[Show abstract][Hide abstract] ABSTRACT: Retinal transplantation experiments have advanced considerably during recent years, but remaining diseased photoreceptor cells in the host retina and inner retinal cells in the transplant physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). Here, we report the surface modification of PGS membranes to promote the attachment of photoreceptor layers, allowing the resulting composite to be handled surgically as a single entity. PGS membranes were chemically modified with peptides containing an arginine-glycine-aspartic acid (RGD) extracellular matrix ligand sequence. PGS membranes were also coated with electrospun nanofiber meshes, containing laminin and poly(epsilon-caprolactone) (PCL). Following in vitro co-culture of biomaterial membranes with isolated embryonic retinal tissue, composites were tested for surgical handling and examined with hematoxylin and eosin staining and immunohistochemical markers. Electrospun nanofibers composed of laminin and PCL promoted sufficient cell adhesion for simultaneous transplantation of isolated photoreceptor layers and PGS membranes. Composites developed large populations of recoverin and rhodopsin labeled photoreceptors. Furthermore, ganglion cells, rod bipolar cells and AII amacrine cells were absent in co-cultured retinas as observed by neurofilament, PKC and parvalbumin labeling respectively. These results facilitate retinal transplantation experiments in which a composite graft composed of a biodegradable membrane adhered to an immature retina dominated by photoreceptor cells may be delivered in a single surgery, with the possibility of improving graft-host neuronal connections.
[Show abstract][Hide abstract] ABSTRACT: Retinal degenerations cause permanent visual loss and affect millions world-wide. Presently, a novel treatment highlights the potential of using biodegradable polymer scaffolds to induce differentiation and deliver retinal progenitor cells for cell replacement therapy. In this study, we engineered and analyzed a micro-fabricated polymer, poly(glycerol sebacate) (PGS) scaffold, whose useful properties include biocompatibility, elasticity, porosity, and a microtopology conducive to mouse retinal progenitor cell (mRPC) differentiation. In vitro proliferation assays revealed that PGS held up to 86,610 (+/-9993) mRPCs per square millimeter, which were retained through simulated transplantations. mRPCs adherent to PGS differentiated toward mature phenotypes as evidenced by changes in mRNA, protein levels, and enhanced sensitivity to glutamate. Transplanted composites demonstrated long-term mRPC survival and migrated cells exhibited mature marker expression in host retina. These results suggest that combining mRPCs with PGS scaffolds for subretinal transplantation is a practical strategy for advancing retinal tissue engineering as a restorative therapy.
[Show abstract][Hide abstract] ABSTRACT: Therapeutic impact of neural stem cells (NSCs) for acute spinal cord injury (SCI) has been limited by the rapid loss of donor cells. Neuroinflammation is likely the cause. As there are close temporal-spatial correlations between the inducible nitric oxide (NO) synthase expression and the donor NSC death after neurotrauma, we reasoned that NO-associated radical species might be the inflammatory effectors which eliminate NSC grafts and kill host neurons. To test this hypothesis, human NSCs (hNSCs: 5 x 10(4) to 2 x 10(6) per milliliter) were treated in vitro with "plain" medium, 20 microM glutamate, or donors of NO and peroxynitrite (ONOO(-); 100 and 400 microM of spermine or DETA NONOate, and SIN-1, respectively). hNSC apoptosis primarily resulted from SIN-1 treatment, showing ONOO(-)-triggered protein nitration and the activation of p38 MAPK, cytochrome c release, and caspases. Therefore, cell death following post-SCI (p.i.) NO surge may be mediated through conversion of NO into ONOO(-). We subsequently examined such causal relationship in a rat model of dual penetrating SCI using a retrievable design of poly-lactic-co-glycolic acid (PLGA) scaffold seeded with hNSCs that was shielded by drug-releasing polymer. Besides confirming the ONOO(-)-induced cell death signaling, we demonstrated that cotransplantation of PLGA film embedded with ONOO(-) scavenger, manganese (III) tetrakis (4-benzoic acid) porphyrin, or uric acid (1 micromol per film), markedly protected hNSCs 24 hours p.i. (total: n = 10). Our findings may provide a bioengineering approach for investigating mechanisms underlying the host microenvironment and donor NSC interaction and help formulate strategies for enhancing graft and host cell survival after SCI.
[Show abstract][Hide abstract] ABSTRACT: Retinal transplantation experiments have advanced considerably during recent years, but remaining diseased photoreceptor cells in the host retina physically obstruct the development of graft-host neuronal contacts that are required for vision. We here report selective removal of photoreceptors using the biodegradable elastomer poly(glycerol sebacate) (PGS). A 1 × 3 mm PGS membrane was implanted in the subretinal space of normal rabbit eyes, and morphologic specimens were examined with hematoxylin and eosin staining and a panel of immunohistochemical markers. Seven days postoperatively, a patent separation of the neuroretina and retinal pigment epithelium was found as well as loss of several rows of photoreceptors in combination with massive terminal transferase-mediated dUTP nick-end labeling (TUNEL) staining for apoptosis in the outer nuclear layer. After 28 days, the neuroretina was reattached, the PGS membrane had degraded, and photoreceptors were absent in the implantation area. Activated Müller cells were found in the entire retina in 7-day specimens, and in the implantation area after 28 days. AII amacrine and rod bipolar cell morphology was not affected, except for disrupted dendritic branching, which was present in rod bipolar cells in 28-day specimens. We conclude that retinal detachment induced by the biodegradable PGS membrane creates a permissive environment in which graft-host neuronal connections may be facilitated in future retinal transplantation experiments.
Tissue Engineering Part A 03/2009; 17(13-14):1675-82. · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diseases that cause photoreceptor cell degeneration afflict millions of people, yet no restorative treatment exists for these blinding disorders. Replacement of photoreceptors using retinal progenitor cells (RPCs) represents a promising therapy for the treatment of retinal degeneration. Previous studies have demonstrated the ability of polymer scaffolds to increase significantly both the survival and differentiation of RPCs. We report the microfabrication of a poly(glycerol-sebacate) scaffold with superior mechanical properties for the delivery of RPCs to the subretinal space. Using a replica molding technique, a porous poly(glycerol-sebacate) scaffold with a thickness of 45 microm was fabricated. Evaluation of the mechanical properties of this scaffold showed that the Young's modulus is about 5-fold lower and the maximum elongation at failure is about 10-fold higher than the previously reported RPC scaffolds. RPCs strongly adhered to the poly(glycerol-sebacate) scaffold, and endogenous fluorescence nearly doubled over a 2-day period before leveling off after 3 days. Immunohistochemistry revealed that cells grown on the scaffold for 7 days expressed a mixture of immature and mature markers, suggesting a tendency towards differentiation. We conclude that microfabricated poly(glycerol-sebacate) exhibits a number of novel properties for use as a scaffold for RPC delivery.
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen and nitrogen radicals produced during metabolic processes, such as respiration and inflammation, combine with DNA to form many lesions primarily at guanine sites. Understanding the roles of the polymerases responsible for the processing of these products to mutations could illuminate molecular mechanisms that correlate oxidative stress with cancer. Using M13 viral genomes engineered to contain single DNA lesions and Escherichia coli strains with specific polymerase (pol) knockouts, we show that pol V is required for efficient bypass of structurally diverse, highly mutagenic guanine oxidation products in vivo. We also find that pol IV participates in the bypass of two spiroiminodihydantoin lesions. Furthermore, we report that one lesion, 5-guanidino-4-nitroimidazole, is a substrate for multiple SOS polymerases, whereby pol II is necessary for error-free replication and pol V for error-prone replication past this lesion. The results spotlight a major role for pol V and minor roles for pol II and pol IV in the mechanism of guanine oxidation mutagenesis.
Journal of Biological Chemistry 05/2007; 282(17):12741-8. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The DNA damage product 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxoG) is a commonly used biomarker of oxidative stress. The mutagenic potential of this DNA lesion is mitigated in Escherichia coli by multiple enzymes. One of these enzymes, MutY, excises an A mispaired with 8-oxoG as part of the process to restore the original G:C base pair. However, numerous studies have shown that 8-oxoG is chemically labile toward further oxidation. Here, we examine the activity of MutY on the 8-oxoG oxidation products guanidinohydantoin (Gh), two diastereomers of spiroiminodihydantoin (Sp1 and Sp2), oxaluric acid (Oa), and urea (Ur). Single-stranded viral genomes containing a site-specific lesion were constructed and replicated in E. coli that are either proficient in DNA repair or that lack MutY. These lesions were found previously to be potently mutagenic in repair competent bacteria, and we report here that these 8-oxoG-derived lesions are equally miscoding when replicated in E. coli lacking MutY; no significant change in mutation identity or frequency is observed. Interestingly, however, in the presence of MutY, Sp1 and Sp2 are more toxic than in cells lacking this repair enzyme.
[Show abstract][Hide abstract] ABSTRACT: The DNA oxidation product 7,8-dihydro-8-oxoguanine (8-oxoG) forms several mutagenic oxidation products, including a metastable oxaluric acid (Oa) derivative. We report here that a synthetic oligonucleotide containing Oa hydrolyzes under simulated "in vivo" conditions to form a mutagenic urea (Ua) lesion. Using the Oa 2'-deoxyribonucleoside as a model, the hydrolysis rate depended strongly upon the concentrations of bicarbonate and divalent magnesium. In buffered solutions containing physiologically relevant levels of these species, the half-life of Oa nucleoside was approximately 40 h at 37 degrees C. The mutagenic properties of Ua in DNA were investigated using a M13mp7L2 bacteriophage genome containing Ua at a specific site. Transfection of the lesion-containing genome into wild-type AB1157 Escherichia coli allowed determination of the mutation frequency and DNA polymerase bypass efficiency from the resulting progeny phage. Ua was bypassed with an efficiency of 11% as compared to a guanine control and caused a 99% G-->T mutation frequency, assuming the lesion originated from G, which is at least an order of magnitude higher than the mutation frequency of 8-oxoG under the same conditions. SOS induction of bypass DNA polymerase(s) in the bacteria prior to transfection caused the mutation frequency and type to shift to 43% G-->T, 46% G-->C, and 10% G-->A mutations. We suggest that Ua is instructional, meaning that the shape of the lesion and its interactions with DNA polymerases influence which nucleotide is inserted opposite the lesion during replication and that the instructional nature of the lesion is modulated by the size of the binding pocket of the DNA polymerase. Replication past Ua, when formed by hydrolysis of the 8-oxoG oxidation product Oa, denotes a pathway that nearly quantitatively generates point mutations in vivo.
Chemical Research in Toxicology 02/2005; 18(1):12-8. · 4.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The in vivo mutagenic properties of 2-aminoimidazolone and 5-guanidino-4-nitroimidazole, two products of peroxynitrite oxidation of guanine, are reported. Two oligodeoxynucleotides of identical sequence, but containing either 2-aminoimidazolone or 5-guanidino-4-nitroimidazole at a specific site, were ligated into single-stranded M13mp7L2 bacteriophage genomes. Wild-type AB1157 Escherichia coli cells were transformed with the site-specific 2-aminoimidazolone- and 5-guanidino-4-nitroimidazole-containing genomes, and analysis of the resulting progeny phage allowed determination of the in vivo bypass efficiencies and mutational signatures of the DNA lesions. 2-Aminoimidazolone was efficiently bypassed and 91% mutagenic, producing almost exclusively G to C transversion mutations. In contrast, 5-guanidino-4-nitroimidazole was a strong block to replication and 50% mutagenic, generating G to A, G to T, and to a lesser extent, G to C mutations. The G to A mutation elicited by 5-guanidino-4-nitroimidazole implicates this lesion as a novel source of peroxynitrite-induced transition mutations in vivo. For comparison, the error-prone bypass DNA polymerases were overexpressed in the cells by irradiation with UV light (SOS induction) prior to transformation. SOS induction caused little change in the efficiency of DNA polymerase bypass of 2-aminoimidazolone; however, bypass of 5-guanidino-4-nitroimidazole increased nearly 10-fold. Importantly, the mutation frequencies of both lesions decreased during replication in SOS-induced cells. These data suggest that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole in DNA are substrates for one or more of the SOS-induced Y-family DNA polymerases and demonstrate that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole are potent sources of mutations in vivo.
Journal of Biological Chemistry 11/2004; 279(42):43568-73. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: [reaction: see text] A convertible nucleoside was synthesized and used to prepare the 2'-deoxynucleoside of 5-guanidino-4-nitroimidazole, a putative in vivo product of the reaction of peroxynitrite with guanine. The convertible nucleoside was incorporated into an oligodeoxynucleotide by the phosphoramidite method and converted postsynthetically to yield an oligodeoxynucleotide containing 5-guanidino-4-nitroimidazole at a specific site. The oligodeoxynucleotide was inserted into a viral genome. Melting temperature analysis revealed that duplexes containing 5-guanidino-4-nitroimidazole were greatly destabilized relative to unmodified duplexes.
[Show abstract][Hide abstract] ABSTRACT: Single-stranded DNA genomes have been constructed that site-specifically contain the 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxoG) oxidation products guanidinohydantoin (Gh) and the two stable stereoisomers of spiroiminodihydantoin (Sp1 and Sp2). The circular viral genomes were transfected into wild-type AB1157 Escherichia coli, and the efficiency of lesion bypass by DNA polymerase(s) was assessed. Viral progeny were analyzed for mutation frequency and type using the recently developed restriction endonuclease and postlabeling (REAP) assay. Gh was bypassed nearly as efficiently as the parent 8-oxoG but was highly mutagenic, causing almost exclusive G --> C transversions. The stereoisomers Sp1 and Sp2 were, in comparison, much stronger blocks to DNA polymerase extension and caused a mixture of G --> T and G --> C transversions. The ratio of G --> T to G --> C mutations for each Sp lesion was dependent on the stereochemical configuration of the base. All observed mutation frequencies were at least an order of magnitude higher than those caused by 8-oxoG. Were these lesions to be formed in vivo, our data show that they are absolutely miscoding and may be refractory to repair after translesion synthesis.
[Show abstract][Hide abstract] ABSTRACT: Single-stranded DNA genomes have been constructed that site-specifically contain the 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG) oxidation products guanidinohyndantoin (Gh), and the two stable stereoisomers of spiroiminodihydantoin (Sp1 and Sp2). The circular viral genomes were transfected into wild-type AB1157 Escherichia coli and the efficiency of lesion bypass by DNA polymerase(s) was assessed. Viral progeny were analyzed for mutation frequency and type using the recently developed restriction endonuclease and post-labeling (REAP) assay. Gh was bypassed nearly as efficiently as the parent 8-oxoG, but was highly mutagenic, causing almost exclusive GC transversions. The stereoisomers Sp1 and Sp2 were, in comparison, much stronger blocks to DNA polymerase extension, and caused a mixture of GT and GC transversions. The ratio of GT to GC mutations for each Sp lesion was dependent on the stereochemical configuration of the base. All observed mutation frequencies were at least an order of magnitude higher than those caused by 8-oxoG. Were these lesions to be formed in vivo, our data show that they are absolutely miscoding, and may be refractory to repair after translesion synthesis.
[Show abstract][Hide abstract] ABSTRACT: Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006. This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. Vita. Includes bibliographical references. The radicals nitric oxide and superoxide are produced endogenously by activated macrophages and neutrophils and combine in a diffusion-limited reaction to form peroxynitrite, a powerful oxidizing and nitrating agent capable of damaging a variety of biomolecules, including DNA. Of the four nucleobases of DNA, guanine has the lowest oxidation potential and thus considerable attention has been given to the study of the oxidation of this base by peroxynitrite. A variety of DNA lesions are generated from guanine including guanidinohydantoin, spiroiminodihydantoin, oxaluric acid, urea, 2-aminoimidazolone, and 5-guanidino-4-nitroimidazole. In order to assess the biological significance and consequences of peroxynitrite-damaged DNA, it is essential that these lesions be characterized for their genotoxic and mutagenic potential. This work focuses on the elucidation of those properties. In the first study, the 2'-deoxynucleoside of 5-guanidino-4-nitroimidazole was chemically synthesized and incorporated into an oligonucleotide by the phosphoramidite method. In the second study, the genotoxic and mutational properties of 2-aminoimidazolone and 5-guanidino-4-nitroimidazole were determined in wild-type uninduced and SOS-induced E. coli. (cont.) In the third study, oxaluric acid was found to hydrolyze to urea in a reaction catalyzed by magnesium cations and bicarbonate. The genotoxic and mutational properties of oxaluric acid and urea were determined in wildtype uninduced and SOS-induced E. coli. In the fourth study, the genotoxic and mutational properties of guanidinohydantoin and spiroiminodihydantoin were determined in wild-type uninduced E. coli. In the fifth study, the genotoxic and mutational properties of guanidinohydantoin, spiroiminodihydantoin, oxaluric acid, urea, 2-aminoimidazolone, and 5-guanidino-4-nitroimidazole were determined in wild-type, polymerase II deficient, polymerase IV deficient, polymerase V deficient, and polymerase II / polymerase IV / polymerase V deficient E. coli under both uninduced and SOS-induced conditions. All of the lesions studied were potent sources of mutations in vivo. Guanidinohydantoin, spiroiminodihydantoin, urea, and 5-guanidino-4-nitroimidazole were significant blocks to replication and were strongly dependent upon induction of the SOS system. Polymerase V was responsible for the majority of translesion synthesis. by William Louis Neeley. Ph.D.