Dexamethasone Controlled Release on TGF-ß1 Treated Vocal Fold Fibroblasts.

OBJECTIVE: Corticosteroids may be beneficial in treating vocal fold scarring. Current drug delivery methods do not permit controlled corticosteroid release. Here we investigate the effects of poly-lactic-co-glycolic acid (PLGA) microparticles loaded with the corticosteroid dexamethasone in reducing collagen synthesis and inflammation in vocal fold fibroblasts treated with and without TGF-ß1. STUDY DESIGN: Experimental, in vitro study. METHODS: PLGA microparticles of differing molecular weight and terminating moieties were synthesized using a hydrogel template method. The release of dexamethasone was characterized from these microparticles over 4 days. Based on the release studies, ester-terminated low molecular weight PLGA microparticles were loaded with dexamethasone and applied to TGF-ß1 treated vocal fold fibroblasts for 4 days. Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assays (ELISAs) were used to assess the effects of released dexamethasone on collagen synthesis and inflammatory mediators. RESULTS: COL3A1 and COL1A2 were significantly down-regulated after exposure to ester-terminated low molecular weight PLGA microparticles loaded with dexamethasone. The loaded microparticles also reduced interleukin-6 synthesis. CONCLUSION: These data show promise in using a PLGA microparticle-based delivery system to control dexamethasone release over 4 days. Our findings lay the groundwork for developing more effective treatments for vocal fold scarring.

RNA interference (RNAi)-dependent and RNAi-independent association of the Chp1 chromodomain protein with distinct heterochromatic loci in fission yeast.

The establishment of centromeric heterochromatin in the fission yeast Schizosaccharomyces pombe is dependent on the RNA interference (RNAi) pathway. Dicer cleaves centromeric transcripts to produce short interfering RNAs (siRNAs) that actively recruit components of heterochromatin to centromeres. Both centromeric siRNAs and the heterochromatin component Chp1 are components of the RITS (RNA-induced initiation of transcriptional gene silencing) complex, and the association of RITS with centromeres is linked to Dicer activity. In turn, centromeric binding of RITS promotes Clr4-mediated methylation of histone H3 lysine 9 (K9), recruitment of Swi6, and formation of heterochromatin. Similar to centromeres, the mating type locus (Mat) is coated in K9-methylated histone H3 and is bound by Swi6. Here we report that Chp1 associates with the mating type locus and telomeres and that Chp1 localization to heterochromatin depends on its chromodomain and the C-terminal domain of the protein. Another protein component of the RITS complex, Tas3, also binds to Mat and telomeres. Tas3 interacts with Chp1 through the C-terminal domain of Chp1, and this interaction is necessary for Tas3 stability. Interestingly, in cells lacking the Argonaute (Ago1) protein component of the RITS complex, or lacking Dicer (and hence siRNAs), Chp1 and Tas3 can still bind to noncentromeric loci, although their association with centromeres is lost. Thus, Chp1 and Tas3 exist as an Ago1-independent subcomplex that associates with noncentromeric heterochromatin independently of the RNAi pathway.

Varying RGD concentration and cell phenotype alters the expression of extracellular matrix genes in vocal fold fibroblasts.

The impact of RGD integrin binding-peptide concentration and cell phenotype on directing extracellular matrix (ECM) gene expression in vocal fold fibroblasts is little understood. Less is known about cell response to RGD concentration on a biomaterial when fibroblasts are in a scar-like environment compared to a healthy environment. We investigated the effects of varying RGD integrin-binding peptide surface concentration on ECM gene expression of elastin, collagen type 3 alpha 1, decorin, fibronectin, hyaluronan synthase 2, and collagen type 1 alpha 2 in scarred and unscarred immortalized human vocal fold fibroblasts (I-HVFFs). Phenotype and RGD concentration affected ECM gene expression. Phenotype change from healthy to myofibroblast-like resulted in ECM gene up-regulation for all genes tested, except for decorin. Systematically altering RGD concentration affected the expression of elastin and collagen type 3 alpha 1 in a myofibroblast phenotype. Specifically greater up-regulation in gene expression was observed with higher RGD concentrations. This research demonstrates that controlling RGD concentration may influence ECM gene expression levels in fibroblasts. Such knowledge is critical in developing the next generation of bioactive materials that, when implanted into sites of tissue damage and scarring, will direct cells to regenerate healthy tissues with normal ECM ratios and morphologies.

Quantifying the effects of altering ambient humidity on ionic composition of vocal fold surface fluid.

OBJECTIVES/HYPOTHESIS: Vocal fold surface fluid (VFSF) is important in hydration and defense of underlying epithelial cells. The objective of this study was to quantify changes in the ionic composition of VFSF after altering the humidity of inhaled air. We tested the hypothesis that low humidity exposure would increase the concentration of VFSF sodium (Na(+)) and chloride (Cl(-)) ions but that high humidity exposure would decrease the concentration of VFSF Na(+) and Cl(-) ions as compared to the low humidity challenge. STUDY DESIGN: Prospective design. METHODS: Eighteen healthy adults participated in this study. VFSF was collected from each subject at baseline and following exposure to low humidity and high humidity environments. VFSF Na(+) concentration was assessed using inductively coupled plasma mass spectrometry. VFSF Cl(-) concentration was measured with indirect potentiometry. All analyses were completed by personnel blinded to the hypothesis being tested. RESULTS: The low humidity environment increased Na(+) concentration in the majority of the subjects. Data for changes in Cl(-) concentrations were variable. Overall the data did not reach statistical significance (P > .05). Subjective impressions suggested that VFSF collection was more difficult in low humidity as compared to the high humidity and baseline conditions. CONCLUSIONS: This study is the first attempt to measure the ionic concentration of VFSF. The results from the current study have important implications for future programmatic research quantifying the effects of pollutants and laryngopharyngeal reflux on VFSF composition, epithelial hydration, and vocal fold defense.

Synthesis and characterization of a poly(lactic-co-glycolic acid) core + poly(N-isopropylacrylamide) shell nanoparticle system.

Poly(lactic-co-glycolic acid) (PLGA) is a popular material used to prepare nanoparticles for drug delivery. However, PLGA nanoparticles lack desirable attributes including active targeting abilities, resistance to aggregation during lyophilization, and the ability to respond to dynamic environmental stimuli. To overcome these issues, we fabricated a nanoparticle consisting of a PLGA core encapsulated within a shell of poly(N-isopropylacrylamide). Dynamic light scattering and transmission electron microscope imaging were used to characterize the nanoparticles, while an MTT assay and ELISA suggested biocompatibility in THP1 cells. Finally, a collagen type II binding assay showed successful modification of these nanoparticles with an active targeting moiety.

Chp1-Tas3 interaction is required to recruit RITS to fission yeast centromeres and for maintenance of centromeric heterochromatin.

The maintenance of centromeric heterochromatin in fission yeast relies on the RNA interference-dependent complexes RITS (RNA-induced transcriptional silencing complex) and RDRC (RNA-directed RNA polymerase complex), which cooperate in a positive feedback loop to recruit high levels of histone H3 K9 methyltransferase activity to centromeres and to promote the assembly and maintenance of centromeric heterochromatin. However, it is unclear how these complexes are targeted to chromatin. RITS comprises Chp1, which binds K9-methylated histone H3; Ago1, which binds short interfering (siRNAs); the adaptor protein Tas3, which links Ago1 to Chp1; and centromeric siRNAs. We have generated mutants in RITS to determine the contribution of the two potential chromatin-targeting proteins Chp1 and Ago1 to the centromeric recruitment of RITS. Mutations in Tas3 that disrupt Ago1 binding are permissive for RITS recruitment and maintain centromeric heterochromatin, but the role of Tas3's interaction with Chp1 is unknown. Here, we define the Chp1 interaction domain of Tas3. A strain expressing a tas3 mutant that cannot bind Chp1 (Tas3(Delta)(10-24)) failed to maintain centromeric heterochromatin, with a loss of centromeric siRNAs, a failure to recruit RITS and RDRC to centromeres, and high levels of chromosome loss. These findings suggest a pivotal role for Chp1 and its association with Tas3 for the recruitment of RITS, RDRC, and histone H3 K9 methyltransferase activity to centromeres.

Functional separation of the requirements for establishment and maintenance of centromeric heterochromatin.

The establishment and maintenance of centromeric heterochromatin in fission yeast require the RITS complex. Comprised of centromeric siRNAs, the chromodomain protein Chp1, Argonaute (Ago1), and Tas3, RITS couples the cellular RNAi pathway with assembly of constitutive heterochromatin. However, the mechanisms governing RITS-dependent establishment versus maintenance of centromeric heterochromatin remain unresolved. Here, we report that a mutant Tas3 protein that cannot bind Ago1 supports the maintenance of centromeric heterochromatin but cannot mediate efficient de novo establishment from cells transiently depleted for the histone H3 lysine 9 methyltransferase Clr4. In contrast, centromeric heterochromatin efficiently assembles in mutant cells transiently depleted for dicer. This mutant therefore allows ordering of the events leading to establishment of centromeric heterochromatin and places lysine 9 methylation of histone H3 upstream of dicer function.