Wuyi Kong

Hypertrophic scar fibroblasts have increased connective tissue growth factor expression after transforming growth factor-beta stimulation.

Background: Hypertrophic scars and keloids respond to dermal disruption with excessive collagen deposition and increased transforming growth factor (TFG)-β expression. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-β activity that is associated with scar and fibrosis. The authors hypothesize that there is increased expression of CTGF by hypertrophic scar and keloid fibroblasts in response to TGF-β stimulation. Methods: Primary fibroblasts were isolated in culture from human hypertrophic scar (n = 2), keloid (n = 2), and normal skin (n = 2). After 18 hours of serum starvation, the cells were stimulated with 10 ng/ml of TGF-β1, TGF-β2, and TGF-β3 for 24 hours. Quantitative real-time polymerase chain reaction was performed on extracted RNA samples to assay for CTGF mRNA expression. Results: Baseline CTGF expression was increased 20-fold in unstimulated hypertrophic scar fibroblasts and 15-fold in keloid fibroblasts compared with normal fibroblasts. CTGF expression increased greater than 150-fold when stimulated with TGF-β1 (p < 0.002) and greater than 100-fold when stimulated by TGF-β2 or TGF-β3 compared with normal fibroblasts (p < 0.02 and p < 0.002, respectively). CTGF expression was greatest after TGF-β1 stimulation in hypertrophic scar fibroblasts compared with TGF-β2 (p < 0.04) and TGF-β3 (p < 0.02). Keloid fibroblast CTGF expression also increased greater than 100-fold after stimulation with TGF-β1 (p = 0.16) and greater than 75-fold after addition of TGF-β2 and TGF-β3 (p = 0.06 and p = 0.22, respectively). Conclusions: Hypertrophic scar fibroblasts have both intrinsic up-regulation of CTGF transcription and an exaggerated capacity for CTGF transcription in response to TGF-β stimulation. These data suggest that blockage of CTGF activity may reduce pathologic scar formation.

Skin wounds in the MRL/MPJ mouse heal with scar

Adult MRL/MpJ mice regenerate cartilage during repair of through‐and‐through ear punch wounds. However, the ability of this mouse strain to heal isolated cutaneous wounds by regeneration or with scar is unknown. The purpose of this study was to characterize the rate of reepithelialization and collagen architecture in dermal wounds from MRL/MpJ mice compared with C57bl/6 and Balb/c strains. Full‐thickness incisional (5 mm) and excisional (2 mm diameter) skin wounds were made on the dorsum of 7‐week‐old MRL/MpJ, C57bl/6, and Balb/c mice. Ear punch wounds were made simultaneously on each animal. Reepithelialization was complete by 48 hours for incisional skin wounds in each strain. All excisional wounds showed incomplete reepithelialization at 24, 48, and 72 hours. At 14 days, all skin wounds had grossly healed. In contrast to the ear wounds made in C57bl/6 and Balb/c mice, MRL/MpJ ear wounds were completely healed by day 28. Dorsal skin wound sections at 14 and 28 days revealed dense collagen deposition and similar degrees of fibrosis between the three strains of mice. In conclusion, in contrast to wound healing in the ear, MRL/MpJ mouse dorsal cutaneous wounds heal similarly to C57bl/6 and Balb/c mice with dermal collagen deposition and scar formation.

Increased CCN2 transcription in keloid fibroblasts requires cooperativity between AP-1 and SMAD binding sites

Objective:We examined the transcriptional response to serum stimulation as an in vitro model of wound healing in keloid fibroblasts to identify molecular mechanisms leading to their aberrant growth. Summary Background Data:Keloids are proliferative dermal growths representing a pathologic wound healing response. Although several groups have shown increased expression of profibrotic factors in keloids, there is little known about why they are expressed at higher levels than normal. Methods:Fibroblasts derived from keloids and normal scar were subjected to serum stimulation as an in vitro model to mimic a component of the wound microenvironment to examine differential gene expression in keloid derived fibroblasts versus normal human fibroblasts. A promoter analysis was performed to identify specific enhancers involved in mediating the differential response of connective tissue growth factor (CTGF, CCN2). Point mutations in the enhancers were performed to confirm their role. Finally, we examined activation of transcription factors known to bind the targeted enhancers. Results:Transcription of CCN2 after serum stimulation was significantly higher in keloid versus normal fibroblasts. Promoter analysis demonstrates the fragment from −625/−140 conferred increased serum responsiveness. Mutational analysis showed an AP-1 and SMAD binding site were both necessary for serum responsiveness. Preventing activation of either transcriptional complex will block CCN2 transcription. Additional experiments suggest that a single complex that includes components of the AP-1 and SMAD binding complexes is responsible for transactivation in response to serum. The key difference between keloid and normal fibroblasts appears to be the degree of activation of c-Jun. Conclusions:We suggest that altered responsiveness to cellular stress, based upon current data using serum stimulation and past data on response to mechanical strain, is a key defect leading to keloid formation.

Cyclophilin C‐associated protein is up‐regulated during wound healing

Cyclophilin C‐associated protein (CyCAP) is identified from macrophages. It locates in intracellular, membrane bound and extracellular, suggesting it has an important role, however both of its regulation and function have not been elucidated. The expression of CyCAP in skin and during wound healing is also unknown. We demonstrate that CyCAP is expressed in both dermal fibroblasts and keratinocytes. In the dermis, the majority of CyCAP protein is located intracellular in a filamentous protein form while a lesser amount is in the extracellular matrix (ECM). CyCAP gene and protein expression is increased 1 day after skin wound healing in both fetal and adult rats and remains elevated level up to 1 week in adult rats. Immunohistochemistry studies demonstrate that the increased CyCAP expression locates mainly to inflammatory cells, including macrophages, monocytes and lymphocytes during wound healing. Interferon‐γ increases CyCAP gene and protein expression in cultured rat fibroblasts. We also found that wound healing is slower and less collagen is expressed in skin of CyCAP null mice. These data are the first observations of CyCAP expression in skin and during wound repair. Our data indicates that CyCAP is regulated by IFNγ and may function on immune defense in macrophages, lymphocytes, dermal fibroblasts and keratinocytes during wound healing. J. Cell. Physiol. 210: 153–160, 2007.

Upregulation of Neurodevelopmental Genes During Scarless Healing

Scarless fetal skin wound healing is a paradigm for ideal skin repair and is dependent on peripheral nerve function.To further explore neurogenic mechanisms influence on the scarless skin repair, fetal rats were wounded on gestational days 16 (E16; n = 24) and 18 (E18; n = 8) and wounds were harvested at 1 and 3 days after injury. Unwounded skin at identical gestational age was used for control comparison. The scarless E16 and scarring E18 wounds underwent macroarray gene expression analysis (1172 genes).During the scarless healing period, 53 (4.5%) genes had a statistically significant upregulation post-injury with at least a 2- to 3-fold change 1 day after wounding and 14 (1.2%) genes 3 days after wounding (P < 0.05). Many neurodevelopmental genes were increased during scarless repair on post-injury days 1 and 3. Neuropeptide Y Receptor type I, cJun related Transcription Factor (junD), Synaptophysin, SNAP 25, Neuronal calcium sensor 1 (NCS1), neural visine-like calcium binding protein 1 (NVP1), nerve growth factor-induced gene A (NGFI-A/EGR1), VGF8A protein, p27kip1, and members of the GABA and serotonin family each had 2- to 3-fold expression increases (P < 0.05).We speculate that fetal skin cells express neurotrophins during skin development that regulate peripheral neuron formation. During injury these factors promote the survival and regeneration of peripheral neurons; this interaction of neuropeptides, neuropeptide receptors, and neurotrophins may modulate the fetal scarless repair mechanisms in response to injury. Identification of these neurodevelopmental candidate genes provides insight for new investigation into mechanisms regulating scarless healing.

Cyclophilin C-associated Protein Is a Mediator for Fibronectin Fragment-induced Matrix Metalloproteinase-13 Expression

The function of cyclophilin C-associated protein (CyC-AP) on expression of extracellular matrix and matrix metalloproteinases (MMPs) was studied in CyC-AP-null mice. Fibronectin showed increased expression of the 53- and 29-kDa fragments in skin and wounds from CyC-AP-null mice. Type I collagen had an initial degraded pattern in the skin of CyC-AP-null mice, which did not occur in wild-type mice. MMP-3, MMP-13, MMP-14, and tumor necrosis factor-α (TNFα) had a higher expression in CyC-AP-null skin. During wound healing, MMP-13 and TNFα were stimulated to an even higher level, suggesting they are regulated by multiple factors. To understand the regulatory mechanisms of the up-regulated MMPs, the direct effects of TNFα, IL-1β, 45-kDa fibronectin fragment (FN-45), and the 70-kDa fibronectin fragments (FN-70) on the expression of MMPs were studied. MMP-13 expression increased significantly in both CyC-AP-null and wild-type dermal fibroblasts after treatment with IL-1β or with TNFα. However, MMP-13 expression did not increase in CyC-AP-null fibroblasts but did increase only in wild-type fibroblasts after FN-45 and FN-70 treatment. MMP-3 activation was induced by FN-45 and did not show a difference between CyC-AP-null and wild-type fibroblasts, suggesting different regulatory pathways for FN-45 on MMP-13 and MMP-3 expression. Our data are the first to demonstrate that deletion of CyC-AP can abolish fibronectin fragment-induced MMP-13 expression through an unknown mechanism. CyC-AP is an important factor for the regulation of MMP-13 expression.

Germ plasm-like Dot cells maintain their wound regenerative function after in vitro expansion.

1. Wounds in fetal skin heal without scarring; however, the mechanism for this is unknown. We have identified a novel group of protein and nucleotides‐positive particles in fetal and adult mouse blood and in human blood, and termed them ‘Dot cells’. Freshly isolated Dot cells regenerate wounds with less scarring and can be cultured without feeder layers.

Cyclophilin C-associated protein/Mac-2 binding protein colocalizes with calnexin and regulates the expression of tissue transglutaminase

Cyclophilin C‐associated protein (CyCAP) or Mac‐2 binding protein has been identified as a binding protein for cyclophilin C in mice and for Mac‐2 (galectin‐3) in human, suggesting its multiple binding activity to proteins. In the present study, using specific anti‐rat‐CyCAP antibody, we found that CyCAP colocalizes with calnexin at the location near the nuclear envelope, however CyCAP does not have colocalization with calreticulin. In senescent fibroblasts and interferon‐gamma (IFNγ) treated fibroblasts, both calnexin and CyCAP form larger polymers and are released from the endoplasmic reticulum (ER) through the cellular membrane to the extracellular area. Immunoprecipitation studies further confirm that the release of calnexin is through binding to CyCAP. Further, we found that tissue transglutaminase (tTG) protein is decreased, however not at the RNA level, in CyCAP null fibroblasts, which suggests that CyCAP is involved in tTG post‐translational modification. Our data give novel evidence that CyCAP regulates the post‐translational modification of tTG through its colocalization with calnexin in ER. J. Cell. Physiol. 223: 151–157, 2010.