Secreted collagen ratios in normal human and osteogenesis imperfecta skin fibroblasts

We examined the effects of several variables on the ratio of type I:type III collagen secreted by human caucasian skin fibroblasts in normal and osteogenesis imperfecta (OI) phenotypes. Isotopically labelled collagen extracted from fibroblast medium was analyzed by DEAE-cellulose chromatography and identified by appropriate methods. Type I procollagen was the major form of collagen secreted into the medium by normal cells cultured from one mid-term fetus, infants (n = 3), children (n = 3), adolescents (n = 2), and adults (n = 3). Interstrain differences in collagen production under standardized conditions were significantly greater than intrastrain variation (anova, p = 0.0051). There was no significant alteration in the type I:type III collagen ratio due to variation in: phase of cell growth, doublings (between 13th and 22nd), rate of isotope incorporation, labelling time (24-72 hrs) in the presence of ascorbic acid (50 micrograms/ml), age of donor (with the possible exception of adolescence), and site of biopsy (genital and non-genital sites). Variable conversion of type I procollagen to collagen did not perturb the type I:type III collagen ratio. Cell strains from OI patients (Sillence classification): type I (one strain); type II, III and IV (3 strains each) had greater interstrain than intrastrain variation in the collagen ratio (p = 0.0149). Interstrain differences were greater in OI cell strains relative to normal cell strains (p less than 0.01). In the aggregate, OI cells had significantly lower type I collagen production relative to type III (I/III ratio = 1.18) when compared with normal cells (I/III ratio = 2.90; t test, p less than 0.0001). These findings imply abnormal synthesis, secretion or stability of type I procollagen and greater phenotypic heterogeneity in OI skin fibroblasts relative to normal cells.     

Differential effects of human granulocytes and lymphocytes on human fibroblasts in vitro

Polymorphonuclear leucocytes (PMN) were found to damage human fibroblast monolayers. Allogeneic and autochthonous PMN were equally efficient and monolayer destruction (plaque-formation) occurred within 24 hr, as a rule, in the absence of agglutinating substances such as phytohaemagglutinin. Living PMN were not necessary for plaque-formation, since cells heated to 56°C for 30 min or disintegrated by ultrasound were still competent to produce plaques. It is suggested that enzymes enclosed in cytoplasmic granules in the PMN are responsible for plaque-formation. Although the monolayer was destroyed, the target cells were not killed after treatment with PMN, but detached from the surface of the culture vessel into the medium and could be recultivated from the supernatant. Heparin, chondroitin sulphate and trypan blue suppressed plaque-formation by intact and disintegrated PMN, while a variety of metabolic inhibitors or X-rays had no effect.

Lymphocytes from non-immunized donors were also competent to cause destruction of fibroblast monolayers, but this reaction required the presence of phytohaemagglutinin. Heating of the lymphocytes to 48·5°C or ultrasound disintegration abolished the plaque-forming ability, suggesting that living lymphocytes were required. In contrast to PMN, lymphocytes were shown to kill the target cells. PHA-treated lymphocytes were equally cytotoxic to allogeneic and autochthonous fibroblasts and it is suggested that actual target destruction is immunologically non-specific and caused by transformed lymphocytes, which acquire the ability to kill all fibroblast targets regardless of their genotype and the event triggering lymphocyte transformation.

     

The mechanism of daunorubicin-induced inhibition of prolidase activity in human skin fibroblasts and its implication to impaired collagen biosynthesis.

One of the recognized side effects accompanying antineoplastic anthracyclines administration is poor wound healing, resulting from impairement of collagen biosynthesis. However, the precise mechanism of anthracyclines-induced inhibition of collagen synthesis has not been established. We have suggested that prolidase, an enzyme involved in collagen metabolism may be one of the targets for anthracyclines-induced inhibition of synthesis of this protein. Prolidase [E.C. 3.4.13.9] cleaves imidodipeptides containing C-terminal proline, providing large amount of proline for collagen synthesis. We have found that daunorubicin (DNR) induced coordinately inhibition of prolidase activity (IC50 = 0.3 microM) and collagen biosynthesis (IC50 = 1 microM) in cultured human skin fibroblasts. The decrease in prolidase activity due to the treatment of confluent cells with DNR was not accompanied by any differences in the amount of the enzyme protein recovered from these cells as shown by western immunoblot analysis. Since prolidase is metaloprotease, requiring manganese for catalytic activity and anthracyclines are known as a chelators of divalent cations we considered that the chelating ability of anthracyclines may be an underlying mechanism for daunorubicin-induced inhibition of prolidase activity. In order to determine the ability of DNR to form complex with manganese (II), potentiometric method was employed based on the measurement of protonation constant by pH-metric titrated assay. We have found that DNR forms stable complex with manganese (II) and the composition of the complex of DNR with Mn (II) was calculated as 3:1. The constant stability value for the investigated complex was calculated as [beta(av) = (1.74 +/- 0.01) 10(23). The strong ability of DNR to chelate manganese may explain the potential mechanism for inhibition of prolidase activity, subsequently collagen biosynthesis and poor wound healing in patients administered DNR.     

细胞因子抑制剂吡非尼酮对体外培养人瘢痕成纤维细胞增殖的影响

背景：有研究表明细胞因子抑制剂吡非尼酮通过调控多种细胞因子,抑制成纤维细胞的生物学活性,其在内脏器官的抗纤维化作用的研究和应用取得了良好的进展,但对于皮肤增生性瘢痕及成纤维细胞是否有影响及其机制尚不清楚。 目的：观察细胞因子抑制剂吡非尼酮对人增生性瘢痕成纤维细胞增殖的影响。 方法：采用组织块法培养人增生性瘢痕成纤维细胞,实验取第3-6代生长状态良好的对数生长期细胞。根据吡非尼酮不同质量浓度分为对照组(吡非尼酮0 g/L),吡非尼酮0.15,0.3,1 g/L组,共干预12,36,48 h。 结果与结论：MTT,反转录-聚合酶链式反应和酶链免疫吸附实验结果显示,与对照组相比,吡非尼酮0.15,0.3,1 g/L组细胞增殖情况、转化生长因子β1 mRNA的表达、Ⅰ、Ⅲ型胶原蛋白的分泌量均降低(P < 0.05),其中吡非尼酮1 g/L组降低最明显(P < 0.05)。干预24,48,72 h后,吡非尼酮0.15,0.3,1 g/L组间细胞增殖抑制率、Ⅰ、Ⅲ型胶原蛋白的分泌量均差异有显著性意义(P < 0.05)。结果证实,细胞因子抑制剂吡非尼酮对体外培养的人增生性瘢痕成纤维细胞胶原蛋白的分泌、转化生长因子β1的表达及细胞增殖活性有明显的抑制作用。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Cross-talk between integrin receptor and insulin-like growth factor receptor in regulation of collagen biosynthesis in cultured fibroblasts

PurposeCellular processes are regulated by signals generated by adhesion receptors and growth factor receptors. IGF-binding protein 1 (IGFBP-1) is a molecule which may affect the both signaling pathways through inactivation of IGF-I (ligand for IGF-IR) and binding to RGD region of integrin receptors. Whether this phenomenon is important in communication between insulin-like growth factor receptor (IGF-IR) and β1-integrin receptor in regulation of prolidase activity and collagen biosynthesis is the aim of this study.Material and MethodWe studied the effects of IGFBP-1, IGF-I, thrombin (integrin activator), echistatin (disintegrin), phosphatidylinositol 3-kinase inhibitor (LY-294002) and ERK 1/2 inhibitors (PD98059 and UO126) on prolidase activity, collagen biosynthesis and expression of proteins participating in pathways generated by these receptors.ResultsStimulation of β1-integrin and IGF-I receptors by standard ligands was proved to up-regulate collagen synthesis in cultured fibroblasts. IGFBP-1, similarly as echistatin and studied inhibitors, contributed to down-regulation of ERK1/2, Akt, mTOR expression and up-regulation of NFκB. It was accompanied by parallel decrease in prolidase activity and collagen biosynthesis.ConclusionThe data suggest that “cross talk” between IGF-I receptor and integrin receptor may play important role in regulation of prolidase activity and collagen biosynthesis.     

Cell morphology, proliferation and collagen synthesis of human fibroblasts cultured on sepiolite-collagen complexes

The growth and morphology as well as collagen biosynthesis of human fibroblasts obtained and cultured on sepiolite-collagen complexes have been studied. No differences on cell morphology and growth properties nor collagen synthesis were observed when compared with standard culture substrates. The type I/type III ratio of biosynthesized collagen by fibroblasts cultured on sepiolite-collagen complexes was about 5-6 with no difference when compared to control conditions. This normal behavior was also observed for the type I/type III procollagens. According to these studies the sepiolite-collagen complexes do not modify the studied features of the fibroblasts.     

Serum stimulation of lysyl hydroxylase activity in cultured human skin fibroblasts

In the absence of ascorbic acid, confluent human skin fibroblasts incubated in 0.5% serum-supplemented medium had one-third of the level of lysyl hydroxylase activity of cells incubated in media containing high serum concentrations (5-20%). This difference appeared to be due to a decline in the enzyme activity following serum deficiency, and was largely abolished by addition of ascorbic acid to the medium. The effect of serum deficiency was slow, manifesting in 48 h at the earliest, and was completely reversed by replenishing the medium with serum. Prolyl hydroxylase activity was independent of serum concentration, both in the absence and in the presence of ascorbic acid in the culture medium.     

Differential effects of estradiol and progesterone on human T cell activation in vitro

Estradiol (E2) and progesterone (P4) are steroid hormones important for the regulation of immune responses during pregnancy. Their increasing levels coincide with an improvement of T cell-mediated diseases such as multiple sclerosis (MS). Although immune-endocrine interactions are involved in this phenomenon, the relative contribution of hormones is not known. We here report a direct comparison of E2- and P4-mediated effects on human CD4⁺ T cells, key cells in immune regulation. T cells were stimulated to obtain different activation levels and exposed to a broad range of hormone concentrations. Activation level was assessed by CD69/CD25 expression by flow cytometry, and secreted proteins (n = 196) were measured in culture supernatants using proximity extension assay and electrochemiluminescence immunoassay. We found that in low activated cells, pregnancy-relevant E2 concentrations increased activation and the secretion of several immune- and inflammation-related proteins. P4, on the other hand, showed a biphasic pattern, where serum-related concentrations upregulated activation and protein secretion while placenta-relevant concentrations induced a prominent dampening irrespective of the initial activation level. Our results demonstrate the importance of P4 as a major hormone in the immune modulation of T cells during pregnancy and emphasize the need to further evaluate its potency in the treatment of diseases like MS.     

The effects of triacetyloleandomycin and oleandomycin phosphate on the glucocorticoid receptor in cultured skin fibroblasts

Triacetyloleandomycin (TAO) has been described as a "steroid-sparing" drug in that poorly controlled asthmatic patients can be stabilized or improved with the addition of TAO despite decreasing dosages of steroids, specifically methylprednisolone (MP). It is not clear whether the beneficial effects of TAO are due to decreased MP clearance or are due to enhanced glucocorticoid effect peripherally. We tested the latter possibility by studying the interaction of TAO and oleandomycin phosphate (OLEO), the active metabolite of TAO in vivo, with glucocorticoid receptors in dispersed, intact cultured human skin fibroblasts. With the use of cells incubated with [3H]dexamethasone at 22 degrees C, we examined the competitive binding properties of TAO and OLEO (at varying concentrations) with and without MP as compared with several other steroids and MP alone. We also studied the effects on cellular glucocorticoid receptor number and affinity when TAO at a concentration of 4 X 10(-5) alone, at 10(-5) M in combination with a receptor saturating concentration of MP (5 X 10(-8) M), with MP alone, or with OLEO (10(-5) M) combined with MP was added to fibroblasts in the growth phase 1 wk before assay. TAO and OLEO did not compete for the binding of [3H]dexamethasone to the fibroblast glucocorticoid receptor, nor did they alter the binding properties of MP. With prolonged cellular exposure, TAO alone did not alter the number of glucocorticoid receptors (per cell) or their affinity for [3H]dexamethasone. Interestingly, prolonged exposure to saturating concentrations of MP alone decreased glucocorticoid receptor density; this effect was not altered by the presence of TAO or OLEO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the flavonoid (+)cyanidanol-3 on procollagen biosynthesis and transport in normal and ataxia telangiectasis cultured skin fibroblasts

The synthesis and secretion of procollagen into the medium of cultures of human skin fibroblasts from normal individuals and from patients with the genetic disorder, ataxia telangiectasia, are markedly inhibited by the flavonoid (+)cyanidanol-3. Those proteins which were secreted into the medium in the presence of cyanidanol were resistant to collagenase treatment (noncollagenous proteins). Polyacrylamide gel electrophoresis revealed the presence of only one noncollagenous protein of 66,000 daltons in the medium of cyanidanol-treated cells as compared with the nine other polypeptides found in the medium of untreated cells.     

Structure and function of the receptor for human atrial natriuretic peptide in cultured human skin fibroblasts

Cultured human skin fibroblasts possessed the high-affinity and low-capacity binding sites for [125I]alpha-human atrial natriuretic peptide (hANP), in which the dissociation constant and maximal binding capacity were computed to 68.7 +/- 11.3 pM and 7.3 +/- 1.2 fmols/mg protein, respectively, from Scatchard plot analysis. The specific [125I] alpha-hANP binding sites of cultured human fibroblast were displaced by unlabeled atriopeptin I, a truncated analogue, to the same extent as the case of alpha-hANP. In human adrenal membrane fractions, [125I] alpha-hANP binding sites were suppressed only by unlabeled alpha-hANP, while the high concentrations of atriopeptin I could slightly inhibit the binding sites for alpha-hANP. As it was reported that atriopeptin I had more significant affinity to the low-molecular weight ANP receptor (60-70 KD) than that to the high-molecular weight form (130-140 KD), the specific bindings may be attributed by the low-molecular weight ANP receptor in cultured human fibroblasts. Furthermore, alpha-hANP up to 10(-8)M failed to induce the significant cGMP formation in cultured human skin fibroblasts. The molecular weight of [125I]alpha-hANP binding sites of human fibroblasts was identified only at the region of 67 KD and no radioactive band was visualized around the region of large molecular weight ANP receptor in the SDS gel electrophoresis of a crosslinked [125I]alpha-hANP-receptor complex. In contrast, the affinity labeling of [125I]alpha-hANP to the human adrenal membrane fractions showed that 135 KD binding sites were responsible to the human adrenal ANP receptor. In conclusion, cultured human skin fibroblasts have a high-affinity low-capacity receptor for ANP. The molecular weight of ANP receptor is approximately 67 KD, and ANP-specific guanylate cyclase may not be linked to the receptor, suggestive that so-called C receptor may be localized in cultured human fibroblasts.     

In vitro growth and differentiated activities of human periodontal ligament fibroblasts cultured on salmon collagen gel

Marine-derived collagen is expected to be a much safer alternative to calf collagen, which in medical applications carries the risk of bovine spongiform encephalopathy. In this study, acid-soluble collagen was extracted from salmon skin and crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide during fibril formation to produce a crosslinked salmon collagen (SC) gel. The growth rates and the differentiated functions of human periodontal ligament fibroblasts (HPdLFs) cultured on the SC gel were investigated. Growth was faster on the SC gel than on porcine collagen (PC) gel. In addition, the HPdLFs cultured on the SC gel exhibited higher alkaline phosphatase (ALP) activity than those cultured on the PC gel. Quantitative RT-PCR revealed higher mRNA expression of type I collagen, ALP, and osteocalcin in the HPdLFs cultured on the SC gel. HPdLFs had a flat shape on the SC gel and a spindle shape on the PC gel, as revealed by observation with scanning electron microscopy and immunostaining with cytoskeletal protein and vinculin. The results showed that HPdLFs could grow and show highly differentiated activity on the SC gel as well as on the PC gel.     

Identification of fibroin-derived peptides enhancing the proliferation of cultured human skin fibroblasts

We previously reported that the fibroin of the silkworm Bombyx mori enhanced the proliferation of cultured human skin fibroblasts. In this work, the fibroin was digested by chymotrypsin, and the resulting peptide fragments were fractionated and assayed for their biological activity. Two peptides that promoted fibroblast growth were isolated and identified to be VITTDSDGNE and NINDFDED. Both sequences are found in the N-terminal region of the fibroin polypeptide and are thought to be the active principle of fibroblast growth-promoting activity.     

Differential effects of tissue processing on human embryonic and fetal skin

To most accurately evaluate quantitative data from studies of developing epidermis, the effects of tissue processing on human embryonic and fetal skin (8-20 weeks gestational age) were examined using two different techniques: 1) EDTA-separated epidermal sheets that were briefly fixed in 2% paraformaldehyde, processed through Permount infiltration, and prepared as whole mounts on glass slides, and 2) skin that was fixed in Karnovsky's fixative and embedded in Epon. Based on en face measurements of surface area before and after tissue processing, both procedures caused differential, age-dependent shrinkage. However, the trend of increasing shrinkage was inversely related to increasing age in the paraformaldehyde-fixed epidermal sheets (y = 57.14 + 1.26x, where x = gestational age in weeks and y = % of original surface area), but directly correlated with aging in the Karnovsky-fixed skin (y = 955.62 - 232.77x + 20.38x2). Shrinkage of epidermal sheets occurred during the dehydration and clearing steps, whereas most of the dimensional changes in whole skin took place during fixation in Karnovsky's. These differences are probably due to greater cross-linking of proteins and longer fixation time in the more concentrated and fast-acting Karnovsky's, as well as the influence of increasing quantities of fibrous proteins in the dermis of whole skin.     

Modulation of collagen synthesis and its gene expression in human skin fibroblasts by tocotrienol-rich fraction

Introduction

Skin aging may occur as a result of increased free radicals in the body. Vitamin E, the major chain-breaking antioxidant, prevents propagation of oxidative stress, especially in biological membranes. In this study, the molecular mechanism of tocotrienol-rich fraction (TRF) in preventing oxidative stress-induced skin aging was evaluated by determining the rate of total collagen synthesis and its gene expression in human skin fibroblasts.

Material and methods

Primary culture of human skin fibroblasts was derived from circumcision foreskin of 9 to 12 year-old boys. Fibroblast cells were divided into 5 different treatment groups: untreated control, hydrogen peroxide (H₂O₂)-induced oxidative stress (20 µM H₂O₂ exposure for 2 weeks), TRF treatment, and pre- and post-treatment of TRF to H₂O₂-induced oxidative stress.

Results

Our results showed that H₂O₂-induced oxidative stress decreased the rate of total collagen synthesis and down-regulated COL I and COL III in skin fibroblasts. Pre-treatment of TRF protected against H₂O₂-induced oxidative stress as shown by increase in total collagen synthesis and up-regulation of COL I and COL III (p<0.05) genes. However, similar protective effects against H₂O₂-induced oxidative stress were not observed in the post-treated fibroblasts.

Conclusions

Tocotrienol-rich fraction protects against H₂O₂-induced oxidative stress in human skin fibroblast culture by modulating the expression of COL I and COL III genes with concomitant increase in the rate of total collagen synthesis. These findings may indicate TRF protection against oxidative stress-induced skin aging.     

N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸对大鼠心脏成纤维细胞胶原蛋白合成及表达的调节作用

目的：探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)对血小板衍生生长因子(PDGF)诱导的大鼠心脏成纤维细胞胶原合成的调节作用。方法：建立新生大鼠心脏成纤维细胞系;采用~3H-脯氨酸掺入法检测心脏成纤维细胞胶原蛋白的合成。采用Western印迹法检测心脏成纤维细胞Ⅰ、Ⅲ型胶原蛋白的表达。结果：PDGF对心脏成纤维细胞胶原蛋白合成的促进作用与浓度相关,并以10 ng/ml时最强。AcSDKP对PDGF介导的心脏成纤维细胞胶原合成有抑制作用,并且在10~(-9)mol/L时作用最强。结论：AcSDKP对PDGF介导的心脏成纤维细胞胶原合成有明显抑制作用,可能与其抗心脏纤维化的作用相关。     

A denatured collagen microfiber scaffold seeded with human fibroblasts and keratinocytes for skin grafting

Biomaterial scaffolds are categorized into artificial or natural polymers, or combinations of the two. Artificial polymers often undergo serum protein adsorption, elicit foreign body and encapsulation immune responses post-implantation. Large pore bovine electrospun collagen I was therefore screened as a candidate for human keratinocyte and fibroblast cell scaffolds. Human HaCaT keratinocyte and dermal fibroblasts were seeded on electrospun denatured collagen I microfiber (DCM) scaffolds and after 72 h Livedead(?) assays performed to determine adhesive cell, survival and scaffold penetration. Both keratinocytes and fibroblasts attached to and survived on DCM scaffolds, however only fibroblasts migrated over and into this biomaterial. HaCaT keratinocytes remained largely stationary on the scaffold surface in discrete islands of monolayered cells. For this reason, normal human epidermal keratinocyte (NHEK) scaffold interactions were assessed using scanning and transmission electron microscopy (EM) that demonstrated DCM scaffolds comprised networks of interlocking and protruding collagen fibers with a mean diameter of 2-5 μm, with a mean inter-fiber pore size of 6.7 μm (range 3-10 μm) and scaffold thickness 50-70 μm. After 72 h the keratinocytes and fibroblasts on DCM scaffolds had attached, flattened and spread over the entire scaffold with assembly of lamellapodia and focal adhesion (FA)-like junctions. Using transmission EM, NHEKs and HaCaT keratinocytes assembled desmosomes, lamellapodia and FA junctions, however, neither hemidesmosomes nor basal lamina were present. In long term (21 day) co-culture fibroblasts migrated throughout the scaffold and primary keratinocytes (and to a lesser extend HaCaTs) stratified on the scaffold surface forming a human skin equivalent (HSE). In vivo testing of these HSEs on immunocompetent (BalbC) and immunodeficient (SCID) excisionally wounded model mice demonstrated scaffold wound biocompatibility and ability to deliver human cells after scaffold biodegradation.     

The biosynthesis of glycoproteins by cultured bovine tendon fibroblasts

Confluent bovine fetal tendon fibroblasts maintained in a chemically defined medium incorporated L-[6-3H]fucose and L-[5-3H]proline in a linear manner into non-diffusible macromolecules for up to 48 hrs. Equilibrium CsCl density gradient centrifugation indicated that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. The [3H]fucose-labelled glycoproteins in the culture medium were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. This technique demonstrated the presence of a number of high mol. wt. fucosylated components, the most notable of which was a glycoprotein of apparent mol. wt. 150,000. Immunological procedures allowed the tentative identification of four glycoproteins including fibronectin which was found in the cell medium and in extracts of the cell layer. Two of the glycoproteins (mol. wts. 150,000 and 270,000) released into the incubation medium were shown to be related to the microfibrillar components of elastic tissue. One or more of the newly synthesized [3H]fucose labelled molecules was shown to be immunologically related to a glycoprotein (mol. wt. 60,000) extracted from bovine Achilles tendon. These studies represent the first demonstration of the synthesis of microfibril-related and tendon glycoprotein-related macromolecules by tendon fibroblasts in culture.