Abelardo Medina

Identification of fibrocytes in postburn hypertrophic scar.

Fibrocytes are a unique leukocyte subpopulation implicated in wound healing. They are derived from peripheral blood mononuclear cells, display fibroblast‐like properties, and synthesize extracellular matrix macromolecules. This study investigated whether fibrocytes are present in healing burn wounds and whether the number of fibrocytes in tissue correlates with the degree of burn injury and the development of hypertrophic scar. Proteins extracted from cultured fibrocytes and nonadherent lymphocytes were found to be similar using two‐dimensional gel electrophoresis and quite distinct from those obtained from fibroblasts. However, one protein, identified as leukocyte‐specific protein 1 using mass spectrometric peptide mapping, was found in significantly larger amounts in fibrocytes than in lymphocytes but was undetectable in fibroblasts. Double immunostaining with antibodies to leukocyte‐specific protein‐1 and to the N‐terminal propeptide of type I collagen was performed on cryosections of hypertrophic scar, mature scar, and normal skin. Fibrocytes were seen in scar tissue as dual‐labeled spindle‐shaped cells but were absent from normal skin. Moreover, the number of fibrocytes was higher in hypertrophic than in mature scar tissue. We conclude that fibrocytes, which have been reported to be antigen‐presenting cells, are recruited to wounds following extensive burn injury and could potentially upregulate the inflammatory response and synthesize collagen and other matrix macromolecules, thus contributing to the development of hypertrophic scarring.

Local Expression of Indoleamine 2,3 Dioxygenase in Syngeneic Fibroblasts Significantly Prolongs Survival of an Engineered Three-Dimensional Islet Allograft

OBJECTIVE The requirement of systemic immunosuppression after islet transplantation is of significant concern and a major drawback to clinical islet transplantation. Here, we introduce a novel composite three-dimensional islet graft equipped with a local immunosuppressive system that prevents islet allograft rejection without systemic antirejection agents. In this composite graft, expression of indoleamine 2,3 dioxygenase (IDO), a tryptophan-degrading enzyme, in syngeneic fibroblasts provides a low-tryptophan microenvironment within which T-cells cannot proliferate and infiltrate islets. RESEARCH DESIGN AND METHODS Composite three-dimensional islet grafts were engineered by embedding allogeneic mouse islets and adenoviral-transduced IDO–expressing syngeneic fibroblasts within collagen gel matrix. These grafts were then transplanted into renal subcapsular space of streptozotocin diabetic immunocompetent mice. The viability, function, and criteria for graft take were then determined in the graft recipient mice. RESULTS IDO-expressing grafts survived significantly longer than controls (41.2 ± 1.64 vs. 12.9 ± 0.73 days; P < 0.001) without administration of systemic immunesuppressive agents. Local expression of IDO suppressed effector T-cells at the graft site, induced a Th2 immune response shift, generated an anti-inflammatory cytokine profile, delayed alloantibody production, and increased number of regulatory T-cells in draining lymph nodes, which resulted in antigen-specific impairment of T-cell priming. CONCLUSIONS Local IDO expression prevents cellular and humoral alloimmune responses against islets and significantly prolongs islet allograft survival without systemic antirejection treatments. This promising finding proves the potent local immunosuppressive activity of IDO in islet allografts and sets the stage for development of a long-lasting nonrejectable islet allograft using stable IDO induction in bystander fibroblasts.

The role of stratifin in fibroblast-keratinocyte interaction

Stratifin is a member of 14-3-3 protein family, a highly conserved group of proteins constituted by seven isoforms. They are involved in numerous crucial intracellular functions such as cell cycle and apoptosis, regulation of signal transduction pathways, cellular trafficking, cell proliferation and differentiation, cell survival, and protein folding and processing, among others. At epidermal level, stratifin (also called 14-3-3 sigma) has been described as molecule with relevant functions. For instance, this isoform is a marker associated with keratinocyte differentiation. In this maturation process, the presence of dominant negative molecules of p53 induces a “stemness condition” of keratinocyte precursor cells and suppression of stratifin expression. In addition, the recently described keratinocyte-releasable form of stratifin is involved in dermal fibroblast MMP-1 over-expression through c-Fos and c-Jun activity. This effect is mediated, at least in part, by p38 mitogen-activated protein kinase (MAPK). Other MMP family members such as stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), neutrophil collagenase (MMP-8), and membrane-type MMP-24 (MT5-MMP) are also up-regulated by stratifin. Within fibroproliferative disorder of skin, hypertrophic scar and keloids exhibit a high content of collagen, proteoglycans, and fibronectin. Thus, the MMP profile induced by stratifin is an interesting starting point to establish new therapeutic tools to control the process of wound healing. In this review, we will focus on site of synthesis and mode of action of stratifin in skin and wound healing.

Identification of different isoforms of 14-3-3 protein family in human dermal and epidermal layers

We have previously demonstrated a high level of stratifin, also known as 14-3-3 sigma (σ) in differentiated keratinocyte cell lysate and conditioned medium (CM). In this study, we asked the question of whether other 14-3-3 isoforms are expressed in human dermal fibroblasts, keratinocytes, intact dermal and epidermal layers of skin. In order to address this question, total proteins extracted from cultured cells or skin layers were subjected to western blot analysis using seven different primary antibodies specific to well-known mammalian isoforms, β, γ, ε, η, σ, τ, and ζ of 14-3-3 protein family. The autoradiograms corresponding to each isoform were then quantified and compared. The results revealed the presence of very high levels of all seven isoforms in cultured keratinocyte and conditioned medium. With the exception of τ isoform, other 14-3-3 isoforms were also present in intact epidermal layer of normal skin. The profile of 14-3-3 proteins in whole skin was similar to that of epidermis. In contrast, only gamma (γ) 14-3-3 isoform, was present in dermal layer obtained from the same skin sample. On the other hand, cultured fibroblasts express a high level of β, ε, γ and η and a low level of ζ and τ, but not σ isoform. However, the levels of 14-3-3 ε, γ and η were barely detectable in fibroblast conditioned medium. Further, we also used immunohistochemical staining to identify the 14-3-3 isoform expressing cells in human skin sections. The finding revealed different expression profile for each of these isoforms mainly in differentiated keratinocytes located within the layer of lucidum. However, fibroblasts located within the dermal layer did not show any detectable levels of these proteins. In conclusion, all members of 14-3-3 proteins are expressed by cells of epidermal but not dermal layer of skins and that these proteins are mainly expressed by differentiated keratinocytes.

Characterization of heterotopic ossification in burn patients.

Heterotopic ossification (HO) is a clinical condition of ectopic bone formation in soft tissue. This clinical entity has been associated with genetic disorders, traumatic injuries, and musculoskeletal surgeries. In this regard, functional impairments secondary to scar contractures seen in burn injuries may be exacerbated with underlying HO. The appropriate prevention or management of this complication is crucial to optimize outcome in burn patients. This clinical study reviews the incidence of HO in our burned patients, diagnostic methods, therapeutic approaches including surgical timing and techniques.

Deep dermal fibroblast profibrotic characteristics are enhanced by bone marrow–derived mesenchymal stem cells

Hypertrophic scars are a significant fibroproliferative disorder complicating deep injuries to the skin. We hypothesize that activated deep dermal fibroblasts are subject to regulation by bone marrow–derived mesenchymal stem cells (BM‐MSCs), which leads to the development of excessive fibrosis following deep dermal injury. We found that the expression of fibrotic factors was higher in deep burn wounds compared with superficial burn wounds collected from burn patients with varying depth of skin injury. We characterized deep and superficial dermal fibroblasts, which were cultured from the deep and superficial dermal layers of normal uninjured skin obtained from abdominoplasty patients, and examined the paracrine effects of BM‐MSCs on the fibrotic activities of the cells. In vitro, deep dermal fibroblasts were found higher in the messenger RNA (mRNA) levels of type 1 collagen, alpha smooth muscle actin, transforming growth factor beta, stromal cell–derived factor 1, and tissue inhibitor of metalloproteinase 1, an inhibitor of collagenase (matrix metalloproteinase 1). As well, deep dermal fibroblasts had low matrix metalloproteinase 1 mRNA, produced more collagen, and contracted collagen lattices significantly greater than superficial fibroblasts. By co‐culturing layered fibroblasts with BM‐MSCs in a transwell insert system, BM‐MSCs enhanced the fibrotic behavior of deep dermal fibroblasts, which suggests a possible involvement of BM‐MSCs in the pathogenesis of hypertrophic scarring.

Circulating monocytes have the capacity to be transdifferentiated into keratinocyte-like cells

Transdifferentiation is a process in which the original commitment of a cell is changed to give rise to unexpected peripheral mature cells. Our previous report showed that circulating stem cells can generate keratinocyte‐like cells (KLCs). However, it remains to be determined whether or not other peripheral blood mononuclear cells (PBMC) subsets have the potential to follow the same cell fate. In this study, the cell transdifferentiation of circulating CD14+ monocytes into KLCs and their regulatory effect on matrix metalloproteinase‐1 (MMP‐1) expression in dermal fibroblasts were evaluated. The results showed that monocytes isolated from peripheral blood mononuclear cells have the capacity to generate KLCs. These transdifferentiated cells exhibited, along with a keratinocyte‐like morphology, a characteristic profile consisting in stratifin+, cytokeratins+ (types I and II), CD14low, and involucrin+ on day 21 in culture. Similar to keratinocyte‐conditioned media, KLC‐derived conditioned media were able to induce an increase in the MMP‐1 expression in dermal fibroblasts. This effect was significantly reduced by using 14‐3‐3 protein‐depleted KLC‐conditioned media. Our findings show the potential transdifferentiation of circulating CD14+ monocytes into KLCs and their regulatory effect on MMP‐1 expression in dermal fibroblasts.

Fibrocytes can be reprogrammed to promote tissue remodeling capacity of dermal fibroblasts

Fibroblasts play a pivotal role in wound healing process participating in both tissue fibrosis and remodeling. However, it remains unclear which factors activate such diversity of fibroblast responses and how this decision-making process is made. Previous reports have demonstrated that wound milieu stimulates the transformation of circulating precursor cells into fibrocytes. These pro-fibrogenic cells promote the collagen production by resident fibroblasts. Conversely, recruited cells with anti-fibrogenic profile that can compete with fibrocytes have not been identified. This report describes a novel transdifferentiation process of fibrocytes induced by changing culture conditions. The reprogrammed fibrocytes markedly increased cell proliferation and MMP-1 expression in dermal fibroblasts. The MMP-1 up-regulation was directly related to the number of fibrocytes that followed this cell transformation. In vitro and in vivo results have confirmed that TGF-β deprivation plays an important role in this novel fibrocyte differentiation pathway. Our findings demonstrate that, changing the fibrocyte commitment, it is possible to exponentially stimulate the tissue remodeling capacity of dermal fibroblasts. These results will open new research approaches to understand the role of cell transdifferentiation and local environment not only in the wound healing process of skin, but also in several other fibrocyte-associated diseases such as lung fibrosis, asthma, liver cirrhosis, chronic pancreatitis, and atherosclerosis.

Reprogrammed fibrocytes induce a mixed Th1/Th2 cytokine response of naïve CD4 + T cells

Naïve CD4+ T cells develop different effector T cells and cytokine profiles after antigenic stimulation. It has been previously documented that fibrocytes function as antigen presenting cells inducing proliferation as well as Th2 cytokine response in naïve CD4+ T cells. Our group has reported that several circulating cell types recruited to the wound site can be transformed into anti-fibrotic profile cells, which subsequently induce MMP-1 stimulation in dermal fibroblasts. Here, we report how similar reprogramming pathway of fibrocytes could modify the CD4+ T cell response. Our findings confirmed that reprogrammed fibrocytes induce CD4+ T cell activation with a mixed Th1/Th2 cytokine response. Since a reciprocal positive feedback between Th2 cells and fibrocytes exist to amplify and perpetuate the pro-fibrotic stimulation in dermal fibroblasts, the novel transdifferentiation of regular mature fibrocytes into reprogrammed fibrocytes appears to be a promising strategy to reverse the Th2 cytokine overproduction, and subsequently control the local fibrogenesis.

Modified Meek Micrografting Technique for Wound Coverage in Extensive Burn Injuries

The modified Meek micrografting technique constitutes a rapid and efficient surgical approach for the skin coverage of extensive full-thickness burn injuries. A total of 10 burn patients (mean 68 ± 9.2% TBSA) admitted to our burn unit required one or more Meek micrografting procedures (mean 2.2 ± 0.5) to cover in average 43.4 ± 11.6% TBSA (range between 10 and 75% TBSA). This goal was achieved using a donor site area ranging between 2.5 and 18% TBSA. All patients developed local infection to Pseudomona aeruginosa (75%), Stenotrophomona maltophilia (25%), methicillin-resistant Staphylococcus aureus (12.5%), and Acinetobacter baumannii (12.5%). Thus, the average of Meek regrafting after graft-take failure was 13.1 ± 6.4% TBSA (median: 9%; range from 0 to 36%). The period to obtain stable definitive wound closure was in average of 67.2 ± 21 days post injury. The modified Meek micrografting provides a reliable and versatile method for the coverage of large burn wounds with limited autograft donor sites and is now routinely used in our institution. Its systematic use improves operating times and overall outcomes reducing the number of surgeries, increasing the percentage of graft take, and decreasing the length of stay.