- Email: [email protected]
Upregulation of WNT-4 expression in keloid fibroblasts after TGF-B1&-B3 stimulation
Vol. 203, No. 3S, September 2006
Plastic Surgery I
CONCLUSIONS: While absence of P-selectin does not appear to alter inflammatory cell recruitment or fibrosis in fetal murine wounds, delayed epithelial maturation is noted in P-selectin deficient mice. This study highlights how specific contributions of various molecules to fetal wound healing can be elucidated using murine knockout/transgenic models.
Closure of mid-gestational excisional wounds in a novel fetal mouse model Stephanie R Goldberg MD, Robert McKinstry MD, David Lanning MD, PhD Virginia Commonwealth University, Medical College of Virginia, Richmond, VA
S57
Upregulation of WNT-4 expression in keloid fibroblasts after TGF-B1&-B3 stimulation Anuja K Antony MD, Amy Colwell MD, Michael T Longaker MD, H Peter Lorenz MD Stanford University Medical Center, Stanford, CA INTRODUCTION: Keloids result from excessive scar formation due to uncontrolled wound healing mechanisms. Wnt proteins,named after the Drosophilia Wingless(Wg) genes,have important roles in embryonic cell proliferation,differentiation,survival,and apoptosis. Thus,Wnt dysregulation may be crucial during development of tissue pathology involving cytokine and growth-factor control. Specifically,Wnt-4 expression has been reported in post-wounded skin(murine model). We hypothesize that differential expression of Wnt-4 occurs in human normal fibroblasts(NF)and keloid fibroblasts(KF)under TGF-beta influence,potent regulators of wound healing.
INTRODUCTION: Studies utilizing mid-gestational wounds have been used to improve our understanding of excisional wound healing with mixed results; rabbit wounds expand while lamb wounds contract. We developed a murine excisional wound model and theorized that mid-gestational wounds would expand with minimal alphasmooth muscle actin(aSMA) expression, a marker for myofibroblasts, when compared with late-gestational wounds.
METHODS: Primary fibroblasts were isolated in culture from human normal skin and keloid skin.After 24hrs serum starvation,cells were stimulated with TGF-beta(10ng/ml) 1,2,and 3 for 24hrs. Quantitative RT-PCR was performed on extracted RNA to assay for Wnt-4 mRNA expression. Experiments were carried out in duplicate in NF and triplicate in KF with separate cell lines for each respectively.
METHODS: Time-dated, pregnant FVB strain mice underwent hysterotomy at gestational days E15, E16, E17, and E18(term⫽20-21 days). Full-thickness, excisional wounds(2.5mm) were made on each fetus, remeasured at 32 hours, and harvested with 1mm of surrounding tissue. aSMA and 18S gene expression was measured by real-time PCR. Control tissue consisted of nearby normal fetal skin and wounded adult skin. ANOVA on Ranks was performed(significance⫽P⬍0.05).
RESULTS: We found TGF-B1,B2 and B3 had no effect on Wnt-4 expression in NF. In contrast,TGF-B1&-B3 caused a 2.8-fold and 3.4-fold increase, respectively, in Wnt-4 expression in KF(p⫽0.0065, p⫽0.0268).TGF-B2 did not significantly change Wnt-4 expression in KF. Baseline expression of Wnt-4 in NF and KF was not statistically different.
RESULTS: The E15 wounds closed more than the E16, E17, and E18 wounds(P⬍0.001, Table). aSMA expression was higher in the E15 group(2.99⫹/⫺0.37, n⫽9) compared to the E16(1.91⫹/⫺0.24, n⫽8), E17(1.58, n⫽1), and E18(2.07⫹/ ⫺0.075, n⫽7) groups, but did not reach statistical significance (P⫽0.1). aSMA levels in the adult wound were 7.51. aSMA expression between wounds and normal fetal skin within groups was not significant. Table. Contraction result per gestational group Fetuses wounded (n) Fetal survival (%) Mean wound contraction (%) SEM (%)
E-15
E-16
E-17
E-18
14 78.6 80.5 4.4
17 94.1 46.6 5.9
16 100 47.4 6.4
16 100ⴱ 1.0 14.3
ⴱ8 fetuses were delivered by 32 hours and were excluded.
CONCLUSIONS: This study demonstrates that mid-gestational excisional wounds in the mouse close signficantly compared to lategestational excisional wounds at the 32 hours. While aSMA expression was higher in the mid-gestational wounds, it remains unlikely that myofibroblasts contributed to wound closure. This model will allow for the use of transgenic mice in further wound healing studies.
CONCLUSIONS: TGF-B1 and TGF-B3,key signaling cytokines during scar formation, upregulate Wnt-4 expression in KF but not NF. In KF, upregulation of Wnt-4 may lead to altered cell proliferation and apoptosis,with eventual keloid production. A more complex and coherent view of embryonic cell signaling pathways,which coordinate cell growth,proliferation,and survival,under the influence of TGF-beta family may lead to improved understanding of uncontrolled keloid scar formation and development of better therapeutic modalities.
bFGF gene transfer through AAV vectors to digital flexor tendons significantly increases healing strength: An in vivo study Jin Bo Tang MD, Yi Cao MD, Ren Guo Xie MD, Bei Zhu MD, Xin Ke-Qin MD, PhD, Wang Xiao Tian MD, Liu Paul MD Nantong University, Jiangsu, China; Roger Williams Medical Center, Providence, RI INTRODUCTION: Tendon injuries are common and weakness in its healing potential causes repair ruptures. In this study, we delivered the bFGF gene to injured tendon in a clinically relevant injury model and found this gene therapy approach significantly enhances healing strength of tendon. METHODS: 36 toes of the 18 leghorn chickens were randomly divided into 3 groups. The flexor digitorum profundus tendons were
Plastic Surgery I
CONCLUSIONS: While absence of P-selectin does not appear to alter inflammatory cell recruitment or fibrosis in fetal murine wounds, delayed epithelial maturation is noted in P-selectin deficient mice. This study highlights how specific contributions of various molecules to fetal wound healing can be elucidated using murine knockout/transgenic models.
Closure of mid-gestational excisional wounds in a novel fetal mouse model Stephanie R Goldberg MD, Robert McKinstry MD, David Lanning MD, PhD Virginia Commonwealth University, Medical College of Virginia, Richmond, VA
S57
Upregulation of WNT-4 expression in keloid fibroblasts after TGF-B1&-B3 stimulation Anuja K Antony MD, Amy Colwell MD, Michael T Longaker MD, H Peter Lorenz MD Stanford University Medical Center, Stanford, CA INTRODUCTION: Keloids result from excessive scar formation due to uncontrolled wound healing mechanisms. Wnt proteins,named after the Drosophilia Wingless(Wg) genes,have important roles in embryonic cell proliferation,differentiation,survival,and apoptosis. Thus,Wnt dysregulation may be crucial during development of tissue pathology involving cytokine and growth-factor control. Specifically,Wnt-4 expression has been reported in post-wounded skin(murine model). We hypothesize that differential expression of Wnt-4 occurs in human normal fibroblasts(NF)and keloid fibroblasts(KF)under TGF-beta influence,potent regulators of wound healing.
INTRODUCTION: Studies utilizing mid-gestational wounds have been used to improve our understanding of excisional wound healing with mixed results; rabbit wounds expand while lamb wounds contract. We developed a murine excisional wound model and theorized that mid-gestational wounds would expand with minimal alphasmooth muscle actin(aSMA) expression, a marker for myofibroblasts, when compared with late-gestational wounds.
METHODS: Primary fibroblasts were isolated in culture from human normal skin and keloid skin.After 24hrs serum starvation,cells were stimulated with TGF-beta(10ng/ml) 1,2,and 3 for 24hrs. Quantitative RT-PCR was performed on extracted RNA to assay for Wnt-4 mRNA expression. Experiments were carried out in duplicate in NF and triplicate in KF with separate cell lines for each respectively.
METHODS: Time-dated, pregnant FVB strain mice underwent hysterotomy at gestational days E15, E16, E17, and E18(term⫽20-21 days). Full-thickness, excisional wounds(2.5mm) were made on each fetus, remeasured at 32 hours, and harvested with 1mm of surrounding tissue. aSMA and 18S gene expression was measured by real-time PCR. Control tissue consisted of nearby normal fetal skin and wounded adult skin. ANOVA on Ranks was performed(significance⫽P⬍0.05).
RESULTS: We found TGF-B1,B2 and B3 had no effect on Wnt-4 expression in NF. In contrast,TGF-B1&-B3 caused a 2.8-fold and 3.4-fold increase, respectively, in Wnt-4 expression in KF(p⫽0.0065, p⫽0.0268).TGF-B2 did not significantly change Wnt-4 expression in KF. Baseline expression of Wnt-4 in NF and KF was not statistically different.
RESULTS: The E15 wounds closed more than the E16, E17, and E18 wounds(P⬍0.001, Table). aSMA expression was higher in the E15 group(2.99⫹/⫺0.37, n⫽9) compared to the E16(1.91⫹/⫺0.24, n⫽8), E17(1.58, n⫽1), and E18(2.07⫹/ ⫺0.075, n⫽7) groups, but did not reach statistical significance (P⫽0.1). aSMA levels in the adult wound were 7.51. aSMA expression between wounds and normal fetal skin within groups was not significant. Table. Contraction result per gestational group Fetuses wounded (n) Fetal survival (%) Mean wound contraction (%) SEM (%)
E-15
E-16
E-17
E-18
14 78.6 80.5 4.4
17 94.1 46.6 5.9
16 100 47.4 6.4
16 100ⴱ 1.0 14.3
ⴱ8 fetuses were delivered by 32 hours and were excluded.
CONCLUSIONS: This study demonstrates that mid-gestational excisional wounds in the mouse close signficantly compared to lategestational excisional wounds at the 32 hours. While aSMA expression was higher in the mid-gestational wounds, it remains unlikely that myofibroblasts contributed to wound closure. This model will allow for the use of transgenic mice in further wound healing studies.
CONCLUSIONS: TGF-B1 and TGF-B3,key signaling cytokines during scar formation, upregulate Wnt-4 expression in KF but not NF. In KF, upregulation of Wnt-4 may lead to altered cell proliferation and apoptosis,with eventual keloid production. A more complex and coherent view of embryonic cell signaling pathways,which coordinate cell growth,proliferation,and survival,under the influence of TGF-beta family may lead to improved understanding of uncontrolled keloid scar formation and development of better therapeutic modalities.
bFGF gene transfer through AAV vectors to digital flexor tendons significantly increases healing strength: An in vivo study Jin Bo Tang MD, Yi Cao MD, Ren Guo Xie MD, Bei Zhu MD, Xin Ke-Qin MD, PhD, Wang Xiao Tian MD, Liu Paul MD Nantong University, Jiangsu, China; Roger Williams Medical Center, Providence, RI INTRODUCTION: Tendon injuries are common and weakness in its healing potential causes repair ruptures. In this study, we delivered the bFGF gene to injured tendon in a clinically relevant injury model and found this gene therapy approach significantly enhances healing strength of tendon. METHODS: 36 toes of the 18 leghorn chickens were randomly divided into 3 groups. The flexor digitorum profundus tendons were