamic but become stable by early adulthood. We demonstrate here that an α5-subunit-containing laminin stabilizes synapses during this developmental transition. Hippocampal neurons deposit laminin α5 at synapses during adolescence as connections stabilize. Disruption of laminin α5 in neurons causes dramatic fluctuations in dendritic spine head size that can be rescued by exogenous α5-containing laminin. Conditional deletion of laminin α5 in vivo increases dendritic spine size and leads to an age-dependent loss of synapses accompanied by behavioral defects. Remaining synapses have larger postsynaptic densities and enhanced neurotransmission. Finally, we provide evidence that laminin α5 acts through an integrin α3β1-Abl2 kinase-p190RhoGAP signaling cascade and partners with laminin β2 to regulate dendritic spine density and behavior. Together, our results identify laminin α5 as a stabilizer of dendritic spines and synapses in the brai...

Journal of ophthalmic & vision research, 2014

Institute (SEI) celebrated its fifth anniversary this past year, and this bold adventure in a wal... more Institute (SEI) celebrated its fifth anniversary this past year, and this bold adventure in a wall-less institute spread across the geographic diversity of the State of New York remains fundamentally healthy. Since its inception as a cross-campus collaboration between the two stand-alone medical centers in the SUNY system, 1 the SEI has grown to an impressive statewide consortium. Our principle scientists, originally drawn from the founding health science centers: the

Experimental eye research, 2014

Overexpression of c-met and suppression of matrix metalloproteinase-10 (MMP-10) and cathepsin F g... more Overexpression of c-met and suppression of matrix metalloproteinase-10 (MMP-10) and cathepsin F genes was previously shown to normalize wound healing, epithelial and stem cell marker patterns in organ-cultured human diabetic corneas. We now examined if gene therapy of limbal cells only would produce similar effects. Eight pairs of organ-cultured autopsy human diabetic corneas were used. One cornea of each pair was treated for 48 h with adenoviruses (Ad) harboring full-length c-met mRNA or a mixture (combo) of Ad with c-met and shRNA to MMP-10 and cathepsin F genes. Medium was kept at the limbal level to avoid transduction of central corneal epithelium. Fellow corneas received control Ad with EGFP gene. After additional 5 (c-met) or 10 days (combo) incubation, central corneal epithelial debridement with n-heptanol was performed, and wound healing times were determined microscopically. Corneal cryostat sections were immunostained for diabetic and putative limbal stem cell markers, α3β...

Journal of ophthalmic & vision research, 2012

Retinal vasculature related pathologies account for a large proportion of global blindness. Choro... more Retinal vasculature related pathologies account for a large proportion of global blindness. Choroidal neovascularization accompanying age-related macular degeneration is the largest cause of blindness in people over the age of 65 years, proliferative diabetic retinopathy is the main cause of acquired blindness in working adults, and retinopathy of prematurity (ROP) is the leading cause of acquired blindness in children. Given the great success in treating the first category of these conditions with anti-vascular endothelial growth factor (anti-VEGF) therapy, there is understandably considerable interest to employ this strategy to other retinal vascular disorders. Anti-VEGF therapy may not be the optimal course of action, as it may compromise neuronal survival; this is of particular concern when treating ROP where retinal neurogenesis is still not complete. Moreover, retinal neovascularization is preceded by alterations in the vascular wall extracellular matrix with concomitant reduc...

Investigative ophthalmology & visual science, 1996

The authors previously hypothesized that laminin beta 2 (S-laminin) plays a role in directing pho... more The authors previously hypothesized that laminin beta 2 (S-laminin) plays a role in directing photoreceptor development. The aim of this study was to examine the temporal and spatial expression pattern of beta 2 laminins in rat retina to test this hypothesis. Retinas from Sprague-Dawley rats were harvested on embryonic days (E) 14, 16, and 21, as well as on postnatal days (P) 2, 5, and 10. Cryostat sections were probed with antibodies directed against beta 2 laminin, laminin-1 (alpha 1-beta 1-gamma 1), and von Willebrand factor. At the onset of rod photoreceptor birth (E14), laminin beta 2 surrounds the cells of the retinal pigmented epithelium (RPE) and is present on the apical surface of the retinal neuroepithelium. At E16, laminin beta 2 persists on the apical surface of the neuroepithelium and the subjacent apical surface of the RPE. At birth, laminin beta 2 fills the matrix between the juxtaposed surfaces of the RPE and neuroepithelium; moreover, laminin beta 2 immunoreactivity...

Results and Problems in Cell Differentiation, 2000

The extracellular matrix (ECM) is a highly organized meshwork of secreted macromolecules that con... more The extracellular matrix (ECM) is a highly organized meshwork of secreted macromolecules that consists of proteins, proteoglycans, and polysaccharides. Components of the ECM provide cells with different signals that are involved in many aspects of the development of multicellular organisms, such as: (1) when to start, continue, or stop dividing; (2) when to differentiate; (3) where to migrate; (4) how to polarize; (5) where to form a synapse; and (6) whether to die or survive. Thus, ECM molecules may be playing many different roles in the development of a complex tissue such as the retina.

Visual Neuroscience, 1998

Previous physiological studies have shown that serotonin (5-HT) reciprocally modulates ON and OFF... more Previous physiological studies have shown that serotonin (5-HT) reciprocally modulates ON and OFF channels in the mammalian retina. This study was undertaken to determine if the serotoninergic system is exclusively associated with the rod pathway. We tested drugs specific to 5-HT3 receptor, a serotonin-gated ion channel, in both dark- and light-adapted retina. Consistent with previous studies, we demonstrated that 5-HT3 receptors modulate the light-evoked responses of ganglion cells in the dark-adapted state. Moreover, we have extended these prior studies and shown that activation of the 5-HT3 receptor is capable of completely blocking the light-evoked response of OFF-center cells whereas inactivation of the 5-HT3 receptor is capable of completely blocking the light-evoked responses of ON-center cells. In contrast, in light-adapted retinae, serotonin agents failed to have any effect on retinal processing. These data suggest that the serotoninergic system in retina is (1) specificall...

Visual Neuroscience, 2005

One clear, crisp November day, Grant returned to the lab after attending a high school sports eve... more One clear, crisp November day, Grant returned to the lab after attending a high school sports event with his family. An hour later an arrhythmia stopped his heart. Those who knew Grant knew of his devotion to his family, his science, and soccer. Few of us knew how many lives he touched until over 1200 people gathered in the early afternoon of November 29, 2004 to remember their husband, father, brother, nephew, friend, colleague, coach, and mentor.

The Journal of Cell Biology, 1999

Laminins are heterotrimeric molecules composed of an α, a β, and a γ chain; they have broad funct... more Laminins are heterotrimeric molecules composed of an α, a β, and a γ chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel laminin, composed of known α and β chains but containing a novel γ chain, γ3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that γ3 contains all the expected domains of a γ chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that γ3-containing laminins are likely to exist in a stable matrix. Studies of the tissue distribution of γ3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, γ3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The γ3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, duct...

Progress in Retinal Research, 1993

Xenopus retina exhibit a kainate release mechanism. In Xenopus some bipolar cells can be shown to... more Xenopus retina exhibit a kainate release mechanism. In Xenopus some bipolar cells can be shown to contain endogenous serotonin immunoreactivity (Sa). Application of lmM kainate causes relase of this endogenous pool of serotonin (b). (Taken from Schiitte and Witkovsky, 1990.

PLoS ONE, 2011

Background: Netrins have been extensively studied in the developing central nervous system as pat... more Background: Netrins have been extensively studied in the developing central nervous system as pathfinding guidance cues, and more recently in non-neural tissues where they mediate cell adhesion, migration and differentiation. Netrin-4, a distant relative of Netrins 1-3, has been proposed to affect cell fate determination in developing epithelia, though receptors mediating these functions have yet to be identified. Methodology/Principal Findings: Using human embryonic pancreatic cells as a model of developing epithelium, here we report that Netrin-4 is abundantly expressed in vascular endothelial cells and pancreatic ductal cells, and supports epithelial cell adhesion through integrins a2b1and a3b1. Interestingly, we find that Netrin-4 recognition by embryonic pancreatic cells through integrins a2b1 and a3b1 promotes insulin and glucagon gene expression. In addition, full genome microarray analysis revealed that fetal pancreatic cell adhesion to Netrin-4 causes a prominent down-regulation of cyclins and upregulation of negative regulators of the cell cycle. Consistent with these results, a number of other genes whose activities have been linked to developmental decisions and/or cellular differentiation are up-regulated. Conclusions/Significance: Given the recognized function of blood vessels in epithelial tissue morphogenesis, our results provide a mechanism by which endothelial-derived Netrin-4 may function as a pro-differentiation cue for adjacent developing pancreatic cell populations expressing adhesion receptors a2b1 and a3b1 integrins.

PLoS ONE, 2011

Background: Glial cells such as retinal Mü ller glial cells are involved in potassium ion and wat... more Background: Glial cells such as retinal Mü ller glial cells are involved in potassium ion and water homeostasis of the neural tissue. In these cells, inwardly rectifying potassium (Kir) channels and aquaporin-4 water channels play an important role in the process of spatial potassium buffering and water drainage. Moreover, Kir4.1 channels are involved in the maintenance of the negative Mü ller cell membrane potential. The subcellular distribution of Kir4.1 and aquaporin-4 channels appears to be maintained by interactions with extracellular and intracellular molecules. Laminins in the extracellular matrix, dystroglycan in the membrane, and dystrophins in the cytomatrix form a complex mediating the polarized expression of Kir4.1 and aquaporin-4 in Mü ller cells. Methodology/Principal Findings: The aim of the present study was to test the function of the b2 and c3 containing laminins in murine Mü ller cells. We used knockout mice with genetic deletion of both b2 and c3 laminin genes to assay the effects on Kir4.1 and aquaporin-4. We studied protein and mRNA expression by immunohistochemistry, Western Blot, and quantitative RT-PCR, respectively, and membrane currents of isolated cells by patch-clamp experiments. We found a downregulation of mRNA and protein of Kir4.1 as well as of aquaporin-4 protein in laminin knockout mice. Moreover, Mü ller cells from laminin b2 and c3 knockout mice had reduced Kir-mediated inward currents and their membrane potentials were more positive than those in age-matched wild-type mice. Conclusion: These findings demonstrate a strong impact of laminin b2 and c3 subunits on the expression and function of both aquaporin-4 and Kir4.1, two important membrane proteins in Mü ller cells.

Molecular and Cellular Neuroscience, 2007

Synapses are formed and stabilized by concerted interactions of pre-, intra-, and post-synaptic c... more Synapses are formed and stabilized by concerted interactions of pre-, intra-, and post-synaptic components; however, the precise nature of the intrasynaptic components in the CNS remains obscure. Potential intrasynaptic components include extracellular matrix molecules such as laminins; here, we isolate β2-containing laminins, including perhaps laminins 13 (α3β2γ3) and 14 (α4β2γ3), from CNS synaptosomes suggesting a role for these molecules in synaptic organization. Indeed, hippocampal synapses that form in vivo in the absence of these laminins are malformed at the ultrastructural level and this malformation is replicated in synapses formed in vitro, where laminins are provided largely by the post-synaptic neuron. This recapitulation of the in vivo function of laminins in vitro suggests that the malformations are a direct consequence of the removal of laminins from the synapse. Together, these results support a role for neuronal laminins in the structural integrity of central synapses.

Investigative Ophthalmology & Visual Science, 2008

Investigative Ophthalmology & Visual Science, 2013

Investigative Ophthalmology & Visual Science, 2010

Investigative Ophthalmology & Visual Science, 2014

Program Number: 3014 Presentation Time: 11:00 AM–11:15 AM Changes in retinal vessel caliber with ... more Program Number: 3014 Presentation Time: 11:00 AM–11:15 AM Changes in retinal vessel caliber with flicker light stimulation in eyes with diabetic retinopathy Laurence S. Lim1, 2, Peng Guan Ong1, E SHyong Tai1, 2, Gemmy C. Cheung1, Wallace S. Foulds1, Tien Y. Wong1, 2. 1Ophthalmology, Singapore National Eye Center, Singapore, Singapore; 2Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. Purpose: Changes in retinal vessel calibre in response to flickering light are believed to be mediated by nitric oxide release from the retinal microvascular endothelium. This study investigated the responses of retinal vessels to flickering light in diabetic patients with various grades of diabetic retinopathy(DR). Methods: This cross-sectional observational study evaluated adult subjects with diabetes mellitus. The Dynamic Vessel Analyser (DVA) was used to measure retinal vascular responses to diffuse illuminance flicker. DR was graded from retinal photography. Each eye was assigned a retinopathy severity score according to the modified Airlie House classification system, and categorized as minimal nonproliferative diabetic retinopathy (NPDR), mild NPDR, moderate NPDR, severe NPDR, or proliferative retinopathy. Eyes were also classified as having any DR (minimal NPDR or worse), moderate DR (moderate NPDR or worse), or vision-threatening DR (severe NPDR or worse, or clinically significant macular edema) according to the Eye Diseases Prevalence Research Group definitions. Results: There were 279 subjects in total, with a mean age of 59.9±9.2 years. The majority were male (73%) and the mean HbA1c level and mean duration of diabetes were 7.7±1.4% and 13.9±10.4 years respectively. After adjustments for age, sex, smoking, duration of diabetes, HbA1c, hypertension and hyperlipidemia, retinal arteriolar and venular dilation responses to flicker stimulation decreased continuously with increasing severity of diabetic retinopathy.(p = 0.008 and <0.001 respectively). Subjects with reduced arteriolar dilation responses were more likely to have any DR [odds ratio (OR) 1.20 (95% confidence interval 1.01 – 1.45) per standard deviation (SD) decrease, p=0.045]. Subjects with reduced venular dilation responses were more likely to have any DR [OR 1.27(1.04 – 1.53) per SD decrease, p=0.02], moderate DR [OR 1.27 (1.06 – 1.49) per SD decrease, p = 0.007] and vision-threatening DR [OR 1.51(1.14 – 1.50) per SD decrease, p = 0.002]. Conclusions: Retinal arteriolar and venular dilation responses to flickering light are diminished in subjects with DR, and decrease progressively with more severe stages of DR. Our findings suggest that the severity of DR is correlated with measurable differences in retinal microvascular endothelial function, supporting a role for the latter in the pathogenesis of DR. Commercial Relationships: Laurence S. Lim, None; Peng Guan Ong, None; E SHyong Tai, None; Gemmy C. Cheung, None; Wallace S. Foulds, None; Tien Y. Wong, None Support: NMRC grant number R710/60/2009 Program Number: 3015 Presentation Time: 11:15 AM–11:30 AM Amacrine cell-derived VEGF is required for development and maintenance of the retinal vasculature in mice Yoshihiko Usui, Toshihide Kurihara, Peter D. Westenskow, Edith Aguilar, Liliana P. Paris, Stacey K. Moreno, Carli M. Wittgrove, Daniel Feitelberg, Martin Friedlander. Cell Biology, The Scripps Research Institute, San Diego, CA. Purpose: The retinal vasculature of many organisms including humans and mice consists of three distinct plexus layers. While it is clear that the inner retinal vascular layer develops over a pre-existing astrocytic network and that development of vascular and neuronal networks are co-dependent, it is unclear how the outer retinal vascular networks form. As retinal neurons populate the retina and mature, oxygen demands change and activation of the oxygen sensing VHL/HIF-α/VEGF pathway in maturing neurons may be a strong driving force for development and maintenance of the outer plexus layers. In this study, we examined the contribution of amacrine and horizontal cells due to their close proximity to the intermediate and outer retinal vascular layers. Methods: Transgenic mice expressing Cre recombinase specifically in amacrine and horizontal cells (Ptf1a-Cre mice) were mated with floxed VHL, HIF-1α, HIF-2α and/or VEGF mice to generate conditional knockouts. Amacrine and horizontal cells were genetically ablated using Ptf1a-Cre and forced expression of diphtheria toxin (DT) receptors. Results: We show that amacrine and horizontal cell processes tightly associate with intermediate and outer plexus retinal capillaries. Pseudo-hypoxia in Ptf1a-Cre; VHL mutants induces formation of a dense intermediate plexus compared to controls, while a dramatically attenuated intermediate plexus is observed in Ptf1a-Cre; VEGF and Ptf1a-Cre; HIF-1α mutants. Co-deletion of HIF-1α, but not HIF-2α, rescued the vascular phenotypes of Ptf1a-Cre; VHL KO…

Investigative Ophthalmology & Visual Science, 2006

Cell reports, Jan 31, 2017

Synapses in the developing brain are structurally dynamic but become stable by early adulthood. W... more Synapses in the developing brain are structurally dynamic but become stable by early adulthood. We demonstrate here that an α5-subunit-containing laminin stabilizes synapses during this developmental transition. Hippocampal neurons deposit laminin α5 at synapses during adolescence as connections stabilize. Disruption of laminin α5 in neurons causes dramatic fluctuations in dendritic spine head size that can be rescued by exogenous α5-containing laminin. Conditional deletion of laminin α5 in vivo increases dendritic spine size and leads to an age-dependent loss of synapses accompanied by behavioral defects. Remaining synapses have larger postsynaptic densities and enhanced neurotransmission. Finally, we provide evidence that laminin α5 acts through an integrin α3β1-Abl2 kinase-p190RhoGAP signaling cascade and partners with laminin β2 to regulate dendritic spine density and behavior. Together, our results identify laminin α5 as a stabilizer of dendritic spines and synapses in the brai...

Journal of ophthalmic & vision research, 2014

Institute (SEI) celebrated its fifth anniversary this past year, and this bold adventure in a wal... more Institute (SEI) celebrated its fifth anniversary this past year, and this bold adventure in a wall-less institute spread across the geographic diversity of the State of New York remains fundamentally healthy. Since its inception as a cross-campus collaboration between the two stand-alone medical centers in the SUNY system, 1 the SEI has grown to an impressive statewide consortium. Our principle scientists, originally drawn from the founding health science centers: the

Experimental eye research, 2014

Overexpression of c-met and suppression of matrix metalloproteinase-10 (MMP-10) and cathepsin F g... more Overexpression of c-met and suppression of matrix metalloproteinase-10 (MMP-10) and cathepsin F genes was previously shown to normalize wound healing, epithelial and stem cell marker patterns in organ-cultured human diabetic corneas. We now examined if gene therapy of limbal cells only would produce similar effects. Eight pairs of organ-cultured autopsy human diabetic corneas were used. One cornea of each pair was treated for 48 h with adenoviruses (Ad) harboring full-length c-met mRNA or a mixture (combo) of Ad with c-met and shRNA to MMP-10 and cathepsin F genes. Medium was kept at the limbal level to avoid transduction of central corneal epithelium. Fellow corneas received control Ad with EGFP gene. After additional 5 (c-met) or 10 days (combo) incubation, central corneal epithelial debridement with n-heptanol was performed, and wound healing times were determined microscopically. Corneal cryostat sections were immunostained for diabetic and putative limbal stem cell markers, α3β...

Journal of ophthalmic & vision research, 2012

Retinal vasculature related pathologies account for a large proportion of global blindness. Choro... more Retinal vasculature related pathologies account for a large proportion of global blindness. Choroidal neovascularization accompanying age-related macular degeneration is the largest cause of blindness in people over the age of 65 years, proliferative diabetic retinopathy is the main cause of acquired blindness in working adults, and retinopathy of prematurity (ROP) is the leading cause of acquired blindness in children. Given the great success in treating the first category of these conditions with anti-vascular endothelial growth factor (anti-VEGF) therapy, there is understandably considerable interest to employ this strategy to other retinal vascular disorders. Anti-VEGF therapy may not be the optimal course of action, as it may compromise neuronal survival; this is of particular concern when treating ROP where retinal neurogenesis is still not complete. Moreover, retinal neovascularization is preceded by alterations in the vascular wall extracellular matrix with concomitant reduc...

Investigative ophthalmology & visual science, 1996

The authors previously hypothesized that laminin beta 2 (S-laminin) plays a role in directing pho... more The authors previously hypothesized that laminin beta 2 (S-laminin) plays a role in directing photoreceptor development. The aim of this study was to examine the temporal and spatial expression pattern of beta 2 laminins in rat retina to test this hypothesis. Retinas from Sprague-Dawley rats were harvested on embryonic days (E) 14, 16, and 21, as well as on postnatal days (P) 2, 5, and 10. Cryostat sections were probed with antibodies directed against beta 2 laminin, laminin-1 (alpha 1-beta 1-gamma 1), and von Willebrand factor. At the onset of rod photoreceptor birth (E14), laminin beta 2 surrounds the cells of the retinal pigmented epithelium (RPE) and is present on the apical surface of the retinal neuroepithelium. At E16, laminin beta 2 persists on the apical surface of the neuroepithelium and the subjacent apical surface of the RPE. At birth, laminin beta 2 fills the matrix between the juxtaposed surfaces of the RPE and neuroepithelium; moreover, laminin beta 2 immunoreactivity...

Results and Problems in Cell Differentiation, 2000

The extracellular matrix (ECM) is a highly organized meshwork of secreted macromolecules that con... more The extracellular matrix (ECM) is a highly organized meshwork of secreted macromolecules that consists of proteins, proteoglycans, and polysaccharides. Components of the ECM provide cells with different signals that are involved in many aspects of the development of multicellular organisms, such as: (1) when to start, continue, or stop dividing; (2) when to differentiate; (3) where to migrate; (4) how to polarize; (5) where to form a synapse; and (6) whether to die or survive. Thus, ECM molecules may be playing many different roles in the development of a complex tissue such as the retina.

Visual Neuroscience, 1998

Previous physiological studies have shown that serotonin (5-HT) reciprocally modulates ON and OFF... more Previous physiological studies have shown that serotonin (5-HT) reciprocally modulates ON and OFF channels in the mammalian retina. This study was undertaken to determine if the serotoninergic system is exclusively associated with the rod pathway. We tested drugs specific to 5-HT3 receptor, a serotonin-gated ion channel, in both dark- and light-adapted retina. Consistent with previous studies, we demonstrated that 5-HT3 receptors modulate the light-evoked responses of ganglion cells in the dark-adapted state. Moreover, we have extended these prior studies and shown that activation of the 5-HT3 receptor is capable of completely blocking the light-evoked response of OFF-center cells whereas inactivation of the 5-HT3 receptor is capable of completely blocking the light-evoked responses of ON-center cells. In contrast, in light-adapted retinae, serotonin agents failed to have any effect on retinal processing. These data suggest that the serotoninergic system in retina is (1) specificall...

Visual Neuroscience, 2005

One clear, crisp November day, Grant returned to the lab after attending a high school sports eve... more One clear, crisp November day, Grant returned to the lab after attending a high school sports event with his family. An hour later an arrhythmia stopped his heart. Those who knew Grant knew of his devotion to his family, his science, and soccer. Few of us knew how many lives he touched until over 1200 people gathered in the early afternoon of November 29, 2004 to remember their husband, father, brother, nephew, friend, colleague, coach, and mentor.

The Journal of Cell Biology, 1999

Laminins are heterotrimeric molecules composed of an α, a β, and a γ chain; they have broad funct... more Laminins are heterotrimeric molecules composed of an α, a β, and a γ chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel laminin, composed of known α and β chains but containing a novel γ chain, γ3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that γ3 contains all the expected domains of a γ chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that γ3-containing laminins are likely to exist in a stable matrix. Studies of the tissue distribution of γ3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, γ3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The γ3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, duct...

Progress in Retinal Research, 1993

Xenopus retina exhibit a kainate release mechanism. In Xenopus some bipolar cells can be shown to... more Xenopus retina exhibit a kainate release mechanism. In Xenopus some bipolar cells can be shown to contain endogenous serotonin immunoreactivity (Sa). Application of lmM kainate causes relase of this endogenous pool of serotonin (b). (Taken from Schiitte and Witkovsky, 1990.

PLoS ONE, 2011

Background: Netrins have been extensively studied in the developing central nervous system as pat... more Background: Netrins have been extensively studied in the developing central nervous system as pathfinding guidance cues, and more recently in non-neural tissues where they mediate cell adhesion, migration and differentiation. Netrin-4, a distant relative of Netrins 1-3, has been proposed to affect cell fate determination in developing epithelia, though receptors mediating these functions have yet to be identified. Methodology/Principal Findings: Using human embryonic pancreatic cells as a model of developing epithelium, here we report that Netrin-4 is abundantly expressed in vascular endothelial cells and pancreatic ductal cells, and supports epithelial cell adhesion through integrins a2b1and a3b1. Interestingly, we find that Netrin-4 recognition by embryonic pancreatic cells through integrins a2b1 and a3b1 promotes insulin and glucagon gene expression. In addition, full genome microarray analysis revealed that fetal pancreatic cell adhesion to Netrin-4 causes a prominent down-regulation of cyclins and upregulation of negative regulators of the cell cycle. Consistent with these results, a number of other genes whose activities have been linked to developmental decisions and/or cellular differentiation are up-regulated. Conclusions/Significance: Given the recognized function of blood vessels in epithelial tissue morphogenesis, our results provide a mechanism by which endothelial-derived Netrin-4 may function as a pro-differentiation cue for adjacent developing pancreatic cell populations expressing adhesion receptors a2b1 and a3b1 integrins.

PLoS ONE, 2011

Background: Glial cells such as retinal Mü ller glial cells are involved in potassium ion and wat... more Background: Glial cells such as retinal Mü ller glial cells are involved in potassium ion and water homeostasis of the neural tissue. In these cells, inwardly rectifying potassium (Kir) channels and aquaporin-4 water channels play an important role in the process of spatial potassium buffering and water drainage. Moreover, Kir4.1 channels are involved in the maintenance of the negative Mü ller cell membrane potential. The subcellular distribution of Kir4.1 and aquaporin-4 channels appears to be maintained by interactions with extracellular and intracellular molecules. Laminins in the extracellular matrix, dystroglycan in the membrane, and dystrophins in the cytomatrix form a complex mediating the polarized expression of Kir4.1 and aquaporin-4 in Mü ller cells. Methodology/Principal Findings: The aim of the present study was to test the function of the b2 and c3 containing laminins in murine Mü ller cells. We used knockout mice with genetic deletion of both b2 and c3 laminin genes to assay the effects on Kir4.1 and aquaporin-4. We studied protein and mRNA expression by immunohistochemistry, Western Blot, and quantitative RT-PCR, respectively, and membrane currents of isolated cells by patch-clamp experiments. We found a downregulation of mRNA and protein of Kir4.1 as well as of aquaporin-4 protein in laminin knockout mice. Moreover, Mü ller cells from laminin b2 and c3 knockout mice had reduced Kir-mediated inward currents and their membrane potentials were more positive than those in age-matched wild-type mice. Conclusion: These findings demonstrate a strong impact of laminin b2 and c3 subunits on the expression and function of both aquaporin-4 and Kir4.1, two important membrane proteins in Mü ller cells.

Molecular and Cellular Neuroscience, 2007

Synapses are formed and stabilized by concerted interactions of pre-, intra-, and post-synaptic c... more Synapses are formed and stabilized by concerted interactions of pre-, intra-, and post-synaptic components; however, the precise nature of the intrasynaptic components in the CNS remains obscure. Potential intrasynaptic components include extracellular matrix molecules such as laminins; here, we isolate β2-containing laminins, including perhaps laminins 13 (α3β2γ3) and 14 (α4β2γ3), from CNS synaptosomes suggesting a role for these molecules in synaptic organization. Indeed, hippocampal synapses that form in vivo in the absence of these laminins are malformed at the ultrastructural level and this malformation is replicated in synapses formed in vitro, where laminins are provided largely by the post-synaptic neuron. This recapitulation of the in vivo function of laminins in vitro suggests that the malformations are a direct consequence of the removal of laminins from the synapse. Together, these results support a role for neuronal laminins in the structural integrity of central synapses.