- Email: [email protected]
Homeobox Genes Hoxd3 and Hoxd8 Are Differentially Expressed in Fetal Mouse Excisional Wounds
Journal of Surgical Research 148, 45– 48 (2008) doi:10.1016/j.jss.2008.02.053
Homeobox Genes Hoxd3 and Hoxd8 Are Differentially Expressed in Fetal Mouse Excisional Wounds Kunoor Jain, M.D.,* Virginia Sykes, B.S.,* Tomasz Kordula, Ph.D.,† and David Lanning, M.D., Ph.D.*,1 *Division of Pediatric Surgery, Department of Surgery; and †Department of Biochemistry and Molecular Biology, Virginia Commonwealth University Health System, Richmond, Virginia Submitted for publication January 4, 2008
groups. Also, Hoxd8 in the mid-gestational near wound controls is significantly greater than that in the lategestational near wound control and control groups. Conclusions. These data suggest that Hoxd3 is constitutively expressed in the skin of mid-gestational mice. However, Hoxd8 expression is increased in the mid-gestational wounds compared with normal control groups and late gestational wounds, suggesting that it may play a role in scarless wound repair. © 2008
Background. Cell signaling pathways underlying wound repair are under extensive investigation; however, there is still a poor understanding of the mechanisms orchestrating these processes. Hox genes, which are a subgroup of homeobox genes, encode for a family of transcription factors that play a critical role in tissue migration and cell differentiation during embryogenesis and may also serve as master regulatory genes of postnatal wound repair. We have developed a fetal excisional wound healing model whereby midgestational wounds heal in a regenerative manner while late-gestational wounds display scar formation. We theorize that Hoxd3 and Hoxd8 will be differentially expressed in mid- and late-gestational wounds compared with normal skin. Materials and methods. Pregnant FVB mice underwent hysterotomy at mid (E15)- or late (E18)-gestational time points, and 3-mm excisional wounds were made on the dorsum of each fetus. Wound samples (w) were collected at the site of injury as well as near wound normal skin (nwc) on the same fetus. Control (c) skin samples were also obtained from unwounded adjacent fetuses. Samples were harvested at 3 and 6 h and real-time polymerase chain reaction was performed for Hoxd3 and Hoxd8 and normalized to glyceraldehyde-3-phosphate dehydrogenase. Data were analyzed by analysis of variance with statistical significance of P < 0.05. Results. Hoxd3 levels were increased in all of the midgestational groups, with a significant increase at 3 h compared with late-gestational control groups. In the 3-h time group, Hoxd8 is increased in mid-gestational wounds compared with late-gestational control skin. This is repeated in the 6-h time group, where Hoxd8 is increased in mid-gestational wounds compared with all
Elsevier Inc. All rights reserved.
Key Words: homeobox genes; wound repair; Hoxd3; Hoxd8. INTRODUCTION
Wounds that heal by excessive or sparse healing present a significant dilemma in health care and patient well being. Various disease processes are affected by flawed wound repair, such as pulmonary and liver fibrosis, contracture scars from burns, keloid formation and pressure, and diabetic ulcers. Wound healing has been researched quite steadily for decades; however, the secondary cell signaling mechanisms underlying wound repair remain unclear. There is even less known regarding the genes that control these processes and the downstream regulation that occurs. Neonatal and adult wound healing are characterized by inflammation, fibrosis, and scarring, whereas these features are absent in fetal wound healing [1]. In fact, the fetal neodermis after wounding is practically indistinguishable from the surrounding non-injured skin, even on a microscopic level [2– 4]. The differences between adult and fetal wound repair are examined in animal models using mid- and late-gestational fetuses. The mid-gestational animals represent regenerative fetal wound healing that lacks scar formation. Lategestational animals, on the other hand, correspond to
1
To whom correspondence and reprint requests should be addressed at Department of Surgery, Division of Pediatric Surgery, Virginia Commonwealth University Health System, P.O. Box 980015, Richmond, VA 23298. E-mail: [email protected].
45
0022-4804/08 $34.00 © 2008 Elsevier Inc. All rights reserved.
46
JOURNAL OF SURGICAL RESEARCH: VOL. 148, NO. 1, JULY 2008
adult wound healing with scar formation [5]. Once we are able to understand the genetic and molecular mechanisms underlying the ability of the fetal wound to regenerate in this capacity, we may apply this knowledge to the vast number of diseases affected by imperfect wound healing. Homeobox genes encode for a large family of developmental transcription factors that have a conserved 180base pair sequence that corresponds to a 60-amino-acid region called the homeodomain [6]. This domain is over 10 9 years old and is responsible for the sequence-specific DNA binding of homeobox proteins [7]. These proteins are critical in morphogenesis, tissue migration, and cell differentiation in the embryo by regulating transcription or repression of target genes [6]. These processes are similar to those important in wound healing and secondary signaling for site-directed tissue repair. Homeobox genes have also been found to play a role in wound repair and regenerative wound healing. For example, the Hoxd-11 gene was found to be up-regulated in the blastema formation of regenerating Axolotl newt forelimbs following amputation, and non-regenerating forelimbs are characterized by an up-regulation of Hoxd-8 and Hoxd-10 [8]. Hox D3 has been found to bind directly to the promoters of the integrin ␣5 and 3 subunits, inducing subunit expression [9, 10]. Also, Hox D3-deficient wounds of diabetic animals display a reduction in expression and deposition of Type 1 collagen [11]. Hoxd8 has been previously researched in rabbits in our laboratory and was found to be differentially expressed in fetal excisional wounds induced to contract with transforming growth factor-3 compared with control wounds [12]. Therefore, our findings as well as those discussed in the literature suggest that homeobox genes may serve as the master genes that control downstream genes and factors responsible for orchestrating secondary signaling and wound repair. We theorize that fetal scarless and scarforming wound repair will be associated with a differential pattern of homeobox gene expression. MATERIALS AND METHODS Using a previously described fetal excisional wound healing model in accordance with Institutional Animal Care and Untilization Com-
FIG. 1.
mittee approval (#0409-3354) time-dated, pregnant FVB mice (Charles River, Wilmington, ME) underwent hysterotomy at mid (E15)- or late (E18)-gestational time points, and 3-mm excisional cutaneous wounds were made on the dorsum of each fetus [5]. Wound samples (w) were collected at the site of injury as well as near wound normal skin (nwc) on the same fetus. Control skin samples (c) were also obtained from unwounded adjacent fetuses. All samples harvested were approximately 5 to 10 mg in weight. As the expression of the clustered homeobox genes can vary along the cranio-caudal axis, all tissue was obtained from the midback area in all animals. Following fetal tissue sample harvest and euthanasia by decapitation at 3 or 6 h, the does were sacrificed per protocol using sodium pentobarbital via intravenous injection. Fetal samples were homogenized, and total RNA in the amount of 1–2 g was extracted and then processed maximally into 10 L of cDNA. Quantitative polymerase chain reaction was performed by using the 7900HT Fast real-time polymerase chain reaction machine (Applied Biosystems, Foster City, CA) on cDNA dilutions at 1:10 in duplicate for the following pre-made gene expression assays: Hoxd3 and Hoxd8. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene. Standard curves were constructed with serial 10-fold dilutions of cDNA and thresholds for detection set at 0.20. GAPDH served as a housekeeping gene, and absolute copy numbers of each gene were normalized against GAPDH. Mean copy number ⫾ standard error of the mean was determined for each gene of interest in each of the following groups at 3- and 6-h time points in E15 and E18 fetuses: wound, near wound control, and control. Hoxd3 3- and 6-h data and Hoxd8 3-h data were analyzed by Kruskal–Wallis analysis of variance with post-hoc analysis by Dunn’s method. Hoxd8 6-h data were analyzed by analysis of variance with post-hoc analysis using the Holm–Sidak method. P ⬍ 0.05 was considered significant.
RESULTS
Hoxd3 levels were increased in all of the midgestational groups, with a significant increase at 3 h compared with late-gestational control groups. However, there did not appear to be any significant differences within the mid-gestational groups. Hoxd8, on the other hand, did show several significant differences. In the 3-h time group, Hoxd8 is increased in midgestational wounds compared with late-gestational control skin. This is repeated in the 6-h time group where Hoxd8 is increased in mid-gestational wounds compared with all groups. Also, Hoxd8 in the midgestational near wound controls is significantly greater than that in the late-gestational near wound control and control groups. These data are shown in Figs. 1 and 2.
Hoxd3 is constitutively expressed in all groups at 3 h (A) and 6 h (B). * and #, P ⬍ 0.05.
JAIN ET AL.: EXPRESSION OF HOMEOBOX GENES
47
FIG. 2. Hoxd8 is increased in mid-gestational (E15) wounds compared with late-gestational (E18) control skin at 3 h (A) and all groups at 6 h (B). * and #, P ⬍ 0.05.
DISCUSSION
Fetal wound healing differs from that seen in the adult in a number of aspects. There is a sparse inflammatory response during fetal healing [13]. Collagen is deposited in a more organized and rapid fashion than the adult and has an increased type 3:1 ratio than the adult [14, 15]. It also is deposited at the fetal wound site in a reticular pattern that is indistinguishable from the surrounding tissue and has greater tensile strength than adult wounds [2– 4]. In our fetal mouse excisional wound model, we have demonstrated that mid-gestational wounds close rapidly and without scar formation, whereas late-gestational wounds close with scar formation. In this study, we found increased levels of Hoxd3 in mid-gestational normal skin and wounds suggesting a role only in embryogenesis. However, Hoxd8 expression is increased in the mid-gestational wounds compared with normal control groups and late gestational wounds, suggesting that it may play a role in scarless wound repair. Therefore, Hoxd8 may serve as a target for up-regulation in late gestational wounds to improve tissue repair. The data also show a slight elevation in the near wound control groups which is not significant. We believe this may have been caused by the proximity of the near wound control tissue to the wound samples. Also, the animals undergoing wounding are the same animals from which the near wound control samples are taken. The increased stress of the surgery over the 3- or 6-h delay may have elevated near wound control Hox levels. To address this concern we plan to perform in situ hybridization and possibly immunohistochemistry in the future to localize Hox transcripts and protein in the tissue samples. We chose early time points for this study as it is likely that Hox transcription factor activation is responsible for directing many of the downstream signaling events necessary for wound repair. The capacity to initiate tissue regeneration has been evidenced by several studies. For example, Weeks and Nath were able to show a moderate elevation in Hoxd-11 in mouse
embryo forelimbs at day 12 of gestation (term ⫽ 21 days) after multiple cross-hatched full-thickness incisions were made prior to placing the amputated limb in culture medium for 24 h [16]. Additional time points before 3 h and after 6 h are under investigation. Whereas downstream targets and transcriptional controls remain unknown and poorly investigated, murine antibodies for Hoxd3 have very recently become available and we are now using these to quantitate changes in protein levels. Unfortunately no murine antibody is yet available for Hoxd8. We are also moving forward to in vitro replication of the data presented here using a scratch model that was recently published [5]. Finally, in an effort to further delineate the role of these specific transcription factors, we are using siRNA techniques to alter their transcipt levels in an in vitro wound model. This study highlights the value of identifying the genes important in initiating the cascade of signaling responsible for wound repair. We plan to use the differential expression of Hox genes by altering their levels to manipulate scarless toward scar-forming wounds and vice versa. We anticipate using the in vitro techniques mentioned to alter gene levels and assess downstream signaling and healing. It is our goal to alter Hox gene regulation to manipulate wounds at a very early stage, up- or down-regulating levels of key signaling genes and mediators to provide the ideal levels for proper wound repair. REFERENCES 1.
Adolph VR, DiSanto SK, Bleacher JC, et al. The potential role of the lymphocyte in fetal wound healing. J Pediatr Surg 1993; 28:1316. 2. Olutoye OO, Cohen IK. Fetal wound healing: an overview. Wound Rep Regen 1996;4:66. 3. Cohen IK, Diegelmann RF, Crossland MC. Wound care and wound healing. In: Schwartz, Ed. Principles of Surgery (6th ed). New York, NY: McGraw-Hill Co., 1994:279 –303. 4. Mast BA, Diegelmann RF, Krummel TM, et al. Scarless wound healing in the mammalian fetus. Surg Gynecol Obstet 1992; 174:441.
48
JOURNAL OF SURGICAL RESEARCH: VOL. 148, NO. 1, JULY 2008
5.
Goldberg RP, McKinstry, V Sykes, et al. Rapid closure of midgestational excisional wounds in a fetal mouse model is associated with altered transforming growth factor-beta isoform and receptor expression. J Pediatr Surg 2007;143:27.
6.
Acampora D, D’Esposito M, Faiella A, et al. The human HOX gene family. Nucl Acid Res 1989;17:10385.
7.
Goldsmith LA, Scott GA. Homeobox genes and skin development: A review. J Invest Dermatol 1993;101:3.
8.
Torok MA, Gardiner DM, Shubin NH, et al. Expression of HoxD genes in developing and regenerating axolotl forelimbs. Dev Biol 1998;200:225.
9.
Boudreau N, Andrews C, Srebrow A, et al. Induction of the angiogenic phenotype by HOXD3. J Cell Biol 1997;139:257.
10.
Boudreau NJ, Varner JA. The homeobox transcription factor Hox D3 promotes integrin alpha5beta1 expression and function during angiogenesis. J Biol Chem 2004;279:4862.
11.
12. 13.
14.
15.
16.
Uyeno LA, Newman-Keagle JA, Cheung I, et al. Hox D3 expression in normal and impaired wound healing. J Surg Res 2001; 100:46. Lanning DA. Excisional Wound Healing and the Role of Homeobox Genes, published Ph.D. dissertation, June 2000. Mast BA, Krummel TM. Acute inflammation in fetal wound healing. In: Adzick NS, Longaker MT, Eds. Fetal Wound Healing. New York, NY: Elsevier, 1992:227– 40. Thomas BL, Krummel TM, Melang M, et al. Collagen synthesis and type expression by fetal fibroblasts in vitro. Surg Forum 1988;39:642. Burd DAR, Longaker MT, Adzick NS, et al. Fetal wound healing in a large animal model: the deposition of collagen is confirmed. Br J Plast Surg 1990;43:571. Nath RK, Jensen J, Weeks PM. Temporally variable induction of homeobox gene D11 during wound repair in the fetal mouse limb. Surg Forum 1991;46:682.
Homeobox Genes Hoxd3 and Hoxd8 Are Differentially Expressed in Fetal Mouse Excisional Wounds Kunoor Jain, M.D.,* Virginia Sykes, B.S.,* Tomasz Kordula, Ph.D.,† and David Lanning, M.D., Ph.D.*,1 *Division of Pediatric Surgery, Department of Surgery; and †Department of Biochemistry and Molecular Biology, Virginia Commonwealth University Health System, Richmond, Virginia Submitted for publication January 4, 2008
groups. Also, Hoxd8 in the mid-gestational near wound controls is significantly greater than that in the lategestational near wound control and control groups. Conclusions. These data suggest that Hoxd3 is constitutively expressed in the skin of mid-gestational mice. However, Hoxd8 expression is increased in the mid-gestational wounds compared with normal control groups and late gestational wounds, suggesting that it may play a role in scarless wound repair. © 2008
Background. Cell signaling pathways underlying wound repair are under extensive investigation; however, there is still a poor understanding of the mechanisms orchestrating these processes. Hox genes, which are a subgroup of homeobox genes, encode for a family of transcription factors that play a critical role in tissue migration and cell differentiation during embryogenesis and may also serve as master regulatory genes of postnatal wound repair. We have developed a fetal excisional wound healing model whereby midgestational wounds heal in a regenerative manner while late-gestational wounds display scar formation. We theorize that Hoxd3 and Hoxd8 will be differentially expressed in mid- and late-gestational wounds compared with normal skin. Materials and methods. Pregnant FVB mice underwent hysterotomy at mid (E15)- or late (E18)-gestational time points, and 3-mm excisional wounds were made on the dorsum of each fetus. Wound samples (w) were collected at the site of injury as well as near wound normal skin (nwc) on the same fetus. Control (c) skin samples were also obtained from unwounded adjacent fetuses. Samples were harvested at 3 and 6 h and real-time polymerase chain reaction was performed for Hoxd3 and Hoxd8 and normalized to glyceraldehyde-3-phosphate dehydrogenase. Data were analyzed by analysis of variance with statistical significance of P < 0.05. Results. Hoxd3 levels were increased in all of the midgestational groups, with a significant increase at 3 h compared with late-gestational control groups. In the 3-h time group, Hoxd8 is increased in mid-gestational wounds compared with late-gestational control skin. This is repeated in the 6-h time group, where Hoxd8 is increased in mid-gestational wounds compared with all
Elsevier Inc. All rights reserved.
Key Words: homeobox genes; wound repair; Hoxd3; Hoxd8. INTRODUCTION
Wounds that heal by excessive or sparse healing present a significant dilemma in health care and patient well being. Various disease processes are affected by flawed wound repair, such as pulmonary and liver fibrosis, contracture scars from burns, keloid formation and pressure, and diabetic ulcers. Wound healing has been researched quite steadily for decades; however, the secondary cell signaling mechanisms underlying wound repair remain unclear. There is even less known regarding the genes that control these processes and the downstream regulation that occurs. Neonatal and adult wound healing are characterized by inflammation, fibrosis, and scarring, whereas these features are absent in fetal wound healing [1]. In fact, the fetal neodermis after wounding is practically indistinguishable from the surrounding non-injured skin, even on a microscopic level [2– 4]. The differences between adult and fetal wound repair are examined in animal models using mid- and late-gestational fetuses. The mid-gestational animals represent regenerative fetal wound healing that lacks scar formation. Lategestational animals, on the other hand, correspond to
1
To whom correspondence and reprint requests should be addressed at Department of Surgery, Division of Pediatric Surgery, Virginia Commonwealth University Health System, P.O. Box 980015, Richmond, VA 23298. E-mail: [email protected].
45
0022-4804/08 $34.00 © 2008 Elsevier Inc. All rights reserved.
46
JOURNAL OF SURGICAL RESEARCH: VOL. 148, NO. 1, JULY 2008
adult wound healing with scar formation [5]. Once we are able to understand the genetic and molecular mechanisms underlying the ability of the fetal wound to regenerate in this capacity, we may apply this knowledge to the vast number of diseases affected by imperfect wound healing. Homeobox genes encode for a large family of developmental transcription factors that have a conserved 180base pair sequence that corresponds to a 60-amino-acid region called the homeodomain [6]. This domain is over 10 9 years old and is responsible for the sequence-specific DNA binding of homeobox proteins [7]. These proteins are critical in morphogenesis, tissue migration, and cell differentiation in the embryo by regulating transcription or repression of target genes [6]. These processes are similar to those important in wound healing and secondary signaling for site-directed tissue repair. Homeobox genes have also been found to play a role in wound repair and regenerative wound healing. For example, the Hoxd-11 gene was found to be up-regulated in the blastema formation of regenerating Axolotl newt forelimbs following amputation, and non-regenerating forelimbs are characterized by an up-regulation of Hoxd-8 and Hoxd-10 [8]. Hox D3 has been found to bind directly to the promoters of the integrin ␣5 and 3 subunits, inducing subunit expression [9, 10]. Also, Hox D3-deficient wounds of diabetic animals display a reduction in expression and deposition of Type 1 collagen [11]. Hoxd8 has been previously researched in rabbits in our laboratory and was found to be differentially expressed in fetal excisional wounds induced to contract with transforming growth factor-3 compared with control wounds [12]. Therefore, our findings as well as those discussed in the literature suggest that homeobox genes may serve as the master genes that control downstream genes and factors responsible for orchestrating secondary signaling and wound repair. We theorize that fetal scarless and scarforming wound repair will be associated with a differential pattern of homeobox gene expression. MATERIALS AND METHODS Using a previously described fetal excisional wound healing model in accordance with Institutional Animal Care and Untilization Com-
FIG. 1.
mittee approval (#0409-3354) time-dated, pregnant FVB mice (Charles River, Wilmington, ME) underwent hysterotomy at mid (E15)- or late (E18)-gestational time points, and 3-mm excisional cutaneous wounds were made on the dorsum of each fetus [5]. Wound samples (w) were collected at the site of injury as well as near wound normal skin (nwc) on the same fetus. Control skin samples (c) were also obtained from unwounded adjacent fetuses. All samples harvested were approximately 5 to 10 mg in weight. As the expression of the clustered homeobox genes can vary along the cranio-caudal axis, all tissue was obtained from the midback area in all animals. Following fetal tissue sample harvest and euthanasia by decapitation at 3 or 6 h, the does were sacrificed per protocol using sodium pentobarbital via intravenous injection. Fetal samples were homogenized, and total RNA in the amount of 1–2 g was extracted and then processed maximally into 10 L of cDNA. Quantitative polymerase chain reaction was performed by using the 7900HT Fast real-time polymerase chain reaction machine (Applied Biosystems, Foster City, CA) on cDNA dilutions at 1:10 in duplicate for the following pre-made gene expression assays: Hoxd3 and Hoxd8. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene. Standard curves were constructed with serial 10-fold dilutions of cDNA and thresholds for detection set at 0.20. GAPDH served as a housekeeping gene, and absolute copy numbers of each gene were normalized against GAPDH. Mean copy number ⫾ standard error of the mean was determined for each gene of interest in each of the following groups at 3- and 6-h time points in E15 and E18 fetuses: wound, near wound control, and control. Hoxd3 3- and 6-h data and Hoxd8 3-h data were analyzed by Kruskal–Wallis analysis of variance with post-hoc analysis by Dunn’s method. Hoxd8 6-h data were analyzed by analysis of variance with post-hoc analysis using the Holm–Sidak method. P ⬍ 0.05 was considered significant.
RESULTS
Hoxd3 levels were increased in all of the midgestational groups, with a significant increase at 3 h compared with late-gestational control groups. However, there did not appear to be any significant differences within the mid-gestational groups. Hoxd8, on the other hand, did show several significant differences. In the 3-h time group, Hoxd8 is increased in midgestational wounds compared with late-gestational control skin. This is repeated in the 6-h time group where Hoxd8 is increased in mid-gestational wounds compared with all groups. Also, Hoxd8 in the midgestational near wound controls is significantly greater than that in the late-gestational near wound control and control groups. These data are shown in Figs. 1 and 2.
Hoxd3 is constitutively expressed in all groups at 3 h (A) and 6 h (B). * and #, P ⬍ 0.05.
JAIN ET AL.: EXPRESSION OF HOMEOBOX GENES
47
FIG. 2. Hoxd8 is increased in mid-gestational (E15) wounds compared with late-gestational (E18) control skin at 3 h (A) and all groups at 6 h (B). * and #, P ⬍ 0.05.
DISCUSSION
Fetal wound healing differs from that seen in the adult in a number of aspects. There is a sparse inflammatory response during fetal healing [13]. Collagen is deposited in a more organized and rapid fashion than the adult and has an increased type 3:1 ratio than the adult [14, 15]. It also is deposited at the fetal wound site in a reticular pattern that is indistinguishable from the surrounding tissue and has greater tensile strength than adult wounds [2– 4]. In our fetal mouse excisional wound model, we have demonstrated that mid-gestational wounds close rapidly and without scar formation, whereas late-gestational wounds close with scar formation. In this study, we found increased levels of Hoxd3 in mid-gestational normal skin and wounds suggesting a role only in embryogenesis. However, Hoxd8 expression is increased in the mid-gestational wounds compared with normal control groups and late gestational wounds, suggesting that it may play a role in scarless wound repair. Therefore, Hoxd8 may serve as a target for up-regulation in late gestational wounds to improve tissue repair. The data also show a slight elevation in the near wound control groups which is not significant. We believe this may have been caused by the proximity of the near wound control tissue to the wound samples. Also, the animals undergoing wounding are the same animals from which the near wound control samples are taken. The increased stress of the surgery over the 3- or 6-h delay may have elevated near wound control Hox levels. To address this concern we plan to perform in situ hybridization and possibly immunohistochemistry in the future to localize Hox transcripts and protein in the tissue samples. We chose early time points for this study as it is likely that Hox transcription factor activation is responsible for directing many of the downstream signaling events necessary for wound repair. The capacity to initiate tissue regeneration has been evidenced by several studies. For example, Weeks and Nath were able to show a moderate elevation in Hoxd-11 in mouse
embryo forelimbs at day 12 of gestation (term ⫽ 21 days) after multiple cross-hatched full-thickness incisions were made prior to placing the amputated limb in culture medium for 24 h [16]. Additional time points before 3 h and after 6 h are under investigation. Whereas downstream targets and transcriptional controls remain unknown and poorly investigated, murine antibodies for Hoxd3 have very recently become available and we are now using these to quantitate changes in protein levels. Unfortunately no murine antibody is yet available for Hoxd8. We are also moving forward to in vitro replication of the data presented here using a scratch model that was recently published [5]. Finally, in an effort to further delineate the role of these specific transcription factors, we are using siRNA techniques to alter their transcipt levels in an in vitro wound model. This study highlights the value of identifying the genes important in initiating the cascade of signaling responsible for wound repair. We plan to use the differential expression of Hox genes by altering their levels to manipulate scarless toward scar-forming wounds and vice versa. We anticipate using the in vitro techniques mentioned to alter gene levels and assess downstream signaling and healing. It is our goal to alter Hox gene regulation to manipulate wounds at a very early stage, up- or down-regulating levels of key signaling genes and mediators to provide the ideal levels for proper wound repair. REFERENCES 1.
Adolph VR, DiSanto SK, Bleacher JC, et al. The potential role of the lymphocyte in fetal wound healing. J Pediatr Surg 1993; 28:1316. 2. Olutoye OO, Cohen IK. Fetal wound healing: an overview. Wound Rep Regen 1996;4:66. 3. Cohen IK, Diegelmann RF, Crossland MC. Wound care and wound healing. In: Schwartz, Ed. Principles of Surgery (6th ed). New York, NY: McGraw-Hill Co., 1994:279 –303. 4. Mast BA, Diegelmann RF, Krummel TM, et al. Scarless wound healing in the mammalian fetus. Surg Gynecol Obstet 1992; 174:441.
48
JOURNAL OF SURGICAL RESEARCH: VOL. 148, NO. 1, JULY 2008
5.
Goldberg RP, McKinstry, V Sykes, et al. Rapid closure of midgestational excisional wounds in a fetal mouse model is associated with altered transforming growth factor-beta isoform and receptor expression. J Pediatr Surg 2007;143:27.
6.
Acampora D, D’Esposito M, Faiella A, et al. The human HOX gene family. Nucl Acid Res 1989;17:10385.
7.
Goldsmith LA, Scott GA. Homeobox genes and skin development: A review. J Invest Dermatol 1993;101:3.
8.
Torok MA, Gardiner DM, Shubin NH, et al. Expression of HoxD genes in developing and regenerating axolotl forelimbs. Dev Biol 1998;200:225.
9.
Boudreau N, Andrews C, Srebrow A, et al. Induction of the angiogenic phenotype by HOXD3. J Cell Biol 1997;139:257.
10.
Boudreau NJ, Varner JA. The homeobox transcription factor Hox D3 promotes integrin alpha5beta1 expression and function during angiogenesis. J Biol Chem 2004;279:4862.
11.
12. 13.
14.
15.
16.
Uyeno LA, Newman-Keagle JA, Cheung I, et al. Hox D3 expression in normal and impaired wound healing. J Surg Res 2001; 100:46. Lanning DA. Excisional Wound Healing and the Role of Homeobox Genes, published Ph.D. dissertation, June 2000. Mast BA, Krummel TM. Acute inflammation in fetal wound healing. In: Adzick NS, Longaker MT, Eds. Fetal Wound Healing. New York, NY: Elsevier, 1992:227– 40. Thomas BL, Krummel TM, Melang M, et al. Collagen synthesis and type expression by fetal fibroblasts in vitro. Surg Forum 1988;39:642. Burd DAR, Longaker MT, Adzick NS, et al. Fetal wound healing in a large animal model: the deposition of collagen is confirmed. Br J Plast Surg 1990;43:571. Nath RK, Jensen J, Weeks PM. Temporally variable induction of homeobox gene D11 during wound repair in the fetal mouse limb. Surg Forum 1991;46:682.