Jian-xing Ma - Academia.edu (original) (raw)

Papers by Jian-xing Ma

RESULTS. The short-wavelength (SWL) cone opsin mRNA was markedly decreased at 2 weeks of age, whe... more RESULTS. The short-wavelength (SWL) cone opsin mRNA was markedly decreased at 2 weeks of age, whereas the decrease in the middle-wavelength (MWL) cone opsin mRNA occurred relatively later in age. In contrast, the rhodopsin mRNA level did not show any significant change at all the ages analyzed. Consistent with the cone opsin changes, the cone transducin -subunit mRNA decreased at

Scientific reports, 2015

4-Hydroxynonenal (4-HNE or HNE) is a main endogenous product of cellular lipid peroxidation in ti... more 4-Hydroxynonenal (4-HNE or HNE) is a main endogenous product of cellular lipid peroxidation in tissues and is reported to play pathogenic roles in eye diseases. Here we investigated the association between 4-HNE and oxidative stress in the corneal epithelium. 4-HNE suppressed the cell viability of human corneal epithelial cells (HCE) in a concentration dependent manner. 4-HNE significantly increased the level of 3-Nitrotyrosine (3-NT), a marker of oxidative stress, in HCE cells and corneal epithelium of rats by immunofluorescent staining and Western blot analysis. To its underlying mechanistic on ROS system, 4-HNE elevated the ROS generation enzyme NADPH oxidase 4 (NOX4) and induced the activation of NF-E2-related factor-2 (NRF2) and its downstream effectors: NAD(P)H dehydrogenase (quinone 1) (NQO1) and glutathione S-transferase P (GSTP). Furthermore, N-acetylcysteine (NAC), an antioxidant and ROS scavenger, antagonized the inhibitory and oxidant effects of 4-HNE on the corneal epit...

Investigative ophthalmology & visual science, 2014

The mechanism for the antiangiogenic activity of peroxisome proliferator-activated receptor alpha... more The mechanism for the antiangiogenic activity of peroxisome proliferator-activated receptor alpha (PPARα) remains incompletely understood. Endothelial progenitor cells (EPC) are known to participate in neovascularization (NV). The purpose of this study was to investigate whether PPARα regulates EPC during retinal NV. Retinal NV was induced by oxygen-induced retinopathy (OIR). Mice with OIR were injected intraperitoneally with the PPARα agonist fenofibric acid (FA) or with adenovirus expressing PPARα (Ad-PPARα). Flow cytometry was used to quantify circulating and retinal EPC. Serum stromal cell-derived factor 1 (SDF-1) levels were measured by ELISA. Hypoxia was induced in primary human retinal capillary endothelial cells (HRCEC) and mouse brain endothelial cells (MBEC) by CoCl2. Levels of SDF-1 and hypoxia-inducible factor 1 alpha (HIF-1α) were measured by Western blotting. Fenofibric acid and overexpression of PPARα attenuated the increase of circulating and retinal EPC, correlating...

The protein RPE65 plays a critical role in retinoid processing in the retinal pigment epithelium ... more The protein RPE65 plays a critical role in retinoid processing in the retinal pigment epithelium (RPE). Previous studies have identified the RPE65 mRNA in salamander cones, but not in rods. The purpose of the present study was to determine whether RPE65 is expressed at the protein level in mammalian cones, as well as in those of amphibians. The specificity of the anti-RPE65 antibody was demonstrated by Western blot analysis. RPE65 cellular localization was determined using immunohistochemistry on flatmounted retinas and retinal sections. RPE65 protein was detected in cones in flatmounted retinas of the mouse, rabbit, and cow, in addition to Xenopus laevis. The morphology and location of labeled cones in the retina were confirmed by double staining of mouse retina sections with the anti-RPE65 antibody and peanut agglutinin (PNA) lectin, which is known to label both types of cones in mouse. The double staining in the flatmounted retinas demonstrated that RPE65 was expressed in both types of the cones in the mouse retina. Under the same double-labeling conditions, however, cones in homozygous RPE65-knockout mouse were labeled by PNA lectin, but not by the anti-RPE65 antibody, indicating that the protein recognized by the anti-RPE65 antibody is encoded by the RPE65 gene rather than by another homologous gene. No RPE65 was detected in rods of any of the species tested. RPE65 is expressed in mammalian cones, but not in rods. These results provide further support for physiological observations that cones may have an alternative retinoid cycle.

Investigative ophthalmology & visual science, 2001

RPE65 is preferentially expressed in the retinal pigment epithelium (RPE) and is essential for re... more RPE65 is preferentially expressed in the retinal pigment epithelium (RPE) and is essential for retinal function. The purpose of the study was to develop methods for the expression of the protein, determine the accurate molecular weight of this expressed protein, and quantitate the amount of RPE65 in the bovine RPE. Human RPE65 was expressed in Sf9 cells using the baculovirus system. The subcellular localization was determined by Western blot analysis and immunocytochemistry. An ELISA was developed for RPE65 and used to measure levels in bovine RPE. Recombinant and native RPE65 were purified by affinity chromatography. Molecular mass was determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Recombinant human (rH)RPE65 was expressed as a major protein associated with cell membrane in Sf9 cells. The recombinant protein was purified to apparent homogeneity from both the membrane and nonmembrane fractions. The identity of the purified protein was confirmed ...

PLOS ONE, 2015

Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid ... more Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined.

Nature genetics, 1998

RPE65, a protein preferentially and abundantly expressed in the RPE (ref. 1), is essential for th... more RPE65, a protein preferentially and abundantly expressed in the RPE (ref. 1), is essential for the maintenance of normal vision. Mutations in RPE65 result in severe forms of early-onset retinal dystrophy, including Leber congenital amaurosis (LCA; OMIM 180069 and 204100; ...

Investigative ophthalmology & visual science, 1996

Salamander photoreceptor cells have been used widely as models in vision research. However, the s... more Salamander photoreceptor cells have been used widely as models in vision research. However, the salamander opsin genes had not been cloned. The purpose of this study was to clone a salamander rhodopsin and to determine its primary structure and cell type-specific expression. Using salamander retina RNA as a template and Xenopus rhodopsin-specific oligonucleotides as primers, reverse transcription and polymerase chain reaction (RT-PCR) were used to amplify and clone a rhodopsin cDNA fragment. This fragment was used as a probe to isolate a full-length cDNA of the rhodopsin from a cDNA library of salamander retina. The dideoxynucleotide chain termination method was used to determine the nucleotide sequence. Single rod and cone cells were isolated by micromanipulation, and the absorbance spectra of the rod outer segments were measured with a photon-counting microspectrophotometer. Individual rod and cone cells were lysed for RT-PCR and Southern blot analysis to detect cell-specific expr...

METHODS. An assay was developed wherein 661W cells, a cone photoreceptor cell line, were stressed... more METHODS. An assay was developed wherein 661W cells, a cone photoreceptor cell line, were stressed with light and percent- age of surviving cells was determined. The degree of cell death was established using the MTT assay. Western blot analysis was used to confirm the activation of multiple proteases. Amounts of retinaldehydes were determined by extraction and HPLC. RESULTS. 661W cells

Diabetes, Jan 3, 2014

Diabetic foot ulcer (DFU), caused by impaired wound healing, is a common vascular complication of... more Diabetic foot ulcer (DFU), caused by impaired wound healing, is a common vascular complication of diabetes. The present study revealed that plasma levels of pigment epithelium-derived factor (PEDF) were elevated in Type 2 diabetic patients with DFU and in db/db mice. To test whether elevated PEDF levels contributes to skin wound healing delay in diabetes, endogenous PEDF was neutralized with an anti-PEDF antibody in db/db mice. Our results showed that neutralization of PEDF accelerated wound healing, increased angiogenesis in the wound skin, and improved functions and numbers of endothelial progenitor cells (EPCs) in the diabetic mice. Further, PEDF deficient mice showed higher baseline blood flow in the skin, higher density of cutaneous micro-vessels, increased skin thickness, improved circulating numbers and functions of EPCs, and accelerated wound healing, compared to the Wt mice. Over-expression of PEDF suppressed the Wnt signaling pathway in the wound skin. Lithium chloride-ind...

Investigative ophthalmology & visual science, 2014

Previous studies have demonstrated that peroxisome proliferator-activated receptor-alpha (PPARα) ... more Previous studies have demonstrated that peroxisome proliferator-activated receptor-alpha (PPARα) agonists have therapeutic effects in diabetic retinopathy, although the mechanism of action remains incompletely understood. The purpose of this study was to evaluate PPARα's protective effects in the ischemic retina, and to delineate its molecular mechanism of action. For the oxygen-induced retinopathy (OIR) model, wild-type (WT), and PPARα knockout (PPARα(-/-)) mice were exposed to 75% O₂ from postnatal day 7 (P7) to P12 and treated with the PPARα agonist fenofibric acid (Feno-FA) from P12 to P16. At P17, the effects of Feno-FA on retinal glial fibrillary acidic protein (GFAP) expression, apoptotic DNA cleavage, and TUNEL labeling were analyzed. Cultured retinal cells were exposed to CoCl₂ to induce hypoxia, and TUNEL staining and 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein dye were used to measure apoptosis and reactive oxygen species (ROS) generation. Western ...

Investigative ophthalmology & visual science, 2002

In the photic visual cycle, retinal G protein-coupled receptor (RGR) isomerizes all-trans retinal... more In the photic visual cycle, retinal G protein-coupled receptor (RGR) isomerizes all-trans retinal to 11-cis retinal in the retinal pigment epithelium (RPE) after illumination. It is unclear, however, how all-trans retinal, the substrate for RGR, is generated in the RPE, because no all-trans retinol dehydrogenase (atRDH) has been identified in the RPE. This study was conducted to identify the atRDH that generates all-trans retinal in the RPE. The full-length cDNA encoding a novel atRDH, RDH10, was cloned by PCR based on an expressed sequence tag (EST). Cellular localization was determined at the mRNA level by Northern blot analysis, RT-PCR, and in situ hybridization and at the protein level by immunohistochemistry with an antibody specific to RDH10. The activity was measured by an RDH activity assay with recombinant RDH10 expressed in COS cells. The full-length RDH10 was cloned from the human, cow, and mouse. These cDNAs encode a protein of 341 amino acids and have significant sequen...

The interface spin state of a ferromagnet (FM) can deviate significantly from its bulk spin state... more The interface spin state of a ferromagnet (FM) can deviate significantly from its bulk spin state and this effect could be strongly orientation-dependent especially in manganites. We have successfully fabricated high-quality (110)-oriented [La0.7Sr0.3MnO3 (t) /SrTiO3 (3ML)]n superlattices (t ranging from 3 to 15 ML), which are characterized by the atomic force microscopy, high-resolution transmission electron microscopy, x-ray diffraction and magnetometry.

Free radical biology & medicine, 2015

The mammalian sirtuin 6 (Sirt6) is a site-specific histone deacetylase that regulates chromatin s... more The mammalian sirtuin 6 (Sirt6) is a site-specific histone deacetylase that regulates chromatin structure and many fundamental biological processes. It inhibits endothelial cell senescence and inflammation, prevents development of cardiac hypertrophy and heart failure, modulates glucose metabolism, and represses tumor growth. The basic molecular mechanisms underlying regulation of Sirt6 enzymatic function are largely unknown. Here we hypothesized that Sirt6 function can be regulated via posttranslational modification, focusing on the role of peroxynitrite, one of the major reactive nitrogen species formed by excessive nitric oxide and superoxide generated during disease processes. We found that incubation of purified recombinant Sirt6 protein with 3-morpholinosydnonimine (SIN-1; a peroxynitrite donor that generates nitric oxide and superoxide simultaneously) increased Sirt6 tyrosine nitration and decreased its intrinsic catalytic activity. Similar results were observed in SIN-1-trea...

Arteriosclerosis, thrombosis, and vascular biology, Jan 5, 2015

The deficiency of very low-density lipoprotein receptor resulted in Wnt signaling activation and ... more The deficiency of very low-density lipoprotein receptor resulted in Wnt signaling activation and neovascularization in the retina. The present study sought to determine whether the very low-density lipoprotein receptor extracellular domain (VLN) is responsible for the inhibition of Wnt signaling in ocular tissues. A plasmid expressing the soluble VLN was encapsulated with poly(lactide-co-glycolide acid) to form VLN nanoparticles (VLN-NP). Nanoparticles containing a plasmid expressing the low-density lipoprotein receptor extracellular domain nanoparticle were used as negative control. MTT, modified Boyden chamber, and Matrigel (™) assays were used to evaluate the inhibitory effect of VLN-NP on Wnt3a-stimulated endothelial cell proliferation, migration, and tube formation. Vldlr(-/-) mice, oxygen-induced retinopathy, and alkali burn-induced corneal neovascularization models were used to evaluate the effect of VLN-NP on ocular neovascularization. Wnt reporter mice (BAT-gal), Western bl...

The Journal of investigative dermatology, 2014

Wound healing, angiogenesis, and hair follicle maintenance are often impaired in the skin of diab... more Wound healing, angiogenesis, and hair follicle maintenance are often impaired in the skin of diabetic patients, but the pathogenesis has not been well understood. Here, we report that circulation levels of kallistatin, a member of the serine proteinase inhibitor (SERPIN) superfamily with antiangiogenic activities, were elevated in type 2 diabetic patients with diabetic vascular complications. To test the hypothesis that elevated kallistatin levels could contribute to a wound-healing deficiency via the inhibition of Wnt/β-catenin signaling, we generated kallistatin-transgenic (KS-TG) mice. KS-TG mice had reduced cutaneous hair-follicle density, microvascular density, and panniculus adiposus layer thickness, as well as altered skin microvascular hemodynamics and delayed cutaneous wound healing. Using Wnt reporter mice, our results showed that Wnt/β-catenin signaling is suppressed in the dermal endothelium and hair follicles in KS-TG mice. Lithium, a known activator of β-catenin via in...

Circulation, 2014

Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy agains... more Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis.

PURPOSE. Human amniotic membrane (HAM) transplantation is commonly used in corneal surface recons... more PURPOSE. Human amniotic membrane (HAM) transplantation is commonly used in corneal surface reconstruction and is known to inhibit neovascularization of this tissue. The purpose of the present study is to reveal the molecular basis underlying antiangiogenic activity of HAM. METHODS. The effects of HAM protein on proliferation of vas- cular endothelial cells and corneal epithelial cells were deter- mined by

Investigative ophthalmology & visual science, Jan 15, 2014

Purpose: To evaluate the anti-neovascularization effects and investigate the possible mechanisms ... more Purpose: To evaluate the anti-neovascularization effects and investigate the possible mechanisms of SERPINA3K, a member of serine proteinase inhibitor family, using a specific rat model of suture-induced corneal neovascularization. Methods: A rat corneal suture model was set up and SERPINA3K was topically administered three times daily for 7 days. The clinical indications were evaluated on day 2, 5 and 7, including area of neovascularization and inflammation index. The eyeballs were collected after day 7 and the following examinations were performed: histological investigation, immunostaining, western blot and quantitative real-time polymerase chain reaction (PCR) assay. Wnt3a, a Wnt pathway ligand, was added to cultured Human Umbilical Vein Endothelial Cells (HUVEC), followed by detecting cell migration and western blot. Meanwhile, an in vitro VEGF165-stimulated HUVEC model was applied and the following measurements were conducted: cell proliferation, cell migration and tube format...

PloS one, 2014

We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity ... more We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity in the cornea. Here we investigated the antioxidant effects of SERPINA3K on the pterygial, which is partially caused by oxidative stress in pathogenesis. The head part of primary pterygial tissue was dissected and then cultured in keratinocyte serum-free defined medium (KSFM). The cultured pterygial epithelial cells (PECs) were treated with SERPINA3K. The cell proliferation and migration of PECs were measured and analyzed. Western blot and quantitative real-time polymerase chain reaction (PCR) assay were performed. It showed that SERPINA3K significantly suppressed the cell proliferation of PECs in a concentration-dependent manner, compared with cultured human conjunctival epithelial cells. SERPINA3K also inhibited the cell migration of PECs. Towards its underlying mechanism, SERPINA3K had antioxidant activities on the PECs by significantly inhibiting NADPH oxidase 4 (NOX4), which is an i...

RESULTS. The short-wavelength (SWL) cone opsin mRNA was markedly decreased at 2 weeks of age, whe... more RESULTS. The short-wavelength (SWL) cone opsin mRNA was markedly decreased at 2 weeks of age, whereas the decrease in the middle-wavelength (MWL) cone opsin mRNA occurred relatively later in age. In contrast, the rhodopsin mRNA level did not show any significant change at all the ages analyzed. Consistent with the cone opsin changes, the cone transducin -subunit mRNA decreased at

Scientific reports, 2015

4-Hydroxynonenal (4-HNE or HNE) is a main endogenous product of cellular lipid peroxidation in ti... more 4-Hydroxynonenal (4-HNE or HNE) is a main endogenous product of cellular lipid peroxidation in tissues and is reported to play pathogenic roles in eye diseases. Here we investigated the association between 4-HNE and oxidative stress in the corneal epithelium. 4-HNE suppressed the cell viability of human corneal epithelial cells (HCE) in a concentration dependent manner. 4-HNE significantly increased the level of 3-Nitrotyrosine (3-NT), a marker of oxidative stress, in HCE cells and corneal epithelium of rats by immunofluorescent staining and Western blot analysis. To its underlying mechanistic on ROS system, 4-HNE elevated the ROS generation enzyme NADPH oxidase 4 (NOX4) and induced the activation of NF-E2-related factor-2 (NRF2) and its downstream effectors: NAD(P)H dehydrogenase (quinone 1) (NQO1) and glutathione S-transferase P (GSTP). Furthermore, N-acetylcysteine (NAC), an antioxidant and ROS scavenger, antagonized the inhibitory and oxidant effects of 4-HNE on the corneal epit...

Investigative ophthalmology & visual science, 2014

The mechanism for the antiangiogenic activity of peroxisome proliferator-activated receptor alpha... more The mechanism for the antiangiogenic activity of peroxisome proliferator-activated receptor alpha (PPARα) remains incompletely understood. Endothelial progenitor cells (EPC) are known to participate in neovascularization (NV). The purpose of this study was to investigate whether PPARα regulates EPC during retinal NV. Retinal NV was induced by oxygen-induced retinopathy (OIR). Mice with OIR were injected intraperitoneally with the PPARα agonist fenofibric acid (FA) or with adenovirus expressing PPARα (Ad-PPARα). Flow cytometry was used to quantify circulating and retinal EPC. Serum stromal cell-derived factor 1 (SDF-1) levels were measured by ELISA. Hypoxia was induced in primary human retinal capillary endothelial cells (HRCEC) and mouse brain endothelial cells (MBEC) by CoCl2. Levels of SDF-1 and hypoxia-inducible factor 1 alpha (HIF-1α) were measured by Western blotting. Fenofibric acid and overexpression of PPARα attenuated the increase of circulating and retinal EPC, correlating...

The protein RPE65 plays a critical role in retinoid processing in the retinal pigment epithelium ... more The protein RPE65 plays a critical role in retinoid processing in the retinal pigment epithelium (RPE). Previous studies have identified the RPE65 mRNA in salamander cones, but not in rods. The purpose of the present study was to determine whether RPE65 is expressed at the protein level in mammalian cones, as well as in those of amphibians. The specificity of the anti-RPE65 antibody was demonstrated by Western blot analysis. RPE65 cellular localization was determined using immunohistochemistry on flatmounted retinas and retinal sections. RPE65 protein was detected in cones in flatmounted retinas of the mouse, rabbit, and cow, in addition to Xenopus laevis. The morphology and location of labeled cones in the retina were confirmed by double staining of mouse retina sections with the anti-RPE65 antibody and peanut agglutinin (PNA) lectin, which is known to label both types of cones in mouse. The double staining in the flatmounted retinas demonstrated that RPE65 was expressed in both types of the cones in the mouse retina. Under the same double-labeling conditions, however, cones in homozygous RPE65-knockout mouse were labeled by PNA lectin, but not by the anti-RPE65 antibody, indicating that the protein recognized by the anti-RPE65 antibody is encoded by the RPE65 gene rather than by another homologous gene. No RPE65 was detected in rods of any of the species tested. RPE65 is expressed in mammalian cones, but not in rods. These results provide further support for physiological observations that cones may have an alternative retinoid cycle.

Investigative ophthalmology & visual science, 2001

RPE65 is preferentially expressed in the retinal pigment epithelium (RPE) and is essential for re... more RPE65 is preferentially expressed in the retinal pigment epithelium (RPE) and is essential for retinal function. The purpose of the study was to develop methods for the expression of the protein, determine the accurate molecular weight of this expressed protein, and quantitate the amount of RPE65 in the bovine RPE. Human RPE65 was expressed in Sf9 cells using the baculovirus system. The subcellular localization was determined by Western blot analysis and immunocytochemistry. An ELISA was developed for RPE65 and used to measure levels in bovine RPE. Recombinant and native RPE65 were purified by affinity chromatography. Molecular mass was determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Recombinant human (rH)RPE65 was expressed as a major protein associated with cell membrane in Sf9 cells. The recombinant protein was purified to apparent homogeneity from both the membrane and nonmembrane fractions. The identity of the purified protein was confirmed ...

PLOS ONE, 2015

Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid ... more Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined.

Nature genetics, 1998

RPE65, a protein preferentially and abundantly expressed in the RPE (ref. 1), is essential for th... more RPE65, a protein preferentially and abundantly expressed in the RPE (ref. 1), is essential for the maintenance of normal vision. Mutations in RPE65 result in severe forms of early-onset retinal dystrophy, including Leber congenital amaurosis (LCA; OMIM 180069 and 204100; ...

Investigative ophthalmology & visual science, 1996

Salamander photoreceptor cells have been used widely as models in vision research. However, the s... more Salamander photoreceptor cells have been used widely as models in vision research. However, the salamander opsin genes had not been cloned. The purpose of this study was to clone a salamander rhodopsin and to determine its primary structure and cell type-specific expression. Using salamander retina RNA as a template and Xenopus rhodopsin-specific oligonucleotides as primers, reverse transcription and polymerase chain reaction (RT-PCR) were used to amplify and clone a rhodopsin cDNA fragment. This fragment was used as a probe to isolate a full-length cDNA of the rhodopsin from a cDNA library of salamander retina. The dideoxynucleotide chain termination method was used to determine the nucleotide sequence. Single rod and cone cells were isolated by micromanipulation, and the absorbance spectra of the rod outer segments were measured with a photon-counting microspectrophotometer. Individual rod and cone cells were lysed for RT-PCR and Southern blot analysis to detect cell-specific expr...

METHODS. An assay was developed wherein 661W cells, a cone photoreceptor cell line, were stressed... more METHODS. An assay was developed wherein 661W cells, a cone photoreceptor cell line, were stressed with light and percent- age of surviving cells was determined. The degree of cell death was established using the MTT assay. Western blot analysis was used to confirm the activation of multiple proteases. Amounts of retinaldehydes were determined by extraction and HPLC. RESULTS. 661W cells

Diabetes, Jan 3, 2014

Diabetic foot ulcer (DFU), caused by impaired wound healing, is a common vascular complication of... more Diabetic foot ulcer (DFU), caused by impaired wound healing, is a common vascular complication of diabetes. The present study revealed that plasma levels of pigment epithelium-derived factor (PEDF) were elevated in Type 2 diabetic patients with DFU and in db/db mice. To test whether elevated PEDF levels contributes to skin wound healing delay in diabetes, endogenous PEDF was neutralized with an anti-PEDF antibody in db/db mice. Our results showed that neutralization of PEDF accelerated wound healing, increased angiogenesis in the wound skin, and improved functions and numbers of endothelial progenitor cells (EPCs) in the diabetic mice. Further, PEDF deficient mice showed higher baseline blood flow in the skin, higher density of cutaneous micro-vessels, increased skin thickness, improved circulating numbers and functions of EPCs, and accelerated wound healing, compared to the Wt mice. Over-expression of PEDF suppressed the Wnt signaling pathway in the wound skin. Lithium chloride-ind...

Investigative ophthalmology & visual science, 2014

Previous studies have demonstrated that peroxisome proliferator-activated receptor-alpha (PPARα) ... more Previous studies have demonstrated that peroxisome proliferator-activated receptor-alpha (PPARα) agonists have therapeutic effects in diabetic retinopathy, although the mechanism of action remains incompletely understood. The purpose of this study was to evaluate PPARα's protective effects in the ischemic retina, and to delineate its molecular mechanism of action. For the oxygen-induced retinopathy (OIR) model, wild-type (WT), and PPARα knockout (PPARα(-/-)) mice were exposed to 75% O₂ from postnatal day 7 (P7) to P12 and treated with the PPARα agonist fenofibric acid (Feno-FA) from P12 to P16. At P17, the effects of Feno-FA on retinal glial fibrillary acidic protein (GFAP) expression, apoptotic DNA cleavage, and TUNEL labeling were analyzed. Cultured retinal cells were exposed to CoCl₂ to induce hypoxia, and TUNEL staining and 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein dye were used to measure apoptosis and reactive oxygen species (ROS) generation. Western ...

Investigative ophthalmology & visual science, 2002

In the photic visual cycle, retinal G protein-coupled receptor (RGR) isomerizes all-trans retinal... more In the photic visual cycle, retinal G protein-coupled receptor (RGR) isomerizes all-trans retinal to 11-cis retinal in the retinal pigment epithelium (RPE) after illumination. It is unclear, however, how all-trans retinal, the substrate for RGR, is generated in the RPE, because no all-trans retinol dehydrogenase (atRDH) has been identified in the RPE. This study was conducted to identify the atRDH that generates all-trans retinal in the RPE. The full-length cDNA encoding a novel atRDH, RDH10, was cloned by PCR based on an expressed sequence tag (EST). Cellular localization was determined at the mRNA level by Northern blot analysis, RT-PCR, and in situ hybridization and at the protein level by immunohistochemistry with an antibody specific to RDH10. The activity was measured by an RDH activity assay with recombinant RDH10 expressed in COS cells. The full-length RDH10 was cloned from the human, cow, and mouse. These cDNAs encode a protein of 341 amino acids and have significant sequen...

The interface spin state of a ferromagnet (FM) can deviate significantly from its bulk spin state... more The interface spin state of a ferromagnet (FM) can deviate significantly from its bulk spin state and this effect could be strongly orientation-dependent especially in manganites. We have successfully fabricated high-quality (110)-oriented [La0.7Sr0.3MnO3 (t) /SrTiO3 (3ML)]n superlattices (t ranging from 3 to 15 ML), which are characterized by the atomic force microscopy, high-resolution transmission electron microscopy, x-ray diffraction and magnetometry.

Free radical biology & medicine, 2015

The mammalian sirtuin 6 (Sirt6) is a site-specific histone deacetylase that regulates chromatin s... more The mammalian sirtuin 6 (Sirt6) is a site-specific histone deacetylase that regulates chromatin structure and many fundamental biological processes. It inhibits endothelial cell senescence and inflammation, prevents development of cardiac hypertrophy and heart failure, modulates glucose metabolism, and represses tumor growth. The basic molecular mechanisms underlying regulation of Sirt6 enzymatic function are largely unknown. Here we hypothesized that Sirt6 function can be regulated via posttranslational modification, focusing on the role of peroxynitrite, one of the major reactive nitrogen species formed by excessive nitric oxide and superoxide generated during disease processes. We found that incubation of purified recombinant Sirt6 protein with 3-morpholinosydnonimine (SIN-1; a peroxynitrite donor that generates nitric oxide and superoxide simultaneously) increased Sirt6 tyrosine nitration and decreased its intrinsic catalytic activity. Similar results were observed in SIN-1-trea...

Arteriosclerosis, thrombosis, and vascular biology, Jan 5, 2015

The deficiency of very low-density lipoprotein receptor resulted in Wnt signaling activation and ... more The deficiency of very low-density lipoprotein receptor resulted in Wnt signaling activation and neovascularization in the retina. The present study sought to determine whether the very low-density lipoprotein receptor extracellular domain (VLN) is responsible for the inhibition of Wnt signaling in ocular tissues. A plasmid expressing the soluble VLN was encapsulated with poly(lactide-co-glycolide acid) to form VLN nanoparticles (VLN-NP). Nanoparticles containing a plasmid expressing the low-density lipoprotein receptor extracellular domain nanoparticle were used as negative control. MTT, modified Boyden chamber, and Matrigel (™) assays were used to evaluate the inhibitory effect of VLN-NP on Wnt3a-stimulated endothelial cell proliferation, migration, and tube formation. Vldlr(-/-) mice, oxygen-induced retinopathy, and alkali burn-induced corneal neovascularization models were used to evaluate the effect of VLN-NP on ocular neovascularization. Wnt reporter mice (BAT-gal), Western bl...

The Journal of investigative dermatology, 2014

Wound healing, angiogenesis, and hair follicle maintenance are often impaired in the skin of diab... more Wound healing, angiogenesis, and hair follicle maintenance are often impaired in the skin of diabetic patients, but the pathogenesis has not been well understood. Here, we report that circulation levels of kallistatin, a member of the serine proteinase inhibitor (SERPIN) superfamily with antiangiogenic activities, were elevated in type 2 diabetic patients with diabetic vascular complications. To test the hypothesis that elevated kallistatin levels could contribute to a wound-healing deficiency via the inhibition of Wnt/β-catenin signaling, we generated kallistatin-transgenic (KS-TG) mice. KS-TG mice had reduced cutaneous hair-follicle density, microvascular density, and panniculus adiposus layer thickness, as well as altered skin microvascular hemodynamics and delayed cutaneous wound healing. Using Wnt reporter mice, our results showed that Wnt/β-catenin signaling is suppressed in the dermal endothelium and hair follicles in KS-TG mice. Lithium, a known activator of β-catenin via in...

Circulation, 2014

Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy agains... more Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis.

PURPOSE. Human amniotic membrane (HAM) transplantation is commonly used in corneal surface recons... more PURPOSE. Human amniotic membrane (HAM) transplantation is commonly used in corneal surface reconstruction and is known to inhibit neovascularization of this tissue. The purpose of the present study is to reveal the molecular basis underlying antiangiogenic activity of HAM. METHODS. The effects of HAM protein on proliferation of vas- cular endothelial cells and corneal epithelial cells were deter- mined by

Investigative ophthalmology & visual science, Jan 15, 2014

Purpose: To evaluate the anti-neovascularization effects and investigate the possible mechanisms ... more Purpose: To evaluate the anti-neovascularization effects and investigate the possible mechanisms of SERPINA3K, a member of serine proteinase inhibitor family, using a specific rat model of suture-induced corneal neovascularization. Methods: A rat corneal suture model was set up and SERPINA3K was topically administered three times daily for 7 days. The clinical indications were evaluated on day 2, 5 and 7, including area of neovascularization and inflammation index. The eyeballs were collected after day 7 and the following examinations were performed: histological investigation, immunostaining, western blot and quantitative real-time polymerase chain reaction (PCR) assay. Wnt3a, a Wnt pathway ligand, was added to cultured Human Umbilical Vein Endothelial Cells (HUVEC), followed by detecting cell migration and western blot. Meanwhile, an in vitro VEGF165-stimulated HUVEC model was applied and the following measurements were conducted: cell proliferation, cell migration and tube format...

PloS one, 2014

We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity ... more We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity in the cornea. Here we investigated the antioxidant effects of SERPINA3K on the pterygial, which is partially caused by oxidative stress in pathogenesis. The head part of primary pterygial tissue was dissected and then cultured in keratinocyte serum-free defined medium (KSFM). The cultured pterygial epithelial cells (PECs) were treated with SERPINA3K. The cell proliferation and migration of PECs were measured and analyzed. Western blot and quantitative real-time polymerase chain reaction (PCR) assay were performed. It showed that SERPINA3K significantly suppressed the cell proliferation of PECs in a concentration-dependent manner, compared with cultured human conjunctival epithelial cells. SERPINA3K also inhibited the cell migration of PECs. Towards its underlying mechanism, SERPINA3K had antioxidant activities on the PECs by significantly inhibiting NADPH oxidase 4 (NOX4), which is an i...