Neonatal Skin Fibroblasts Research Articles

The Human Neonatal Skin Fibroblast, an Available Cell Source for Tissue Production and Transplantation, Exhibits Low Risk of Immunogenicity In Vitro.

The immunogenicity of allogeneic skin fibroblasts in transplantation has been controversial. Whether this controversy comes from a natural heterogeneity among fibroblast subsets or species-specific differences between human and mouse remains to be addressed. In this study, we sought to investigate whether fibroblasts derived from either adult or neonatal human skin tissues could induce different immune responses toward phagocytosis and T cell activation using in vitro co-culture models. Our results indicate that both phagocytosis and T cell proliferation are reduced in the presence of neonatal skin fibroblasts compared to adult skin fibroblasts. We also show that neonatal skin fibroblasts secrete paracrine factors that are responsible for reduced T cell proliferation. In addition, we show that neonatal skin fibroblasts express less class II human leukocyte antigen (HLA) molecules than adult skin fibroblasts after interferon gamma priming, which might also contribute to reduced T cell proliferation. In conclusion, this study supports the use of allogeneic neonatal skin fibroblasts as a readily available cell source for tissue production and transplantation to treat patients with severe injuries.

Abstract PO012: In silico drug repurposing identifies mebendazole as a treatment option for chemoresistant hepatoblastoma

Abstract Resistance to conventional chemotherapy remains a huge challenge in the clinical management and treatment of hepatoblastoma, the most common liver tumor of childhood. The in silico repurposing of drugs with known pharmacokinetics and safety profiles, which have been approved for other indications, represents a promising strategy to identify new drugs effective in chemoresistant hepatoblastoma. In this study, we integrated RNA sequencing-derived gene expression data into the pharmacologic perturbation prediction tool Connectivity Map (CMap) and identified the clinical widely used anthelmintic drug mebendazole as a putative drug to circumvent chemoresistance in hepatoblastoma. Consequently, we experimentally validated the efficacy of mebendazole in permanent and patient-derived xenograft cell lines that are resistant to cisplatin, the therapeutic backbone of hepatoblastoma treatment. Viability assays clearly indicated a potent reduction of tumor cell growth upon mebendazole treatment in a dose-dependent manner. In contrast, similar mebendazole concentrations had little inhibitory effect on the growth of normal neonatal and adult skin fibroblasts. The combination of mebendazole and cisplatin revealed a strong synergistic effect, which was comparable to the one seen with cisplatin and doxorubicin (cardiotoxic), the current standard treatment for high-risk hepatoblastoma patients. Moreover, mebendazole treatment resulted in reduced colony formation capability, induction of apoptosis, and cell cycle arrest of hepatoblastoma cells. Immunofluorescent staining of alpha-tubulin showed that mebendazole causes blockage of microtubule formation and subsequent RNA sequencing analyses confirmed the transcriptional downregulation of tubulins as the major mode of action of mebendazole treatment. Most importantly, mebendazole significantly reduced tumor growth in a subcutaneous xenograft transplantation mouse model. In conclusion, our results strongly support the clinical use of mebendazole as a replacement for doxorubicin in the treatment of chemoresistant hepatoblastoma. This could potentially not only improve outcome but also reduce severe long-term side effects. Citation Format: Qian Li, Salih Demir, Xuanwen Bao, Alexandra Wagner, Yanxin Fan, Stefano Cairo, Roland Kappler. In silico drug repurposing identifies mebendazole as a treatment option for chemoresistant hepatoblastoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO012.

No mitigation of IFN-β and HLA class I expression in early sub-cultured human neonatal skin fibroblasts but both molecules are overexpressed in starved cells

Allogeneic fibroblasts containing-dermal substitutes are known as neutral allografts. There is evidence that successive subculturing of fibroblasts leads to a decrease in HLA class I expression. The aim of this study was to investigate this downregulation and its correlation with the expression of interferon-beta (IFN- β) as a HLA class I regulating cytokine. This correlation was also measured under serum starvation that is prevalent in wounds, especially in burn wounds, and that is also used in pre-transplantation fibroblasts culture to manufacture bioengineered skin substitutes on an industrial scale. Fibroblasts isolated from neonatal foreskin were cultured for eight successive passages in DMEM supplemented with bovine serum (n = 3). Those cells were also cultured for different time points in DMEM without serum (n = 4). Flow cytometry, Elisa, and real-time PCR methods were used to determine the levels of HLA class I expression, IFN-β protein and IFN-β mRNA respectively. The expression of HLA class I molecules was not significantly altered by cell subculturing (p > 0.05). Interestingly, these cells also produced substantial levels of IFN-β -mRNA and -protein with non-significant alterations upon successive sub-culturing (p > 0.05). However, HLA expression in starved fibroblasts was significantly raised in a timely-dependent manner (r2 = 0.84, p < 0.001), with strong correlation with IFN-β concentrations (r2 = 0.86, p < 0.0001).These data reveal that sub-cultured allogeneic fibroblasts are not HLA negative cells supported by cells spontaneous production of IFN-β, a multifunctional immunoregulatory cytokine. Even the levels of this antigen can be increased in association with IFN-β up-regulation in certain stressful conditions, such as growth factor deprivation.These findings open a new window in transplantation immunology for immuneregulatory roles of metabolic stress- IFN-β-HLA axis leading to successful allogeneic HLA-unmatched organ transplantation in immunocompetence recipients. Moreover these data can be applied in the optimization of conditions for pre-clinical fibroblasts culture to the better designation of human dermal substitutes.

The Expression and Degradation of SM22-Alpha/Transgelin are Regulated by Mechanical Tension in the Cytoskeleton

SM22-alpha, also named transgelin, encoded by the gene TAGLN is a calponin-related protein found in smooth muscle, fibroblast and cancer cells. SM22-alpha was discovered three decades ago but its biological function remains unclear. In addition to an application as a differentiation marker for smooth muscle cells, SM22-alpha has been reported to regulate the structure and dynamics of actin cytoskeleton and cell motility in fibroblasts and cancer cells. Here we report a novel finding that the expression and degradation of SM22-alpha/transgelin are both regulated by mechanical tension. Mass spectrometry detected that SM22-alpha was significantly decreased in mouse aortic rings after incubation under low mechanical tension. Using specific monoclonal antibodies developed against chicken gizzard SM22-alpha, we found high levels of SM22-alpha in human fetal lung fibroblast cells line MRC-5 and primary neonatal mouse skin fibroblasts. Similar to that of calponin 2, the level of SM22-alpha is positively dependent on the mechanical tension in the cytoskeleton as determined by the stiffness of culture substrate. Quantitative RT-PCR demonstrated a transcriptional regulation of TAGLN gene expression by mechanical tension in the cytoskeleton. The cellular localization of SM22-alpha overlaps with that of myosin IIA and blebbistatin inhibition of myosin motor decreased the expression of SM22-alpha. The level of SM22-alpha is decreased in skin fibroblasts isolated from calponin 2 knockout mice compared to that in calponin 2-positive wild type cells, suggesting their correlated functions. With the close phylogenetic relationship between TAGLN and the calponin genes, SM22-alpha is identified as a calponin-like cytoskeleton regulatory protein. These findings laid a groundwork for understanding the physiological function of SM22-alpha in mechanoregulation of cytoskeleton and cell motility and its relationship with calponins.

Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity.

ObjectiveProduction of alpha-1,3-galactosyltransferase (αGT)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous αGT knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce αGT-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts.MethodsMiniature pig fibroblasts were transfected with αGT gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous αGT gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-α-1,3-galactose, an epitope produced by αGT. Using magnetic activated cell sorting, cells with monoallelic disruption of αGT were removed. Remaining cells with LOH carrying biallelic disruption of αGT were used for the second round NT to produce homozygous αGT gene-targeted piglets.ResultsMonoallelic mutation of αGT gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous αGT gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous αGT knockout piglets.ConclusionThe present study demonstrates that the time required for the production of αGT-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.

MUC1 Is Expressed by Human Skin Fibroblasts and Plays a Role in Cell Adhesion and Migration

The mucin MUC1 is expressed by normal and cancerous epithelial cells and some nonepithelial cells in which it plays roles in regulating adhesion, migration, and cell signaling. In the present studies we found that MUC1 is expressed by normal human neonatal and adult skin fibroblasts. Fibroblasts are usually considered negative for MUC1 expression. Reverse-transcription polymerase chain reaction and Western blot analyses indicate the presence of full-length MUC1, and immunofluorescence and subcellular fractionation studies show that the protein is expressed on the plasma membrane. Immunohistochemical analyses confirmed the expression of MUC1 by fibroblasts in cryosections of normal human skin. Silencing MUC1 expression in fibroblasts using MUC1 shRNA increased the adhesion of cells to collagen and laminin. Transfection with MUC1 shRNA also increased fibroblast migration on collagen as measured in a wound-healing assay. The expression of α2-integrin was increased in MUC1 shRNA-transfected fibroblasts in which it was localized to membrane ruffles, providing a possible explanation for the increased cell migration on collagen. These results extend the range of expression of MUC1 to skin fibroblasts and suggest a functional role for MUC1 in fibroblast adhesion and motility.

39 EFFECT OF FOLLICULAR SIZE OF CYTOPLAST DONOR ON CLONING EFFICIENCY IN GOATS

In goats, oocytes derived from small follicles (&lt;3 mm in diameter) have low developmental competence after IVF (Crozet et al. 1995). However, their performance is compatible to oocytes derived from large follicles (&gt;3 mm) when intracytoplasmic sperm injection is used (Jimenez-Macedo et al. 2006). No data are currently available on the ability of oocytes derived from small follicles to support development to term after somatic cell nuclear transfer (SCNT) in goats. Here we evaluated the effect of follicular size of cytoplast donor on cloning efficiency in goats. Seven rounds of oocyte maturation and nuclear transfer were carried out on separate days. During each replicate, oocytes were collected separately from large follicles (LF, n = 378) and small follicles (SF, n = 981) from the same group of ovaries. First, only large follicles (&gt;3 mm) were dissected and rinsed, and then an ovary was moved to another dish to dissect the rest of the follicles. The groups of oocytes derived from LF and SF were treated separately throughout the process including maturation, SCNT, embryo culture and embryo transfers. Neonatal skin fibroblasts derived from transgenic goats were used as nuclear donors in this study. Nine embryo transfers were conducted for LF group and 12 for the SF group. Oocytes from SF had significantly lower rate of nuclear maturation compared to oocytes derived from LF (33.5% ± 3.7 v. 54.3% ± 5.4; P &lt; 0.01). There was no difference in fusion rate between these 2 groups (66 v. 69%; 215/325 v. 138/199; SF v. LF). Additionally, we found no statistical difference in Day 30 pregnancy rate (42% v. 33%; SF v. LF group) or birth rate (2 healthy kids produced from each of the groups, 16.7% v. 22.2%; SF v. LF). The results demonstrate that oocytes from small follicles have similar competence to support development to term as oocytes derived from large follicles following SCNT in goats. This work was supported by the Utah Multidisciplinary Arrhythmia Consortium and Utah Agricultural Experiment Station project #1100.

Paracrine anti‐fibrotic effects of neonatal cells and living cell constructs on young and senescent human dermal fibroblasts

Senescent cells observed in the area of chronic wounds have been proposed to affect wound healing. Therapeutic approaches against chronic wounds include, among others, the local application of living cell constructs (LCCs), containing fibroblasts and/or keratinocytes. Accordingly, the aim of the present work was to examine the effects of factors secreted by early passage neonatal fibroblasts and LCCs--in the form of a conditioned medium (CM)--on senescent adult dermal fibroblasts regarding functions related to the healing process, i.e., cell proliferation, alpha-smooth muscle actin and metalloproteinase expression, and collagen synthesis. Target cells were fibroblasts senescent either due to subsequent divisions (replicative senescence) or due to an exogenous stress (stress-induced premature senescence). No effect on the proliferation of senescent fibroblasts was observed, as expected. All CMs were found to inhibit overall collagen synthesis both in early passage and in senescent fibroblasts. The LCC-derived CM was found to be more potent than fibroblast-derived CMs and, furthermore, to inhibit alpha-smooth muscle actin expression. In conclusion, these results may indicate anti-contractile and anti-fibrotic activities of factor(s) secreted by neonatal skin fibroblasts, and more intensely by LCCs on adult donor-derived fibroblasts. These activities seem to persist during senescence of the target cells.

41 RAPID PRODUCTION OF α-1,3-GALACTOSYLTRANSFERASE-DEFICIENT MINIATURE PIGS BY NUCLEAR TRANSFER FOLLOWING NEONATAL SKIN BIOPSY AND LOSS OF HETEROZYGOSITY

Production of α-1,3-galactosyltransferase (αGT)-deficient pigs is a fundamental task to overcome hyperacute rejection in pig-to-human xenotransplantation. However, the procedure to produce such pigs requires a great deal of cost, time, and labor. Heterozygote αGT knockout (KO) pigs should be bred at least for 2 generations to ultimately produce homozygote progenies. Here, we show that αGT-deficient miniature pigs could be produced in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Miniature pig fetal fibroblasts were transfected with αGT-targeting vector and treated with neomycin. Eighty-seven colonies were screened by PCR, and 2 among them were identified as homologous recombinants with monoallelic disruption of αGT (targeting efficiency = 2.3%). One of the two colonies was chosen and used for subsequent NT. In total, 559 nuclear transfer embryos reconstructed with αGT gene-targeted fetal fibroblasts were transferred to 5 surrogate gilts. Three gilts became pregnant and developed to term with 3 live piglets delivered. Being analyzed by PCR, all piglets born were found to be αGT gene targeted. Fibroblasts were isolated from ear skin biopsies of a 10-day-old αGT gene-targeted miniature pig. To increase the number of mitotic recombination that occurs spontaneously during cell proliferation, fibroblasts were cultured for 6 to 8 passages. Then, these cells were treated with IB4-conjugated Dynabeads; IB4 lectin binds to galactose-α-1,3-galactose (αGal), the antigen formed by αGT, and is responsible for hyperacute rejection (HAR). Using a magnetic column, fibroblasts bound to IB4 lectin were removed. Remaining cells lacking αGal were cultured further until colony formation. A total of 22 fibroblast colonies were screened by PCR, and 15 among them were confirmed as loss of heterozygosity (LOH) with biallelic disruption of αGT by mitotic recombination (LOH efficiency = 68%). One of these clonal cultures of αGT-deficient fibroblasts was chosen and used for subsequent NT. In total, 729 nuclear transfer embryos reconstructed with αGT-deficient neonatal ear skin fibroblasts were transferred to 4 surrogate gilts. Two gilts became pregnant, and 3 live piglets were produced. Based on PCR, both αGT alleles were targeted in all piglets born. Only 8 months were required from the production of heterozygote to homozygote KO in contrast to approximately 2 years consumed for conventional breeding of heterozygotes. The present study demonstrates that the time required for the production of αGT-deficient miniature pigs could be reduced significantly by neonatal skin biopsies and subsequent selection of mitotic recombinants. Such a procedure may be beneficial for the production of other homozygote KO animals, especially in species that require substantial periods of gestation such as pigs.

Melanogenesis Inhibitory and Fibroblast Proliferation Accelerating Effects of Noroleanane- and Oleanane-Type Triterpene Oligoglycosides from the Flower Buds of Camellia japonica

A 28-noroleanane-type triterpene oligoglycoside, camellioside E (4), an oleanane-type triterpene oligoglycoside, camellioside F (5), and the known compounds camelliosides A (1) and D (3) were isolated from a 50% EtOH extract of Camellia japonica flower buds from Korea. The principal constituents (1 and 5) significantly inhibited melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. Camellioside B (2), a major constituent of C. japonica grown in Japan, showed potent inhibition of melanogenesis [95.0 ± 1.0% (p < 0.01) at 20 μM]. The inhibitory effects of 1, 2, and 5 were stronger than that of the reference compound, arbutin. We believe the melanogenesis inhibitory effects of 2 and 5 are partly related to the proliferation inhibitory effects in B16 melanoma 4A5 cells. Conversely, camelliosides tended to enhance proliferation in normal human neonatal skin fibroblasts. Interestingly, camellioside B (2) significantly accelerated fibroblast proliferation. This biological selectivity could make camellioside B useful for treating skin disorders. Herein, we report the first scientific investigation of a triterpene that displays an inhibitory effect on melanogenesis, but that also has an enhancing effect on fibroblast proliferation.

Human Endometrial Cells Express Elevated Levels of Pluripotent Factors and Are More Amenable to Reprogramming into Induced Pluripotent Stem Cells

The human endometrium is a tissue with remarkable plasticity and regenerative capacity. Additionally, endometrial cells can be retrieved using minimally invasive procedures, which makes them an ideal source for reprogramming into a pluripotent state. Endometrial cells were obtained from donors in their fifth decade and reprogrammed into induced pluripotent stem (iPS) cells using retroviral transduction with SOX2, OCT4, KLF4, and MYC. The human endometrial cells displayed accelerated expression of endogenous NANOG and OCT4 during reprogramming compared with neonatal skin fibroblasts. As a result, iPS cell colonies that could be subcultured and propagated were established as early as 12 d after transduction rather than the usually reported 3-4 wk for other cell types. After 3 wk of reprogramming, the human endometrial cells also yielded significantly higher numbers of iPS colonies in comparison with the neonatal skin fibroblasts. Although the efficiency of iPS colony formation varied depending on the donor, the basal level of endogenous expression of the defined factors was positively correlated with reprogramming efficiency. The reprogramming resulted in an average colony-forming efficiency of 0.49 ± 0.10%, with a range from 0.31-0.66%, compared with the neonatal skin fibroblasts, resulting in an average efficiency of 0.03 ± 0.00% per transduction, with a range from 0.02-0.03%. Our studies show that the human endometrium expresses elevated levels of pluripotent factors, which with additional defined factors, results in significantly more efficient and accelerated generation of induced pluripotent stem cells compared with conventional somatic cells.

Stress-induced epinephrine levels compromise murine dermal fibroblast activity through β-adrenoceptors

Stress-induced catecholamine impairs the formation of granulation tissue acting directly in fibroblast activity; however, the mechanism by which high levels of catecholamines alter the granulation tissue formation is still unclear. Thus, the aim of this study was to investigate how high levels of epinephrine compromise the activity of murine dermal fibroblasts. Dermal fibroblasts isolated from the skin of neonatal Swiss mice were preincubated with α- or β-adrenoceptor antagonists. Thereafter, cells were exposed to physiologically elevated levels of epinephrine or epinephrine plus α- or β-adrenoceptor antagonists, and fibroblast activity was evaluated. The blockade of β1- and β2-adrenoceptors reversed the increase in fibroblast proliferation, ERK 1/2 phosphorylation, myofibroblastic differentiation and the reduction of collagen deposition induced by epinephrine. In addition, the blockade of β3-adrenoceptors reversed the increase in fibroblast proliferation and nitric oxide synthesis as well as the reduction of fibroblast migration, AKT phosphorylation and active matrix metalloproteinase-2 expression induced by epinephrine. However, the blockade of α1- and α2-adrenoceptors did not alter the effects of epinephrine on the activity of murine dermal fibroblasts. In conclusion, high levels of epinephrine directly compromise the activity of neonatal mouse skin fibroblasts through the activation of β1-, β2- and β3-adrenoceptors, but not through α1- and α2-adrenoceptors.

Direct Conversion of Skin Cells into Blood: Alchemy or Science?

Our neighborhood blood bank just announced an emergency Thanksgiving-season blood drive. For years, the field of stem cell biology has described an elusive but commonly understood goal: the elimination of a need for blood drives. Many years of work have led to methods for generation of bona fide hematopoietic cells from human and mouse embryonic stem cells, but problems with efficiency as well as maturation of the blood cells persist, impeding the utility of these methods in manufacturing blood. Now, in the era of “direct reprogramming,” we can imagine a goal of converting an adult cell, such as a skin cell, into a hematopoietic stem cell that can be used for autologous blood replacement (Figure 1). In an article recently published in Nature, Szabo et al. report a huge step toward that goal.1 The authors describe the generation of differentiated hematopoietic cells following the enforced expression of the POU-domain transcription factor OCT4 in adult or neonatal human skin fibroblasts.

Liposomes Encapsulating Aloe vera Leaf Gel Extract Significantly Enhance Proliferation and Collagen Synthesis in Human Skin Cell Lines

Aloe vela leaf gel extract (AGE) are widely used as cosmetic and pharmaceutical ingredients because of its versatile skin care properties. In order to enhance the bioavailability of AGE, liposomes encapsulating AGE were prepared and examined for their interfacial and biochemical properties. The liposomes prepared from a soybean lecithin (SLP-WHITE, 1.0 wt%) by the Bangham method gave relatively a good trapping efficiency up to the AGE concentration of 0.5 wt%. The stable liposomes were then prepared from 1.0 wt% of SLP-WHITE and different concentrations of AGE by the mechanochemical method using a homogenizer and microfluidizer. The liposomes obtained from 0.25 wt% of AGE were confirmed to be small unilamellar vesicles with a diameter of less than 200 nm, and remained well dispersed for at least two weeks. The obtained liposomes encapsulating AGE were further examined for the effects on proliferation and type I collagen synthesis in normal human neonatal skin fibroblasts, NB1RGB cells. Liposomal AGE clearly showed higher proliferation rate than that of AGE alone. In addition, compared to the control, liposomal AGE significantly increased the collagen synthesis by 23%, while AGE alone showed a small effect. Liposomal AGE was also assayed for the effect on proliferation in normal human epidermal keratinocytes, NHEK(F) cells. Interestingly, liposomal AGE fractions containing 4 and 20 microg/mL of the extract considerably increased the proliferation rate by 77% and 101%, respectively. In contrast, AGE alone fractions containing 4 and 20 microg/mL of the extract increased the rate by 41% and 60%, respectively. Accordingly, the bioavailability and skin care properties of AGE will be significantly enhanced by liposome encapsulation, and the present liposomal AGE should have a great potential as an effective skin care formulation.

Latent TGF-β-binding protein 2 binds to DANCE/fibulin-5 and regulates elastic fiber assembly

Elastic fibers play the principal roles in providing elasticity and integrity to various types of human organs, such as the arteries, lung, and skin. However, the molecular mechanism of elastic fiber assembly that leads to deposition and crosslinking of elastin along microfibrils remains largely unknown. We have previously shown that developing arteries and neural crest EGF-like protein (DANCE) (also designated fibulin-5) is essential for elastogenesis by studying DANCE-deficient mice. Here, we report the identification of latent transforming growth factor-beta-binding protein 2 (LTBP-2), an elastic fiber-associating protein whose function in elastogenesis is not clear, as a DANCE-binding protein. Elastogenesis assays using human skin fibroblasts reveal that fibrillar deposition of DANCE and elastin is largely dependent on fibrillin-1 microfibrils. However, downregulation of LTBP-2 induces fibrillin-1-independent fibrillar deposition of DANCE and elastin. Moreover, recombinant LTBP-2 promotes deposition of DANCE onto fibrillin-1 microfibrils. These results suggest a novel regulatory mechanism of elastic fiber assembly in which LTBP-2 regulates targeting of DANCE on suitable microfibrils to form elastic fibers.

Role of Cathepsin K in the Turnover of the Dermal Extracellular Matrix during Scar Formation

Cathepsins are a group of cysteine proteinases that are involved in various aspects of extracellular matrix turnover. The collagenolytic activity of cathepsin K plays a pivotal role in bone resorption and lung matrix homeostasis, but so far has not been described in skin. To study the role of cathepsin K in the turnover of the cutaneous extracellular matrix, we studied the expression of cathepsin K in human skin and in cultured primary neonatal skin fibroblasts. Normal skin exhibited only low levels or no expression of cathepsin K. In contrast, dermal fibroblasts in surgical scars showed strong cytoplasmic cathepsin K expression. Cathepsin K expression was most prominent in young scars and declined with time. Cultured neonatal primary fibroblasts showed strong cathepsin K staining in the perinuclear endosomal compartment, consistent with intracellular degradation of internalized collagen in lysosomes. Cathepsin K was also found to be strongly expressed in keloids and dermatofibromas, but not in sclerotic areas of morphea. Our data suggest that cathepsin K may play an important role in the homeostasis of the dermal extracellular matrix and the dynamic equilibrium between matrix synthesis and proteolytic degradation, by counteracting deposition of matrix proteins during scar formation with its matrix-degrading activity.

Guidance of engineered tissue collagen orientation by large-scale scaffold microstructures

The tensile strength and stiffness of load-bearing soft tissues are dominated by their collagen fiber orientation. While microgrooved substrates have demonstrated a capacity to orient cells and collagen in monolayer tissue culture, tissue engineering (TE) scaffolds are structurally distinct in that they consist of a three-dimensional (3-D) open pore network. It is thus unclear how the geometry of these open pores might influence cell and collagen orientation. In the current study we developed an in vitro model system for quantifying the capacity of large scale (∼200 μm), geometrically well-defined open pores to guide cell and collagen orientation in engineered tissues. Non-degradable scaffolds exhibiting a grid of 200 μm wide rectangular pores (1:1, 2:1, 5:1, and 10:1 aspect ratios) were fabricated from a transparent epoxy resin via high-resolution stereolithography. The scaffolds ( n = 6 per aspect ratio) were surface modified to support cell adhesion by covalently grafting GRGDS peptides, sterilized, and seeded with neonatal rat skin fibroblasts. Following 4 weeks of static incubation, the resultant collagen orientation was assessed quantitatively by small angle light scattering (SALS), and cell orientation was evaluated by laser confocal and scanning electron microscopy. Cells adhered to the struts of the pores and proceeded to span the pores in a generally circumferential pattern. While the cell and collagen orientations within 1:1 aspect ratio pores were effectively random, higher aspect ratio rectangular pores exhibited a significant capacity to guide global cell and collagen orientation. Preferential alignment parallel to the long strut axis and decreased spatial variability were observed to occur with increasing pore aspect ratio. Intra-pore variability depended in part on the spatial uniformity of cell attachment around the perimeter of each pore achieved during seeding. Evaluation of diamond-shaped pores [Sacks, M.S. et al., 1997. J. Biomech. Eng. 119(1), 124–127] suggests that they are less sensitive to initial conditions of cell attachment than rectangular pores, and thus more effective in guiding engineered tissue cell and collagen orientation.

Regulation of collagen synthesis in mouse skin fibroblasts by distinct angiotensin II receptor subtypes.

We examined the possibility of whether angiotensin (Ang) II type 1 (AT1) and type 2 (AT2) receptor stimulation differentially regulates collagen production in mouse skin fibroblasts. Both AT1 and AT2 receptors were expressed in neonatal skin fibroblasts prepared from wild-type mice to a similar degree, and the AT1a receptor was exclusively expressed as opposed to the AT1b receptor. In wild-type fibroblasts, Ang II increased collagen synthesis accompanied by an increase in expression of tissue inhibitor of metalloproteinase (TIMP)-1, and these increases were inhibited by valsartan, an AT1 receptor blocker, but augmented by PD123319, an AT2 receptor antagonist. Ang II decreased basal and IGF-I-induced collagen production and inhibited TIMP-1 expression in neonatal skin fibroblasts prepared from AT1a knockout (KO) mice. These Ang II-mediated inhibitory effects on collagen production and TIMP-1 expression observed in AT1a KO fibroblasts were attenuated by the addition of PD123319 or a tyrosine phosphatase inhibitor, sodium orthovanadate, but not affected by a serine/threonine phosphatase inhibitor, okadaic acid. Moreover, we demonstrated that transfection of a catalytically inactive, dominant negative SHP-1 (Src homology 2-containing protein-tyrosine phosphatase-1) mutant inhibited the Ang II-mediated inhibitory effect on both collagen synthesis and TIMP-1 expression in AT1a KO fibroblasts. These results suggest that AT1a receptor stimulation increases collagen production in skin fibroblasts at least in part due to the inhibition of collagen degradation via the increase in TIMP-1 expression, whereas AT2 receptor stimulation exerts inhibitory effects on TIMP-1 expression, which is mediated at least partially by the activation of SHP-1, thereby possibly inhibiting collagen production.

The effect of phosphorylation by casein kinase 2 on the activity of insulin-like growth factor-binding protein-3.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is known to be secreted as a phosphoprotein, constitutively phosphorylated at casein kinase 2 (CK2) sites. To examine the effect of phosphorylation by CK2 on the properties of glycosylated human IGFBP-3, we phosphorylated plasma-derived IGFBP-3, containing less than 1 mol/mol phosphoserine, in vitro. As judged by incorporated 32P, enzymatic deglycosylation did not decrease the phosphate content of phospho-IGFBP-3. Phosphorylation had no effect on IGF-I or IGF-II binding, but was inhibitory to acid-labile subunit binding in the presence of either IGF. Determined in simian virus 40-transformed human fibroblasts, cell association by phospho-IGFBP-3 was inhibited approximately 50% compared with that of the nonphosphorylated preparation. Phospho-IGFBP-3 showed significant resistance to proteolysis by plasmin and a cysteine protease secreted by MCF-7 cells. However, no difference was seen between the two preparations in their inhibition of IGF-I-stimulated DNA synthesis when coincubated with IGF-I in neonatal skin fibroblasts or MCF-7 breast cancer cells, and little difference was found in their ability to potentiate IGF-I-stimulated DNA synthesis when preincubated with fibroblasts. These results indicate that IGFBP-3 interaction with acid-labile subunit and with the cell surface, both of which involve basic carboxyl-terminal residues, may be modulated by phosphorylation. Relative resistance to proteolysis and poor binding to cells suggest that CK2-phospho-IGFBP-3 may be a significant inhibitor of IGF activity in the extracellular environment.

The proliferative capacity of neonatal skin fibroblasts is reduced after exposure to venous ulcer wound fluid: A potential mechanism for senescence in venous ulcers

Purpose: We have previously shown that fibroblasts cultured from venous ulcers display characteristics of senescence and have reduced growth rates. Susceptibility of young fibroblasts to the microcirculatory changes associated with venous ulcers, such as macrophage trapping and activation, could explain the prevalence of senescent fibroblasts in these wounds. Methods: We tested the in vitro effect of venous ulcer wound fluid (VUWF), as well as pro-inflammatory cytokines known to be present in VUWF (TNF-α, IL-1β, and TGF-β1), on neonatal foreskin fibroblasts (NFFs). NFF growth rates, cellular morphology, and senescence-associated β-galactosidase (SA-β-Gal) activity were determined in the presence or absence of VUWF and the above cytokines. VUWF TNF-α concentration and the effect of anti–TNF-α antibody on VUWF inhibitory activity were determined in samples obtained from four patients with venous ulcers. Results: NFF growth rates were significantly reduced by VUWF (42,727 ± 6301 vs 3902 ± 2191 P = .006). TNF-α also significantly reduced NFF growth rates in a dose-dependent manner ( P = .01). No significant growth-inhibitory activity was seen for IL-1α or TGF-β. Incubation with VUWF significantly increased the percentage of SA-β-Gal–positive fibroblasts in vitro on culture day 12 ( P = .02). TNF-α and TGF-β1 had similar effects. TNF-α was detected in all VUWF tested, with a mean of 254 ± 19 pg/mL. Conclusion: These data suggest that the venous ulcer microenvironment adversely affects young, rapidly proliferating fibroblasts such as NFFs and induces fibroblast senescence. Pro-inflammatory cytokines such as TNF-α and TGF-β1 might be involved in this process. The role of other unknown inhibitory mediators, as well as pro-inflammatory cytokines, in venous ulcer development and impaired healing must be considered. (J Vasc Surg 1999;30:734-43.)