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1.  Targeting miR-146a to Treat Delayed Wound Healing in Human Diabetic Organ-Cultured Corneas 
PLoS ONE  2014;9(12):e114692.
Limbal epithelial stem cells (LESC) residing at the corneal periphery are largely responsible for maintaining corneal optical transparency by continuously supplying new corneal epithelial cells, which mature during their radial migration to the central cornea. Diabetes mellitus (DM) affects all the structures of the eye including the cornea. Frequent epithelial erosions, delayed wound healing, and microbial infections are common alterations of the diabetic eye that can result in vision loss. MicroRNAs (miRNAs) are short non-coding oligonucleotides that regulate gene expression by repressing translation. Our purpose was to understand the role of miR-146a in the human limbal versus central corneal epithelial compartment in normal and pathological conditions such as diabetes mellitus. Using quantitative real-time PCR (QPCR) we found miR-146a enrichment in the limbal corneal compartment. This miRNA was also expressed at higher levels in the diabetic vs. normal limbus. Cell migration and wound closure were significantly delayed in normal and diabetic primary limbal epithelial cells (LEC) transfected with miR-146a. Cells treated with miR-146a had decreased levels of phosphorylated (activated) p38 and EGFR, mediators of epithelial wound healing. Conversely, inhibition of miR-146a significantly enhanced cell migration in both normal and diabetic primary LEC and in diabetic organ-cultured corneas by nearly 40% vs. scrambled miRNA control, accompanied by increased phosphorylated signaling intermediates. Transfection of miR-146a in cultured LEC resulted in an increased immunoreactivity for putative LEC markers Frizzled-7 and K15, whereas inhibition of miR-146a decreased their expressions. These data suggest that miR-146a plays a role in LEC maintenance at the corneal periphery, and its expression is downregulated during their migration towards the central cornea and accompanying terminal differentiation. Furthermore, abnormal miR-146a upregulation may be an important mechanism of delayed wound healing in the diabetic cornea.
PMCID: PMC4260878  PMID: 25490205
2.  Enhanced Wound Healing, Kinase and Stem Cell Marker Expression in Diabetic Organ-Cultured Human Corneas Upon MMP-10 and Cathepsin F Gene Silencing 
Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression.
Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections.
Proteinase silencing reduced epithelial wound healing time versus rAV–enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α3β1 integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain.
Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.
Adenovirus-driven shRNA silencing of select proteinases upregulated in diabetic corneas restored normal wound healing time, the expression of diabetes-altered markers including limbal stem cell markers, and patterns of activated EGFR and Akt in human diabetic corneal organ cultures. The maximum effect was obtained combining proteinase shRNA with c-met overexpression.
PMCID: PMC3867183  PMID: 24255036
diabetic cornea; limbal stem cell; wound healing; MMP-10; cathepsin F; EGFR; Akt; p-38; keratin; organ culture; c-met; gene therapy
3.  Differentially Expressed Wound Healing-Related microRNAs in the Human Diabetic Cornea 
PLoS ONE  2013;8(12):e84425.
MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples. HCEC were transfected with human pre-miRTMmiRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.
PMCID: PMC3869828  PMID: 24376808
4.  A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture 
PLoS ONE  2013;8(11):e79632.
Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.
PMCID: PMC3827346  PMID: 24236148
Diabetic corneas display altered basement membrane and integrin markers, increased expression of proteinases, decreased hepatocyte growth factor receptor, c-met proto-oncogene, and impaired wound healing. Recombinant adenovirus (rAV) driven c-met overexpression in human organ-cultured corneas was tested for correction of diabetic abnormalities.
Forty-six human autopsy corneas from twenty-three long-term diabetic donors (five with diabetic retinopathy) were organ-cultured and transduced with rAV expressing c-met gene (rAV-cmet) under the cytomegalovirus promoter at about 108 plaque-forming units per cornea for 48 hr. Each control fellow cornea received control rAV (rAV expressing β-galactosidase gene or vector alone). After additional 4-5 days incubation, 5-mm epithelial wounds were created with n-heptanol, and healing was monitored. Corneas were analyzed afterwards by immunohistochemistry and Western blotting. Signaling molecule expression and role was examined by immunostaining, phosphokinase antibody arrays, Western blotting, and inhibitor analysis.
rAV-cmet transduction led to increased epithelial staining for c-met (total, extracellular and phosphorylated) and normalization of the patterns of select diabetic markers compared to rAV-vector transduced control fellow corneas. Epithelial wound healing time in c-met transduced diabetic corneas decreased two-fold compared to rAV-vector transduced corneas and became similar to normal. C-met action apparently involved increased activation of p38 mitogen-activated protein kinase. C-met transduction did not change tight junction protein patterns suggesting unaltered epithelial barrier function.
rAV-driven c-met transduction into diabetic corneas appears to restore HGF signaling, normalize diabetic marker patterns, and accelerate wound healing. C-met gene therapy could be useful for correcting human diabetic corneal abnormalities.
PMCID: PMC2846188  PMID: 19933191
gene therapy; adenovirus; c-met; diabetes; cornea; organ culture; wound healing; corneal epithelium; hepatocyte growth factor; p38MAPK
Brain research bulletin  2009;81(2-3):262.
Our previous data suggested the involvement of matrix metalloproteinase-10 (MMP-10) and cathepsin F (CTSF) in the basement membrane and integrin changes occurring in diabetic corneas. These markers were now examined in normal human organ-cultured corneas upon recombinant adenovirus (rAV)-driven transduction of MMP-10 and CTSF genes.
Fifteen pairs of normal autopsy human corneas were used. One cornea of each pair was transduced with rAV expressing either CTSF or MMP-10 genes. 1–2 × 108 plaque forming units of rAV per cornea were added to cultures for 48 hr with or without sildenafil citrate. The fellow cornea of each pair received control rAV with vector alone. After 6–10 days incubation without rAV, corneas were analyzed by Western blot or immunohistochemistry, or tested for healing of 5-mm circular epithelial wounds caused by topical application of n-heptanol.
Sildenafil significantly increased epithelial transduction efficiency, apparently through stimulation of rAV endocytosis through caveolae. Corneas transduced with CTSF or MMP-10 genes or their combination had increased epithelial immunostaining of respective proteins compared to fellow control corneas. Staining for diabetic markers integrin α3β1, nidogen-1, nidogen-2, and laminin γ2 chain became weaker and irregular upon proteinase transduction. Expression of phosphorylated Akt was decreased in proteinase-transduced corneas. Joint overexpression of both proteinases led to significantly slower corneal wound healing that became similar to that observed in diabetic ones.
The data suggest that MMP-10 and CTSF may be responsible for abnormal marker patterns and impaired wound healing in diabetic corneas. Inhibition of these proteinases in diabetic corneas may alleviate diabetic keratopathy symptoms.
PMCID: PMC2815249  PMID: 19828126
diabetic cornea; organ culture; MMP-10; cathepsin F; Akt; sildenafil
7.  Alterations of epithelial stem cell marker patterns in human diabetic corneas and effects of c-met gene therapy 
Molecular Vision  2011;17:2177-2190.
We have previously identified specific epithelial proteins with altered expression in human diabetic central corneas. Decreased hepatocyte growth factor receptor (c-met) and increased proteinases were functionally implicated in the changes of these proteins in diabetes. The present study examined whether limbal stem cell marker patterns were altered in diabetic corneas and whether c-met gene overexpression could normalize these patterns.
Cryostat sections of 28 ex vivo and 26 organ-cultured autopsy human normal and diabetic corneas were examined by immunohistochemistry using antibodies to putative limbal stem cell markers including ATP-binding cassette sub-family G member 2 (ABCG2), N-cadherin, ΔNp63α, tenascin-C, laminin γ3 chain, keratins (K) K15, K17, K19, β1 integrin, vimentin, frizzled 7, and fibronectin. Organ-cultured diabetic corneas were studied upon transduction with adenovirus harboring c-met gene.
Immunostaining for ABCG2, N-cadherin, ΔNp63α, K15, K17, K19, and β1 integrin, was significantly decreased in the stem cell-harboring diabetic limbal basal epithelium either by intensity or the number of positive cells. Basement membrane components, laminin γ3 chain, and fibronectin (but not tenascin-C) also showed a significant reduction in the ex vivo diabetic limbus. c-Met gene transduction, which normalizes diabetic marker expression and epithelial wound healing, was accompanied by increased limbal epithelial staining for K17, K19, ΔNp63α, and a diabetic marker α3β1 integrin, compared to vector-transduced corneas.
The data suggest that limbal stem cell compartment is altered in long-term diabetes. Gene therapy, such as with c-met overexpression, could be able to restore normal function to diabetic corneal epithelial stem cells.
PMCID: PMC3159681  PMID: 21866211
8.  Normalization of Wound Healing and Diabetic Markers in Organ Cultured Human Diabetic Corneas by Adenoviral Delivery of c-Met Gene 
The paper describes successful gene therapy for diabetic corneas with an adenovirus-delivered c-met gene. Overexpressed c-met reversed diabetic marker alterations and accelerated epithelial wound healing to normal rates in human corneal organ cultures. This is one of the first examples of correcting disease abnormalities in the cornea with gene therapy.
Diabetic corneas display altered basement membrane and integrin markers, increased expression of proteinases, decreased hepatocyte growth factor (HGF) receptor, c-met proto-oncogene, and impaired wound healing. Recombinant adenovirus (rAV)–driven c-met overexpression in human organ-cultured corneas was tested for correction of diabetic abnormalities.
Forty-six human corneas obtained postmortem from 23 donors with long-term diabetes (5 with diabetic retinopathy) were organ cultured and transduced with rAV-expressing c-met gene (rAV-cmet) under the cytomegalovirus promoter at approximately 108 plaque-forming units per cornea for 48 hours. Each control fellow cornea received control rAV (rAV expressing the β-galactosidase gene or vector alone). After an additional 4 to 5 days of incubation, 5-mm epithelial wounds were created with n-heptanol, and healing was monitored. The corneas were analyzed afterward by immunohistochemistry and Western blot analysis. Signaling molecule expression and role was examined by immunostaining, phosphokinase antibody arrays, Western blot analysis, and inhibitor analysis.
rAV-cmet transduction led to increased epithelial staining for c-met (total, extracellular, and phosphorylated) and normalization of the patterns of select diabetic markers compared with rAV-vector–transduced control fellow corneas. Epithelial wound healing time in c-met–transduced diabetic corneas decreased twofold compared with rAV-vector–transduced corneas and became similar to normal. c-Met action apparently involved increased activation of p38 mitogen-activated protein kinase. c-Met transduction did not change tight junction protein patterns, suggesting unaltered epithelial barrier function.
rAV-driven c-met transduction into diabetic corneas appears to restore HGF signaling, normalize diabetic marker patterns, and accelerate wound healing. c-Met gene therapy could be useful for correcting human diabetic corneal abnormalities.
PMCID: PMC2846188  PMID: 19933191
9.  Involvement of Protein Kinase CK2 in Angiogenesis and Retinal Neovascularization 
The purpose of the study was to characterize signaling intermediates involved in angiogenic responses of retinal endothelial cells (RECs) to the extracellular matrix and growth factors, by using specific inhibitors.
Tubelike structure formation and the development of secondary sprouts on a basement membrane (BM) matrix, cell proliferation, and cell migration were studied in cultures of bovine and human RECs. Specific inhibitors were tested for inhibition of retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR).
In initial experiments, the broad-spectrum protein kinase inhibitors, H7 and H89, stabilized REC tubes on BM matrix and inhibited secondary sprouting, cell migration, and cell proliferation. Among more specific kinase inhibitors tested, only inhibitors of protein kinase CK2 (formerly, casein kinase II), such as emodin and DRB, were able to duplicate the effects of H7 and H89. Actinomycin D caused only minor changes in angiogenic assays, suggesting that CK2’s effects on REC did not involve its known impact on transcription. The extent of retinal neovascularization in a mouse OIR model was reduced >70% (versus untreated or vehicle-treated groups) after treatment with emodin (6 days at 60 mg/kg per day) and by approximately 60% after treatment at the same dose with TBB, the most specific CK2 inhibitor known. In the treated retinas, the main vascular tree had minimal changes, but the neovascular tufts were greatly reduced in number or absent.
This is the first demonstration of the involvement of ubiquitous protein kinase CK2 in angiogenesis. Naturally derived CK2 inhibitors may be useful for treatment of proliferative retinopathies.
PMCID: PMC2917328  PMID: 15557471
10.  Alterations of Extracellular Matrix Components and Proteinases in Human Corneal Buttons With INTACS for Post–Laser In Situ Keratomileusis Keratectasia and Keratoconus 
Cornea  2008;27(5):565-573.
To perform an immunohistochemical evaluation of corneas with INTACS for post–laser in situ keratomileusis (LASIK) keratectasia and keratoconus, obtained after corneal transplantation.
Corneas from 1 patient with INTACS for post-LASIK keratectasia and 2 patients with INTACS for keratoconus were obtained within 3 hours after penetrating keratoplasty, and cryostat sections were analyzed by immunostaining for 35 extracellular matrix (ECM) components and proteinases.
In the stroma of all corneas next to an INTACS implant, ECM components typically associated with fibrosis were observed. These included tenascin-C, fibrillin-1, and types III, IV (α1/α2 chains), and XIV collagen. Also, significant deposition of perlecan, nidogen-2, and cellular fibronectin was revealed in the same locations. The keratoconus cases displayed typical Bowman layer breaks and subepithelial fibrosis with deposition of various ECM components. In all cases, some keratocytes around INTACS were positive for specific proteinases associated with stromal remodeling, including cathepsins F and H, matrix metalloproteinase (MMP)-1, MMP-3, and MMP-10. Staining for MMP-7 was variable; MMP-2 and MMP-9 were mostly negative. Patterns of type IV collagen α3, α4, and α6 chains; types VI and VIII collagen; laminin-332,α4, α5,β1, β2, and γ1 laminin chains; vitronectin; thrombospondin-1; urokinase; EMMPRIN; and cathepsins B and L were unchanged around INTACS in all 3 cases compared with normal.
Abnormal accumulation of fibrotic ECM components and proteinases near INTACS suggests ongoing lysis and remodeling of corneal stroma. Specific changes observed in each case may be related to underlying pathology.
PMCID: PMC2746565  PMID: 18520507
INTACS; keratoconus; laser in situ keratomileusis; cornea; extracellular matrix; fibrosis; matrix metalloproteinase; tenascin-C; cathepsin; nidogen
11.  Compositional Differences between Infant and Adult Human Corneal Basement Membranes 
Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. In the infant DM, type IV collagen α1-α6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.
The distribution of laminin γ3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
PMCID: PMC2151758  PMID: 17962449
12.  Proteinase and Growth Factor Alterations Revealed by Gene Microarray Analysis of Human Diabetic Corneas 
PURPOSE. To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I.
METHODS. RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agi-lent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10.
RESULTS. More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin α4 chain, and thymosin β4 genes were down-regulated. The data were corroborated by QPCR and immuno-histochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin α3β1, whereas in normal corneas MMP-10 decreased laminin-10 and integrin α3β1 expression.
CONCLUSIONS. Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.
PMCID: PMC1459105  PMID: 16186340
13.  Ocular Changes in TgF344-AD Rat Model of Alzheimer's Disease 
Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive decline in learning, memory, and executive functions. In addition to cognitive and behavioral deficits, vision disturbances have been reported in early stage of AD, well before the diagnosis is clearly established. To further investigate ocular abnormalities, a novel AD transgenic rat model was analyzed.
Transgenic (Tg) rats (TgF344-AD) heterozygous for human mutant APPswe/PS1ΔE9 and age-matched wild type (WT) rats, as well as 20 human postmortem retinal samples from both AD and healthy donors were used. Visual function in the rodent was analyzed using the optokinetic response. Immunohistochemistry on retinal and brain sections was used to detect various markers including amyloid-β (Aβ) plaques.
As expected, Aβ plaques were detected in the hippocampus, cortex, and retina of Tg rats. Plaque-like structures were also found in two AD human whole-mount retinas. The choroidal thickness was significantly reduced in both Tg rat and in AD human eyes when compared with age-matched controls. Tg rat eyes also showed hypertrophic retinal pigment epithelial cells, inflammatory cells, and upregulation of complement factor C3. Although visual acuity was lower in Tg than in WT rats, there was no significant difference in the retinal ganglion cell number and retinal vasculature.
Further studies are needed to elucidate the significance and mechanisms of this pathological change and luminance threshold recording from the superior colliculus.
In this study, we observed pathological changes in the choroid and in RPE cells in the TgF344-AD rat model; choroidal thinning was further observed in human AD retina. Along with Aβ deposition, the inflammatory response was manifested by microglial recruitment and complement activation.
PMCID: PMC3907137  PMID: 24398104
Alzheimer disease; choroidal thickness; visual acuity; retinal pigment epithelium
14.  Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment 
Journal of drug targeting  2013;21(10):956-967.
Engineered nanoparticles are widely used for delivery of drugs but frequently lack proof of safety for cancer patient's treatment. All-in-one covalent nanodrugs of the third generation have been synthesized based on a poly(β-L-malic acid) (PMLA) platform, targeting human triple-negative breast cancer (TNBC). They significantly inhibited tumor growth in nude mice by blocking synthesis of epidermal growth factor receptor, and α4 and β1 chains of laminin-411, the tumor vascular wall protein and angiogenesis marker. PMLA and nanodrug biocompatibility and toxicity at low and high dosages were evaluated in vitro and in vivo. The dual-action nanodrug and single-action precursor nanoconjugates were assessed under in vitro conditions and in vivo with multiple treatment regimens (6 and 12 treatments). The monitoring of TNBC treatment in vivo with different drugs included blood hematologic and immunologic analysis after multiple intravenous administrations. The present study demonstrates that the dual-action nanoconju-gate is highly effective in preclinical TNBC treatment without side effects, supported by hematologic and immunologic assays data. PMLA-based nanodrugs of the Polycefin™ family passed multiple toxicity and efficacy tests in vitro and in vivo on preclinical level and may prove to be optimized and efficacious for the treatment of cancer patients in the future.
PMCID: PMC4043297  PMID: 24032759
Hematologic; immunogenicity; in vivo treatment; nanoconjugate drugs; polymalic acid; toxicity; triple-negative breast cancer
Laminins are the major constituents of blood vessel basement membranes (BMs). Each laminin is a trimer consisting of three assembled polypeptide chains, α, β and γ. More than 15 laminin isoforms are known to date and the expression of specific isoforms may change in certain pathological conditions. Here we show that during progression of glial tumors laminin-9 (α4β2γ1) is switched to laminin-8 (α4β1γ1), which is dramatically increased in glial brain tumors. Laminin-8 overproduction by glial tumor cells facilitates spread of glioma. Brain tumors with laminin-8 overexpression recur faster after standard treatment and patients have shorter survival time. Laminin-8 may be thus used as a predictor of tumor recurrence, patient survival and as a potential molecular target for glioma therapy.
PMCID: PMC3506377  PMID: 16146715
Laminin-8; Laminin-9; Basement Membrane; Extracellular Matrix; Angiogenesis; Human; Cancer; Tumor; Neoplasm; Glioma; Glioblastoma Multiforme; Recurrence; Survival; Invasion; Morpholino antisense; Review
16.  Inhibition of laminin-8 in vivo using a novel poly(malic acid)-based carrier reduces glioma angiogenesis 
Angiogenesis  2006;9(4):183-191.
We have previously shown that laminin-8, a vascular basement membrane component, was over-expressed in human glioblastomas multiforme and their adjacent tissues compared to normal brain. Increased laminin-8 correlated with shorter glioblastoma recurrence time and poor patient survival making it a potential marker for glioblastoma diagnostics and prediction of disease outcome. However, laminin-8 therapeutic potential was unknown because the technology of blocking the expression of multi-chain complex proteins was not yet developed. To inhibit the expression of laminin-8 constituents in glioblastoma in vitro and in vivo, we used Polycefin, a bioconjugate drug delivery system based on slime-mold Physarum polycephalum-derived poly(malic acid). It carries an attached transferrin receptor antibody to target tumor cells and to deliver two conjugated morpholino antisense oligonucleotides against laminin-8 α4 and β1 chains. Polycefin efficiently inhibited the expression of both laminin-8 chains by cultured glioblastoma cells. Intracranial Polycefin treatment of human U87MG glioblastoma-bearing nude rats reduced incorporation of both tumor-derived laminin-8 chains into vascular basement membranes. Polycefin was thus able to simultaneously inhibit the expression of two different chains of a complex protein. The treatment also significantly reduced tumor microvessel density (p < 0.001) and area (p < 0.001) and increased animal survival (p < 0.0004). These data suggest that laminin-8 may be important for glioblastoma angiogenesis. Polycefin, a versatile nanoscale drug delivery system, was suitable for in vivo delivery of two antisense oligonucleotides to brain tumor cells causing a reduction of glioblastoma angiogenesis and an increase of animal survival. This system may hold promise for future clinical applications.
PMCID: PMC3487708  PMID: 17109197
Tumor angiogenesis; Glioma; Laminin-8; Multiple drug targeting; Poly(malic acid)
17.  Focus on Molecules: Protein kinase CK2 
Experimental eye research  2010;101:111-112.
PMCID: PMC3437250  PMID: 21194531
protein kinase; CK2; cancer; apoptosis; angiogenesis
18.  Polymalic Acid–Based Nanobiopolymer Provides Efficient Systemic Breast Cancer Treatment by Inhibiting both HER2/neu Receptor Synthesis and Activity 
Cancer research  2011;71(4):1454-1464.
Biodegradable nanopolymers are believed to offer great potential in cancer therapy. Here, we report the characterization of a novel, targeted, nanobiopolymeric conjugate based on biodegradable, nontoxic, and nonimmunogenic PMLA [poly(β-l-malic acid)]. The PMLA nanoplatform was synthesized for repetitive systemic treatments of HER2/neu-positive human breast tumors in a xenogeneic mouse model. Various moieties were covalently attached to PMLA, including a combination of morpholino antisense oligonucleotides (AON) directed against HER2/neu mRNA, to block new HER2/neu receptor synthesis; anti-HER2/neu antibody trastuzumab (Herceptin), to target breast cancer cells and inhibit receptor activity simultaneously; and transferrin receptor antibody, to target the tumor vasculature and mediate delivery of the nanobiopolymer through the host endothelial system. The results of the study showed that the lead drug tested significantly inhibited the growth of HER2/neu-positive breast cancer cells in vitro and in vivo by enhanced apoptosis and inhibition of HER2/neu receptor signaling with suppression of Akt phosphorylation. In vivo imaging analysis and confocal microscopy demonstrated selective accumulation of the nanodrug in tumor cells via an active delivery mechanism. Systemic treatment of human breast tumor-bearing nude mice resulted in more than 90% inhibition of tumor growth and tumor regression, as compared with partial (50%) tumor growth inhibition in mice treated with trastuzumab or AON, either free or attached to PMLA. Our findings offer a preclinical proof of concept for use of the PMLA nanoplatform for combination cancer therapy.
PMCID: PMC3428373  PMID: 21303974
19.  Identification of Amyloid Plaques in Retinas from Alzheimer’s Patients and Noninvasive In Vivo Optical Imaging of Retinal Plaques in a Mouse Model 
NeuroImage  2010;54S1:S204-S217.
Noninvasive monitoring of β-amyloid (Aβ) plaques, the neuropathological hallmarks of Alzheimer's disease (AD), is critical for AD diagnosis and prognosis. Current visualization of Aβ plaques in brains of live patients and animal models is limited in specificity and resolution. The retina as an extension of the brain portrays an appealing target for a live, noninvasive optical imaging of AD if disease pathology is manifested there. We identified retinal Aβ plaques in postmortem eyes from AD patients (n=8) and in suspected early stage cases (n=5), consistent with brain pathology and clinical reports; plaques were undetectable in age-matched non-AD individuals (n=5). In APPSWE/PS1ΔE9 transgenic mice (AD-Tg; n=18) and not in non-Tg wt mice (n=10), retinal Aβ plaques were detected following systemic administration of curcumin, a safe plaque-labeling fluorochrome. Moreover, retinal plaques were detectable earlier than in the brain and accumulated with disease progression. An immune-based therapy effective in reducing brain plaques, significantly reduced retinal Aβ plaque burden in immunized versus non-immunized AD mice (n=4 mice per group). In live AD-Tg mice (n=24), systemic administration of curcumin allowed noninvasive optical imaging of retinal Aβ plaques in vivo with high resolution and specificity; plaques were undetectable in non-Tg wt mice (n=11). Our discovery of Aβ specific plaques in retinas from AD patients, and the ability to noninvasively detect individual retinal plaques in live AD mice establish the basis for developing high resolution optical imaging for early AD diagnosis, prognosis assessment and response to therapies.
PMCID: PMC2991559  PMID: 20550967
human retina; Aβ deposit; Aβ plaque; Alzheimer’s disease; mild cognitive impairment; vaccination; curcumin; in vivo optical imaging; fluorescence; spectral classification
20.  Exacerbation of corneal scarring in HSV-1 gK immunized mice correlates with elevation of CD8+CD25+ T cells in corneas of ocularly infected mice 
Virology  2010;399(1):11-22.
We have shown previously that exacerbation of corneal scarring (CS) in HSV-1 glycoprotein K (gK) immunized mice was associated with CD8+ T cells. In this study, we investigated the type and the nature of the immune responses that are involved in the exacerbation of CS in gK-immunized animals. BALB/c mice were vaccinated with baculovirus expressed gK, gD, or mock-immunized. Twenty-one days after the third immunization, mice were ocularly infected with 2×105 PFU/eye of virulent HSV-1 strain McKrae. Infiltration of the cornea by CD4+, CD8+, CD25+, CD4+CD25+, CD8+CD25+, CD19+, CD40+, CD40L+, CD62L+, CD95+, B7-1+, B7-2+, MHC-I+, and MHC-II+ cells were monitored by immunohistochemistry, qRT-PCR and FACS at various times post infection (PI). This study demonstrated for the first time that the presence of CD8+CD25+ T cells in the cornea is correlated with exacerbation of CS in the gK-immunized group.
PMCID: PMC2830294  PMID: 20079918
Immunostaining; TaqMan qRT-PCR; Cornea; FACS
21.  Human diabetic corneas preserve wound healing, basement membrane, integrin and MMP-10 differences from normal corneas in organ culture 
Experimental eye research  2003;77(2):211-217.
The authors have previously documented decreased epithelial basement membrane (BM) components and α3β1 epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar–collagen gel at the air–liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 α5 chain, nidogen-1/entactin, integrin α3β1, and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of α3β1 integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized α3β1 integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas.
PMCID: PMC2909880  PMID: 12873452
diabetic retinopathy; cornea; organ culture; basement membrane; integrin; laminin; nidogen; stromelysin; matrix metalloproteinase; MMP-10; tenascin-C; fibrillin-1; α-enolase; keratin 3
22.  High Glucose Suppresses Epidermal Growth Factor Receptor/Phosphatidylinositol 3-Kinase/Akt Signaling Pathway and Attenuates Corneal Epithelial Wound Healing 
Diabetes  2009;58(5):1077-1085.
Patients with diabetes are at an increased risk for developing corneal complications and delayed wound healing. This study investigated the effects of high glucose on epidermal growth factor receptor (EGFR) signaling and on epithelial wound healing in the cornea.
Effects of high glucose on wound healing and on EGFR signaling were investigated in cultured porcine corneas, human corneal epithelial cells, and human corneas using Western blotting and immunofluorescence. Effects of high glucose on reactive oxygen species (ROS) and glutathione levels and on EGFR pathways were assessed in porcine and primary human corneal epithelial cells, respectively. The effects of EGFR ligands and antioxidants on high glucose–delayed epithelial wound healing were assessed in cultured porcine corneas.
High glucose impaired ex vivo epithelial wound healing and disturbed cell responses and EGFR signaling to wounding. High glucose suppressed Akt phosphorylation in an ROS-sensitive manner and decreased intracellular glutathione in cultured porcine corneas. Exposure to high glucose for 24 h resulted in an increase in ROS-positive cells in primary human corneal epithelial cells. Whereas heparin-binding EGF-like growth factor and antioxidant N-acetylcysteine had beneficial effects on epithelial wound closure, their combination significantly accelerated high glucose–delayed wound healing to a level similar to that seen in control subjects. Finally, Akt signaling pathway was perturbed in the epithelia of human diabetic corneas, but not in the corneas of nondiabetic, age-matched donors.
High glucose, likely through ROS, impairs the EGFR–phosphatidylinositol 3-kinase/Akt pathway, resulting in delayed corneal epithelial wound healing. Antioxidants in combination with EGFR ligands may be promising potential therapeutics for diabetic keratopathy.
PMCID: PMC2671049  PMID: 19188434
23.  Phosphodiesterase Type 5 Inhibitors Increase Herceptin Transport and Treatment Efficacy in Mouse Metastatic Brain Tumor Models 
PLoS ONE  2010;5(4):e10108.
Chemotherapeutic drugs and newly developed therapeutic monoclonal antibodies are adequately delivered to most solid and systemic tumors. However, drug delivery into primary brain tumors and metastases is impeded by the blood-brain tumor barrier (BTB), significantly limiting drug use in brain cancer treatment.
Methodology/Principal Findings
We examined the effect of phosphodiesterase 5 (PDE5) inhibitors in nude mice on drug delivery to intracranially implanted human lung and breast tumors as the most common primary tumors forming brain metastases, and studied underlying mechanisms of drug transport. In vitro assays demonstrated that PDE5 inhibitors enhanced the uptake of [14C]dextran and trastuzumab (Herceptin®, a humanized monoclonal antibody against HER2/neu) by cultured mouse brain endothelial cells (MBEC). The mechanism of drug delivery was examined using inhibitors for caveolae-mediated endocytosis, macropinocytosis and coated pit/clathrin endocytosis. Inhibitor analysis strongly implicated caveolae and macropinocytosis endocytic pathways involvement in the PDE5 inhibitor-enhanced Herceptin uptake by MBEC. Oral administration of PDE5 inhibitor, vardenafil, to mice with HER2-positive intracranial lung tumors led to an increased tumor permeability to high molecular weight [14C]dextran (2.6-fold increase) and to Herceptin (2-fold increase). Survival time of intracranial lung cancer-bearing mice treated with Herceptin in combination with vardenafil was significantly increased as compared to the untreated, vardenafil- or Herceptin-treated mice (p<0.01). Log-rank survival analysis of mice bearing HER2-positive intracranial breast tumor also showed a significant survival increase (p<0.02) in the group treated with Herceptin plus vardenafil as compared to other groups. However, vardenafil did not exert any beneficial effect on survival of mice bearing intracranial breast tumor with low HER2 expression and co-treated with Herceptin (p>0.05).
These findings suggest that PDE5 inhibitors may effectively modulate BTB permeability, and enhance delivery and therapeutic efficacy of monoclonal antibodies in hard-to-treat brain metastases from different primary tumors that had metastasized to the brain.
PMCID: PMC2856671  PMID: 20419092
24.  ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina 
To characterize the ZBED4 cDNA identified by subtractive hybridization and microarray of retinal cone degeneration (cd) adult dog mRNA from mRNA of normal dog retina.
The cDNA library obtained from subtractive hybridization was arrayed and screened with labeled amplicons from normal and cd dog retinas. Northern blot analysis was used to verify ZBED4 mRNA expression in human retina. Flow cytometry sorted peanut agglutinin (PNA)-labeled cones from dissociated mouse retinas, and quantitative RT-PCR (QPCR) was used to measure ZBED4 mRNA levels in these cone cells. Immunohistochemistry localized ZBED4 in human retinas. Expression of ZBED4 mRNA transiently transfected into HEK293 cells was analyzed by immunofluorescence. ZBED4 subcellular localization was determined with Western blot analysis.
One of 80 cDNAs differentially expressed in normal and cd dog retinas corresponded to a novel gene, ZBED4, which is also expressed in human and mouse retinas. ZBED4 mRNA was found to be present in cone photoreceptors. When ZBED4 cDNA was transfected into HEK293 cells, the expressed protein showed nuclear localization. However, in human retinas, ZBED4 was localized to cone nuclei, inner segments, and pedicles, as well as to Müller cell endfeet. Confirming these immunohistochemical results, the 135-kDa ZBED4 was found in both the nuclear and cytosolic extracts of human retinas. ZBED4 has four predicted DNA-binding domains, a dimerization domain, and two LXXLL motifs characteristic of coactivators/corepressors of nuclear hormone receptors.
ZBED4 cellular/subcellular localization and domains suggest a regulatory role for this protein, which may exert its effects in cones and Müller cells through multiple ways of action.
PMCID: PMC2848067  PMID: 19369242
25.  Poly(malic acid) nanoconjugates containing various antibodies and oligonucleotides for multitargeting drug delivery 
Nanomedicine (London, England)  2008;3(2):247-265.
Nanoconjugates are emerging as promising drug-delivery vehicles because of their multimodular structure enabling them to actively target discrete cells, pass through biological barriers and simultaneously carry multiple drugs of various chemical nature. Nanoconjugates have matured from simple devices to multifunctional, biodegradable, nontoxic and nonimmunogenic constructs, capable of delivering synergistically functioning drugs in vivo. This review mainly concerns the Polycefin family of natural-derived polymeric drug-delivery devices as an example. This type of vehicle is built by hierarchic conjugation of functional groups onto the backbone of poly(malic acid), an aliphatic polyester obtained from the microorganism Physarum polycephalum. Particular Polycefin variants target human brain and breast tumors implanted into animals specifically and actively and could be detected easily by noninvasive imaging analysis. Delivery of antisense oligonucleotides to a tumor-specific angiogenic marker using Polycefin resulted in significant inhibition of tumor angiogenesis and increase of animal survival.
PMCID: PMC2734385  PMID: 18373429
biodegradable; brain cancer; breast cancer; imaging analysis; multiple antibodies; multiple drug delivery; multitargeting; Polycefin; poly(malic acid); tumor angiogenesis

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