- Email: [email protected]
BMP antagonism via noggin in adult dura mater
Vol. 203, No. 3S, September 2006
tors. We believe aberrant responses to cellular stresses including mechanical stress is a significant part of keloid pathobiology. Future work is directed at elucidating the molecular mechanisms for this enhanced stress response.
BMP antagonism via noggin in adult dura mater Matthew D Kwan MD, Derrick Wan MD, Michael Longaker MD, MBA, FACS Stanford University School of Medicine, Stanford, CA INTRODUCTION: Elucidating mechanisms that underlie the unique ability of young animals to heal calvarial defects is important for developing alternative approaches to repairing criticalsized calvarial defects. One such mechanism may be the balance between bone morphogenetic proteins (BMP) and its antagonist, Noggin, in dura mater. Dura mater, especially from juvenile animals, provides strong paracrine signals directing osteogenesis and may also be a source of new bone. We hypothesize that the regenerative potential of juvenile animals may be due in part to decreased BMP antagonism, in the form of Noggin, within the dura mater. METHODS: Dura mater from non-suture associated regions of the parietal bone was harvested from young (6 days old;n⫽10) and adult (60 days old;n⫽5) mice and immediately snap frozen. RNA extraction was performed using the Trizol method. RNA was subjected to reverse transcription and subsequent quantitative polymerase chain reaction (QRT-PCR), to measure expression levels of Noggin and BMP-2. RESULTS: Consistent with previous microarray data, QRT-PCR revealed a 2.5 ⫾0.2 (p⬍0.05) fold increase in Noggin expression in dura mater from adult mice as compared to young mice. BMP-2 expression in young dura mater was also 4.1 ⫾0.3 (p⬍0.05) fold higher than adults, by QRT-PCR. CONCLUSIONS: These data confirm that Noggin is elevated in adult dura mater, providing a possible explanation for their decreased ability to reossify calvarial defects. These results facilitate further studies on the suppression of Noggin, via RNA interference, in adult dura mater with subsequent evaluation of its ability for osteogenic differentiation.
Effects of circulating factors in diabetic wound healing Giorgio Pietramaggiori MD, Saja Scherer MD, Michael Alperovich Bs, Amy Wagers PhD, Dennis Orgill MD, PhD Brigham and Women’s Hospital, Harvard Medical School, Boston, MA INTRODUCTION: Fifteen percent of diabetics suffer from foot ulcers – chronic, non-healing wounds whose treatment is often unsuccessful, necessitating lower extremity amputation. The mechanisms by which wound healing is impaired in diabetics remain unclear, and we have hypothesized that blood circulating factors can influence the rate of diabetic wound healing.
Plastic Surgery I
S55
METHODS: Db/db mice were joined surgically to wild-type control mice, such that the two develop a shared blood circulation, with rapid exchange of blood-borne factors across the vascular junction. Pairs consisting of two wild type or two diabetic mice were also created as controls. Both animals in the couples were wounded on the dorsal region with a 0.8 cm2 dermal punch. The kinetics of cutaneous wound healing of the dorsal and the joining wound were assessed on in these pairs by several criteria, including wound closure rate, tensile strength measurement, and immunohistochemistry. RESULTS: Diabetic mice joined with wild type showed faster rates of wound closure, compared to control couples consisting of two diabetic mice. The joining wound between a diabetic and a wild type animal healed without complications in 60% of the cases, while wounds between two diabetic animals healed only in 12% of the cases. CONCLUSIONS: Circulating cells or factors provided by the wildtype mouse may have functioned to either directly promote wound closure, or to dilute or inhibit negative regulators of wound repair in diabetic animals. This study established a novel model to test the mechanisms of diabetic wound healing possibly leading to new treatment targets for diabetic ulcers.
In-vivo gene silencing using topical delivery of siRNA Vishal Thanik MD, Matthew Greives BA, Natalie Seiser PhD, Oren Lerman MD, Alexes Hazen MD, Jamie Levine MD, Pierre Saadeh MD New York University School of Medicine, New York, NY INTRODUCTION: The ability to effect gene alteration via topical therapy has protean implications in the potential treatment of conditions ranging from tumors to wound healing. We created a technique of RNA interference to modulate gene expression in-vivo. Specifically, a novel use of small interfering RNA (siRNA) was developed to knockdown several ubiquitous housekeeping genes (MAPK-1 and lamin A/C) of a wound bed. METHODS: Mice were wounded using our described model of excisional wound healing. MAPK-1/lamin siRNA and transfection reagent were delivered to the wounds via an agarose gel suspension. The treatment protocol was optimized and wounds were harvested at varying time points. MAPK-1/lamin protein expression was evaluated (immunohistochemistry and Western blotting). MAPK-1/ lamin gene expression was evaluated (in-situ hybridization). RESULTS: Immunohistochemistry of treated wounds revealed almost complete knockdown of MAPK-1/lamin versus controls. Additionally, the tissue directly beyond the treatment area revealed normal MAPK-1/lamin staining. Western blot analysis similarly showed marked decrease of MAPK-1/lamin in treated wounds. Gene expression paralleled protein findings. At later time points, the effect waned with a return of MAPK-1/lamin to baseline. CONCLUSIONS: We demonstrated effective gene silencing via topical delivery of siRNA in-vivo. Advantages of this powerful new
tors. We believe aberrant responses to cellular stresses including mechanical stress is a significant part of keloid pathobiology. Future work is directed at elucidating the molecular mechanisms for this enhanced stress response.
BMP antagonism via noggin in adult dura mater Matthew D Kwan MD, Derrick Wan MD, Michael Longaker MD, MBA, FACS Stanford University School of Medicine, Stanford, CA INTRODUCTION: Elucidating mechanisms that underlie the unique ability of young animals to heal calvarial defects is important for developing alternative approaches to repairing criticalsized calvarial defects. One such mechanism may be the balance between bone morphogenetic proteins (BMP) and its antagonist, Noggin, in dura mater. Dura mater, especially from juvenile animals, provides strong paracrine signals directing osteogenesis and may also be a source of new bone. We hypothesize that the regenerative potential of juvenile animals may be due in part to decreased BMP antagonism, in the form of Noggin, within the dura mater. METHODS: Dura mater from non-suture associated regions of the parietal bone was harvested from young (6 days old;n⫽10) and adult (60 days old;n⫽5) mice and immediately snap frozen. RNA extraction was performed using the Trizol method. RNA was subjected to reverse transcription and subsequent quantitative polymerase chain reaction (QRT-PCR), to measure expression levels of Noggin and BMP-2. RESULTS: Consistent with previous microarray data, QRT-PCR revealed a 2.5 ⫾0.2 (p⬍0.05) fold increase in Noggin expression in dura mater from adult mice as compared to young mice. BMP-2 expression in young dura mater was also 4.1 ⫾0.3 (p⬍0.05) fold higher than adults, by QRT-PCR. CONCLUSIONS: These data confirm that Noggin is elevated in adult dura mater, providing a possible explanation for their decreased ability to reossify calvarial defects. These results facilitate further studies on the suppression of Noggin, via RNA interference, in adult dura mater with subsequent evaluation of its ability for osteogenic differentiation.
Effects of circulating factors in diabetic wound healing Giorgio Pietramaggiori MD, Saja Scherer MD, Michael Alperovich Bs, Amy Wagers PhD, Dennis Orgill MD, PhD Brigham and Women’s Hospital, Harvard Medical School, Boston, MA INTRODUCTION: Fifteen percent of diabetics suffer from foot ulcers – chronic, non-healing wounds whose treatment is often unsuccessful, necessitating lower extremity amputation. The mechanisms by which wound healing is impaired in diabetics remain unclear, and we have hypothesized that blood circulating factors can influence the rate of diabetic wound healing.
Plastic Surgery I
S55
METHODS: Db/db mice were joined surgically to wild-type control mice, such that the two develop a shared blood circulation, with rapid exchange of blood-borne factors across the vascular junction. Pairs consisting of two wild type or two diabetic mice were also created as controls. Both animals in the couples were wounded on the dorsal region with a 0.8 cm2 dermal punch. The kinetics of cutaneous wound healing of the dorsal and the joining wound were assessed on in these pairs by several criteria, including wound closure rate, tensile strength measurement, and immunohistochemistry. RESULTS: Diabetic mice joined with wild type showed faster rates of wound closure, compared to control couples consisting of two diabetic mice. The joining wound between a diabetic and a wild type animal healed without complications in 60% of the cases, while wounds between two diabetic animals healed only in 12% of the cases. CONCLUSIONS: Circulating cells or factors provided by the wildtype mouse may have functioned to either directly promote wound closure, or to dilute or inhibit negative regulators of wound repair in diabetic animals. This study established a novel model to test the mechanisms of diabetic wound healing possibly leading to new treatment targets for diabetic ulcers.
In-vivo gene silencing using topical delivery of siRNA Vishal Thanik MD, Matthew Greives BA, Natalie Seiser PhD, Oren Lerman MD, Alexes Hazen MD, Jamie Levine MD, Pierre Saadeh MD New York University School of Medicine, New York, NY INTRODUCTION: The ability to effect gene alteration via topical therapy has protean implications in the potential treatment of conditions ranging from tumors to wound healing. We created a technique of RNA interference to modulate gene expression in-vivo. Specifically, a novel use of small interfering RNA (siRNA) was developed to knockdown several ubiquitous housekeeping genes (MAPK-1 and lamin A/C) of a wound bed. METHODS: Mice were wounded using our described model of excisional wound healing. MAPK-1/lamin siRNA and transfection reagent were delivered to the wounds via an agarose gel suspension. The treatment protocol was optimized and wounds were harvested at varying time points. MAPK-1/lamin protein expression was evaluated (immunohistochemistry and Western blotting). MAPK-1/ lamin gene expression was evaluated (in-situ hybridization). RESULTS: Immunohistochemistry of treated wounds revealed almost complete knockdown of MAPK-1/lamin versus controls. Additionally, the tissue directly beyond the treatment area revealed normal MAPK-1/lamin staining. Western blot analysis similarly showed marked decrease of MAPK-1/lamin in treated wounds. Gene expression paralleled protein findings. At later time points, the effect waned with a return of MAPK-1/lamin to baseline. CONCLUSIONS: We demonstrated effective gene silencing via topical delivery of siRNA in-vivo. Advantages of this powerful new