Tatyana Yufit

Low oxygen tension stimulates collagen synthesis and COL1A1 transcription through the action of TGF-β1

Recent findings point to low oxygen tension (hypoxia) as an important mechanism for the expression of several eukaryotic genes. We have previously shown that hypoxia (2% O2), when compared to standard oxygen tension (20% O2), upregulates the mRNA levels of the human α1(I) (COL1A1) procollagen gene and transforming growth factor‐beta1 (TGF‐β1) in human dermal fibroblasts. In this report, we determined the effect of hypoxia on collagen synthesis and transcription. Exposure of human dermal fibroblasts to hypoxia for 24–72 h led to a threefold, dose‐dependent increase in collagenous protein (P < 0.0001; r = 0.9794) and to enhanced type I procollagen deposition, as shown by direct immunofluorescence. Transient transfections with a series of luciferase‐ and CAT‐promoter constructs of the human COL1A1 gene (spanning from − 2.5 kb to + 113 bp) showed that hypoxia increases the transcriptional activity of constructs having 5′ endpoints between − 804 bp and − 107 bp, with loss of stimulation at − 84 bp. Maximal increase in promoter activity in hypoxia was observed between − 190 and − 174 bp of the proximal promoter, once a cKrox repressor site (− 199 to − 224 bp) was deleted. Upregulation of COL1A1 mRNA levels in hypoxia was blocked by a TGF‐β1 anti‐sense oligonucleotide, and failed to occur in fibroblasts from TGF‐β1 knock‐out mice. Co‐transfection and overexpression with a Smad7 construct abrogated the increase in COL1A1 promoter activity observed in hypoxia. Upregulated transcriptional activity of the TGF‐β1 promoter in hypoxia was found to be maximal between − 453 and − 175 bp from the transcriptional start site. Since hypoxia is a critical feature of the early phases of wound repair, we conclude that it may act as a potent physiologic stimulus for collagen synthesis. TGF‐β1 appears to be a critical component of this response. J. Cell. Physiol. 191: 42–50, 2002.

Fibroblasts from chronic wounds show altered TGF-β-signaling and decreased TGF-β Type II Receptor expression†

Chronic wounds are characterized by failure to heal in a defined time frame. However, the pathogenic steps leading from the etiological factors to failure to heal are unknown. Recently, increasing evidence suggests that resident cells in chronic wounds display a number of critical abnormalities, including senescence and unresponsiveness to the stimulatory action of transforming growth factor‐β1 (TGF‐β1). In this study, we have determined some of the mechanisms that might be responsible for unresponsiveness to TGF‐β1. Using Northern analysis and affinity labeling, we show that venous ulcer fibroblasts have decreased TGF‐β Type II receptor expression. This finding is not the result of genetic mutation, as shown by experiments with Type II receptor satellite instability. Decreased Type II receptor expression was accompanied by failure of ulcer fibroblasts to phosphorylate Smad 2, Smad 3, and p42/44 mitogen activating protein kinase (MAPK), and was associated with a slower proliferative rate in response to TGF‐β1. We conclude that venous ulcer fibroblasts show decreased Type II receptor expression and display abnormalities in the downstream signaling pathway involving MAPK and the early Smad pathway. These findings suggest ways to address and treat the abnormal cellular phenotype of cells in chronic wounds.

Modulation of cellular tryptophan metabolism in human fibroblasts by transforming growth factor-β: Selective inhibition of indoleamine 2,3- dioxygenase and tryptophanyl-tRNA synthetase gene expression

Alterations in the rate of cellular tryptophan metabolism are involved in mediating important biological activities associated with cytokines and growth factors. Indoleamine 2,3‐dioxygenase (IDO) and tryptophanyl‐tRNA synthetase are enzymes of tryptophan metabolism whose expression in a variety of cells and tissues is highly inducible by interferon‐γ (IFN‐γ). Transforming growth factor‐β (TGF‐β) antagonizes many cellular responses to IFN‐γ. The interaction of these two cytokines plays an important role in maintaining homeostasis during inflammation and repair. In human skin and synovial fibroblasts in vitro, TGF‐β caused time‐ and dose‐dependent abrogation of IFN‐γ‐stimulated expression of IDO and tryptophanyl‐tRNA synthetase mRNAs. The inhibition was selective and did not appear to be due to down‐regulation of IFN‐γ signaling by TGF‐β. In parallel with its effect on IDO mRNA expression, TGF‐β caused a marked reduction in intracellular IDO protein levels and abrogated IDO activity and tryptophan catabolism in these cells induced by IFN‐γ. IFN‐γ caused a rapid and striking increase in the amount of IDO heterogeneous nuclear pre‐mRNA and induced transcription of the IDO gene, as demonstrated by transient transfection assays. TGF‐β partially reversed this stimulation. IFN regulatory factor (IRF)‐1 and stat1 are cellular intermediates in IFN signaling. Both are implicated in activation of IDO transcription in response to IFN‐γ. The stimulation by IFN‐γ of IRF‐1 protein and mRNA expression was not prevented by treatment of fibroblasts with TGF‐β. Furthermore, gel mobility shift assays indicated that TGF‐β did not inhibit the induction of stat1 and IRF‐1 binding activity to their cognate DNA recognition sites in the IDO gene promoter. In contrast, the stability of IDO mRNA transcripts was reduced in fibroblasts treated with TGF‐β, as shown by determination of mRNA half‐lives following blockade of transcription with 5,6‐dichlorobenzimidazole riboside. The findings indicate that TGF‐β prevents the induction of IDO and tryptophanyl‐tRNA synthetase gene expression in fibroblasts. The repression of IDO expression by TGF‐β is mediated at both transcriptional and posttranscriptional levels. These results implicate TGF‐β in the negative regulation of tryptophan metabolism, provide evidence for the molecular basis of this regulation, and indicate that cellular tryptophan metabolism is under tight immunological control. J. Cell. Physiol. 177:174–186, 1998.

Slow release iodine preparation and wound healing: in vitro effects consistent with lack of in vivo toxicity in human chronic wounds.

Summary Background Antiseptic agents, particularly slow‐release preparations, are increasingly being used in the management of chronic wounds. One such agent, cadexomer iodine, carries iodine (0·9% weight/weight) immobilized in beads of dextrin and epichlorhydrin and has been demonstrated to be highly effective in promoting healing of exudative wounds. However, there have been no studies directly assessing the potential lack of toxicity of cadexomer iodine on human cutaneous tissues.

Negative modulation of α1(I) procollagen gene expression in human skin fibroblasts: Transcriptional inhibition by interferon-γ

Interferon‐γ (IFN‐γ), a multifunctional cytokine produced by activated Th1 lymphocytes, exerts potent effects on the extracellular matrix by regulating fibroblast function. In this study, we examined the modulation of α1(I) procollagen gene (COL1A1) expression by recombinant IFN‐γ. The results showed that IFN‐γ stimulated the rapid accumulation of interferon regulated factor (IRF)‐1 mRNA, followed by a delayed and dose‐dependent inhibition of α1(I) procollagen mRNA expression in skin fibroblasts from several different donors. The inhibitory response was abrogated in fibroblasts stably expressing IRF‐1 in the antisense orientation. A marked decrease in the amount of heterogeneous nuclear pre‐mRNA preceded the inhibition of COL1A1 mRNA expression. In fibroblasts transiently transfected with COL1A1 promoter–chloramphenicol acetyltransferase reporter gene plasmids, IFN‐γ selectively inhibited promoter activity and abrogated its stimulation induced by TGF‐β. The inhibition by IFN‐γ was not due to downregulation of TGF‐β receptor mRNA expression in the fibroblasts or decreased ligand binding to the receptor. IFN‐α and IFN‐β by themselves had little effect on promoter activity, but IFN‐α augmented the inhibitory effect of IFN‐γ. Using a series of 5′ deletion constructs, a proximal region of the COL1A1 promoter was shown to function as an IFN‐γ response element. This region of the gene harbors overlapping binding sites for transcription factors Sp1, Sp3, and NF‐1 but no homologs of previously characterized IFN‐γ response elements. The putative IFN‐γ response region was sufficient to confer inhibition of reporter gene expression by treatment with IFN‐γ. Gel mobility shift analysis showed that two distinct and specific DNA‐protein complexes were formed when fibroblast nuclear extracts were incubated with oligonucleotides spanning the IFN‐γ response region. IFN‐γ did not modify the ability of nuclear proteins to bind to this region. The results indicate that IFN‐γ inhibits COL1A1 expression in fibroblasts principally at the level of gene transcription. Inhibition involves IRF‐1 and is mediated through a short proximal promoter segment but without an apparent change in promoter occupancy. The findings provide novel insight into the mechanism of IFN‐γ regulation of fibroblast function. J. Cell. Physiol. 179:97–108, 1999.

Human β-defensin-2 expression is increased in chronic wounds

First identified in psoriatic epidermis and subsequently in other inflammatory cutaneous lesions, human β‐defensin‐2 (hβD‐2) is one of two endogenous antimicrobial peptides related to defensins in plants and animals. Our objective was to determine the expression of hβD‐2 after injury and in chronic wounds. Biopsies of normal ipsilateral thigh skin and wound edges were taken from nine consecutive patients with venous leg ulcers (day 1) and from the same biopsy sites 2 days later (day 3). Sequential samples were also obtained from intact or meshed bilayered bioengineered skin consisting of neonatal human keratinocytes and dermal fibroblasts in a collagen matrix. Specimens were processed and immunostained for hβD‐2 using a polyclonal rabbit antibody. In both human tissues and bioengineered skin, staining for hβD‐2 was confined to the upper epidermal layers, sparing the basal cells. Analysis of 26 tissue samples from patients showed that normal skin had no hβD‐2 expression but that marked up‐regulation occurred after wounding by day 3. Conversely, chronic ulcers showed moderate‐to‐strong immunostaining for hβD‐2 at baseline on day 1, with little or no change in intensity after wounding by day 3. In vitro, bioengineered skin showed increased distribution of cytoplasmic hβD‐2 immunostaining after meshing. We conclude that the expression of hβD‐2 is up‐regulated after injury. Chronic wounds uniformly show a constitutively high baseline expression of hβD‐2, possibly due to ongoing tissue injury and bacterial colonization.

Control of Extracellular Matrix Degradation by Interferon-γ

Interleukin-1β (IL-1β) is a potent signal for the induction of the matrix-degrading enzymes collagenase and stromelysin. These metalloproteinases (MMP) play a critical role in physiologic and pathologic connective tissue remodeling, and are potential targets for therapeutic manipulation. Treatment of human dermal fibroblasts with interferon-γ inhibited Type I collagen gene expression, and abrogated the effect of IL-1β on MMP expression. Interferon-γ also caused a dramatic dose-dependent increase in indoleamine 2,3-dioxygenase mRNA, with consequent depletion of tryptophan and accumulation of kynurenine in the culture media. To examine the role of tryptophan metabolism in the effects of interferon-γ on matrix-degrading enzymes, exogenous tryptophan was added to tryptophan-depleted media, followed by stimulation of the cultures with IL-1β. Supplementation with tryptophan completely overcame the inhibitory effects of interferon-γ on MMP mRNA expression and metalloproteinase secretion into the media. In contrast, mRNA levels for Type I collagen remained profoundly depressed in interferon-γ-treated cultures in spite of addition of exogenous tryptophan. These results indicate that oxidative tryptophan metabolism mediates the effects of interferon-γ on MMP gene expression in human fibroblasts.

Differential keratin expression during epiboly in a wound model of bioengineered skin and in human chronic wounds.

Epiboly represents the process by which keratinocytes migrate to envelop a surface. The authors have been investigating a living bilayered skin construct (BSC) that is used in the treatment of lower extremity wounds due to venous insufficiency and diabetes. The construct demonstrates epiboly after injury and incubation in vitro, and this model may be useful for studying epidermal migration and the process of skin maturation. Punch biopsies of the construct in vitro were cultured and immunostained for specific keratins at baseline and at 24 to 72 hours. For comparison, skin biopsy specimens from human chronic venous ulcers and acute healing wounds were similarly processed. The authors found that K1 and K10 were fully expressed in the epidermis of the fully epibolized surface on BSC. K1 was also present in the migrating edge of specimens, whereas K10 was not detectable. K16 and K6 were evident in normal skin and the epibolized area of the construct; K6 expression was very prominent in the migrating edge. Importantly, K17 was distinctly limited to the epibolized surface and the migrating edge, and its expression was very similar to that observed in healing human wounds. In conclusion, differential expression of keratins in this epiboly model closely reflects in vivo studies and supports keratin specificity in the processes of migration and differentiation of new epidermis. Therefore, these findings provide further and important validity for the study of epithelialization and the hope of developing prognostic markers for venous ulcer healing.

Bone Marrow Cell Mobilization by the Systemic Use of Granulocyte Colony-Stimulating Factor (GCSF) Improves Wound Bed Preparation

Innovative approaches are needed to accelerate the healing of human chronic wounds not responding to conventional therapies. An evolving and promising treatment is the use of stem cells. Our group has previously described the use of expanded (in vitro) autologous stem cells aspirated from human bone marrow and applied topically in a fibrin spray to human acute and chronic wounds. More recently, we have sought ways to mobilize stem cells directly from the bone marrow, without in vitro expansion. In this report, we show that systemic injections of granulocyte colony-stimulating factor (GCSF) can mobilize stem cells from bone marrow into the peripheral blood and then to the wound site. Our objectives were to optimize parameters for this method by using mouse models and proof of principle in a human chronic wound situation. Mice were injected for 5 days with 2 different formulations of GCSF and compared to control saline. To monitor stem cell mobilization, flow cytometric measurements of Sca-1 and c-Kit and colony-forming cell assays were performed. Full-thickness tail wounds in mice were created and monitored for healing, and polyvinyl alcohol sponges were implanted dorsally to assess collagen accumulation. To determine bone marrow stem cell homing to the wound site, chimeric mice transplanted with Green Fluorescent Protein bone marrow cells were scanned by live imaging. Additionally, as proof of principle, we tested the systemic GCSF approach in a patient with a nonhealing venous ulcer. Our findings lay the ground work and indicate that the systemic administration of GCSF is effective in mobilizing bone marrow stem cells into the peripheral blood and to the wound site. These findings are associated with an increased accumulation of collagen and promising results in terms of wound bed preparation and healing.

Topical delivery of cultured stem cells to human non-healing wounds: GMP facility development in an academic setting and FDA requirements for an IND and human testing.

With increasing emphasis on translational research, the need for appropriate regulatory oversight and approval has become essential. The requirements of the Food and Drug Administration (FDA) for Investigational New Drug (IND) exemption in studies that are investigator-initiated have become increasingly stringent. Moreover, academic institutions have not had substantial experience in establishing Good Manufacturing Practice (GMP) facilities required for manipulating human cells in vitro and for chemical or biochemical manufacturing. GMP regulations are established by the FDA under the authority of the Federal Food, Drug and Cosmetic Act. In this report, the authors outline the general strategy and some critical steps that an investigator and the institution may find helpful in developing a GMP facility, especially in an academic center. Also, more specifically and as proof of principle, we describe our approach to culturing autologous bone marrow-derived human mesenchymal stem cells (MSCs) and delivering them to non healing wounds. The lessons learned in this often lengthy and challenging process may be helpful to other academic institutions and investigators embarking on manipulating and delivering viable cells for human experimentation.