4-Hydroxynonenal induces dysfunction and apoptosis of cultured endothelial cells.

Lipolytic products of triglyceride-rich lipoproteins, i.e., free fatty acids, may cause activation and dysfunction of the vascular endothelium. Mechanisms of these effects may include lipid peroxidation. One of the major and biologically active products of peroxidation of n-6 fatty acids, such as linoleic acid or arachidonic acid, is the aldehyde 4-hydroxynonenal (HNE). To study the hypothesis that HNE may be a critical factor in endothelial cell dysfunction caused by free fatty acids, human umbilical endothelial cells (HUVEC) were treated with up to160 microM of linoleic or arachidonic acid. HNE formation was detected by immunocytochemistry in cells treated for 24 h with either fatty acid, but more markedly with arachidonic acid. To study the cellulareffects of HNE, HUVEC were treated with different concentrations of this aldehyde, and several markers of endothelial cell dysfunction were determined. Exposure to HNE for 6 and 9 h resulted in increased cellular oxidative stress. However, short time treatment with HNE did not cause activation of nuclear factor-kappaB (NF-kappaB). In addition, HUVEC exposure to HNE caused a dose-dependent decrease in production of both interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1). On the other hand, HNE exerted prominent cytotoxic effects in cultured HUVEC, manifested by morphological changes, diminished cellular viability, and impaired endothelial barrier function. Furthermore, HNE treatment induced apoptosis of HUVEC. These data provide evidence that HNE does not contribute to NF-kappaB-related mechanisms of the inflammatory response in HUVEC, but rather to endothelial dysfunction, cytotoxicity, and apoptotic cell death.     

Novel molecular approaches for improving enzymatic and nonenzymatic detoxification of 4-hydroxynonenal: toward the discovery of a novel class of bioactive compounds

4-Hydroxy-trans-2-nonenal (HNE), an α,β-unsaturated aldehyde generated endogenously by the radical-mediated peroxidation of ω-6 polyunsaturated fatty acids, is a bioactive molecule acting in several physiopathological mechanisms and most of its activity is due to the covalent modification of biomolecules. Although at low and physiological levels HNE acts as an endogenous signaling molecule, a growing bulk of evidence indicates that at high and toxic concentrations, HNE is involved in the onset and propagation of several human diseases. To get more conclusive evidence of HNE as a pathogenetic factor, a pharmacological tool able to inhibit the HNE-induced cellular response is required. Such compound is currently not available, although several molecular strategies have so far been reported with the aim of inhibiting HNE formation or catalyzing its removal. Although most of these are not selective, such strategies have been found to induce several biological responses and would merit further investigation. In this review the various strategies are reported and discussed together with their limits and potentials.     

4-Hydroxynonenal, a Lipid Peroxidation Product, Rapidly Accumulates Following Traumatic Spinal Cord Injury and Inhibits Glutamate Uptake

Abstract: Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult. One such event is the accumulation of free radicals that damage lipids, proteins, and nucleic acids. A major reactive product formed following lipid peroxidation is the aldehyde, 4-hydroxynonenal (HNE), which cross-links to side chain amino acids and inhibits the function of several key metabolic enzymes. In the present study, we used immunocytochemical and immunoblotting techniques to examine the accumulation of protein-bound HNE, and synaptosomal preparations to study the effects of spinal cord injury and HNE formation on glutamate uptake. Protein-bound HNE increased in content in the damaged spinal cord at early times following injury (1–24 h) and was found to accumulate in myelinated fibers distant to the site of injury. Immunoblots revealed that protein-bound HNE levels increased dramatically over the same postinjury interval. Glutamate uptake in synaptosomal preparations from injured spinal cords was decreased by 65% at 24 h following injury. Treatment of control spinal cord synaptosomes with HNE was found to decrease significantly, in a dose-dependent fashion, glutamate uptake, an effect that was mimicked by inducers of lipid peroxidation. Taken together, these findings demonstrate that the lipid peroxidation product HNE rapidly accumulates in the spinal cord following injury and that a major consequence of HNE accumulation is a decrease in glutamate uptake, which may potentiate neuronal cell dysfunction and death through excitotoxic mechanisms.     

4-Hydroxynonenal-protein adducts: A reliable biomarker of lipid oxidation in liver diseases

The aldehyde 4-hydroxynonenal (HNE) is a major end-product of peroxidation of membrane n-6-polyunsaturated fatty acids. Primary reactants for HNE are the amino acids cysteine, histidine and lysine, and quantitatively, proteins and peptides represent the most important group of HNE-targeted biomolecules. HNE-protein adducts actually elude the metabolism of the aldehyde, particularly active in the liver, so that they can be easily detected in the hepatic tissue itself and in peripheral blood, and quantified by using immunoassays. Since consistently detectable in various liver disease processes and well related to the intensity of necro-inflammation, HNE-protein adducts may be considered a particularly good marker of lipid oxidation during liver injury. In addition, the demonstrated adduction reaction of HNE with important signalling proteins strongly suggests a pathogenetic role for this lipid aldehyde in the progression of liver diseases.     

From a computational point of view: deciphering the molecular synergism between oxidative stress-induced lipid peroxidation products and metabolic dysfunctionality of human liver mitochondrial aldehyde dehydrogenase-2

Accumulation of oxidative stress-induced lipid peroxidation products; 4-hydroxynonenal (4HNE) and 4-oxononenal (4ONE), inactivates the metabolic activity of human liver aldehyde dehydrogenase 2 (ALDH2), an enzyme that converts acetaldehyde to carboxylic acids during alcohol metabolism. Previous reports showed that 4HNE and 4ONE covalently target the catalytic Cys302 residue and inactivate ALDH2, thereby preventing the metabolism of acetaldehyde (ACE), its primary substrate. However, the molecular basis of these reactions remains elusive. Therefore, in this study, we investigated the inactivation mechanism of 4HNE and 4ONE on ALDH2 using advanced computational tools. Interestingly, our findings revealed that both inhibitors significantly distorted ALDH2 oligomerization and co-enzyme binding domains, which are crucial to its metabolic activity. The resulting structural alterations could disrupt co-factor binding and enzymatic oligomerization mechanisms. In contrast to the acetaldehyde, 4HNE and 4ONE were bound to ALDH2 with high affinity, coupled with high energy contributions by catalytic site residues and could indicate the possible mechanism by which acetaldehyde is displaced from ALDH2 binding by 4HNE and 4ONE. These findings will be useful in the design of novel compounds that either mop up or block the binding of these endogenous compounds to ALDH2 thereby preventing the development of associated cancers and neurodegenerative diseases.     

The inclusion complex of 4-hydroxynonenal with a polymeric derivative of β-cyclodextrin enhances the antitumoral efficacy of the aldehyde in several tumor cell lines and in a three-dimensional human melanoma model

4-Hydroxynonenal (HNE) is the most studied end product of the lipoperoxidation process, by virtue of its relevant biological activity. The antiproliferative and proapoptotic effects of HNE have been widely demonstrated in a great variety of tumor cell types in vitro. Thus, it might represent a promising new molecule in anticancer therapy strategies. However, the extreme reactivity of this aldehyde, as well as its insolubility in water, a limiting factor for drug bioavailability, and its rapid degradation by specific enzymes represent major obstacles to its possible in vivo application. Various strategies can used to overcome these problems. One of the most attractive strategies is the use of nanovehicles, because loading drugs into nanosized structures enhances their stability and solubility, thus improving their bioavailability and their antitumoral effectiveness. Several natural or synthetic polymers have been used to synthesize nanosized structures and, among them, β-cyclodextrin (βCD) polymers are playing a very important role in drug formulation by virtue of the ability of βCD to form inclusion compounds with a wide range of solid and liquid molecules by molecular complexation. Moreover, several βCD derivatives have been designed to improve their physicochemical properties and inclusion capacities. Here we report that the inclusion complex of HNE with a derivative of βCD, the βCD–poly(4-acryloylmorpholine) conjugate (PACM-βCD), enhances the aldehyde stability. Moreover, the inclusion of HNE in PACM-βCD potentiates its antitumor effects in several tumor cell lines and in a more complex system, such as a human reconstructed skin carrying melanoma tumor cells.     

4-hydroxynonenal as a bioactive marker of pathophysiological processes

The review is focused on the currently major aspect of 4-hydroxynonenal (HNE) research--studies that combine biological activities of the aldehyde together with the methods of its identification in cells and tissues. Because there were some excellent reviews on HNE published in recent years, starting in 1990 and 1991 with supreme reviews done by Hermann Esterbauer, who discovered the aldehyde, and colleagues from the Institute of Biochemistry in Graz, this article pays most of attention to the most recent articles, published in the last 15 months. Additionally, an overview on the relevance of HNE is given with respect to the research and publication trends in the period of 10 years (1993-2002) according to the data in the Current Contents and Medline data bases. It is obvious that HNE started in 1993 as a "toxic product of lipid peroxidation" and "second toxic messenger of free radicals", to become in 2002 a reliable marker of oxidative stress, a possible causative agent of several diseases (such as Alzheimer's disease), growth modulating factor and a signaling molecule. Novel analytical methods developed suitable pathways for HNE to become a clinically applicable marker of lipid peroxidation on one side and on the other a standardized parameter of food quality control. As it is also present physiologically in various cells and tissues, it is likely that HNE will soon become one of the most attractive factors for those who search for a small and reactive molecular link between genomics and proteomics.     

4-Hydroxynonenal-induced MEL cell differentiation involves PKC activity translocation

4-Hydroxynonenal (HNE) is a highly reactive aldehyde, produced by cellular lipid peroxidation, able to inhibit proliferation and to induce differentiation in MEL cells at concentrations similar to those detected in several normal tissues. Inducer-mediated differentiation of murine erythroleukemia (MEL) cells is a multiple step process characterized by modulation of several genes as well as by a transient increase in the amount of membrane-associated protein kinase C (PKC) activity. Here we demonstrate that a rapid translocation of PKC activity from cytosol to the membranes occurs during the differentiation induced by HNE. When PKC is completely translocated by phorbol-12-myristate-13-acetate (TPA), the degree of HNE-induced MEL cells differentiation is highly decreased. However, if TPA is washed out from the culture medium before the exposition to the aldehyde, HNE gradually resumes its differentiative ability. The incubation of cells with a selective inhibitor of PKC activity, bisindolylmaleimide GF 109203X, partially prevents the HNE-induced differentiation in MEL cells. In conclusion, our results demonstrate that HNE-induced MEL cell differentiation is preceded by a rapid translocation of PKC activity, and that the inhibition of this phenomenon prevents the onset of terminal differentiation.     

Fate of 4-hydroxynonenal in vivo: disposition and metabolic pathways

Due to the cytotoxicity of 4-hydroxynonenal (HNE), and to the fact that this major product of lipid peroxidation is a rather long-living compound compared with reactive oxygen species, the capability of organisms to inactivate and eliminate HNE has received increasing attention during the last decade. Several recent in vivo studies have addressed the issue of the diffusion, kinetics, biotransformation and excretion of HNE. Part of these studies are primarily concerned with the toxicological significance of HNE biotransformation and more precisely with the metabolic pathways by which HNE is inactivated and eliminated. The other aim of in vivo metabolic study is the characterisation of end-metabolites, especially in urine, in order to develop specific and non-invasive biomarkers of lipid peroxidation. When HNE is administered intravenously or intraperitoneally, it is mainly excreted into urine and bile as conjugated metabolites, in a proportion that is dependent on the administration route. However, biliary metabolites undergo an enterohepatic cycle that limits the final excretion of faecal metabolites. Only a very low amount of metabolites is found to be bound to macromolecules. The main urinary metabolites are represented by two groups of compounds. One comes from the mercapturic acid formation from (i) 1,4 dihydroxynonene-glutathione (DHN-GSH) which originates from the conjugation of HNE with GSH by glutathione-S-transferases and the subsequent reduction of the aldehyde by a member of aldo-keto reductase superfamily; (ii) the lactone of 4-hydroxynonanoic-GSH (HNA-lactone-GSH) which originates from the conjugation of HNE followed by the oxidation of the aldehyde by aldehyde dehydrogenase; (iii) HNA-GSH which originates from the hydrolysis of the corresponding lactone. The other one is a group of metabolites issuing from the omega-hydroxylation of HNA or HNA-lactone by cytochromes P450 4A, followed eventually, in the case of omega-oxidized-HNA-lactone, by conjugation with GSH and subsequent mercapturic acid formation. Biliary metabolites are GSH or mercapturic acid conjugates of DHN, HNE and HNA. Stereochemical aspects of HNE metabolism are also discussed.     

Odorant binding protein has the biochemical properties of a scavenger for 4-hydroxy-2-nonenal in mammalian nasal mucosa

Odorant binding proteins (OBP) are soluble lipocalins produced in large amounts in the nasal mucosa of several mammalian species. Although OBPs can bind a large variety of odorous compounds, direct and exclusive involvement of these proteins in olfactory perception has not been clearly demonstrated. This study investigated the binding properties and chemical resistance of OBP to the chemically reactive lipid peroxidation end-product 4-hydroxy-2-nonenal (HNE), in an attempt to establish a functional relationship between this protein and the molecular mechanisms combating free radical cellular damage. Experiments were carried out on recombinant porcine and bovine OBPs and results showed that both forms were able to bind HNE with affinities comparable with those of typical OBP ligands (K(d) = 4.9 and 9.0 microm for porcine and bovine OBP, respectively). Furthermore, OBP functionality, as determined by measuring the binding of the fluorescent ligand 1-aminoanthracene, was partially lost only when incubating HNE levels and exposure time to HNE exceeded physiological values in nasal mucosa. Finally, preliminary experiments in a simplified model resembling nasal epithelium showed that extracellular OBP can preserve the viability of an epithelial cell line derived from bovine turbinates exposed to toxic amounts of the aldehyde. These results suggest that OBP, which is expressed at millimolar levels, might reduce HNE toxicity by removing from the nasal mucus a significant fraction of the aldehyde that is produced as a consequence of direct exposure to the oxygen present in inhaled air.     

Down-modulation of nuclear localisation and pro-fibrogenic effect of 4-hydroxy-2,3-nonenal by thiol- and carbonyl-reagents

Among the oxidative breakdown products of ω-6 unsaturated fatty acids, the aldehyde 4-hydroxy-2,3-nonenal (HNE) is receiving increasing attention for its potential pathophysiological implication, which at least partly lies on the demonstrated ability to modulate gene expression of a number of genes. Here we show that a marked down-modulation of HNE nuclear localisation in cells of a macrophage line (J774-A1) can be afforded by treatment with sulfydryl and carbonyl reagents without significantly interfering with cell viability. As regards the addition of thiol-group reagents to the cell suspension, N-ethylmaleimide (NEM) led to a sustained decrease of HNE nuclear localisation, while 4-(chloromercuri)-benzene-sulfonic acid (PCMBS) gave a similar but more transient effect. Hydroxylamine (HYD), a carbonyl-group reagent, was also able to inhibit HNE nuclear localisation. The actual efficacy of the inhibitors used was then tested on the HNE-induced stimulation of transforming growth factor β1 (TGFβ1) production by J774-A1 cells. Indeed, the thiol reagents NEM and PCMBS, both markedly down-modulating HNE nuclear localisation, were able to inhibit HNE-induced increase of TGFβ1 protein synthesis. The carbonyl reagent HYD was less effective on this respect, producing strong but incomplete protection against HNE-induced TGFβ1 increase. Taken together, the results indicate that sulfydryl groups are involved in the process of HNE cellular internalisation, while both sulfydryl and carbonyl groups are involved in the process of HNE nuclear translocation, and consequently in the modulation of gene expression by the aldehyde. Further, an actual demonstration is provided that HNE-induced effect on gene regulation can be efficiently counteracted by suitable interference with HNE biochemistry.     

Reactions of 4-hydroxynonenal with proteins and cellular targets

Peroxidative degradation of lipids yields the aldehyde 4-hydroxy-2-nonenal (4HNE) as a major product. The lipid aldehyde is an electrophile, and reactivity of 4HNE toward protein nucleophiles (i.e., Cys, His, and Lys) has been characterized. Through the use of purified enzymes and isolated cells, various pathways for biotransformation of the lipid aldehyde have been identified and include enzyme-mediated oxidation, reduction, and glutathione conjugation. Uncontrolled oxidative stress can yield excessive lipid peroxidation and 4HNE generation, however, and overwhelm these cellular defenses. Indeed, in vitro and in vivo production of 4HNE in response to pro-oxidant exposure has been demonstrated using antibodies to protein adducts of the lipid aldehyde. Recent evidence suggests a role for protein modification by 4HNE in the pathogenesis of several diseases (e.g., alcohol-induced liver disease); however, the precise mechanism(s) is currently unknown but likely results from adduction of proteins involved in cellular homeostasis or biological signaling.     

Basic aspects of the biochemical reactivity of 4-hydroxynonenal

4-hydroxynonenal (HNE), a major lipid peroxidation product of n-6 polyunsaturated fatty acids, which was discovered by the late Hermann Esterbauer, is a remarkable trifunctional molecule. Both the hydroxy group and the conjugated system consisting of a C=C double bond and a carbonyl group contribute to the high reactivity of HNE. Most of the biochemical effects of HNE can be explained by its rapid reactions with thiol and amino groups. Among the primary reactants for HNE are the amino acids cysteine, histidine and lysine, which--either free or protein-bound--undergo readily Michael additions to the C=C bond. After this primary reaction, which confers rotational freedom to the C2-C3 bond, secondary reactions may occur involving the carbonyl and the hydroxy group. Primary amines may alternatively react with the carbonyl group to form Schiff bases. Reactions which do not fit into this scheme are the oxidation and the reduction respective of the carbonyl group and the epoxidation of the C=C double bond. Examples will be presented for the interaction of HNE with various classes of biomolecules such as proteins and peptides, lipids and nucleic acids and the biochemical consequences will be discussed.     

Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal

The hepatic enzyme bile acid CoA:amino acid N-acyltransferase (BAT) catalyzes the formation of amino acid-conjugated bile acids. In the present study, protein carbonylation of BAT, consistent with modification by reactive oxygen species and their products, was increased in hepatic homogenates of apolipoprotein E knock-out mice. 4-Hydroxynonenal (4HNE), an electrophilic lipid generated by oxidation of polyunsaturated long-chain fatty acids, typically reacts with the amino acids Cys, His, Lys, and Arg to form adducts, some of which (Michael adducts) preserve the aldehyde (i.e., carbonyl) moiety. Because two of these amino acids (Cys and His) are members of the catalytic triad of human BAT, it was proposed that 4HNE would cause inactivation of this enzyme. As expected, human BAT (1.6 microM) was inactivated by 4HNE in a dose-dependent manner. To establish the sites of 4HNE's reaction with BAT, peptides from proteolysis of 4HNE-treated, recombinant human BAT were analyzed by peptide mass fingerprinting and by electrospray ionization-tandem mass spectrometry using a hybrid linear ion trap Fourier transform-ion cyclotron resonance mass spectrometer. The data revealed that the active-site His (His362) dose-dependently formed a 4HNE adduct, contributing to loss of activity, although 4HNE adducts on other residues may also contribute.     

A Role for 4-Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, in Disruption of Ion Homeostasis and Neuronal Death Induced by Amyloid β-Peptide

Abstract: Peroxidation of membrane lipids results in release of the aldehyde 4-hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid β-peptide (Aβ) can promote free radical production, we tested the hypothesis that HNE mediates Aβ25-35-induced disruption of neuronal ion homeostasis and cell death. Aβ induced large increases in levels of free and protein-bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time- and concentration-dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na⁺,K⁺-ATPase activity and induced an increase of neuronal intracellular free Ca²⁺ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both Aβ and HNE. The antioxidant propyl gallate protected neurons against Aβ toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates Aβ-induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.     

Apoptosis signalling by 4-hydroxynonenal: a role for JNK-c-Jun/AP-1 pathway

4-Hydroxynonenal (HNE) is one of the most abundant aldehyde components of ox-LDL and it exerts various effects on intracellular and extracellular signaling cascades. In this mini-review, a brief synopsis of HNE-modulated signaling pathways will be presented mainly focused on cell death, including recent studies from our laboratory. The results of a number of studies demonstrate the ability of HNE to induce apoptosis and ROS formation in a dose-dependent manner. Several signaling pathways have been shown to be modulated by HNE, including MAP kinases, PKC isoforms, cell-cycle regulators, receptor tyrosine kinases and caspases. In order to get insight into the mechanisms of apoptotic response by HNE, MAP kinase and caspase activation pathways have been studied in 3T3 fibroblasts; HNE induced early activation of JNK and p38 proteins but down-regulated the basal activity of ERK-1/2. We have shown that HNE-induced release of cytochrome c from mitochondria, caspase-9 and caspase-3 activation. Activation of AP-1 along with increased c-Jun and phospho-c-Jun levels could be inhibited by pretreatment of cells with certain molecules such as resveratrol. Additionally, overexpression of dominant negative c-Jun and JNK1 in 3T3 fibroblasts prevented HNE-induced apoptosis, which indicated a role for JNK-c-Jun/AP-1 pathway. JNK-dependent induction of c-Jun/AP-1 activation data in the literature indicates a critical potential role for JNK in the cellular response against toxic products of lipid peroxidation.     

Changes in IL-8 release and intracellular content in DMSO-differentiated HL-60 cells after treatment with 4-hydroxynonenal

4-Hydroxynonenal (HNE), a chemotactic aldehyde produced by lipid peroxidation, has been shown to trigger exocytosis in HL-60 cells induced to differentiate toward the granulocytic cell line by DMSO. In this work we studied HNE effects on the intracellular content of IL-8 and its release in DMSO-differentiated HL-60 cells. Cell incubation at 37 degrees C in the presence of 0.1 microM HNE induced a significant increase of IL-8 release after 30 min; the degree of HNE-induced IL-8 secretion became quite strong after 1 h, whereas the intracellular content showed no statistically significant changes. By contrast, 1 microM HNE induced a low decrease of the chemokine release; however, the used HNE concentrations failed to increase the release of lactate dehydrogenase (LDH), a test used to assay cell viability. The addition of 0.1 microM IL-8 to DMSO-differentiated HL-60 cells induced a strong increase of exocytosis, measured by beta-glucuronidase secretion. Exocytosis stimulation by IL-8 was much higher than that given by the aldehyde; the addition of various HNE concentrations to cells incubated in the presence of IL-8 decreased the secretion given by the cytokine alone. However, HNE-induced exocytosis was likely to be a direct action of the aldehyde and was not mediated through the stimulation of IL-8 release since HNE was unable to modify IL-8 secretion during the short time of 10 min used in the exocytosis assay.     

Influence of 4-hydroxynonenal on chemiluminescence production by unstimulated and opsonized zymosan-stimulated human neutrophils

The lipid peroxidation product 4-hydroxy-2, 3-trans-nonenal (HNE) has a spectrum of biological effects on different cell types depending on the concentrations tested. In particular micromolar HNE concentrations stimulate neutrophil migration and polarization whereas higher doses inhibit. In our experimental conditions, fMet-Leu-Phe (fMLP) increased CL production of both unstimulated and zymosan-stimulated neutrophils, whereas cell stimulation with low HNE concentrations as well as zymosan addition to HNE incubated cells did not enhance light emission. In contrast 10(-4) M HNE reduced CL emission by unstimulated cells nearly to background values, completely depressed CL production by zymosan-stimulated cells and reduced phagocytosis. Cysteine was found to be able to counteract the HNE effect by about 70 per cent. The possibility that this aldehyde could exert its inhibitory effect through the alkylation of NADPH-oxidase SH-groups is postulated. Moreover, our present data on differences observed between fMLP and HNE indicate a different chemotactic mechanism induced by these two classes of compounds and lead to the conclusion that the local functional features of the attracted cells may be different.