The biomechanics of the umbilical cord Wharton Jelly: Roles in hemodynamic proficiency and resistance to compression.

The umbilical cord is a complex structure containing three vessels, one straight vein and two coiled arteries, encased by the Wharton Jelly (WJ) a spongy structure made of collagen and hydrated macromolecules. Fetal blood reaches the placenta through the arteries and flows back to the fetus through the vein. The role of the WJ in maintaining cord circulation proficiency and the ultimate reason for arterial coiling still lack of reasonable mechanistic interpretations. We performed biaxial tension tests and evidenced significant differences in the mechanical properties of the core and peripheral WJ. The core region, located between the arteries and the vein, resulted rather stiffer close to the fetus. Finite element modelling and optimization based inverse method were used to create 2D and 3D models of the cord and to simulate stress distribution in different hemodynamic conditions, compressive loads and arterial coiling. We recorded a facilitated stress transmission from the arteries to the vein through the soft core of periplacental WJ. This condition generates a pressure gradient that boosts the venous backflow circulation towards the fetus. Peripheral WJ allows arteries to act as pressure buffering chambers during the cardiac diastole and helps to dissipate compressive forces away from vessels. Altered WJ biomechanics may represent the structural basis of cord vulnerability in many high-risk clinical conditions.

Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms.

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.

Membranes modification of differentiating proerythroblasts. Variation of 1,6-diphenyl-1,3,5-hexatriene lifetime distributions by multifrequency phase and modulation fluorimetry.

The fluorescence emission of 1,6-diphenyl-1,3,5-hexatriene (DPH) in K562 cell membranes has been studied using multifrequency phase and modulation fluorimetry. The DPH decay data collected at various modulation frequencies were analysed by assuming either a model of discrete exponential components or a model of continuous lifetime distribution. The fits showed smaller values of the reduced chi square using the model of continuous lifetime distribution. The K562 cell membranes dynamics were investigated during the cell differentiation along the erythroid pathway. By using the continuous lifetime distribution method for the analysis of the DPH decay, marked variations were observed during the four initial days of the erythroid differentiation. Namely, the width of the DPH lifetime distribution increased by a factor of about two, while the center value of the distribution remained constant. By using the discrete exponential components model for the analysis of the DPH decay no variations were observed during the K562 differentiation.

Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids.

Changes in membrane properties during the differentiation process in K562 cells have been investigated. A decrease of lectin-induced agglutination has been detected. The agglutination assay revealed to be an early and sensitive test to monitor the induced differentiation of the K562 cells. Naturally occurring fluorescent fatty acids (cis- and trans-parinaric acids) and the recently developed multifrequency phase and modulation technique were used to study cell membrane properties. Changes in fluorescence lifetime and polarization are clearly associated with cell differentiation, suggesting the involvement of the cellular plasma membrane in the differentiation process.

Surface properties of cholesterol-containing membranes detected by Prodan fluorescence.

The fluorescent membrane probe 6-propionyl-2-dimethylaminonaphthalene (Prodan) displays a high sensitivity to the polarity and packing properties of lipid membrane. Contrary to 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), Prodan can also monitor the properties of the membrane surface, i.e., the polar-head pretransition. In bilayers composed of coexisting gel and liquid-crystalline phases, Prodan shows a preferential partitioning in the latter, so that the detected membrane properties mainly belong to fluid domains. In the presence of cholesterol, the packing properties of the gel phase phospholipids are modified in such a way that Prodan can penetrate and label the membrane. Although Prodan labeling of the gel phase is a function of cholesterol concentration, 3 mol percent cholesterol is sufficient for a 60% Prodan labeling with respect to the maximum labeling reached at 15 mol percent cholesterol. We present steady-state and dynamical fluorescence measurements of Prodan in bilayers in the presence of cholesterol. Our results fit the liquid-ordered/liquid-disordered phase model for cholesterol-containing membranes and show that the presence of cholesterol, in addition to modification to the phase state of the hydrophobic portion of the bilayer, strongly affects the packing and the polarity of the membrane hydrophobic-hydrophilic interface.

Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types.

Fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) are used to explore gel and liquid-crystalline phase domains coexistence in membranes of various cell types and in erythrocyte ghosts. Experiments and simulations were performed using liposomes composed of equimolar gel and liquid-crystalline phases in the absence and in the presence of 30 mol% cholesterol. In this model system two distinct coexisting phases can be easily recognized in the absence of cholesterol. When cholesterol is added to this phospholipid mixture, Laurdan parameters characteristic of the gel and of the liquid-crystalline phase are no longer resolvable. Coexisting domains of gel and liquid-crystalline phase were not detected in any of the examined cell membranes as judged by Laurdan excitation and emission Generalized Polarization (GP) spectra. Both in liposomes and in cell membranes, the behaviour of GP values as a function of excitation and emission wavelength corresponds to a homogeneous liquid-crystalline phase, despite the absolute GP values being relatively high, closer to the values observed in gel phase phospholipids. The presence of cholesterol appears to be the major cause for the homogeneity of phospholipids' dynamical properties in natural membranes, properties that appear close to the liquid-ordered phase state, defined to describe model systems with cholesterol concentration > or = 30 mol%.

Cholesterol protects the phospholipid bilayer from oxidative damage.

The measurement of fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene is used for the detection of oxidative damage produced in phospholipid membranes by ionizing radiation. The recently developed method is based on the linear relationship between the width of the probe lifetime distribution and the logarithm of the dose. The molecular origin of the damage resides in the production of hydroperoxide residues at the level of acyl chains double bonds. A chemiluminescence assay was used to quantitate the amount of produced hydroperoxides. Consequences of the produced damages include an increased disorder in the upper portion of the bilayer, accompanied by the penetration of water molecules. In the presence of the physiological concentration of cholesterol in phopholipid bilayers, the amount of hydroperoxides produced by ionizing radiation is dramatically reduced. The packing effect of cholesterol in phopholipid bilayers is well recognized, as well as its influence on the reduction of water concentration in the bilayer. The dramatic reduction of hydroperoxides concentration observed when irradiation is performed in the presence of cholesterol probably originates from a steric hindrance to the radical chain reaction through the unsaturated lipids due to the presence of cholesterol.

Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides.

Oxidatively modified LDL mimics several aspects of atherogenesis. In this disease, degradation of the matrix proteins' network also occurs. By a new morphological ex vivo approach, not requiring sample processing, we explored the relationship between the degradation of matrix protein and oxidatively modified LDL. Two-photon excitation fluorescence microscopy images of fresh cross-section rings of rat aorta, acquired while the sample was maintained in a glucose- and oxygen-supplemented buffer, showed straight, parallel, thick, long extracellular matrix proteins. Traditional microscopic examination, requiring sample fixation and staining, shows smaller and curved fibers. Instead, we observed curved and broken fibers after a 30-min incubation of aorta with either LDL containing lipid hydroperoxides, or tert-butyl-hydroperoxide. The adhesion of LDL to the endothelium and its internalization was directly visualized by using a lipid fluorophore. The damage to aorta matrix proteins induced by LDL and tert-butyl-hydroperoxide was fully prevented by antioxidants, such as ascorbate or Trolox C, or inhibitors of proteases. The image spectroscopy of the fibers' autofluorescence (polarization and lifetime) revealed an increased mobility of the fluorescent cross-link in fibers. Damaged matrix proteins were also imaged in aorta samples from apolipoprotein E knock-out mice. Our ex vivo images directly visualized the activation of a fast redox-sensitive proteolytic process in the arterial wall triggered by lipid hydroperoxides in LDL.

Loss of apoB-100 secondary structure and conformation in hydroperoxide rich, electronegative LDL(-).

A subpopulation of low-density lipoproteins (LDL) is present in human plasma that contains lipid hydroperoxides and is more negatively charged (LDL(-)) than normal native LDL. By circular dichroism and tryptophan lifetime measurements we found that apoB-100 secondary structure is markedly decreased and its conformation is severely altered in LDL(-). The low tryptophan fluorescence intensity confirms the oxidative degradation of the lipoprotein, and the very long lifetime value of one of its decay components indicates a low polarity environment for the remaining unbleached residues. Either a peculiar folding or, most likely, a sinking of the apoB-100 into the lipid core can account for the observed long lifetime component. Oxidation in vitro produces a similar unfolding of the apolipoprotein but the lifetime of tryptophan fluorescence is shifted to lower values, indicating that the denatured apoprotein remains at the hydrophilic surface of the lipoprotein particle. A disordering and an increased polarity of the LDL(-) surface lipids was demonstrated by measuring the generalized polarization of 2-dimethylamino-6-lauroylnaphthalene (Laurdan). The looser monolayer packing apparently favors the new conformation of apoB-100 and its sinking into a more hydrophobic environment, possibly accounting for it reduced receptor binding properties.

In K562 and HL60 cells membrane ageing during cell growth is associated with changes in cholesterol concentration.

In a cell culture model of aging we have previously shown that there is an age-related decrease in the lipid dynamics of the proerythropoetic K562 cell membranes, as determined by the generalized polarization (GP) of the phase-sensitive lipid probe 2-dimethylamino-6-lauroylnaphthalene (Laurdan) (T. Parasassi, M. Di Stefano, G. Ravagnan, O. Sapora and E. Gratton. Exp. Cell Res., 202 (1992) 432-439). In the present study we also extended our observations to the lymphoblastoid HL60 cell line. In both K562 and HL60 cells during the four days after the last cell culture medium renewal the GP Laurdan value increased in a linear fashion indicating a time-dependent decrease in lipid dynamics. The initial membrane physical properties were almost completely restored upon renewal of the cell culture medium. We measured lipid composition, including individual and total phospholipids, free and esterified cholesterol at the first ('young') and at the fourth ('aged') day after culture medium renewal. We found that the decreased membrane lipid 'fluidity' at the fourth day of cell growth was associated with a 40% increase in cholesterol concentration in both cell lines. This increase in cholesterol concentration was reversible 24 h following the culture medium change. We conclude that in K562 and HL60 cells the 'age-related' decrease in membrane lipid dynamics is mediated by an 'age-related' increase in cell cholesterol content.

Abscisic acid-induced microheterogeneity in phospholipid vesicles. A fluorescence study.

Changes in the thermal behavior of DMPC (dimyristoyl-L-phosphatidylcholine) and an equimolar mixture of DMPC and DMPE (dimyristoyl-L-phosphatidylethanolamine) induced by the plant hormone abscisic acid (ABA) have been investigated using fluorescent probes. The fluorescence decay of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in these vesicles has been measured using frequency-domain fluorometry, and has been analyzed using both models of discrete exponential components and continuous lifetime distributions. In the DMPC vesicles, using the distributional approach, higher center and width values were observed in the presence of abscisic acid (ABA), indicating a decrease in the dielectric constant of the lipid phase that we attribute to a decrease in the water concentration within the bilayer. Moreover, the presence of ABA in the liposomes increased the phospholipid phase transition temperature. The addition of ABA to the DMPC/DMPE mixture strongly increased the microheterogeneity of the system as reported by the FWHM (full-width at half-maximum) of the distributional approach.

Abscisic acid-induced microheterogeneity in phospholipid vesicle: a fluorescence study.

Changes in the thermal behavior of DMPC (dimyristoyl-r-phosphatidylcholine) and an equimolar mixture of DMPC and DMPE (dimyristoyl-L-phosphatidylethanolamine) induced by the plant hormone abscisic acid (ABA) have been investigated using fluorescent probes. The fluorescence decay of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in these vesicles has been measured using frequency-domain fluorometry, and has been analyzed using both models of discrete exponential components and continuous lifetime distributions. In the DMPC vesicles, using the distributional approach, higher center and width values were observed in the presence of abscisic acid (ABA), indicating a decrease in the dielectric constant of the lipid phase that we attribute to a decrease in the water concentration within the bilayer. Moreover, the presence of ABA in the liposomes increased the phospholipid phase transition temperature. The addition of ABA to the DMPC/DMPE mixture strongly increased the microheterogeneity of the system as reported by the FWHM (full-width at half-maximum) of the distributional approach.

Modulation and dynamics of phase properties in phospholipid mixtures detected by Laurdan fluorescence.

Steady-state and dynamic fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) have been used to ascertain the coexistence of separate phase domains and their dynamic properties in phospholipid vesicles composed of different mole ratios of dilauroyl- and dipalmitoyl-phosphatidylcholine (DLPC and DPPC, respectively). The recently introduced generalized polarization together with time-resolved emission spectra have been utilized for detecting changes. The results indicate the coexistence of phospholipid phase domains in vesicle compositions in the range between 30 mol% and 70 mol% DPPC in DLPC. Below and above these concentrations a homogeneous phase is observed, with averaged properties. In the case of coexisting phase domains, the properties of each individual phase are largely influenced by the presence of the other phase. Implications on fluctuations between the coexisting phases and on the size and shape of domains are discussed.

A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties.

Although 6-lauroyl-2-(N,N-dimethylamino)naphthalene (LAURDAN) is now widely used as a probe for lipid systems, most studies focus on the effect of the lipid environment on its emission properties but not on the excitation properties. The present study is intended to investigate the excitation properties of LAURDAN in diverse lipid environments. To this end, the fluorescence properties of LAURDAN were studied in synthetic ester and ether phosphatidylcholines and sphingomyelin vesicles below, at and above the corresponding lipid main phase-transition temperature. The excitation spectra of LAURDAN in these environments always show at least two well-resolved bands. In the different lipid vesicles the behavior of the red band in the LAURDAN excitation spectra is sensitive to the lipid chemical environment near the probe fluorescent moiety and to the packing of the different lipid phases (gel and liquid crystalline). We propose that the interaction between the LAURDAN dimethylamino group and the ester linkage of ester phospholipids is responsible for the strong stabilization of LAURDAN's red excitation band in the gel phase of ester phospholipid vesicles. We discuss the consequence of these proposed ground-state interactions on LAURDAN's emission generalized polarization function. In the context of variable excitation wavelengths, information concerning solvent dipolar relaxation through excitation generalized polarization function is also discussed.

Giant phospholipid vesicles: comparison among the whole lipid sample characteristics using different preparation methods: a two photon fluorescence microscopy study.

Several methods for the preparation of giant unilamellar vesicles (GUVs) using synthetic phosphatidylcholine phospholipids were evaluated. We compared the physical characteristics--in terms of lamellarity and morphology--of the whole lipid sample for each different lipid preparation using the sectioning capability of the two-photon excitation fluorescence microscope. From the evaluation of the entire lipid sample we determined that vesicle size, internal shape and shell thickness distributions depend on the vesicle's preparation method. Our results show that the preparation of giant unilamellar vesicles by the application of external electric fields offers several advantages among the other methods tested here. Using this method a high yield (approximately 95%) of giant unilamellar vesicles with a narrow size distribution was obtained. Independently of the preparation method, some lipid structures, which are held together by lipid tethers, were identified and resolved. These particular lipid structures show shell thickness and size heterogeneity. Labeling the lipid samples with 6-lauroyl-2-(N,N-dimethylamino)naphtalene (LAURDAN) and using the LAURDAN generalized polarization function we show that the lipid packing in these tethers or tubes is similar to those found in the phospholipid vesicles. The fact that both vesicles and tethers are found in the lipid preparations indicates similar stability between these structures.

Membrane oxidative damage induced by ionizing radiation detected by diphenylhexatriene fluorescence lifetime distributions.

The sensitivity of the fluorescence lifetime of 1,6-diphenyl- 1,3,5-hexatriene (DPH) to the dielectric constant of its environment has been used to detect oxidative damage to phospholipid membranes induced by ionizing radiation. The DPH fluorescence decay in phospholipid vesicles is described well by a continuous distribution of lifetime values, reflecting the various DPH depths in the bilayer and related to the gradient of the dielectric constant. Ionizing radiation oxidizes unsaturated acyl residues of phospholipids, altering the dielectric constant across the bilayer, sharpening the distribution of DPH lifetimes and increasing the centre of the distribution. Ionizing radiation doses between 22 and 110 Gy were used, and were effective only in the presence of oxygen. A model based on the formation of packing defects in the bilayer describes the phenomenon.

Human cell membrane oxidative damage induced by single and fractionated doses of ionizing radiation: a fluorescence spectroscopy study.

PURPOSE: To investigate the production and repair of lipid oxidative damage in two human cell lines exposed to acute and fractionated dose of ionizing radiation. Radiation dose was in the range from 0.1 to 44 Gy. MATERIALS AND METHODS: K562 and HL60 human cell lines have been used, 24 and 96 h after seeding. Membrane lipid oxidative damage has been detected by the measurement of the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH), its polarization value and the conjugated dienes concentration. The modification of DPH decay has been previously reported to be directly related to the lipid hydroperoxide concentration. RESULTS: A modification of the DPH decay has been observed as a linear function of the logarithm of the radiation dose and only when the irradiation was performed in the presence of oxygen. The amount of the damage is related to the time after the cell medium change. By exposing the cells to fractionated radiation doses for several days (10 cGy day(-1)), the oxidative damage has been found to be cumulative. After a single acute dose, evidence of repair of the lipid oxidative damage was not obtained. CONCLUSIONS: Following a previously developed method, the membrane damage was attributed to the production of hydroperoxide residues in the lipid acyl chains with the consequence of water penetration into the external portion of the bilayer, from the aqueous environment to the position of hydroperoxides. This damage is not repaired. The results obtained by measuring the DPH fluorescence decay have been compared with those obtained using other current optical and biochemical methods. None of these techniques could detect membrane oxidative damage at doses < 10 Gy. Finally, the different sensitivity of 'young' and 'old' cells to the oxidative damage can be related to different cholesterol concentrations.

Alterations in erythrocyte membrane lipids induced by low doses of ionizing radiation as revealed by 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime.

Damage in membrane lipids induced by low doses of ionizing radiation in the presence of oxygen has been detected in rabbit erythrocyte ghosts labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH). Multifrequency phase and modulation fluorometry was used to measure DPH fluorescence lifetime. This technique is particularly suited for the observation of heterogeneous fluorescence decays. DPH decay in erythrocyte membranes is described by a two-component continuous distribution of lifetimes. The value of the distribution width of the long-lived component is found to be affected by radiation-induced membrane lipid damage at doses as low as 0.5 Gy, well within the dose range used to measure cell survival. The width of the DPH lifetime distribution decreases when the ghosts are irradiated in the presence of oxygen. Such a decrease is a linear function of the logarithm of the dose. After a dose of 110 Gy and above, the fractional intensity of the short-lived component of the DPH decay increases linearly, indicating severe membrane damage. Experiments performed in the absence of oxygen do not show any change in the fluorescence parameters up to a dose of 550 Gy. The molecular identification of the produced damage has not been accomplished, but the necessity of oxygen to observe the damage suggests that hydroperoxides and lipids crosslinks are produced.

Evidence for an increase in water concentration in bilayers after oxidative damage of phospholipids induced by ionizing radiation.

The two membrane fluorescent probes 2-dimethyl-amino-6-lauroyl-naphthalene (Laurdan) and 2-dimethylamino-6-propionyl-naphthalene (Prodan) have been used to study the molecular basis of the damage induced in phospholipid membranes by ionizing radiation. Laurdan and Prodan display a spectral sensitivity to the polarity of their environment, showing a red shift of both excitation and emission spectra with increase of the polarity of their environment. Owing to their chemical differences, the two probes are anchored in the membrane with different strengths. In aqueous environments Laurdan is not fluorescent while Prodan shows appreciable fluorescence. Laurdan and Prodan show an opposite response to oxidative damage produced in phospholipid bilayers by ionizing radiation. The results support the model recently developed of water penetration in the bilayer as a consequence of oxidative damage.

Phototoxic effect of fluoroquinolones on two human cell lines.

Photosensitization induced by the fluoroquinolone ofloxacin (OFLX) has been studied using two human cell lines, HL60 and K562, two UV wavelengths, 290 and 330 nm, and two different exposure protocols, acute and protracted. The examined endpoints are the cellular lethality and recovery and the membrane changes produced by the oxidative damage, studied using cloning and counting techniques and the measurement of the generalized polarization (GP) of the fluorescent membrane probe 2-dimethylamino-6-lauroyl-naphthalene (Laurdan). The results show that: (i) the photosensitizing effect is detectable at concentrations similar to those found in patients treated with OFLX only when the cells are irradiated with 330 nm; (ii) the amount of photodamage is a function of the drug concentration and of UV dose and persists also after the removal of the drug; (iii) during the first 24 h after OFLX treatment, a large decrease of the cell number can be observed due to cell lysis; (iv) the OFLX is inserted in the cell membranes at concentrations directly related with the drug concentration and incubation time; (v) the OFLX produces an increase in the GP values similar to that produced by membrane lipid oxidation which persists for hours after the removal of the drug. The overall results suggest the cell membrane as the main target of the OFLX adverse action, with a possible mechanism involving the formation of reactive oxygen species (ROS), which triggers, in turn, the lipid peroxidation chain reaction.