The Laryngoscope C 2014 The American Laryngological, V
Rhinological and Otological Society, Inc.
Smad3: An Emerging Target for Vocal Fold Fibrosis Benjamin C. Paul, MD; Benjamin Y. Rafii, MD; Sonate Gandonu, BS; Renjie Bing, MD; Hayley Born, BS; Milan R. Amin, MD; Ryan C. Branski, PhD Objectives/Hypothesis: To determine the efficacy of small interfering RNA (siRNA) targeting Smad3 to mediate fibroplasia in vitro, to investigate the temporal regulation of Smad3 following vocal fold (VF) injury, and to determine the local and distal effects of Smad3 siRNA VF injection. Study Design: In vitro and in vivo. Methods: In vitro, Smad3 regulation was examined at both the level of transcription and translation in a human VF cell line in response to Smad3 siRNA 6 transforming growth factor b (TGF-b). Collagen transcription was also examined. In vivo, Smad3 messenger RNA (mRNA) expression was quantified as a function of time following rabbit VF injury. Also, the effects of injected Smad3 siRNA were assessed at local and distal sites. Results: Smad3 siRNA knocked down Smad3 transcription and translation and limited TGF-b-mediated collagen mRNA expression with minimal cytotoxicity in vitro. In vivo, Smad3 mRNA increased 1 day following VF injury and remained elevated through day 7. Smad3 siRNA injection into the uninjured vocal fold had no local or distant effect on Smad3 mRNA at multiple organ sites. Conclusions: These data provide a foundation for further investigation regarding the development of novel RNA-based therapeutics for the VF, specifically locally delivered siRNA for challenging fibrotic conditions of the VF. Key Words: Voice, vocal fold, dysphonia, siRNA, Smad3, fibrosis, dysphonia, scarring, inflammation. Level of Evidence: NA Laryngoscope, 124:2327–2331, 2014
INTRODUCTION Although insult to the vocal folds may be generated by varied mechanisms, architectural disturbances to the vibratory margin of the musculomembranous vocal folds are causative of dysphonia for many. In cases of discrete, benign vocal fold lesions, behavioral and/or surgical treatments have been shown to be efficacious at improving phonatory physiology. In contrast, the treatment of vocal fold scar remains suboptimal. The one exception may be localized steroid injections, which have been reported to show modest, unreliable success in limited case series.1–3 We posit that an ideal therapy for vocal From the Department of Otolaryngology, NYU Voice Center, New York University School of Medicine, New York, New York, U.S.A. Editor’s Note: This Manuscript was accepted for publication April 14, 2014. Portions of the data were presented at the 2013 American Bronchoesophageal Association/Combined Otolaryngology Spring Meetings, Orlando, Florida, U.S.A., April 10–14, 2013. This study was performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act (7 U.S.C. et seq.); the animal use protocol was approved by the Institutional Animal Care and Use Committee of New York University School of Medicine. This work was partially funded through the American Academy of Otolaryngology–Head and Neck Surgery Foundation Centralized Otolaryngology Research Efforts Grant (principal investigator: Benjamin C. Paul, MD). The authors have no other funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Ryan C. Branski, PhD, Associate Director, NYU Voice Center, 345 East 37th Street, Suite 306, New York, NY 10016. E-mail: [email protected] DOI: 10.1002/lary.24723
Laryngoscope 124: October 2014
fold fibrosis must have targeted and predictable control of both the inflammatory and subsequent fibroplastic phenotype. As such, we hypothesize that the transforming growth factor (TGF)-b pathway is an ideal target for therapies for vocal fold scar, as it is a regulator of wound healing and the development of fibrosis through its interactions with mesenchymal cells.4 TGF-b transcription has been previously shown to be upregulated as early as 8 hours following vocal fold injury.5 Upon ligand binding, TGF-b activates a unique pathway through the Smad family of proteins. Smad3 is a receptor-activated or pathway-restricted protein directly activated by TGF-b1 receptor ligands. Smad3, mapped to chromosome 15q22.33, heterodimerizes with Smad2 and translocates to the nucleus as a regulator of transcription. In vivo, Smad3-null mice are resistant to radiation induced cutaneous fibrosis, bleomycin-induced pulmonary fibrosis, carbon tetrachloride-induced hepatic fibrosis, and glomerular fibrosis.6–10 Based on this previous body of work, we hypothesized that RNA-based therapeutics targeting Smad3 are likely to yield favorable outcomes. In the skin, localized application of Smad3 small interfering RNA (siRNA) inhibited radiation-induced fibrosis.11 More globally, a recent review article described over 50 current clinical trials employing RNAbased therapeutics, 23 of which were funded by industry12; however, none involved diseases of the larynx. Specifically, siRNA has several characteristics that are ideal for localized delivery, including a temporary effect and direct targeting of aberrant gene activity while Paul et al.: Smad3 and Vocal Fold Fibrosis
2327
limiting off-target effects. In addition, siRNA is accompanied by a relatively low toxicity profile, high sequence specificity, and the ability to induce RNA interference at relatively low concentrations.13 Though Smad3 siRNA is postulated to provide directed, localized modulation of fibrosis, potential issues related to off-target effects and cellular stability must be addressed.13,14 Systemic inhibition of Smad3 may have profound morbidity. Animals deficient in Smad3 have been shown to have poor wound healing and increased risk of aortic aneurysms.15 Though a single dose of Smad3 siRNA injected into the larynx is unlikely to have the same impact as a chronic gene deficiency, we believe that limited localized therapy is a critical approach to minimize systemic toxicity. In the current investigation, we sought to obtain several critical pieces of information to assess the potential of siRNA as a relevant therapeutic in the larynx to safely manipulate wound healing in the vocal folds. Specifically, we hypothesized that Smad3 would be upregulated in the vocal folds following injury. With regard to siRNA, we hypothesize that Smad3 siRNA would limit the fibrotic phenotype mediated by TGF-b in vitro, with limited cytotoxicity, and in vivo, localized injection of Smad3 siRNA to the vocal folds would result in no alterations in Smad3 messenger RNA (mRNA) expression in relevant distal and proximal tissue. Data obtained in this study provide the fundamental building blocks to consider a novel therapeutic agent to prevent, modulate, and/or treat vocal fold scarring. Such treatment has broad clinical applications.
buffered saline. For both tissue and cells, RNA extraction was performed using the RNeasy Kit (Qiagen Inc., Valencia, CA) following the manufacturer’s protocol. RNA was quantified using the NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, DE) according to the manufacturer’s protocol. Quantitative Reverse Transcriptase-Polymerase Chain Reaction. The Taqman RNA-to-Ct 1-Step kit (Applied Biosystems, Grand Island, NY) was used to perform quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The primers and probes for the Smad3, Collagen IA1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were acquired in the form of Taqman gene expression assays (Applied Biosystems). Quantitative RT-PCR (qRT-PCR) was run on the Applied Biosystems ViiA 7 Real-Time PCR System, as recommended by the manufacturer. All data were normalized to GAPDH expression, which was used as an endogenous control. Expression levels were presented as fold expression and were calculated using the formula 2^(ddCt). Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western blotting. Protein was extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run using 8% loading and 5% stacking gels. Antibody dilutions were as follows: b-actin (1:1000) and Smad3 (1:1000). Primary antibody incubation was performed overnight; secondary antibody (1:2000, anti-rabbit immunoglobulin G, horseradish peroxidase-linked antibody) was incubated for 1 hour at room temperature. All antibodies were purchased from Cell Signaling (Danvers, MA). Gels were examined qualitatively. Statistical analysis. All experiments were performed in triplicate, at least. For each outcome, a one-way analysis of variance (ANOVA) was employed (P
Rhinological and Otological Society, Inc.
Smad3: An Emerging Target for Vocal Fold Fibrosis Benjamin C. Paul, MD; Benjamin Y. Rafii, MD; Sonate Gandonu, BS; Renjie Bing, MD; Hayley Born, BS; Milan R. Amin, MD; Ryan C. Branski, PhD Objectives/Hypothesis: To determine the efficacy of small interfering RNA (siRNA) targeting Smad3 to mediate fibroplasia in vitro, to investigate the temporal regulation of Smad3 following vocal fold (VF) injury, and to determine the local and distal effects of Smad3 siRNA VF injection. Study Design: In vitro and in vivo. Methods: In vitro, Smad3 regulation was examined at both the level of transcription and translation in a human VF cell line in response to Smad3 siRNA 6 transforming growth factor b (TGF-b). Collagen transcription was also examined. In vivo, Smad3 messenger RNA (mRNA) expression was quantified as a function of time following rabbit VF injury. Also, the effects of injected Smad3 siRNA were assessed at local and distal sites. Results: Smad3 siRNA knocked down Smad3 transcription and translation and limited TGF-b-mediated collagen mRNA expression with minimal cytotoxicity in vitro. In vivo, Smad3 mRNA increased 1 day following VF injury and remained elevated through day 7. Smad3 siRNA injection into the uninjured vocal fold had no local or distant effect on Smad3 mRNA at multiple organ sites. Conclusions: These data provide a foundation for further investigation regarding the development of novel RNA-based therapeutics for the VF, specifically locally delivered siRNA for challenging fibrotic conditions of the VF. Key Words: Voice, vocal fold, dysphonia, siRNA, Smad3, fibrosis, dysphonia, scarring, inflammation. Level of Evidence: NA Laryngoscope, 124:2327–2331, 2014
INTRODUCTION Although insult to the vocal folds may be generated by varied mechanisms, architectural disturbances to the vibratory margin of the musculomembranous vocal folds are causative of dysphonia for many. In cases of discrete, benign vocal fold lesions, behavioral and/or surgical treatments have been shown to be efficacious at improving phonatory physiology. In contrast, the treatment of vocal fold scar remains suboptimal. The one exception may be localized steroid injections, which have been reported to show modest, unreliable success in limited case series.1–3 We posit that an ideal therapy for vocal From the Department of Otolaryngology, NYU Voice Center, New York University School of Medicine, New York, New York, U.S.A. Editor’s Note: This Manuscript was accepted for publication April 14, 2014. Portions of the data were presented at the 2013 American Bronchoesophageal Association/Combined Otolaryngology Spring Meetings, Orlando, Florida, U.S.A., April 10–14, 2013. This study was performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act (7 U.S.C. et seq.); the animal use protocol was approved by the Institutional Animal Care and Use Committee of New York University School of Medicine. This work was partially funded through the American Academy of Otolaryngology–Head and Neck Surgery Foundation Centralized Otolaryngology Research Efforts Grant (principal investigator: Benjamin C. Paul, MD). The authors have no other funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Ryan C. Branski, PhD, Associate Director, NYU Voice Center, 345 East 37th Street, Suite 306, New York, NY 10016. E-mail: [email protected] DOI: 10.1002/lary.24723
Laryngoscope 124: October 2014
fold fibrosis must have targeted and predictable control of both the inflammatory and subsequent fibroplastic phenotype. As such, we hypothesize that the transforming growth factor (TGF)-b pathway is an ideal target for therapies for vocal fold scar, as it is a regulator of wound healing and the development of fibrosis through its interactions with mesenchymal cells.4 TGF-b transcription has been previously shown to be upregulated as early as 8 hours following vocal fold injury.5 Upon ligand binding, TGF-b activates a unique pathway through the Smad family of proteins. Smad3 is a receptor-activated or pathway-restricted protein directly activated by TGF-b1 receptor ligands. Smad3, mapped to chromosome 15q22.33, heterodimerizes with Smad2 and translocates to the nucleus as a regulator of transcription. In vivo, Smad3-null mice are resistant to radiation induced cutaneous fibrosis, bleomycin-induced pulmonary fibrosis, carbon tetrachloride-induced hepatic fibrosis, and glomerular fibrosis.6–10 Based on this previous body of work, we hypothesized that RNA-based therapeutics targeting Smad3 are likely to yield favorable outcomes. In the skin, localized application of Smad3 small interfering RNA (siRNA) inhibited radiation-induced fibrosis.11 More globally, a recent review article described over 50 current clinical trials employing RNAbased therapeutics, 23 of which were funded by industry12; however, none involved diseases of the larynx. Specifically, siRNA has several characteristics that are ideal for localized delivery, including a temporary effect and direct targeting of aberrant gene activity while Paul et al.: Smad3 and Vocal Fold Fibrosis
2327
limiting off-target effects. In addition, siRNA is accompanied by a relatively low toxicity profile, high sequence specificity, and the ability to induce RNA interference at relatively low concentrations.13 Though Smad3 siRNA is postulated to provide directed, localized modulation of fibrosis, potential issues related to off-target effects and cellular stability must be addressed.13,14 Systemic inhibition of Smad3 may have profound morbidity. Animals deficient in Smad3 have been shown to have poor wound healing and increased risk of aortic aneurysms.15 Though a single dose of Smad3 siRNA injected into the larynx is unlikely to have the same impact as a chronic gene deficiency, we believe that limited localized therapy is a critical approach to minimize systemic toxicity. In the current investigation, we sought to obtain several critical pieces of information to assess the potential of siRNA as a relevant therapeutic in the larynx to safely manipulate wound healing in the vocal folds. Specifically, we hypothesized that Smad3 would be upregulated in the vocal folds following injury. With regard to siRNA, we hypothesize that Smad3 siRNA would limit the fibrotic phenotype mediated by TGF-b in vitro, with limited cytotoxicity, and in vivo, localized injection of Smad3 siRNA to the vocal folds would result in no alterations in Smad3 messenger RNA (mRNA) expression in relevant distal and proximal tissue. Data obtained in this study provide the fundamental building blocks to consider a novel therapeutic agent to prevent, modulate, and/or treat vocal fold scarring. Such treatment has broad clinical applications.
buffered saline. For both tissue and cells, RNA extraction was performed using the RNeasy Kit (Qiagen Inc., Valencia, CA) following the manufacturer’s protocol. RNA was quantified using the NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, DE) according to the manufacturer’s protocol. Quantitative Reverse Transcriptase-Polymerase Chain Reaction. The Taqman RNA-to-Ct 1-Step kit (Applied Biosystems, Grand Island, NY) was used to perform quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The primers and probes for the Smad3, Collagen IA1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were acquired in the form of Taqman gene expression assays (Applied Biosystems). Quantitative RT-PCR (qRT-PCR) was run on the Applied Biosystems ViiA 7 Real-Time PCR System, as recommended by the manufacturer. All data were normalized to GAPDH expression, which was used as an endogenous control. Expression levels were presented as fold expression and were calculated using the formula 2^(ddCt). Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western blotting. Protein was extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run using 8% loading and 5% stacking gels. Antibody dilutions were as follows: b-actin (1:1000) and Smad3 (1:1000). Primary antibody incubation was performed overnight; secondary antibody (1:2000, anti-rabbit immunoglobulin G, horseradish peroxidase-linked antibody) was incubated for 1 hour at room temperature. All antibodies were purchased from Cell Signaling (Danvers, MA). Gels were examined qualitatively. Statistical analysis. All experiments were performed in triplicate, at least. For each outcome, a one-way analysis of variance (ANOVA) was employed (P
