Results of the Phase 1 Open-Label Safety Study of Umbilical Cord Lining Mesenchymal Stromal/Stem Cells (Corlicyte®) to Heal Chronic Diabetic Foot Ulcers.

BACKGROUND: Mesenchymal stromal/stem cells (MSCs) play a critical role in wound healing. Corlicyte® is an MSC product derived from allogeneic umbilical cord tissue donated under an institutional review board-approved protocol and processed in accordance with section 501(a)(2)(B) of the Federal Food, Drug, and Cosmetic Act. This open-label phase 1 trial was performed under a United States Food and Drug Administration Investigational New Drug Application to establish the safety and tolerability of Corlicyte® in patients with diabetes and chronic diabetic foot ulcer (DFU). METHODS: Escalating doses were applied topically twice a week for up to 8 weeks after ulcer debridement, wound photography, and measurement. Subjects were followed for 4 weeks after the treatment phase. Adverse events were assessed at every visit. RESULTS: Nine subjects in 2 dosing cohorts completed the trial. No subjects experienced a serious adverse reaction to Corlicyte® or the development of anti-human leukocyte antigen (HLA) antibodies. Sixty percentage of subjects in the lower dose cohort experienced ulcer closure by Day 70 of follow-up, while the mean ulcer size was reduced by 54-67% in the other subjects. CONCLUSIONS: Topical administration of Corlicyte®, a novel biologic therapy consisting of allogeneic umbilical cord lining MSCs, appeared safe and tolerable and resulted in a significant decrease in ulcer area, demonstrating its potential as a therapy for healing of chronic DFU.

Improvement in Skin Elasticity Using Red Deer Umbilical Cord Lining Mesenchymal Stem Cell Conditioned Media.

BACKGROUND: Significant improvement in skin tone was reported after topical application of a facial cream (CALECIM® Professional Multi-Action Cream, CALECIM Cosmeceuticals, Singapore) containing conditioned media (CM) derived from Red Deer Umbilical Cord Lining Mesenchymal Stem Cell (RD-CLMSC) culture. This study investigates the paracrine effects of RD-CLMSC-CM on human dermal fibroblasts (HDF) to understand how it may increase skin turgor and elasticity. Skin aging is associated with lower levels of extracellular matrix components such as hyaluronic acid (HA) and elastin, resulting in poor skin turgor and elasticity. Histochemical staining followed by photocolorimetry demonstrated that RD-CLMSC-CM upregulated HDF expression of elastin by 56% and HA by 83% compared with DMEM/10% Fetal Calf Serum (FCS).To further quantify the effects of CM, a proliferation assay was used to assess HDF response to RD-CLMSC-CM exposure. Exposure to RD-CLMSC-CM resulted in the highest increase in HDF proliferation over DMEM/10% FCS (113%) followed by Human (H)-CLMSC-CM (112%), then Human Foreskin Fibroblast (FSF)-CM (16%).These experimental results demonstrate both the cross-species efficacy and lack of toxicity of RD-CLMSC-CM on HDF. These pre-clinical studies also suggest the clinical effects of RD-CLMSC-CM on skin turgor may be related to increased HA and elastin production by HDF, as well as enhanced proliferation. J Drugs Dermatol. 2023;21(1):82-89. doi:10.36849/JDD.6906.

Intraperitoneally Delivered Umbilical Cord Lining Mesenchymal Stromal Cells Improve Survival and Kidney Function in Murine Lupus via Myeloid Pathway Targeting.

To determine the therapeutic efficacy of human umbilical cord lining mesenchymal stromal cells (CL-MSCs) (US Patent number 9,737,568) in lupus-prone MRL/lpr (Faslpr) mice and elucidate its working mechanisms. A total of 4 doses of (20-25) × 106 cells/kg of CL-MSCs was given to 16-week-old female Faslpr mice by intraperitoneal injection. Three subsequent doses were given on 17 weeks, 18 weeks, and 22 weeks, respectively. Six-week-old Faslpr mice were used as disease pre-onset controls. Mice were monitored for 10 weeks. Mouse kidney function was evaluated by examining complement component 3 (C3) deposition, urinary albumin-to-creatinine ratio (ACR), and lupus nephritis (LN) activity and chronicity. Working mechanisms were elucidated by flow cytometry, Luminex/ELISA (detection of anti-dsDNA and isotype antibodies), and RNA sequencing. CL-MSCs improved mice survival and kidney function by reducing LN activity and chronicity and lymphocyte infiltration over 10 weeks. CL-MSCs also reduced urinary ACR, renal complement C3 deposition, anti-dsDNA, and isotype antibodies that include IgA, IgG1, IgG2a, IgG2b, and IgM. Immune and cytokine profiling demonstrated that CL-MSCs dampened inflammation by suppressing splenic neutrophils and monocytes/macrophages, reducing plasma IL-6, IL-12, and CXCL1 and stabilizing plasma interferon-γ and TNF-α. RNA sequencing further showed that CL-MSCs mediated immunomodulation via concerted action of pro-proinflammatory cytokine-induced chemokines and production of nitric oxide in macrophages. CL-MSCs may provide a novel myeloid (neutrophils and monocytes/macrophages)-targeting therapy for SLE.

Preclinical Evaluation of Tegaderm™ Supported Nanofibrous Wound Matrix Dressing on Porcine Wound Healing Model.

Objective: Nanofibers for tissue scaffolding and wound dressings hold great potential in realizing enhanced healing of wounds in comparison with conventional counterparts. Previously, we demonstrated good fibroblast adherence and growth on a newly developed scaffold, Tegaderm™-Nanofiber (TG-NF), made from poly ɛ-caprolactone (PCL)/gelatin nanofibers electrospun onto Tegaderm (TG). The purpose of this study is to evaluate the performance and safety of TG-NF dressings in partial-thickness wound in a pig healing model. Approach: To evaluate the rate of reepithelialization, control TG, human dermal fibroblast-seeded TG-NF(+) and -unseeded TG-NF(-) were randomly dressed onto 80 partial-thickness burns created on four female and four male pigs. Wound inspections and dressings were done after burns on day 7, 14, 21, and 28. On day 28, full-thickness biopsies were taken for histopathological evaluation by Masson-Trichrome staining for collagen and hematoxylin-eosin staining for cell counting. Results: No infection and severe inflammation were recorded. Wounds treated with TG-NF(+) reepithelialized significantly faster than TG-NF(-) and control. Wound site inflammatory responses to study groups were similar as total cell counts on granulation tissues show no significant differences. Most of the wounds completely reepithelialized by day 28, except for two wounds in control and TG-NF(-). A higher collagen coverage was also recorded in the granulation tissues treated with TG-NF(+). Innovation and Conclusion: With better reepithelialization achieved by TG-NF(+) and similar rates of wound closure by TG-NF(-) and control, and the absence of elevated inflammatory responses to TG-NF constructs, TG-NF constructs are safe and demonstrated good healing potentials that are comparable to Tegaderm.

Epithelial and mesenchymal stem cells from the umbilical cord lining membrane.

Intense scientific research over the past two decades has yielded much knowledge about embryonic stem cells, mesenchymal stem cells from bone marrow, as well as epithelial stem cells from the skin and cornea. However, the billions of dollars spent in this research have not overcome the fundamental difficulties intrinsic to these stem cell strains related to ethics (embryonic stem cells), as well as to technical issues such as accessibility, ease of cell selection and cultivation, and expansion/mass production, while maintaining consistency of cell stemness (all of the stem cell strains already mentioned). Overcoming these technical hurdles has made stem cell technology expensive and any potential translational products unaffordable for most patients. Commercialization efforts have been rendered unfeasible by this high cost. Advanced biomedical research is on the rise in Asia, and new innovations have started to overcome these challenges. The Nobel Prize-winning Japanese development of iPSCs has effectively introduced a possible replacement for embryonic stem cells. For non-embryonic stem cells, cord lining stem cells (CLSCs) have overcome the preexisting difficulties inherent to mesenchymal stem cells from the bone marrow as well as epithelial stem cells from the skin and cornea, offering a realistic, practical, and affordable alternative for tissue repair and regeneration. This novel CLSC technology was developed in Singapore in 2004 and has 22 international patents granted to date, including those from the US and UK. CLSCs are derived from the umbilical cord outer lining membrane (usually regarded as medical waste) and is therefore free from ethical dilemmas related to its collection. The large quantity of umbilical cord lining membrane that can be collected translates to billions of stem cells that can be grown in primary stem cell culture and therefore very rapid and inexpensive cell cultivation and expansion for clinical translational therapies. Both mesenchymal and epithelial stem cells can be isolated from the umbilical cord lining membrane, usefully regenerating not only mesenchymal tissue, such as bone, cartilage, and cardiac and striated muscle, but also epithelial tissue, such as skin, cornea, and liver. Both mesenchymal and epithelial CLSCs are immune privileged and resist rejection. Clinically, CLSCs have proved effective in the treatment of difficult-to-heal human wounds, such as diabetic ulcers, recalcitrant chronic wounds, and even persistent epithelial defects of the cornea. Heart and liver regeneration has been shown to be successful in animal studies and await human trials. CLSCs have also been shown to be an effective feeder layer for cord blood hematopoietic stem cells and, more recently, has been recognized as an abundant and high-quality source of cells for iPSC production. Banking of CLSCs by cord blood banks in both private and public settings is now available in many countries, so that individuals may have their personal stores of CLSCs for future translational applications for both themselves and their families. Cord lining stem cells are strongly positioned to be the future of cell therapy and regenerative medicine.

The role of hepatocyte growth factor/c-Met system in keloid pathogenesis.

BACKGROUND: Keloid scar is a fibroproliferative disorder characterized by increased deposition of extracellular matrix components. Hepatocyte growth factor (HGF), also known as the "scatter factor," and its receptor, a product of the Met oncogene, play multiple roles in regulating cell behavior. However, the role of this system in pathogenic fibrosis is still unclear. Our aim was to investigate and to clarify the role of HGF and its receptor c-Met in pathogenesis of keloid scars. METHODS: This study investigated the expression profile of HGF and c-Met in keloid and normal skin tissue. In addition, the role of normal and keloid keratinocytes in modulating the expression of fibroblast HGF (epithelial-mesenchymal interactions) was examined using a two-chamber serum-free coculture model. The effect of serum stimulation on fibroblast expression of HGF and c-Met was also studied. RESULTS: Increased levels of HGF and c-Met were observed in tissue extracts obtained from keloid tissue. Increased levels of HGF and c-Met localization were observed in the basal epidermis and in the dermis of keloid tissue compared with normal skin. Serum stimulation seemed to upregulate the expression of both HGF and c-Met in fibroblasts. Finally, coculture of keloid keratinocytes with keloid fibroblasts upregulated levels of both HGF and c-Met in keratinocyte cell lysates and conditioned media obtained from fibroblast culture. CONCLUSIONS: These findings emphasize the importance of the HGF/c-Met system in keloid biology and pathogenesis and suggest a possible target for therapeutic intervention in the prevention and treatment of keloids.

Syndecan-2 and decorin: proteoglycans with a difference--implications in keloid pathogenesis.

BACKGROUND: Growth factors and cytokines involved in the wound healing process seem to be immobilized at the cell surface and extracellular matrix via binding with proteoglycans, making them important modulators of cell dynamics. Our aim was to investigate the expression of two proteoglycans, namely syndecan-2 and decorin, and to elucidate their role in the pathogenesis of an aberrant wound healing process leading to keloid scar. METHODS: Intrinsic expression of syndecan-2, fibroblast growth factor (FGF)-2, and decorin in keloid tissue was investigated using Western blotting and immunohistochemistry. Normal and keloid fibroblasts were treated with serum to see the effects of serum growth factors on the expression of syndecan-2 and decorin. The role of epithelial-mesenchymal interactions in modulating syndecan-2, FGF-2, and decorin expression was investigated using an established two-chamber serum-free coculture model. Finally, the antifibrotic effect of decorin was investigated by studying its effect on the expression of extracellular matrix components. RESULTS: Syndecan-2 and FGF-2 were upregulated in keloid tissue; decorin was downregulated. Normal and keloid fibroblasts treated with serum led to increase in syndecan-2 and decrease in decorin expression. Under coculture conditions, syndecan-2 was shed in the conditioned media. FGF-2 was also upregulated under coculture conditions and, when added to fibroblast monocultures, increased shedding of syndecan-2. Decorin levels were upregulated under coculture conditions only in normal cocultures. Decorin was also able to decrease extracellular matrix proteins, highlighting its importance as an antifibrotic agent. CONCLUSION: Syndecan-2 and FGF-2 are not only overexpressed in keloid tissues but may interact with each other resulting in the shedding of syndecan-2, which in turn might activate a whole cascade of events responsible for a keloidic phenotype. In addition, decorin had an antifibrotic effect and could well be used as a potential therapeutic agent for keloids.

Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts.

We previously reported an increase in signal transducer and activator of transcription 3 (Stat3) activation in keloid fibroblasts, which contributes to collagen production, cell proliferation, and migration. We further investigated the effect of epithelial-mesenchymal interaction on Stat3 in normal and keloid fibroblasts in noncoculture and coculture conditions. pY705 Stat3 was higher in keloid fibroblasts compared to normal fibroblasts in noncoculture. However, a more drastic decrease in pY705 Stat3 was observed in keloid fibroblasts compared to normal fibroblasts when cocultured with their respective keratinocytes over 5 days. To explore this paracrine effect, we examined the secretion of cytokines by cytokine arrays. Altered cytokine production was detected in keloid fibroblasts and keratinocytes, either in noncoculture or coculture conditions. IL-6, IL-8, monocyte chemoattractant protein-1, tissue inhibitor of metalloproteinases (TIMPs)-1, and TIMP-2 were major cytokines detected. Angiogenin, oncostatin M (OSM), vascular endothelial cell growth factor, IGF-binding protein-1, osteoprotegerin, and transforming growth factor-beta2 were present in keloid keratinocyte-fibroblast coculture, but absent in normal keratinocyte-fibroblast coculture. Only IL-6 and OSM stimulated strong pY705 Stat3 and cell proliferation in both normal and keloid fibroblasts. Other cytokines increased proliferation of keloid fibroblasts, but not normal fibroblasts, suggesting an altered state in keloid fibroblasts. Multiple cytokines likely contribute to keloid pathogenesis and a combinatorial neutralizing antibody/cytokine therapy may be effective in ameliorating keloid scars.

Innervation of the Face Studied Using Modifications to Sihler's Technique in a Primate Model.

BACKGROUND: There has been no reliable technique with which to display the innervation within whole-tissue specimens of the face. Such a technique preserves the architecture of the facial muscles and provides new data on intramuscular and sensory neural networks. Sihler's technique preferentially stains myelinated nerves within whole tissue, which is rendered transparent. On transillumination, entire neural networks can be studied in situ without the need for dissection and histologic examination. The purpose of this study was to apply Sihler's technique to study innervation patterns of the face, define end points, and improve specimen transillumination. METHODS: Eight macaque fascicularis monkeys were studied. The mimetic muscles of the face with intact facial nerve and sensory nerves were harvested as whole tissue composites. Sihler's technique was modified with formalin fixation before dissection to minimize autolysis of the myelin sheath. Prolonged immersion in glycerin improved tissue transparency. A replica of the skull was made with silicone and a light source embedded to restore three-dimensional configuration and provide transillumination. RESULTS: The facial nerve and sensory nerves were clearly seen up to their terminations in the transparent muscle and soft tissue. Observations were made with regard to the extramuscular and intramuscular innervation patterns of the facial nerve and sensory nerve patterns. CONCLUSIONS: Sihler's technique is a simple and reliable method with which to study the innervation of the face. This process may be applied to the human face to provide a much-needed roadmap to surgery, and the primate model may be developed for the study of facial reinnervation, facial reanimation, and dynamic facial transplantation.

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.

The role of the activin system in keloid pathogenesis.

Keloid scars represent a pathological response to cutaneous injury under the regulation of many growth factors. Activin-A, a dimeric protein and a member of the transforming growth factor-beta superfamily, has been shown to regulate various aspects of cell growth and differentiation in the repair of the skin mesenchyme and the epidermis. Thus our aim was to study the role of activin and its antagonist, follistatin, in keloid pathogenesis. Increased mRNA expression for activin was observed in keloid scar tissue by performing RNase protection assay. Immunohistochemistry showed increased localization of both activin-A and follistatin in the basal layer of epidermis of keloid tissue compared with normal tissue. ELISA demonstrated a 29-fold increase in concentration of activin-A and an approximately 5-fold increase in follistatin in conditioned media in keloid fibroblasts compared with normal fibroblasts. Although keloid keratinocytes produced 25% more follistatin than normal keratinocytes, the amounts of activin-A, in contrast, was approximately 77% lower. Proliferation of fibroblasts was stimulated when treated with exogenous activin-A (46% increase in keloids fibroblasts) or following co-culture with hbetaAHaCaT cells (66% increase). Activin-A upregulated key extracellular matrix components, namely collagen, fibronectin, and alpha-smooth muscle actin, in normal and keloid fibroblasts. Co-treatment of follistatin with activin-A blocked the stimulatory effects of activin on extracellular matrix components. These findings emphasize the importance of the activin system in keloid biology and pathogenesis and suggest a possible therapeutic potential of follistatin in the prevention and treatment of keloids.

Upregulation of secretory connective tissue growth factor (CTGF) in keratinocyte-fibroblast coculture contributes to keloid pathogenesis.

Connective tissue growth factor (CTGF) plays a critical role in keloid pathogenesis by promoting collagen synthesis and deposition. Previous work suggested epithelial-mesenchymal interactions as a plausible factor affecting the expression of various growth factors and cytokines by both the epithelial and dermal mesenchymal cells. The aim of this study is to explore the role of epithelial-mesenchymal interactions in modulating CTGF expression. Immunohistochemistry was employed to check CTGF localization in skin tissue. Western blot assay was performed on total protein extracts from skin tissue, cell lysates and conditioned media to detect the basal/expression levels of CTGF. Study groups were subjected to serum stimulation (fibroblast-single cell culture) and pharmacological inhibitors targeted against mTOR (Rapamycin), Sp1 (WP631 and Mitoxanthrone), Smad3 (SB431542), and PI3K (LY294002). Increased localization of CTGF in the basal layer of keloid epidermis and higher expression of CTGF was observed in the keloid tissue extract. Interestingly, lower basal levels of CTGF was observed in fibroblast cell lysates cocultured with keloid keratinocytes compared to normal keratinocytes, while the conditioned media from the former culture consistently demonstrated a higher expression of secreted CTGF as compared to the latter group. These results demonstrate an important role of epithelial-mesenchymal interactions in the regulation of CTGF expression. Fibroblasts treated with inhibitors against mTOR, Sp1, Smad3, and PI3K demonstrated a reduced expression of CTGF, suggesting these signaling pathways to be important in the regulation of CTGF expression. Thus, revealing the therapeutic potentials for inhibitors that are selective for these factors in controlling CTGF expression in fibrotic conditions.

Differential transcriptional responses of keloid and normal keratinocytes to serum stimulation.

BACKGROUND: Keloids are benign tumors that occur only in response to injury, for which there is no effective treatment. We demonstrated previously that keloid keratinocytes (KKs) promote fibroblast proliferation more than normal keratinocytes (NKs) and that transforming growth factor (TGF)-beta is a component of that signal. We used the transcriptional response to serum stimulation to examine how TGF-beta expression is stimulated in KKs. MATERIALS AND METHODS: Quiescent KKs and NKs were stimulated using serum; harvested using RNA at 0, 1, 6, 12, and 24 h; and analyzed using quantitative real-time polymerase chain reaction. TGF-beta activity in the conditioned medium was measured with an MLEC/PAI-luciferase assay. Inhibition of ERK1/2, p38 kinase, and JNK pathways was performed with PD98059, SB203580, and SP600125, respectively. RESULTS: Increased transcription of TGF-beta2 occurs within 1 h of serum stimulation in KKs but not in NKs. In contrast, TGF-beta3 transcription was suppressed in KKs compared with NKs. No significant differences were observed in the transcriptional response of TGF-beta1. Increased TGF-beta2 mRNA correlated with increased TGF-beta biological activity in the conditioned medium. Inhibition of the ERK, p38 kinase or JNK signal transduction pathways blocked the transcriptional up-regulation of TGF-beta2, TbetaR1, and TbetaR2 in KKs. CONCLUSIONS: KKs produce more TGF-beta2 mRNA than NKs in response to serum stimulation, resulting in increased TGF-beta activity in conditioned medium. Combining these results with our previous data lead us to propose a model of keloid formation characterized by an exaggerated response to cellular stress and abnormal epithelial-mesenchymal signaling promoting keloid formation.

Increased transcriptional response to mechanical strain in keloid fibroblasts due to increased focal adhesion complex formation.

Clinicians have observed that keloids preferentially form in body areas subject to increased skin tension. We hypothesized a difference exists in the transcriptional response of keloid fibroblasts to mechanical strain compared with normal fibroblasts. Normal and keloid fibroblasts were seeded in a device calibrated to deliver a known level of equibiaxial strain. We examined the transcriptional response of TGF-beta isoforms and collagen Ialpha, genes differentially expressed in keloids. Keloid fibroblasts produced more mRNA for TGF-beta1, TGF-beta2, and collagen Ialpha after mechanical strain compared to normals, and this was correlated with protein production. Inhibiting the major mechanical signal transduction pathway with the ERK inhibitor, U0126, blocked upregulation of gene expression. In addition, keloid fibroblasts formed more focal adhesion complexes as measured by immunofluorescence for focal adhesion kinase, integrin beta1, and vinculin. Finally, there is increased activation of focal adhesion kinase when we detected the phosphorylated form of focal adhesion kinase with immunofluorescence and immunoblotting. In summary, keloid fibroblasts have an exaggerated response to mechanical strain compared to normal fibroblasts leading to increased production of pro-fibrotic growth factors. This may be one molecular mechanism for the development of keloids.

Suppression of transforming growth factor beta/smad signaling in keloid-derived fibroblasts by quercetin: implications for the treatment of excessive scars.

BACKGROUND: Keloids are characterized by abnormal proliferation and overproduction of extracellular matrix. Quercetin, a dietary compound, has strong antioxidant and anticancer properties. Previous studies by the authors have shown that quercetin inhibits fibroblast proliferation, collagen production, and contraction of keloid and hypertrophic scar-derived fibroblasts. Quercetin also blocks the signal transduction of insulin-like growth factor-1 in keloid fibroblasts. This study assessed the effects of quercetin on the transforming growth factor (TGF)-beta/Smad-signaling pathway in keloid-derived fibroblasts, which may be an important biologic mechanism of this proliferative scarring. METHODS: Keloid fibroblasts were isolated from keloid tissue specimens. Cells were treated with quercetin at different concentrations, then harvested, and subjected to immunoblotting analysis. RESULTS: Quercetin significantly inhibited the expression of TGF-beta receptors 1 and 2 in keloid fibroblasts at three concentrations (low, medium, and high). Quercetin also strongly suppressed the basal expression of Smad2, Smad3, and Smad4 as well as the phosphorylation of Smad2 and Smad3 and the formation of the Smad2-Smad3-Smad4 complex. CONCLUSIONS: Taken together, these data suggest that quercetin effectively blocks the TGF-beta/Smad-signaling pathway in keloid fibroblasts. These data indicate that quercetin-based therapies for keloids should be investigated further.

Complex epithelial-mesenchymal interactions modulate transforming growth factor-beta expression in keloid-derived cells.

Keloids are proliferative dermal growths representing a pathologic wound healing response. We have previously demonstrated that coculture of fibroblasts derived from either keloid or normal skin have an elevated proliferation rate when cocultured with keloid-derived keratinocytes vs. normal keratinocytes. In these studies, we examined the contribution of transforming growth factor-beta (TGF-beta) to this phenomenon using a two-chamber coculture system. Fibroblast proliferation in coculture was slower with the addition of a pan-TGF-beta neutralizing antibody. Keloid keratinocytes in coculture expressed more TGF-beta1, -beta3, and TGF-beta receptor 1 than normal keratinocytes. Keloid fibroblasts cocultured with keloid keratinocytes expressed more mRNA for TGF-beta1, -beta2, TGF-beta receptor 1, and Smad2. Keloid fibroblasts also produced more type I collagen, connective tissue growth factor, and insulin-like growth factor-II/mannose-6-phosphate receptor when cocultured with keloid keratinocytes vs. normal keratinocytes. Levels of total and activated TGF-beta activity increased when fibroblasts were cocultured with keratinocytes, correlating with the changes in transcriptional activity of TGF-beta. In conclusion, we find a complex paracrine interaction regulates TGF-beta mRNA expression and activation between keratinocytes and fibroblasts. These data suggest that keloid pathogenesis may result from both an increased TGF-beta production and activation by the keloid keratinocyte, and elevated TGF-beta expression, utilization, and signaling in keloid fibroblasts.

From scarless fetal wounds to keloids: molecular studies in wound healing.

Surgical researchers were among the first to describe the different phases of wound healing and the events in tissue repair and regeneration that were taking place during each phase. The understanding of these events has been significantly enhanced in recent years by modern techniques in molecular and cellular biology. In this article, we discuss new findings in scarless fetal repair, angiogenesis in wound healing, and keloid pathogenesis. This serves to highlight the advances that have been made and also how much remains to be understood.

Synchronous activation of ERK and phosphatidylinositol 3-kinase pathways is required for collagen and extracellular matrix production in keloids.

Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2-5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I-III, laminin beta2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I-III production and significantly decreased laminin beta2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I-III and laminin beta2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.

Role of IGF system of mitogens in the induction of fibroblast proliferation by keloid-derived keratinocytes in vitro.

Keloids are proliferative dermal growths representing a pathological wound-healing response. We report high proliferation rates in normal (NF) and keloid-derived fibroblasts (KF) cocultured with keloid-derived keratinocytes (KK). IGF binding protein (IGFBP)-3 mRNA and secreted IGFBP-3 in conditioned media were increased in NF cocultured with KK compared with NF but markedly reduced in KF cocultured with KK or normal keratinocytes (NK). IGFBP-2 and IGFBP-4 mRNA levels were elevated, whereas IGFBP-5 mRNA was decreased in KF cocultured with KK or NK. Significant increases in IGFBP-2 and -4 mRNA in KF cocultured with KK did not correlate with protein secretion. Downstream IGF signaling cascade components, phospho-Raf, phospho-MEK1/2, phospho-MAPK, PI-3 kinase, phospho-Akt, and phospho-Elk-1, were elevated in KF cocultured with KK. Addition of recombinant human IGFBP-3 or antibodies against IGF-I or IGF-IR significantly inhibited proliferation of KF. The bioavailability of IGF-I may be related to the levels of IGFBP-3 produced, which in turn influences KF proliferation, suggesting that modulation of IGF-I, IGF-IR, and IGFBP-3, individually or in combination, may represent novel approaches to the treatment of keloids.

Fibroblasts cocultured with keloid keratinocytes: normal fibroblasts secrete collagen in a keloidlike manner.

Keloid scars represent a pathological response to cutaneous injury, reflecting a new set point between synthesis and degradation biased toward extracellular matrix (ECM) collagen accumulation. Using a serum-free two-chamber coculture model, we recently demonstrated a significant increase in normal fibroblast proliferation when cocultured with keloid-derived keratinocytes. We hypothesized that similar keratinocyte-fibroblast interactions might influence fibroblast collagen production and examined conditioned media and cell lysate from coculture for collagen I and III production by Western blot, allied with Northern analysis for procollagen I and III mRNA. Normal fibroblasts cocultured with keloid keratinocytes produced increased soluble collagen I and III with a corresponding increase in procollagen I and III mRNA transcript levels. This was associated with decreased insoluble collagen from cell lysate. When keloid fibroblasts were cocultured with keloid keratinocytes, both soluble and insoluble collagen were increased with associated procollagen III mRNA upregulation. Transmission electron microscopy of normal fibroblasts cocultured with keloid keratinocytes showed an ECM appearance similar to in vivo keloid tissue, an appearance not seen when normal fibroblasts were cocultured with normal keratinocytes.