Abdi Ghaffari

Role of keratinocyte–fibroblast cross‐talk in development of hypertrophic scar

The ability to generate or repair injured tissue is essential to the continuity of human life. As in all other organs, wound healing in the skin is a dynamic process involving tissue response to different types of insults. This process involves a continuous sequence of signals and responses in which platelets, fibroblasts, epithelial, endothelial, and immune cells come together outside their usual domains to orchestrate a very complex event that results in tissue repair. These signals, which are mainly growth factors and cytokines, orchestrate the initiation, continuation, and termination of wound healing. An imbalance in the synthesis and release of these cytokines and growth factors at the wound site, therefore, may result in either retarded wound healing, as is seen in diabetic patients and the elderly population, or overhealing wounds such as fibroproliferative disorders frequently seen following surgical incision, traumatic wounds, and severe electrical and thermal injury. In general, regardless of the site of injury, in any phase of the dynamic healing process, a fine balance between synthesis of extracellular matrix and degradation by a large family of enzymes, known as matrix metalloproteinases, is required for maintaining the structural integrity of healing tissue. The availability of new models such as organotypic co‐culture systems have allowed us to gain new insight into the cell–cell interactions at both cellular and molecular levels. Recent evidence indicates that mesenchymal–epithelial interactions play a critical role in regulation of skin homeostasis and this cross‐talk is mediated by soluble factors acting as autocrine/paracrine regulators of fibroblast and keratinocyte growth, function, and differentiation. In this review we address the question of how keratinocyte–fibroblast interaction plays a role in controlling the expression of key extracellular matrix molecules such as matrix metalloproteinases, which are critical in the healing process following any types of insults to the skin.

Gaseous nitric oxide bactericidal activity retained during intermittent high-dose short duration exposure

Previously, we have shown that gaseous Nitric oxide (gNO) has great potential as an effective topical anti-infective agent for non-healing wounds due to its non-specific antimicrobial properties. These same antimicrobial attributes may be useful for pulmonary infections. However, gNO would have limited usefulness as an inhaled antimicrobial agent as continuous exposure to the concentration required for a bactericidal effect (160-200 ppm) leads to methemoglobinemia. To overcome this problem, we investigated whether a thirty minute exposure of 160 ppm every four hours would retain the same antimicrobial effect as continuous delivery. In vitro, exposure of clinical multi-drug resistant Staphylococcus aureus and Escherichia coli strains isolated from the lungs of nosocomial pneumonia patients and a lethal antibiotic-resistant strain of Pseudomonas aeruginosa, isolated from a deceased cystic fibrosis patient resulted in over a 5 log(10) reduction in bacterial load after multiple thirty minute treatments (4 cycles) every four hours to 160 ppm gNO. The intermittent regimen required 320 (SD=0)ppm h for 100% lethality whereas the continuous exposure required 800 (SD=160)ppm h. We have also shown that selection for a gNO resistant phenotype did not lead to decrease sensitivity to gNO therapy (p>0.05). In addition, no host cellular toxicity was observed in human THP-1 monocytes and macrophages following intermittent delivery of a high concentration of gNO, and the proliferation and migration of pulmonary epithelial cells was not adversely affected by the administration of intermittent high-dose gNO. These results justify further studies that should focus on whether intermittent delivery of 160 ppm of gNO every four hours can technically be administered while keeping inhaled NO(2) levels less than 2 ppm and methemoglobin saturation less than 2.5 percent.

Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin.

Termination of wound‐healing process requires a fine balance between connective tissue deposition and its hydrolysis. Previously, we have demonstrated that keratinocyte‐releasable stratifin, also known as 14‐3‐3 σ protein, stimulates collagenase (MMP‐1) expression in dermal fibroblasts. However, role of extracellular stratifin in regulation of extracellular matrix (ECM) factors and other matrix metalloproteinases (MMPs) in dermal fibroblast remains unexplored. To address this question, large‐scale ECM gene expression profile were analyzed in human dermal fibroblasts co‐cultured with keratinocytes or treated with recombinant stratifin. Superarray pathway‐specific microarrays were utilized to identify upregulation or downregulation of 96 human ECM and adhesion molecule genes. RT‐PCR and Western blot were used to validate microarray expression profiles of selected genes. Comparison of gene profiles with the appropriate controls showed a significant (more than twofold) increase in expression of collagenase‐1, stromelysin‐1 and ‐2, neutrophil collagenase, and membrane type 5 MMP in dermal fibroblasts treated with stratifin or co‐cultured with keratinocytes. Expression of type I collagen and fibronectin genes decreased in the same fibroblasts. The results of a dose–response experiment showed that stratifin stimulates the expression of stromelysin‐1 (MMP‐3) mRNA by dermal fibroblasts in a concentration‐dependent fashion. Furthermore, Western blot analysis of fibroblast‐conditioned medium showed a peak in MMP‐3 protein levels 48 h following treatment with recombinant stratifin. In a lasting‐effect study, MMP‐3 protein was detected in fibroblast‐condition medium for up to 72 h post removal of stratifin. In conclusion, our results suggest that keratinocyte‐releasable stratifin plays a major role in induction of ECM degradation by dermal fibroblasts through stimulation of key MMPs, such as MMP‐1 and MMP‐3. Therefore, stratifin protein may prove to be a useful target for clinical intervention in controlling excessive wound healing in fibrotic conditions. J. Cell. Biochem. 98: 383–393, 2006.

Keratinocyte-Conditioned Media Regulate Collagen Expression in Dermal Fibroblasts

Excessive extracellular matrix (ECM) production during dermal wound healing often leads to fibrotic conditions such as keloids and hypertrophic scarring (HSc). Type I collagen is the predominant form of collagen in the human skin and is produced mainly by dermal fibroblasts. It has been suggested that abnormalities in epidermal-dermal interaction can lead to excessive production of collagen by fibroblasts. To identify and further characterize any possible keratinocyte-derived collagen-inhibitory factors (KD-CIFs), we investigated the expression of pro-alpha1(I) collagen at the level of mRNA and protein in human fibroblasts that had been either co-cultured with keratinocytes or treated with keratinocyte-conditioned medium (KCM). Fibroblasts in both groups demonstrated a significant reduction in the steady-state levels of collagen mRNA and protein. Further characterization of KD-CIFs revealed a high-molecular-weight factor (>30 kDa) that showed stable activity at high temperature (56 degrees C) and acidic pH (pH 2). Keratinocyte differentiation did not alter the release of KD-CIFs into KCM. These results provide further evidence that type I collagen expression and synthesis in fibroblasts are regulated by a keratinocyte-releasable factor(s) with an apparent molecular weight between 30 and 50 kDa.

Efficacy of gaseous nitric oxide in the treatment of skin and soft tissue infections

Bacterial burden significantly interferes with the healing process in chronic ulcers. Nitric oxide (NO) plays a key role in regulating skins response to infection and wound healing. In previous studies, we demonstrated that exogenous NO gas (gNO) at 200u2003 parts per million (ppm) exhibits potent antimicrobial effects against a representative range of pathogens. The aim of the present study is to explore the antimicrobial properties of gNO in vivo and to determine skin cells sensitivity to the cytotoxic effects of gNO. To test gNOs antimicrobial effects, full‐thickness wounds were infected with Staphylococcus aureus on the dorsal skin surface of New Zealand White rabbit and treated with 200u2003ppm gNO for 8 hours/day for 3 consecutive days. Significant reduction in wound bacterial content was observed in the presence of gNO. In a separate experiment, primary cultures of human fibroblasts, keratinocytes, and endothelial cells were established to test gNOs cytotoxicity in the skin. Methyl thiazolyl tetrazolium proliferation assays demonstrated that human skin cells, unlike bacterial cells, exhibited significant resistance toward gNO cytotoxicity. In vitro migration studies on keratinocytes and endothelial cells revealed that gNO treatment does not seem to interfere with reepithelialization and angiogenesis during the process of wound healing. Following 24 hours of gNO treatment, fibroblasts expressed significantly higher levels of procollagen and, to a lesser degree, a decrease in matrix metalloproteinase ‐1 mRNA. In conclusion, the present study provides evidence for the potential application of high doses of gNO as an antimicrobial agent for the treatment of infection in chronic nonhealing ulcers or burn patients, without compromising the viability, and function of skin cells.

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.

14-3-3σ associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1

Matrix metalloproteinases (MMPs) are implicated in the degradation of the extracellular matrix during development and tissue repair, as well as in pathological conditions such as tumor invasion and fibrosis. MMP expression by stromal cells is partly regulated by signals from the neighboring epithelial cells. Keratinocyte-releasable 14-3-3σ, or stratifin, acts as a potent MMP-1-stimulatory factor in fibroblasts. However, its mechanism of transmembrane signaling remains unknown. Ectodomain biotin labeling, serial affinity purification and mass spectroscopy analysis revealed that the stratifin associates with aminopeptidase N (APN), or CD13, at the cell surface. The transient knockdown of APN in fibroblasts eliminated the stratifin-mediated p38 MAP kinase activation and MMP-1 expression, implicating APN in a receptor-mediated transmembrane signaling event. Stratifin deletion studies implicated its C-terminus as a potential APN-binding site. Furthermore, the dephosphorylation of APN ectodomains reduced its binding affinity to the stratifin. The presence of a phosphorylated serine or threonine residue in APN has been implicated. Together, these findings provide evidence that APN is a novel cell surface receptor for stratifin and a potential target in the regulation of MMP-1 expression in epithelial–stromal cell communication.

SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase‐1) and type I collagen in fibroblasts. The first one, we called it keratinocyte‐derived anti‐fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14‐3‐3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte‐derived collagen‐inhibiting factor(s) (KD‐CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte‐conditioned media. Using co‐immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid. J. Cell. Biochem. 113: 2622–2632, 2012.

Microarray‐based identification of aminopeptidase N target genes in keratinocyte conditioned medium‐stimulated dermal fibroblasts

Of the many processes that affect the outcome of wound repair, epidermal–dermal interactions are essential to extracellular matrix (ECM) remodeling and in particular, soluble factors released by keratinocytes are known to have a direct impact on the production of ECM by dermal fibroblasts. Aminopeptidase N (APN) has recently been proposed as a cell‐surface receptor for stratifin and is responsible for the stratifin‐mediated matrix metalloproteinase‐1 (MMP‐1) upregulation in fibroblasts. The present study examines whether modulation of APN gene expression has any impact on the fibroblast ECM gene expression profile. The result reveals that in the presence of keratinocyte‐derived soluble factors, transient knockdown of APN in dermal fibroblasts affects the expression of key ECM components such as fibronectin, tenascin‐C, MMP‐1, MMP‐3, and MMP‐12. The regulatory effects of APN on fibronectin and selective MMPs appear to be associated with receptor‐mediated signal transduction independently of its peptidase activity. On the contrary, inhibition of the APN enzymatic activity by bestatin significantly reduces the tenascin‐C expression and enhances the contraction of fibroblast‐populated collagen gel, suggesting an activity‐dependent regulation of fibroblast contractility by APN. The overall effects of APN on the expression of fibronectin, tenascin‐C, and MMPs in fibroblasts propose an important role for APN in the regulation of keratinocyte‐mediated ECM remodeling and fibroblast contractile activity. J. Cell. Biochem. 113: 1061–1068, 2012.

Paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin†

As wound healing proceeds into the tissue remodeling phase, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in the skin, which is largely mediated through paracrine signaling and greatly affects the molecular constitution of the extracellular matrix. We have recently identified aminopeptidase N (APN)/CD13 as a potential fibroblast receptor for 14‐3‐3 sigma (also known as stratifin), a keratinocyte‐releasable protein with potent matrix metalloproteinase 1 (MMP1) stimulatory activity. The present study demonstrates that the expression of APN on dermal fibroblasts is regulated through paracrine signaling by keratinocyte‐derived soluble factors. By using an in vitro keratinocyte‐fibroblast co‐culture system, we showed that APN expression in dermal fibroblasts is induced in the presence of keratinocytes or in response to keratinocyte‐conditioned medium. Conditioned medium collected from differentiated keratinocytes further increases APN protein production, suggesting an amplified stimulatory effect by keratinocyte differentiation. Recombinant stratifin potently induces APN synthesis in a dose‐dependent manner. A consistent correlation between the protein expression levels of APN and MMP1 was also observed. These results confirm paracrine regulation of APN expression in dermal fibroblasts by keratinocyte‐derived stimuli, in particular stratifin, and provide evidence that APN may serve as a target in the regulation of MMP1 expression in epidermal–mesenchymal communication. J. Cell. Physiol. 226: 3114–3120, 2011.