Alpha-melanocyte stimulating hormone cytoprotective biology in human dermal fibroblast cells

Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory peptide effective in various tissues including skin. It acts by inhibiting the production and action of several pro-inflammatory stimuli including TNF-alpha, IL-1beta and LPS in a number of cell types. The role of such stimuli in inducing cellular apoptosis is also well described; however the precise role of alpha-MSH in apoptosis is presently unclear, with studies reporting both anti- and pro-apoptotic activity. The present study demonstrates that cultured human dermal fibroblasts respond to serum depletion and TNF-alpha, IL-1beta and LPS with an increase in membrane permeability, a decrease in viability and an increase in phosphatidylserine externalization (indicative of apoptosis) over 48-96 h. alpha-MSH (at 10(-6) M, but not 10(-9) M) was found to inhibit the serum free and pro-inflammatory mediated reduction in membrane permeability and cellular viability and also inhibited increases in apoptosis. In conclusion, data support a cytoprotective and anti-apoptotic role of the alpha-MSH peptide in human dermal fibroblast cells.     

Melanocyte stimulating hormone peptides inhibit TNF-alpha signaling in human dermal fibroblast cells

Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory in various tissues including the skin. It has previously been shown in skin cell keratinocytes and melanocytes/melanoma cells that MSH peptides inhibit TNF-alpha stimulated NF-kappaB activity and intercellular adhesion molecule-1 (ICAM-1) upregulation. However, the precise anti-inflammatory role of MSH peptides in dermal fibroblasts is unclear. Some studies report on pro-inflammatory responses, while others on anti-inflammatory responses. The present study confirms MC1R expression in cultured human dermal fibroblasts and reports that the MSH peptides alpha-MSH and KP(-D-)V inhibit TNF-alpha stimulated NF-kappaB activity and ICAM-1 upregulation, consistent with an anti-inflammatory role. However, involvement of IkappaB-alpha regulation by either peptide was not confirmed, supporting a mechanism independent of the NF-kappaB inhibitor. In conclusion, alpha-MSH and KP(-D-)V peptides have an anti-inflammatory action on dermal fibroblast signaling by inhibiting the pro-inflammatory activity of TNF-alpha in vitro.     

Cooling rate dependent biophysical and viability response shift with attachment state in human dermal fibroblast cells

While studies on the freezing of cells in suspension have been carried out extensively, corresponding studies with cells in the attached state and in tissue or tissue-equivalents are less developed. As attachment is a hallmark of the tissue state it is important to understand its impact on biophysics and viability to better apply freezing towards tissue preservation. The current study reports on observed biophysical response changes observed during freezing human dermal fibroblasts in suspension, attached cell, and fibrin tissue-equivalent models. Specifically, intracellular ice formation is shown to increase and dehydration is inferred to increase from suspension to attached systems. Biophysical model parameters fit to these experimental observations reflect the higher kinetics in the attached state. Post-thaw viability values from fast cooling rates were higher for suspension systems, and correlated well with the amount of IIF observed. On the other hand, viability values from slow cooling rates were higher for attached systems, although the degree of dehydration was predicted to be comparable to suspension cells. This disconnect between biophysics and viability predictions at slow rates clearly requires further investigation as it runs counter to our current understanding of dehydration injury in cells. This may suggest a possible protective effect of the attachment state on cell systems.     

Cooling rate dependent biophysical and viability response shift with attachment state in human dermal fibroblast cells

While studies on the freezing of cells in suspension have been carried out extensively, corresponding studies with cells in the attached state and in tissue or tissue-equivalents are less developed. As attachment is a hallmark of the tissue state it is important to understand its impact on biophysics and viability to better apply freezing towards tissue preservation. The current study reports on observed biophysical response changes observed during freezing human dermal fibroblasts in suspension, attached cell, and fibrin tissue-equivalent models. Specifically, intracellular ice formation is shown to increase and dehydration is inferred to increase from suspension to attached systems. Biophysical model parameters fit to these experimental observations reflect the higher kinetics in the attached state. Post-thaw viability values from fast cooling rates were higher for suspension systems, and correlated well with the amount of IIF observed. On the other hand, viability values from slow cooling rates were higher for attached systems, although the degree of dehydration was predicted to be comparable to suspension cells. This disconnect between biophysics and viability predictions at slow rates clearly requires further investigation as it runs counter to our current understanding of dehydration injury in cells. This may suggest a possible protective effect of the attachment state on cell systems.     

Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: important role of uric acid

Oxidized LDL is present within atherosclerotic lesions, demonstrating a failure of antioxidant protection. A normal human serum ultrafiltrate of Mr below 500 was prepared as a model for the low Mr components of interstitial fluid, and its effects on LDL oxidation were investigated. The ultrafiltrate (0.3%, v/v) was a potent antioxidant for native LDL, but was a strong prooxidant for mildly oxidized LDL when copper, but not a water-soluble azo initiator, was used to oxidize LDL. Adding a lipid hydroperoxide to native LDL induced the antioxidant to prooxidant switch of the ultrafiltrate. Uric acid was identified, using uricase and add-back experiments, as both the major antioxidant and prooxidant within the ultrafiltrate for LDL. The ultrafiltrate or uric acid rapidly reduced Cu2+ to Cu+. The reduction of Cu2+ to Cu+ may help to explain both the antioxidant and prooxidant effects observed. The decreased concentration of Cu2+ would inhibit tocopherol-mediated peroxidation in native LDL, and the generation of Cu+ would promote the rapid breakdown of lipid hydroperoxides in mildly oxidized LDL into lipid radicals. The net effect of the low Mr serum components would therefore depend on the preexisting levels of lipid hydroperoxides in LDL. These findings may help to explain why LDL oxidation occurs in atherosclerotic lesions in the presence of compounds that are usually considered to be antioxidants.     

Fluid pressure in human dermal fibroblast aggregates measured with micropipettes

Previous studies indicated that connective tissue cells in dermis are involved in control of interstitial fluid pressure (P_if). We wanted to develop and characterize an in vitro model representative of loose connective tissue to study dynamic changes in fluid pressure (P_f) over a time course of a few minutes. P_f was measured with micropipettes in human dermal fibroblast cell aggregates of varying size (<100- and >100-µm diameter) and age (days 1-4) kept at different temperatures (15, 25, and 35°C). Pressures were measured at different depths of micropipette penetration and after treatment with prostaglandin E₁ isopropyl ester (PGE₁), latanoprost (PGF₂), and ouabain. P_f was positive (more than +2 mmHg) during control conditions and increased with increasing aggregate size (day 2), age (day 4 vs. day 1), temperature, and depth of micropipette penetration. P_f decreased from 2.9 to 2.0 mmHg during the first 10 min after application of 10 µl of 1 mM PGE₁ (P < 0.001). P_f increased from 3.0 to 4.8 mmHg (P < 0.01) after administration of 10 µl of 1.4 µM ouabain and from 3.1 to 4.4 mmHg after addition of 5 µl of 1.42 mM PGF₂ (P > 0.05). In conclusion, we have developed and validated a new in vitro method for studying fluid pressure in loose connective tissue elements with the advantage of allowing reliable and rapid screening of substances that have a potential to modify P_f and studying in more detail specific cell types involved in control of P_f. This study also provides evidence that fibroblasts in the connective tissue can actively modulate P_f. micropuncture; prostaglandin E₁; prostaglandin F₂; ouabain; integrins     

Lipoic acid plays a role in scleroderma: insights obtained from scleroderma dermal fibroblasts

Introduction

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and organs. Increase in oxidative stress and platelet-derived growth factor receptor (PDGFR) activation promote type I collagen (Col I) production, leading to fibrosis in SSc. Lipoic acid (LA) and its active metabolite dihydrolipoic acid (DHLA) are naturally occurring thiols that act as cofactors and antioxidants and are produced by lipoic acid synthetase (LIAS). Our goals in this study were to examine whether LA and LIAS were deficient in SSc patients and to determine the effect of DHLA on the phenotype of SSc dermal fibroblasts. N-acetylcysteine (NAC), a commonly used thiol antioxidant, was included as a comparison.

Methods

Dermal fibroblasts were isolated from healthy subjects and patients with diffuse cutaneous SSc. Matrix metalloproteinase (MMPs), tissue inhibitors of MMPs (TIMP), plasminogen activator inhibitor 1 (PAI-1) and LIAS were measured by enzyme-linked immunosorbent assay. The expression of Col I was measured by immunofluorescence, hydroxyproline assay and quantitative PCR. PDGFR phosphorylation and α-smooth muscle actin (αSMA) were measured by Western blotting. Student’s t-tests were performed for statistical analysis, and P-values less than 0.05 with two-tailed analysis were considered statistically significant.

Results

The expression of LA and LIAS in SSc dermal fibroblasts was lower than normal fibroblasts; however, LIAS was significantly higher in SSc plasma and appeared to be released from monocytes. DHLA lowered cellular oxidative stress and decreased PDGFR phosphorylation, Col I, PAI-1 and αSMA expression in SSc dermal fibroblasts. It also restored the activities of phosphatases that inactivated the PDGFR. SSc fibroblasts produced lower levels of MMP-1 and MMP-3, and DHLA increased them. In contrast, TIMP-1 levels were higher in SSc, but DHLA had a minimal effect. Both DHLA and NAC increased MMP-1 activity when SSc cells were stimulated with PDGF. In general, DHLA showed better efficacy than NAC in most cases.

Conclusions

DHLA acts not only as an antioxidant but also as an antifibrotic because it has the ability to reverse the profibrotic phenotype of SSc dermal fibroblasts. Our study suggests that thiol antioxidants, including NAC, LA, or DHLA, could be beneficial for patients with SSc.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0411-6) contains supplementary material, which is available to authorized users.     

Quantification of uric acid, xanthine and hypoxanthine in human serum by HPLC for pharmacodynamic studies

A simple HPLC method was developed and validated for the determination of uric acid (UA), xanthine (X) and hypoxanthine (HX) concentrations in human serum to support pharmacodynamic (PD) studies of a novel xanthine oxidase inhibitor during its clinical development. Serum proteins were removed by ultrafiltration. The hydrophilic analytes and the I.S. were eluted by 100% aqueous phosphate buffer mobile phase. The hydrophobic matrix components (late peaks) were eluted with a step gradient of a higher organic mobile phase. Validation on linearity, sensitivity, precision, accuracy, stability, and robustness of the method for PD biomarkers (UA, X, and HX) was carried out in a similar manner to that for pharmacokinetic (PK) data where applicable. Issues of selectivity for endogenous biomarker analytes and individual concentration variations were addressed during method validation. Standards were prepared in analyte-free phosphate buffer. Quality control samples were prepared in control serum from individuals not dosed with the xanthine oxidase inhibitor. The method was simple and robust with good accuracy and precision for the measurement of serum UA, X, and HX concentrations.     

Multi-oscillatory control of eclosion and oviposition rhythms in Drosophila melanogaster: evidence from limits of entrainment studies

The eclosion and oviposition rhythms of flies from a population of Drosophila melanogaster maintained under constant conditions of the laboratory were assayed under constant light (LL), constant darkness (DD), and light/dark (LD) cycles of 10:10h (T20), 12:12h (T24), and 14:14h (T28). The mean (+/- 95% confidence interval; CI) free-running period (tau) of the oviposition rhythm was 26.34 +/- 1.04h and 24.50 +/- 1.77h in DD and LL, respectively. The eclosion rhythm showed a tau of 23.33 +/- 0.63 h (mean +/- 95% CI) in DD, and eclosion was not rhythmic in LL. The tau of the oviposition rhythm in DD was significantly greater than that of the eclosion rhythm. The eclosion rhythm of all 10 replicate vials entrained to the three periodic light regimes, T20, T24, and T28, whereas the oviposition rhythm of only about 24 and 41% of the individuals entrained to T20 and T24 regimes, respectively, while about 74% of the individuals assayed in T28 regimes showed entrainment. Our results thus clearly indicate that the tau and the limits of entrainment of eclosion rhythm are different from those of the oviposition rhythm, and hence this reinforces the view that separate oscillators may regulate these two rhythms in D. melanogaster.     

Methylselenocysteine treatment leads to diselenide formation in human cancer cells: evidence from X-ray absorption spectroscopy studies

The selenoamino acids methylselenocysteine (MeSeCys) and selenomethionine (SeMet) have disparate efficacies as anticancer agents. Herein, we use X-ray absorption spectroscopy to determine the chemical form of selenium in human neuroblastoma cells. Cells treated with MeSeCys contain a significant diselenide component, which is absent from SeMet-treated cells and suggests that metabolites of MeSeCys are capable of altering the redox status of the cells. The differences in the speciation of Se in the selenoamino acid-treated cells may provide insight into the differing anticancer activities of MeSeCys and SeMet.     

Formation of ursodeoxycholic acid from chenodeoxycholic acid in the human colon: studies of the role of 7-ketolithocholic acid as an intermediate

The formation of ursodeoxycholic acid from chenodeoxycholic acid and the role of 7-ketolithocholic acid as an intermediate in this biotransformation were studied in vitro in fecal incubations as well as in vivo in the human colon. [24-14C]-Labeled 7-ketolithocholic and chenodeoxycholic acids were studied at various concentrations, and the biotransformation products were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry. There was rapid colonic conversion of 7-ketolithocholic acid to ursodeoxycholic acid and, to a lesser extent, to chenodeoxycholic acid. The reduction of 7-ketolithocholic to ursodeoxycholic acid proceeded significantly faster anaerobically and at acid pH than under aerobic and alkaline conditions. When chenodeoxycholic acid was incubated in vitro or instilled into the colon, various amounts of 7-ketolithocholic and ursodeoxycholic acids were formed. The formation of 7-ketolithocholic acid was favored by alkaline conditions. Isotope dilution studies, in which trace amounts of labeled 7-ketolithocholic acid were incubated with unlabeled chenodeoxycholic acid, indicate 7-ketolithocholic acid to be the major intermediate in the intestinal bacterial conversion of chenodeoxycholic to ursodeoxycholic acid.     

Parabiosis and transplantation models show no evidence of circulating dermal fibroblast progenitors in bleomycin-induced skin fibrosis

To test the hypothesis of an extra-dermal origin of dermal fibroblasts, parabiosis, and transplantation models were developed utilizing a collagen promoter green fluorescent protein (GFP) reporter transgene expressed in dermal fibroblasts. Parabiotic pairs were treated with bleomycin to induce the skin fibrosis that was evaluated for a dense deposition of collagen and inflammatory cell infiltrates in the thickened dermis in comparison with parabiotic pairs treated with saline. Although, in all cases, repeated injection of bleomycin for 4 weeks induced skin fibrosis, only a few GFP positive cells were detected in skin samples from some of the treated non-transgenic mice. Unexpectedly, similar results were observed in saline treated controls. Furthermore, bone marrow chimeras were created in which non-transgenic recipient mice received injections of bone marrow cell preparations isolated from pOBCol3.6GFP transgenic mice. After bone marrow chimerism had been successfully established, fibrotic lesions in the skin were induced by local bleomycin injections. Donor GFP expressing cells were observed in the skin from all recipient mice. However, no difference in the presence of GFP expressing cells was observed between non-treated mice or mice treated with bleomycin or saline. A large number of GFP expressing cells were observed in the lung preparations from all chimeric mice. Mac-3 antibody immunostaining confirmed a macrophage phenotype for these GFP expressing cells suggesting the expression of the pOBCol3.6GFP transgene in a non-collagen producing cell. Based on these observations, we found no evidence of circulating dermal fibroblast progenitors that participate in the development of bleomycin-induced skin fibrosis.     

The role of xanthine oxidoreductase and uric acid in metabolic syndrome

Xanthine oxidoreductase (XOR) could contribute to the pathogenesis of metabolic syndrome through the oxidative stress and the inflammatory response induced by XOR-derived reactive oxygen species and uric acid. Hyperuricemia is strongly linked to hypertension, insulin resistance, obesity and hypertriglyceridemia. The serum level of XOR is correlated to triglyceride/high density lipoprotein cholesterol ratio, fasting glycemia, fasting insulinemia and insulin resistance index. Increased activity of endothelium-linked XOR may promote hypertension. In addition, XOR is implicated in pre-adipocyte differentiation and adipogenesis. XOR and uric acid play a role in cell transformation and proliferation as well as in the progression and metastatic process. Collected evidences confirm the contribution of XOR and uric acid in metabolic syndrome. However, in some circumstances XOR and uric acid may have anti-oxidant protective outcomes. The dual-face role of both XOR and uric acid explains the contradictory results obtained with XOR inhibitors and suggests caution in their therapeutic use.     

Double-tether extraction from human umbilical vein and dermal microvascular endothelial cells

Multiple tethers are very likely extracted when leukocytes roll on the endothelium under high shear stress. Endothelial cells have been predicted to contribute more significantly to simultaneous tethers and thus to the overall rolling stabilization. We therefore extracted and quantified double tethers from endothelial cells with the micropipette aspiration technique. We show that the constitutive parameters (threshold force (F0) and effective viscosity (etaeff)) for double-tether extraction are twice those for single-tether extraction and are remarkably similar for human neonatal (F0=105+/-5 pN; etaeff=1.0+/-0.1 pN.s/microm) and adult (F0=118+/-13 pN; etaeff=1.3+/-0.2 pN.s/microm) dermal microvascular, and human umbilical vein (F0=99+/-3 pN; etaeff=1.0+/-0.1 pN.s/microm) endothelial cells. Additionally, these parameters are also independent of surface receptor type, cytokine stimulation, and attachment state of the endothelial cell. We also introduce a novel correlation between the cell-substrate contact stress and gap width, with which we can predict the apparent cell-substrate separation range to be 0.01-0.1 microm during leukocyte rolling. With a biomechanical model of leukocyte rolling, we calculate the force history on the receptor-ligand bond during tether extraction and predict maximum stabilization for the double simultaneous tether extraction case.     

Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures

Normal human dermal fibroblasts have a limited life-span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase-3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.