- Email: [email protected]
08:50: Differential Expression of the SKI Oncogene in Hemangiomas
P96
Otolaryngology-Head and Neck Surgery, Vol 137, No 2S, August 2007
is continually responding to potentially injurious mechanical forces. The role of these forces on turnover of the ECM is poorly understood. The authors’ laboratory has been using a rabbit model to study phonation-related molecular alterations in vivo. In this experiment, the effects of three hours of experimental induced phonation on mRNA expression of matrix metalloproteinase (MMP)-1, -9, and IL-1 beta, were studied. METHODS: An in vivo rabbit model was used to study the effect of three hours of experimental induced phonation (77 ⫹/- 3 dB; 429 ⫹/-141 Hz) on mRNA expression of the normal vocal fold. The mRNA expression of MMP-1, MMP-9, and IL-1 beta was measured using real time reverse transcribed polymerase chain reaction (RT-PCR) from phonated (n⫽10) and nonphonated control vocal folds (n⫽10). RESULTS: Independent samples t-Tests were used to compare means. Alpha was adjusted to account for multiple tests (alpha ⫽ 0.10/3). Phonation resulted in upregulated gene ex-
pression of MMP-1 (p⫽0.030) and MMP-9 (p⫽0.029) in phonated control folds. IL-1 beta expression was also elevated in the phonation group, but not significantly (p⫽0.053). CONCLUSION: Degradation of connective tissue is controlled by MMPs and tissue inhibitors of MMPs. MMP levels are usually low in normal tissue. However, expression is elevated when the system is disturbed. Results from this study showed increased MMP-1 and MMP-9 gene expression following three hours of experimental induced phonation in normal vocal fold. Increased MMP expression may provide evidence of mild vocal fold injury associated with phonation. SIGNIFICANCE: Phonotrauma has been implicated in the development of vocal pathology. Normal daily voice use may also contribute to injury. The study of cellular responses to experimental induced phonation may provide clues on how overuse, misuse, and normal daily use contribute to vocal pathology. SUPPORT: NIH-NIDCD R03 DC008400
8:40 AM to 9:20 AM RESEARCH FORUM WCC 208AB
CONCLUSION: Vascularity does not appear to be different between groups of tonsils removed for infections or obstructive symptoms. SIGNIFICANCE: Diagnosis of obstructing versus infected tonsils does not seem to determine tonsil bed vascularity. Age may be a factor in increased vascularity, and possibly in increased post-tonsillectomy hemorrhage.
䡲
Research Forum: Pediatrics Moderators: David L. Mandell, MD; Tulio Alberto Valdez, MD
08:50
Vascularity in Obstructing vs. Infected Tonsils Justin Gull, MD (presenter); James Batti, MD; Fabiola Balarezo, MD
Differential Expression of the SKI Oncogene in Hemangiomas Teresa Min-Jung O, MD (presenter); Mark Tarango AA; Yupo Ma MD, PhD; Melin Tan, MD; Milton Waner, MD
PROBLEM: Chronic tonsillitis appears to be a risk factor for post-tonsillectomy hemorrhage. Increased vascularity in the tonsil bed may be the underlying pathology. The objective is to compare the vascularity of tonsils removed for obstructive symptoms versus recurrent infections. METHODS: A total of 75 tonsils were examined prospectively at an academic pediatric hospital. A single pathologist examined them. Vessels per high power field (v/hpf) were reported for each tonsil and the data were analyzed according to diagnosis (hypertrophy vs. recurrent infection). RESULTS: The mean vascularity for the recurrent infection group was 11.3 v/hpf, while that for the hypertrophy group was 11.6 v/hpf. Average, maximum and total vascularity showed no significant difference between diagnoses (p⫽0.40, 0.38, 0.52, respectively). No significant difference in vascularity was noted for sex (p⫽0.30). The ages of patients was different, with the average in the hypertrophy group being 5.0 years old, and 7.6 years old in the infections group (p⬍0.001).
PROBLEM: The pathogenesis for benign tumorigenesis in juvenile hemangiomas is unknown. Oncogene proteins may be influential in this process. SKI proteins have been previously described in melanomas, esophageal and breast cancers. This study investigated the differential expression of the SKI (sarcoma viral oncogene) protein in hemangioma tissues. METHODS: Paraffin-embedded tissue specimens of surgical hemangioma tissues was obtained from the senior author at a tertiary care hospital from 2005-2006. The first vascular tissue array was created in order to eliminate slide-to-slide variability; it was composed of 12 hemangioma specimens at various stages of growth and anatomic location. Two cores were taken from each sample and placed in varying locations on the array. Control tissues were included in the same array. The block was processed and immunohistochemical studies were performed using the SKI antibodies CD31, and Ki67. RESULTS: All 12 hemangioma tissues overexpressed the SKI protein. The staining pattern was perinuclear within the endo-
08:40
thelial cells only. The intensity of staining was inversely proportional to the growth stage: immature or proliferating tissues showed a higher concentration of SKI, whereas, mature lesions stained less intensely. The endothelial cells which were SKIpositive were involved in active cell division. CONCLUSION: The SKI oncogene protein is differentially and specifically expressed in hemangioma tissues. SKI acts as a transcriptional co-repressor and inhibits the TGF-b pathway, thus leading to uncontrolled cellular proliferation and transformation. All controls were negative for SKI staining. SIGNIFICANCE: The SKI oncogene protein is a new hemangioma-specific marker and is involved in hemangioma tumorigenesis. SUPPORT: Vascular Birthmark Foundation.
09:00 RNA Interference Knockdown of TGF-B2 in Human Fibroblasts Andrew R. Gilbert, MD (presenter); Jon M. Robitschek, MD; Benjamin B Cable, MD PROBLEM: 1. Introduce RNA Interference (RNAi) technology as a viable means of targeted gene silencing in cultured human respiratory fibroblasts. 2. Demonstrate transfection of cultured human respiratory fibroblasts with RNAi constructs utilizing microsomal lipids. METHODS: This was a nonrandomized, controlled, in vitro study performed from June through December 2006. RNA Interference (RNAi) constructs targeted to the TGF-B2 transcript were encapsulated in microsomal lipids and applied to human respiratory fibroblasts in cell culture. Transfection efficiency and cell viability were measured by fluorescence microscopy. Messenger RNA for TGF-B2 was measured 48hrs post-transfection using real time quantitative PCR. RESULTS: Transfection efficiency of over 80% was achieved with minimal induced cell death. Treated cells showed a selective knockdown of 80% of TGF-B2 mRNA, which was confirmed with negative controls. CONCLUSION: RNA Interference technology is an effective means of localized and transient gene silencing in cultured human fibroblasts. Transfection can be achieved using microsome encapsulated RNAi with minimal induced cell death. SIGNIFICANCE: This preliminary result shows great promise for future in vivo investigation utilizing topical RNAi therapy as an adjunct to surgical repair, reducing scarring in respiratory tissues after surgical or intubation trauma.
09:10 The Role of VEGF in Isolated Cleft Palate Sarah L Rohde, MD (presenter); Steven L Goudy, MD PROBLEM: Although the role of vascular endothelial growth factor (VEGF) in the early stages of vascular assembly has
P97 been studied extensively, its role in vascular remodeling and patterning remains unknown. It has recently been suggested that VEGF is a modifier of birth defects in del22q11 (DiGeorge) syndrome. A selected deletion of the VEGF gene in Wnt1 cells creates a facial phenotype consisting of an isolated cleft palate in mice. Using Wnt1 Cre VEGF mice, the role of VEGF in vascular patterning of the palate will be examined. METHODS: Examination of VEGF expression in the wildtype mouse was examined using in situ hybridization. Wnt1 Cre mice were mated with mice carrying loxP sites within the VEGF gene site to create mice lacking expression of VEGF in neural crest cells expressing Wnt1. Using PECAM immunohistochemical staining, vascular patterning throughout palate development was examined in wild-type and Wnt1 Cre f/f mice. RESULTS: VEGF is widely expressed in the developing palate. Normal vascular development in the palate shows development of large vessels lateral to the palate, and a fine network of vascular channels leading up to the edge of the palate fusion plane. In Wnt1 Cre VEGF f/f mice there is decreased small vessel development along the developing palate shelves, but maintenance of the architecture of the large central vessel. CONCLUSION: Vascular patterning through VEGF signaling appears to play a crucial role in palate development, the absence of which leads to cleft palate. SIGNIFICANCE: This study is the first demonstration of altered vascular development in an animal model of cleft palate. Maintenance of the central large vessel is clinically relevant as cleft palates show normal development of the greater palatine arteries, but it is clear that the fine network of vascular channels is necessary for normal palate development.
09:20 Branchio-Oto-Renal Syndrome: A Translational Research Model Amit Kochhar, BS (presenter); Jessica Lynn Sorensen, BS; Tao Yang; Dana Jo Orten, PhD; William J Kimberling, PhD; Richard J H Smith, MD PROBLEM: Investigation into Branchio-Oto-Renal (BOR) syndrome (OMIM No: 113650) provides a powerful model for translational research. BOR syndrome, an autosomal dominant form of syndromic hearing impairment, is phenotypically characterized by hearing loss, malformations of the pinnae, the presence of branchial arch remnants, and various renal abnormalities. Mutations in two interacting genes, EYA1 and SIX1, are known to be associated with this phenotype in approximately 40% of affected individuals. Providing genetic screening of these genes offers improved healthcare to affected individuals and their families. However, in over
TUESDAY
Research Forum—Tuesday
Otolaryngology-Head and Neck Surgery, Vol 137, No 2S, August 2007
is continually responding to potentially injurious mechanical forces. The role of these forces on turnover of the ECM is poorly understood. The authors’ laboratory has been using a rabbit model to study phonation-related molecular alterations in vivo. In this experiment, the effects of three hours of experimental induced phonation on mRNA expression of matrix metalloproteinase (MMP)-1, -9, and IL-1 beta, were studied. METHODS: An in vivo rabbit model was used to study the effect of three hours of experimental induced phonation (77 ⫹/- 3 dB; 429 ⫹/-141 Hz) on mRNA expression of the normal vocal fold. The mRNA expression of MMP-1, MMP-9, and IL-1 beta was measured using real time reverse transcribed polymerase chain reaction (RT-PCR) from phonated (n⫽10) and nonphonated control vocal folds (n⫽10). RESULTS: Independent samples t-Tests were used to compare means. Alpha was adjusted to account for multiple tests (alpha ⫽ 0.10/3). Phonation resulted in upregulated gene ex-
pression of MMP-1 (p⫽0.030) and MMP-9 (p⫽0.029) in phonated control folds. IL-1 beta expression was also elevated in the phonation group, but not significantly (p⫽0.053). CONCLUSION: Degradation of connective tissue is controlled by MMPs and tissue inhibitors of MMPs. MMP levels are usually low in normal tissue. However, expression is elevated when the system is disturbed. Results from this study showed increased MMP-1 and MMP-9 gene expression following three hours of experimental induced phonation in normal vocal fold. Increased MMP expression may provide evidence of mild vocal fold injury associated with phonation. SIGNIFICANCE: Phonotrauma has been implicated in the development of vocal pathology. Normal daily voice use may also contribute to injury. The study of cellular responses to experimental induced phonation may provide clues on how overuse, misuse, and normal daily use contribute to vocal pathology. SUPPORT: NIH-NIDCD R03 DC008400
8:40 AM to 9:20 AM RESEARCH FORUM WCC 208AB
CONCLUSION: Vascularity does not appear to be different between groups of tonsils removed for infections or obstructive symptoms. SIGNIFICANCE: Diagnosis of obstructing versus infected tonsils does not seem to determine tonsil bed vascularity. Age may be a factor in increased vascularity, and possibly in increased post-tonsillectomy hemorrhage.
䡲
Research Forum: Pediatrics Moderators: David L. Mandell, MD; Tulio Alberto Valdez, MD
08:50
Vascularity in Obstructing vs. Infected Tonsils Justin Gull, MD (presenter); James Batti, MD; Fabiola Balarezo, MD
Differential Expression of the SKI Oncogene in Hemangiomas Teresa Min-Jung O, MD (presenter); Mark Tarango AA; Yupo Ma MD, PhD; Melin Tan, MD; Milton Waner, MD
PROBLEM: Chronic tonsillitis appears to be a risk factor for post-tonsillectomy hemorrhage. Increased vascularity in the tonsil bed may be the underlying pathology. The objective is to compare the vascularity of tonsils removed for obstructive symptoms versus recurrent infections. METHODS: A total of 75 tonsils were examined prospectively at an academic pediatric hospital. A single pathologist examined them. Vessels per high power field (v/hpf) were reported for each tonsil and the data were analyzed according to diagnosis (hypertrophy vs. recurrent infection). RESULTS: The mean vascularity for the recurrent infection group was 11.3 v/hpf, while that for the hypertrophy group was 11.6 v/hpf. Average, maximum and total vascularity showed no significant difference between diagnoses (p⫽0.40, 0.38, 0.52, respectively). No significant difference in vascularity was noted for sex (p⫽0.30). The ages of patients was different, with the average in the hypertrophy group being 5.0 years old, and 7.6 years old in the infections group (p⬍0.001).
PROBLEM: The pathogenesis for benign tumorigenesis in juvenile hemangiomas is unknown. Oncogene proteins may be influential in this process. SKI proteins have been previously described in melanomas, esophageal and breast cancers. This study investigated the differential expression of the SKI (sarcoma viral oncogene) protein in hemangioma tissues. METHODS: Paraffin-embedded tissue specimens of surgical hemangioma tissues was obtained from the senior author at a tertiary care hospital from 2005-2006. The first vascular tissue array was created in order to eliminate slide-to-slide variability; it was composed of 12 hemangioma specimens at various stages of growth and anatomic location. Two cores were taken from each sample and placed in varying locations on the array. Control tissues were included in the same array. The block was processed and immunohistochemical studies were performed using the SKI antibodies CD31, and Ki67. RESULTS: All 12 hemangioma tissues overexpressed the SKI protein. The staining pattern was perinuclear within the endo-
08:40
thelial cells only. The intensity of staining was inversely proportional to the growth stage: immature or proliferating tissues showed a higher concentration of SKI, whereas, mature lesions stained less intensely. The endothelial cells which were SKIpositive were involved in active cell division. CONCLUSION: The SKI oncogene protein is differentially and specifically expressed in hemangioma tissues. SKI acts as a transcriptional co-repressor and inhibits the TGF-b pathway, thus leading to uncontrolled cellular proliferation and transformation. All controls were negative for SKI staining. SIGNIFICANCE: The SKI oncogene protein is a new hemangioma-specific marker and is involved in hemangioma tumorigenesis. SUPPORT: Vascular Birthmark Foundation.
09:00 RNA Interference Knockdown of TGF-B2 in Human Fibroblasts Andrew R. Gilbert, MD (presenter); Jon M. Robitschek, MD; Benjamin B Cable, MD PROBLEM: 1. Introduce RNA Interference (RNAi) technology as a viable means of targeted gene silencing in cultured human respiratory fibroblasts. 2. Demonstrate transfection of cultured human respiratory fibroblasts with RNAi constructs utilizing microsomal lipids. METHODS: This was a nonrandomized, controlled, in vitro study performed from June through December 2006. RNA Interference (RNAi) constructs targeted to the TGF-B2 transcript were encapsulated in microsomal lipids and applied to human respiratory fibroblasts in cell culture. Transfection efficiency and cell viability were measured by fluorescence microscopy. Messenger RNA for TGF-B2 was measured 48hrs post-transfection using real time quantitative PCR. RESULTS: Transfection efficiency of over 80% was achieved with minimal induced cell death. Treated cells showed a selective knockdown of 80% of TGF-B2 mRNA, which was confirmed with negative controls. CONCLUSION: RNA Interference technology is an effective means of localized and transient gene silencing in cultured human fibroblasts. Transfection can be achieved using microsome encapsulated RNAi with minimal induced cell death. SIGNIFICANCE: This preliminary result shows great promise for future in vivo investigation utilizing topical RNAi therapy as an adjunct to surgical repair, reducing scarring in respiratory tissues after surgical or intubation trauma.
09:10 The Role of VEGF in Isolated Cleft Palate Sarah L Rohde, MD (presenter); Steven L Goudy, MD PROBLEM: Although the role of vascular endothelial growth factor (VEGF) in the early stages of vascular assembly has
P97 been studied extensively, its role in vascular remodeling and patterning remains unknown. It has recently been suggested that VEGF is a modifier of birth defects in del22q11 (DiGeorge) syndrome. A selected deletion of the VEGF gene in Wnt1 cells creates a facial phenotype consisting of an isolated cleft palate in mice. Using Wnt1 Cre VEGF mice, the role of VEGF in vascular patterning of the palate will be examined. METHODS: Examination of VEGF expression in the wildtype mouse was examined using in situ hybridization. Wnt1 Cre mice were mated with mice carrying loxP sites within the VEGF gene site to create mice lacking expression of VEGF in neural crest cells expressing Wnt1. Using PECAM immunohistochemical staining, vascular patterning throughout palate development was examined in wild-type and Wnt1 Cre f/f mice. RESULTS: VEGF is widely expressed in the developing palate. Normal vascular development in the palate shows development of large vessels lateral to the palate, and a fine network of vascular channels leading up to the edge of the palate fusion plane. In Wnt1 Cre VEGF f/f mice there is decreased small vessel development along the developing palate shelves, but maintenance of the architecture of the large central vessel. CONCLUSION: Vascular patterning through VEGF signaling appears to play a crucial role in palate development, the absence of which leads to cleft palate. SIGNIFICANCE: This study is the first demonstration of altered vascular development in an animal model of cleft palate. Maintenance of the central large vessel is clinically relevant as cleft palates show normal development of the greater palatine arteries, but it is clear that the fine network of vascular channels is necessary for normal palate development.
09:20 Branchio-Oto-Renal Syndrome: A Translational Research Model Amit Kochhar, BS (presenter); Jessica Lynn Sorensen, BS; Tao Yang; Dana Jo Orten, PhD; William J Kimberling, PhD; Richard J H Smith, MD PROBLEM: Investigation into Branchio-Oto-Renal (BOR) syndrome (OMIM No: 113650) provides a powerful model for translational research. BOR syndrome, an autosomal dominant form of syndromic hearing impairment, is phenotypically characterized by hearing loss, malformations of the pinnae, the presence of branchial arch remnants, and various renal abnormalities. Mutations in two interacting genes, EYA1 and SIX1, are known to be associated with this phenotype in approximately 40% of affected individuals. Providing genetic screening of these genes offers improved healthcare to affected individuals and their families. However, in over
TUESDAY
Research Forum—Tuesday