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.     

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.     

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.     

The nerve-dependency of Merkel cell proliferation in cultured human fetal glabrous skin.

Merkel cells are thought to function as slowly adapting mechanoreceptors and are known as targets for sensory nerves. However, the nerve-dependency of Merkel cells remains controversial. In this respect, some investigators have found interregional differences between hairy and glabrous skin and others have shown intraregional differences within denervated rat touch domes. Differences between species have also been reported. This study was performed to determine whether Merkel cells proliferate in vitro in the absence of the systemic factors, blood vessels and the intact nerves in human skin. Suspension organ culture was performed using fetal digits to investigate their in vitro proliferation. Merkel cells and cutaneous nerves were identified using antibodies to cytokeratin 20 and protein gene product 9.5 (PGP 9.5), respectively. Fetal digits of 56-82 day gestational age were cultured in serum free medium in a high O2 (45%) environment. Tissues were harvested before starting culture (D0) and 1, 4, 7, 14, 28 d after culture. Merkel cells were observed in the volar pads and dorsal nail matrices at D0. After 28 d of suspension organ culture, digits looked healthy structurally and the number of Merkel cells had increased. However, PGP 9.5-immunoreactive nerves were markedly diminished after 1 day of culture and almost disappeared after 4 days. Merkel cell proliferation in vitro suggested that Merkel cell development is probably nerve-independent in human fetal glabrous skin.     

Identification of genes differentially expressed in cultured human osteoblasts versus human fibroblasts by DNA microarray analysis

Little is known about patterns of gene expression from cells populating the connective tissues. This study investigated the possible variance of gene expression profile between human osteoblasts (HO) and human fibroblasts (HF) in vitro, using DNA microarray technology. Clustering identification was used to compare expression patterns between HO and HF for biological significance. Our results showed that genes encoding the extracellular matrix or apoptosis-related proteins tended to be expressed in greater abundance in HO, while more proteolysis-related proteins were expressed in higher level in HF. Significant differences in expression were also noted with genes related to signaling pathways. To confirm the array results, three genes (periostin, MFG-E8, MMP-10) were selected and analyzed independently by RT-PCR and northern blot. The results were found consistent with the array data in HO and HF. The present findings suggest that HO and HF differ not only phenotypically but in the expression level of tissue specific genes to assure the turnover and homeostasis of their respective tissues.     

The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro

The effects of chitin [(1 --> 4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the proliferation of human dermal fibroblasts and keratinocytes were examined in vitro. Chitosans with relatively high degrees of deacetylation strongly stimulated fibroblast proliferation while samples with lower levels of deacetylation showed less activity. Fraction, CL313A, a shorter chain length, 89% deacetylated chitosan chloride was further evaluated using cultures of fibroblasts derived from a range of human donors. Some fibroblast cultures produced a positive mitogenic response to CL313A treatment with proliferation rates being increased by approximately 50% over the control level at an initial concentration of 50 microg/ml, whilst others showed no stimulation of proliferation or even a slight inhibition (< 10%). The stimulatory effect on fibroblast proliferation required the presence of serum in the culture medium suggesting that the chitosan may be interacting with growth factors present in the serum and potentiating their effect. In contrast to the stimulatory effects on fibroblasts, fraction CL313A inhibited human keratinocyte mitogenesis with up to 40% inhibition of proliferation being observed at 50 microg/ml. In general highly deacetylated chitosans were more active than those with a lower degree of deacetylation. These data demonstrate that highly deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Analysis of their effects on cells in culture may be useful as a screen for their potential activity in vivo as wound healing agents, although in the case of fibroblasts it is important to select appropriate strains of cells for use in the screen.     

Ropivacaine and lidocaine inhibit proliferation of non-transformed cultured adult human fibroblasts,endothelial cells and keratinocytes

Local anaesthetics are known to affect a variety of cell functions, many of which are involved in the inflammatory response. Local anaesthetics have also been shown to influence cell proliferation. The aim of this study was to examine the effect of two local anaesthetics (ropivacaine and lidocaine) on cell proliferation of cultured human fibroblasts, vascular endothelial cells and epithelial cells, i.e. keratinocytes, as earlier studies have not included primary human cell types. Significant inhibition of fibroblast proliferation was observed with concentrations of 50 M ropivacaine or 100 M lidocaine in 1% newborn calf serum and 500 M ropivacaine or lidocaine in 10% newborn calf serum. The proliferation of endothelial cells was significantly inhibited by 1 mM ropivacaine in 5% human serum and 500 M ropivacaine or 100 M lidocaine in 40% human serum. Significant inhibition was not obtained with lidocaine when these cells were cultured in 5% HS. Significant inhibition of keratinocytes was obtained with 100 M ropivacaine and 500 M lidocaine. The effective concentrations are within the range of therapeutical concentrationsin vivo and there seems to be a general correlation between the local anaesthetic potency and the inhibiting effect on cell proliferation. This suggest a mechanism by which local anaesthetics may exhibit anti-hyperproliferative effects in clinical situations.     

Inhibition of androgen receptor binding by natural and synthetic steroids in cultured human genital skin fibroblasts

Summary The ability of various natural and synthetic steroids (some of which are widely used in clinical practice) to compete with dihydrotestosterone receptor binding in human genital skin fibroblasts was studied. Binding was assessed in fibroblast monolayers after incubation for 1 h at 37 °C with 2 nM ³H-dihydrotestosterone in the presence or absence of increasing concentrations of the steroid to be tested. Inhibition constants (K_i) were determined as the concentration of competitor-required for 50% inhibition of³H-dihydrotestosterone binding. In addition, relative binding activity (RBA) of each test compound was calculated. Each competitor was tested in at least two different cell strains. The concentrations of unlabeled methyltrienolone (a synthetic nonmetabolizable androgen) and dihydrotestosterone for 50% inhibition of³H-dihydrotestosterone binding were in the same order of magnitude, namely, 2 nM (2.2 respectively, 2.4 nM), whereas the affinity of testosterone was approximately one-fifth that of dihydrotestosterone.Other potent competitors for dihydrotestosterone binding were three progestins (norgestrel, gestoden, and medroxyprogesterone acetate) which have K_i values similar to testosterone. An order of magnitude lower K_i values (around 10^–7 M) were found for the androgen 17-propylmesterolone, the antiandrogen cyproterone acetate, and the progestin norethisterone acetate. Binding affinities of all other steroids to the androgen receptor were markedly lower and showed the following order of potency: estrogens (estradiol, ethinyl estradiol, diethylstilbestrol) > glucocorticoids as well as aromatase inhibitors and potassium canrenoate. We conclude that (a) among the compounds tested some progestins are very potent in their ability to interact with human skin fibroblast receptors and thus may affect endogenous androgen action; (b) estrogens are relatively weak androgen receptor binders; and (c) this receptor assay in combination with pharmacokinetic and metabolic studies appears to be a useful screening test to evaluate the potency of various steroids for androgen and antiandrogen therapy.Abbreviations DHT 5-dihydrotestosterone - EMM Eagle's minimum essential medium - FCS Fetal calf serum - RBA Relative binding activity Supported by Deutsche Forschungsgemeinschaft Schw 168/5–7Dedicated to Prof. Dr. F. Krück on the occasion of his 65th birthdayThe following trivial names have been used: see page 737     

Fidelity of histone synthesis in cultured human fibroblasts

The results in the literature to support Orgel's general error hypothesis of ageing only provide indirect evidence that errors in protein synthesis increase during senescence. This study attempts to provide direct evidence of errors in protein synthesis by measuring the misincorporation of 35S-methionine into histone H1 obtained from young and old fibroblasts (MRC-5). The conclusions that can be drawn from this study are: (a) the error level for the misincorporation of methionine into histone H1 is less than 7 methionines/10(5) amino acids and 2-3 methionines/10(4) amino acids in young and old cells respectively; (b) a methionine containing fraction associated with H1 is obtained after the final purification. The amount of this fraction increases with the age of the cell culture as does the number of methionine residues; (c) there is a variation in the complexity of H1 polypeptide chains, the complexity increasing with the age of cultured cells.     

Biosynthesis of embryonic prealbumin by cultured human fibroblasts

Embryonic prealbumin (EPA) was found in human fibroblasts by immunodiffusion and immunofluorescence analysis. No quantitative or qualitative differences in the content and localization of this antigen were found between embryonic and adult human fibroblasts. It is concluded that human fibroblasts synthesize EPA, which can be used as a marker for cells of this type.Department of Biochemistry and Problem Laboratory for Immunochemistry of Malignant and Embryonic Tissues, N. I. Pirogov Second Moscow Medical Institute. Laboratory of General Cytogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. P. Bochkov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 2, pp. 176–179, February, 1980.     

α2-Macroglobulin production by cultured human fibroblasts

Alpha₂-macroglobulin (₂M), a high-molecular-weight plasma protease inhibitor has been shown, by both immunological and functional methods, to be produced by cultured adult lung fibroblasts. Cultured skin fibroblasts synthesized approximately one tenth as much ₂M as lung fibroblasts. This quantitative difference in ₂M production was also demonstrated in fibroblasts of isogenic origin. There was no difference in the amount of ₂M produced between adult and fetal fibroblasts of the same tissue type (i.e., of lung or of skin origin). ₂M was produced in culture during log-phase growth as well as at confluence. Two other plasma protease inhibitors, C1-esterase inhibitor and a substance immunologically cross-reacting with human inter--trypsin inhibitor, were also made by the cultured fibroblasts. Plasma protease inhibitors not detectable in culture supernatants were ₁-antitrypsin, ₁-antichymotrypsin, and antithrombin III.     

Characterization of angiotensin receptors on human skin fibroblasts

 Angiotensin II is involved in blood pressure regulation, cell growth and angioneogenesis. The angiotensin receptors which mediate the intracellular effects of angiotensin II are expressed in numerous tissues and cell types. We studied the expression of angiotensin II receptors in cultured human skin fibroblasts derived from a skin biopsy. Angiotensin II binding characteristics were analyzed by radioligand binding assays. The DNA synthesis was assessed by [H]thymidine incorporation assays. Intracellular calcium concentrations were measured by fura-2 spectrofluorometry, and mRNA expression levels were analyzed by northern blot technology. Two distinct angiotensin receptors were detectable on human skin fibroblasts: the AT1 receptor with K _d=1.0± 0.7 nmol/l and B _max=17.9±0.9 fmol/mg protein, and an angiotensin_(1–7) binding site with K _d=26±6.6 nmol/l and B _max=80.4±3.5 fmol/mg protein, as shown by competition binding assays using selective angiotensin II receptor antagonists and the heptapeptide angiotensin_(1–7). The angiotensin AT1 receptor mRNA was substantially expressed in human skin fibroblasts and was subjected to homologous downregulation. In human skin fibroblasts angiotensin II caused a profound increase in intracellular calcium which was blocked by angiotensin AT1 receptor antagonists such as Exp-3174. Furthermore, both angiotensin II and angiotensin_(1–7) led to increased DNA synthesis in human skin fibroblasts. In conclusion, cultured human skin fibroblasts express angiotensin AT1 receptors and a putatively new angiotensin receptor activated by angiotensin_(1–7), both coupled to signaling pathways involved in DNA synthesis. Received: 5 July 1996 / Accepted: 29 November 1996     

Angiotensin II stimulates proliferation of adventitial fibroblasts cultured from rat aortic explants

It has been proposed that the local renin-angiotensin system is activated in the adventitia after vascular injury. However, the physiological role of Angiotensin II (Ang II) in the adventitia has not been studied at a cellular level. This study was designed to assess the role of Ang II in the growth response of cultured adventitial fibroblasts (AFs). Adventitial explants of the rat thoracic aorta showed outgrowth of AFs within 5-7 days. Ang II caused hyperplastic response of AF cultures. The Ang II-induced mitogenic response of AFs was mediated primarily by the AT1 receptor. Ang II caused a rapid induction of immediate early genes (c-fos, c-myc and jun B). Induction of c-fos expression was fully blocked by an AT1 receptor antagonist but not by an AT2 receptor antagonist. Epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) induced DNA synthesis in AFs. Co-stimulation of AFs with the growth factors and Ang II potentiated the incorporation of 3H-thymidine into DNA. Results from this study indicate that Ang II causes mitogenesis of AFs via AT1 receptor stimulation and potentiates the responses to other mitogens. These data suggest that the Ang II may play an important role in regulating AF function during vascular remodeling following arterial injury.     

Low density lipoprotein receptors in cultured skin fibroblasts from psoriasis patients

Low density lipoprotein (LDL) receptor activity, measured as 125I-LDL association and degradation at 37 degrees C, was determined in cultured fibroblasts from involved as well as uninvolved skin obtained from 20 psoriasis patients. The same analyses were conducted in fibroblasts from two reference groups consisting of 19 heterozygotes for familial hypercholesterolemia and 16 normal subjects, respectively. Psoriasis patients had significantly lower LDL receptor activity than normals, and it was comparable to that of the heterozygotes for familial hypercholesterolemia. The reduced LDL receptor activity was not accompanied by an increase in total serum cholesterol. The psoriasis patients had a significant reduction in apo-B concentration, but did not differ from the normals in the other serum lipid or lipoprotein parameters. There was no difference in LDL receptor activity between involved and uninvolved skin from psoriasis patients. These results suggest that there is an abnormal cell membrane in dermal fibroblasts from psoriasis patients. Since their total serum cholesterol is normal, their low LDL receptor activity may be confined to dermal cells, leaving the hepatic lipid metabolism normal. The pathogenetic significance of this finding is unknown.     

Properties of sulfatases in cultured skin fibroblasts of multiple sulfatase deficient patients

Various sulfatase activities were assayed in cultured skin fibroblasts from patients with multiple sulfatase deficiency (MSD). MSD cell lines displayed deficiencies of arylsulfatase A and iduronate sulfatase, but activities of arylsulfatase B, N-acetylgalactosamine 6-sulfate sulfatase and N-acetylglucosamine 6-sulfate sulfatase were within normal ranges, but not consistently. Arylsulfatase A, minor anionic arylsulfatase and N-acetylgalactosamine 6-sulfate sulfatase in MSD cell lines had similar Km, pH optima, inhibitory or activator sensitivity to that of normal skin fibroblasts. Arylsulfatase B in MSD cell lines also had properties similar to that of normal skin fibroblasts, except an abnormal heat stability. From our results, we conclude that properties of arylsulfatase A, minor anionic arylsulfatase and N-acetylgalactosamine 6-sulfate sulfatase in MSD fibroblasts were intact. On the other hand, arylsulfatase B in MSD might be a functionally abnormal enzyme.