Fatty acid chlorohydrins and bromohydrins are cytotoxic to human endothelial cells

Reaction of unsaturated lipids with the hypohalous acids (hypochlorous acid and hypobromous acid) results in the addition of the halide (X) across double bonds to form halohydrins (-CH2CH(OH)CH(X)CH2-). These modified lipids could be potentially destabilising to cell membranes due to their increased polarity. We have investigated the effect of pre-formed halohydrins on human umbilical vein endothelial cells (HUVEC) by incubating cultured cells with oleic acid micelles containing chlorohydrins or bromohydrins. Cell detachment and necrotic death were observed with increasing doses of halohydrins, whereas the cells were unaffected by equivalent doses of oleic acid. Bromohydrins caused more lysis than did chlorohydrins at equivalent doses. Complete lysis was seen with 200 microM fatty acid/chlorohydrin micelles and with 50 microM fatty acid/bromohydrin micelles. Chlorohydrin uptake was much less than the oleic acid control whereas bromohydrins were incorporated into the endothelial cells similarly to oleic acid. This difference or the bulkier nature of the bromohydrins could account for their increased toxicity. This study has demonstrated the potential toxicity of the halohydrins, and implications for their formation in inflammation are discussed.     

Arachidonic acid randomizes endothelial cell motion and regulates adhesion and migration

Cell adhesion and migration are essential for the evolution, organization, and repair of living organisms. An example of a combination of these processes is the formation of new blood vessels (angiogenesis), which is mediated by a directed migration and adhesion of endothelial cells (ECs). Angiogenesis is an essential part of wound healing and a prerequisite of cancerous tumor growth. We investigated the effect of the amphiphilic compound arachidonic acid (AA) on EC adhesion and migration by combining live cell imaging with biophysical analysis methods. AA significantly influenced both EC adhesion and migration, in either a stimulating or inhibiting fashion depending on AA concentration. The temporal evolution of cell adhesion area was well described by a two-phase model. In the first phase, the spreading dynamics were independent of AA concentration. In the latter phase, the spreading dynamics increased at low AA concentrations and decreased at high AA concentrations. AA also affected EC migration; though the instantaneous speed of individual cells remained independent of AA concentration, the individual cells lost their sense of direction upon addition of AA, thus giving rise to an overall decrease in the collective motion of a confluent EC monolayer into vacant space. Addition of AA also caused ECs to become more elongated, this possibly being related to incorporation of AA in the EC membrane thus mediating a change in the viscosity of the membrane. Hence, AA is a promising non-receptor specific regulator of wound healing and angiogenesis.     

Fatty acid modulation of tumor cell adhesion to microvessel endothelium and experimental metastasis

Tumor cell interaction with the endothelium of the vessel wall is a rate limiting step in metastasis. The fatty acid modulation of this interaction was investigated in low (LM) and high (HM) metastatic B16 amelanotic melanoma (B16a) cells. 12(S)-HETE increased the adhesion of LM cells to endothelium derived from pulmonary microvessels. All other monohydroxy and dihydroxy fatty acids were ineffective. LTB₄ induced a modest stimulation but LTC₄, LTD₄, LTE₄ as well as LXA₄ and LXB₄ were ineffective. The 12(S)-HETE enhanced adhesion of B16a cells was inhibited by pretreatment with 13(S)-HODE but not by 13(R)-, 9(S)-HODE or 13-OXO-ODE. 13(S)-HODE decreased adhesion of HM B16a cells to endothelium. 12(S)-HETE enhanced surface expression of integrin αIIbβ₃ and monoclonal antibodies against this integrin but not against α₅β₁, blocked enhanced but not basal adhesion to endothelium. Intravenous injection of 12(S)-HETE treated LM cells resulted in increased lung colonization (experimental metastasis). This effect was specific for 12(S)-HETE and was inhibited by 13(S)-HODE but not by other HODE's. 12(S)-HETE also enhanced lung colonization by HM cells and 13(S)-HODE decreased lung colonization by HM cells. Our results suggest a highly specific bidirectional modulation of metastatic phenotype and lung colonization by 12(S)-HETE and 13(S)-HODE.     

Fatty acid contamination and dielectric relaxation in phospholipid vesicle suspensions

Aqueous vesicle or micelle suspensions from various synthetic lecithins or surfactants - most of them purified by a simple ion-exchange procedure in methanol - were investigated, some with ionic admixtures. The dielectric permittivity '(nu) between 5 kHz and 100 MHz was determined by different time-and frequency-domain methods, with attention given to electrode polarization below 1 MHz. Pure ether lecithins (used to reduce hydrolysis during preparation) as well as ester lecithins showed no dielectric dispersion below 10 MHz (Delta' 3). In contrast, even dilute colloidal solutions containing about 1 mol% (with respect to solute) ionic amphiphiles normally exhibited large dielectric dispersion (10 < Delta' < 700), especially with electrolyte present. This low-frequency dispersion is sensitive to vesicle coagulation or fusion. Underlying relaxation mechanisms are discussed, and the main relaxation is shown to be the same as for other charged colloids. This conclusion suggest a new interpretation of measurements, previously reported by other authors, who gave an interpretation in terms of correlated zwitterionic head group orientation in multilamellar lecithin liposomes. Possible effects from traces of impurities in lipids are discussed.     

Fatty acid modulation of tumor cell adhesion to microvessel endothelium and experimental metastasis.

Tumor cell interaction with the endothelium of the vessel wall is a rate limiting step in metastasis. The fatty acid modulation of this interaction was investigated in low (LM) and high (HM) metastatic B16 amelanotic melanoma (B16a) cells. 12(S)-HETE increased the adhesion of LM cells to endothelium derived from pulmonary microvessels. All other monohydroxy and dihydroxy fatty acids were ineffective. LTB4 induced a modest stimulation but LTC4, LTD4, LTE4 as well as LXA4 and LXB4 were ineffective. The 12(S)-HETE enhanced adhesion of B16a cells was inhibited by pretreatment with 13(S)-HODE but not by 13(R)-, 9(S)-HODE or 13-OXO-ODE. 13(S)-HODE decreased adhesion of HM B16a cells to endothelium. 12(S)-HETE enhanced surface expression of integrin alpha IIb beta 3 and monoclonal antibodies against this integrin but not against alpha 5 beta 1, blocked enhanced but not basal adhesion to endothelium. Intravenous injection of 12(S)-HETE treated LM cells resulted in increased lung colonization (experimental metastasis). This effect was specific for 12(S)-HETE and was inhibited by 13(S)-HODE but not by other HODE's. 12(S)-HETE also enhanced lung colonization by HM cells and 13(S)-HODE decreased lung colonization by HM cells. Our results suggest a highly specific bidirectional modulation of metastatic phenotype and lung colonization by 12(S)-HETE and 13(S)-HODE.     

Phospholipid chlorohydrins cause ATP depletion and toxicity in human myeloid cells

Chlorohydrins of stearoyl-oleoyl phosphatidylcholine (SOPC), stearoyl-linoleoyl phosphatidylcholine, and stearoyl-arachidonyl phosphatidylcholine were incubated with cultured myeloid cells (HL60) for 24 h, and the cellular ATP level was measured using a bioluminescent assay. The chlorohydrins caused significant depletion of cellular ATP in the range 10–100 μM. The ATP depletion by the phospholipid chlorohydrins was slightly less than that of 4-hydroxy-2-nonenal, but greater than that of hexanal, trans-2-nonenal, and autoxidised palmitoyl-arachidonoyl phosphatidylcholine. SOPC chlorohydrin was also found to cause loss of viability in U937 cells, and thus phospholipid chlorohydrins could contribute to the formation of a necrotic core in advanced atherosclerotic lesions.     

Modification of red cell membrane lipids by hypochlorous acid and haemolysis by preformed lipid chlorohydrins

Summary

Hypochlorous acid (HOCI), a strong oxidant generated by the myeloperoxidase system of neutrophils and monocytes, has been implicated in inflammatory tissue damage by these cells. Reaction of HOCI with the double bonds of unsaturated lipids produces α, β-chlorohydrin isomers. We have expose red cell membranes to HOCI and used thin layer chromatography (TLC) of the extracted lipids and enzyme-linked immunosorbent assay (ELISA), using an antichlorohydrin monoclonal antibody, to show that fatty acyl chlorohydrins are formed. The ELISA was approximately 25 fold more sensitive than TLC, and chlorohydrins were detected when membranes from 10⁶ cells were treated with ≥ 0.16 nmoles HOCI. Lipid chlorohydrins are more polar and bulky than their parent lipids and as such could affect membrane stability and function. To determine the effect of incorporation of lipid chlorohydrins into cell membranes, preformed fatty acid and cholesterol chlorohydrins were incubated with red cells. Lysis was measured as release of haemoglobin and incorporation of lipids was determined by ¹⁴C scintillation counting. Addition of HOCI-treated oleic acid to red cells resulted in rapid lysis ofa fraction of the cells in a concentration dependent manner. HOCI-treated cholesterol also caused a small amount of cell lysis that was predominantly due to chlorohydrin 3, one of the three major cholesterol chlorohydrin products. Chlorohydrin 3, which has a decreased planarity and polarity, was also primarily responsible for altering the critical micelle concentration of HOCI-treated cholesterol-containing liposomes.     

The LINC complex is required for endothelial cell adhesion and adaptation to shear stress and cyclic stretch

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a structure consisting of nesprin, SUN, and lamin proteins. A principal function of the LINC complex is anchoring the nucleus to the actin, microtubule, and intermediate filament cytoskeletons. The LINC complex is present in nearly all cell types, including endothelial cells. Endothelial cells line the innermost surfaces of blood vessels and are critical for blood vessel barrier function. In addition, endothelial cells have specialized functions, including adaptation to the mechanical forces of blood flow. Previous studies have shown that depletion of individual nesprin isoforms results in impaired endothelial cell function. To further investigate the role of the LINC complex in endothelial cells we utilized dominant negative KASH (DN-KASH), a dominant negative protein that displaces endogenous nesprins from the nuclear envelope and disrupts nuclear–cytoskeletal connections. Endothelial cells expressing DN-KASH had altered cell–cell adhesion and barrier function, as well as altered cell–matrix adhesion and focal adhesion dynamics. In addition, cells expressing DN-KASH failed to properly adapt to shear stress or cyclic stretch. DN-KASH–expressing cells exhibited impaired collective cell migration in wound healing and angiogenesis assays. Our results demonstrate the importance of an intact LINC complex in endothelial cell function and homeostasis.     

Fatty acid composition in native and cultured human endothelial cells

Summary Endothelial cells from human umbilical veins were isolated by collagenase treatment. Cells were cultured in the presence of either 20% fetal bovine serum (FBS) or 20% human serum (HS). At confluency, endothelial cell lipids were labeled with tracer concentrations of tritiated arachidonic acid, then extracted and separated into lipid subclasses by thin layer chromatography. The fatty acid composition of each lipid class was determined by glass capillary gas-liquid chromatography analysis and compared to that of cells freshly isolated from the cord (NC cells). The fatty acid compositions differed only in phospholipids. Polyunsaturated fatty acids (PFAs), arachidonic, and linoleic acids were depleted in FBS cell phospholipids and replaced by both stearic and oleic acids. No significant difference could be observed between NC cell and HS cell phospholipids. We conclude that PFAs might be decreased in FBS cells because of the relative paucity of PFAs in FBS as compared to HS. It seems therefore more convenient to cultivate endothelial cells in the presence of HS, especially in respect to their phospholipid content of arachidonic acid, which is the physiological reservoir for prostacyclin synthesis. This work was supported by a grant from the Délégation Générale à la Recherche Scientifique et Technique, Paris, France (79.7.0091).     

Fatty acid composition in native and cultured human endothelial cells

Endothelial cells from human umbilical veins were isolated by collagenase treatment. Cells were cultured in the presence of either 20% fetal bovine serum (FBS) or 20% human serum (HS). At confluency, endothelial cell lipids were labeled with tracer concentrations of tritiated arachidonic acid, then extracted and separated into lipid subclasses by thin layer chromatography. The fatty acid composition of each lipid class was determined by glass capillary gas-liquid chromatography analysis and compared to that of cells freshly isolated from the cord (NC cells). The fatty acid compositions differed only in phospholipids. Polyunsaturated fatty acids (PFAs), arachidonic, and linoleic acids were depleted in FBS cell phospholipids and replaced by both stearic and oleic acids. No significant difference could be observed between NC cell and HS cell phospholipids. We conclude that PFAs might be decreased in FBS cells because of the relative paucity of PFAs in FBS as compared to HS. It seems therefore more convenient to cultivate endothelial cells in the presence of HS, especially in respect to their phospholipid content of arachidonic acid, which is the physiological reservoir for prostacyclin synthesis.     

Fatty acid synthesis and metabolism of phospholipid acyl groups in strain L mouse fibroblasts

Strain L mouse fibroblasts grown in medium supplemented with 2.5% delipidized horse serum were found capable of desaturating oleic and linoleic acid to dienoic and trienoic acid(s), respectively. Although 40-60% of de novo fatty acid synthesis from [2-3H]acetate was inhibited by the administration of exogenous oleic or linoleic acid, sterole synthesis was only slightly affected. Within 24-48 h after incorporation, phospholipid fatty acyl groups could undergo active exchange between phospholipids. After this dynamic transition period was over, not only were the phospholipid acyls retained but some vicinal fatty acyl pairs of phospholipid also appeared to be stable and remained together throughout the depletion period. At any time in the experiment, however, introduction of exogenous fatty acid perturbed this phospholipid acyl retention, delayed the time at which the phospholipid acyl groups no longer moved between phospholipids and also decreased the ultimate number of phospholipid acyl groups retained by strain L mouse fibroblasts.     

Glycated collagen and altered glucose increase endothelial cell adhesion strength

Cell adhesion strength is important to cell survival, proliferation, migration, and mechanotransduction, yet changes in endothelial cell adhesion strength have not yet been examined in diseases such as diabetes with high rates of cardiovascular complications. We therefore investigated porcine aortic endothelial cell adhesion strength on native and glycated collagen‐coated substrates and in low, normal, and high glucose culture using a spinning disc apparatus. Adhesion strength increased by 30 dynes/cm² in cells on glycated collagen as compared to native collagen. Attachment studies revealed that cells use higher adhesion strength α_vβ₃ integrins to bind to glycated collagen instead of the typical α₂β₁ integrins used to bind to native collagen. Similarly, endothelial cells cultured in low and high glucose had 15 dynes/cm² higher adhesion strength than cells in normal glucose after 2 days. Increased adhesion strength was due to elevated VEGF release and intracellular PKC in low and high glucose cells, respectively. Thus glucose increased endothelial cell adhesion strength via different underlying mechanisms. These adhesion strength changes could contribute to diabetic vascular disease, including accelerated atherosclerosis and disordered angiogenesis. J. Cell. Physiol. 228: 1727–1736, 2013. © 2013 Wiley Periodicals, Inc.     

Methyl mercury triggers endothelial leukocyte adhesion and increases expression of cell adhesion molecules and chemokines

Cardiovascular disease is the leading cause of morbidity, mortality, and health care costs in the USA, and around the world. Among the various risk factors of cardiovascular disease, environmental and dietary exposures to methyl mercury, a highly toxic metal traditionally labeled as a neurotoxin, have been epidemiologically linked to human cardiovascular disease development. However, its role in development and promotion of atherosclerosis, an initial step in more immediately life-threatening cardiovascular diseases, remains unclear. This study was conducted to examine the role that methyl mercury plays in the adhesion of monocytes to human microvascular endothelial cells (HMEC-1), and the underlying mechanisms. Methyl mercury treatment significantly induced the adhesion of monocyte to HMEC-1 endothelial cells, a critical step in atherosclerosis, while also upregulating the expression of proinflammatory cytokines interleukin-6, interleukin-8. Further, methyl mercury treatment also upregulated the chemotactic cytokine monocyte chemoattractant protein-1 and intercellular adhesion molecule-1. These molecules are imperative for the firm adhesion of leukocytes to endothelial cells. Additionally, our results further demonstrated that methyl mercury stimulated a significant increase in NF-κB activation. These findings suggest that NF-κB signaling pathway activation by methyl mercury is an important factor in the binding of monocytes to endothelial cells. Finally, by using flow cytometric analysis, methyl mercury treatment caused a significant increase in necrotic cell death only at higher concentrations without initiating apoptosis. This study provides new insights into the molecular actions of methyl mercury that can lead to endothelial dysfunction, inflammation, and subsequent atherosclerotic development.     

Fatty acid synthase modulates homeostatic responses to myocardial stress

Fatty acid synthase (FAS) promotes energy storage through de novo lipogenesis and participates in signaling by the nuclear receptor PPARα in noncardiac tissues. To determine if de novo lipogenesis is relevant to cardiac physiology, we generated and characterized FAS knockout in the myocardium (FASKard) mice. FASKard mice develop normally, manifest normal resting heart function, and have normal cardiac PPARα signaling as well as fatty acid oxidation. However, they decompensate with stress. Most die within 1 h of transverse aortic constriction, probably due to arrhythmia. Voltage clamp measurements of FASKard cardiomyocytes show hyperactivation of L-type calcium channel current that could not be reversed with palmitate supplementation. Of the classic regulators of this current, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) but not protein kinase A signaling is activated in FASKard hearts, and knockdown of FAS in cultured cells activates CaMKII. In addition to being intolerant of the stress of acute pressure, FASKard hearts were also intolerant of the stress of aging, reflected as persistent CaMKII hyperactivation, progression to dilatation, and premature death by ～1 year of age. CaMKII signaling appears to be pathogenic in FASKard hearts because inhibition of its signaling in vivo rescues mice from early mortality after transverse aortic constriction. FAS was also increased in two mechanistically distinct mouse models of heart failure and in the hearts of humans with end stage cardiomyopathy. These data implicate a novel relationship between FAS and calcium signaling in the heart and suggest that FAS induction in stressed myocardium represents a compensatory response to protect cardiomyocytes from pathological calcium flux.     

Fatty acid oxidation and isoprostanes: Oxidative strain and oxidative stress

Oxidative stress is implicated as one of the key causes underlying many diseases. Free radicals are important constituents of basal physiology. Assessment of free radicals or the end products of their action has proved to be difficult. Consequently, authentication of the contribution of free radicals to physiology and pathology has usually been equivocal. Isoprostanes are biosynthesized in vivo, predominantly through free radical attack on arachidonic acid and are now regarded as robust biomarkers of oxidative stress in vivo. Isoprostanes are associated with many human diseases, and their concentration is altered over the course of normal human pregnancy, but their (patho)physiological roles have not yet been clearly defined. Measurement of F₂-isoprostanes in body fluids could offer a unique analytical opportunity to study the role of free radicals in physiology and pathophysiology in order to comprehend both oxidative strain and oxidative stress.     

Fatty acid and phospholipid composition of five psychrotrophic Pseudomonas spp. grown at different temperatures

The free fatty acid and phospholipid composition of 5 psychrotrophic marine Pseudomonas spp. have been determined in chemostat culture with glucose as the limiting substrate over the range 0–20°C. The predominant fatty acid present in all the isolates was hexadecenoic acid (C16:1) together with lesser quantities of octadecenoic acid (C 18:1) whilst none contained acids with chain lengths exceeding 18 carbon atoms. Decreasing the growth temperature from 20°C to 0°C resulted in little significant change in fatty acid composition. The principal phospholipid components of the five psychrotrophic pseudomonads have been identified as phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Decreasing the growth temperature did not elicit significant changes either in the total quantities of phospholipid synthesized or in the concentration of individual phospholipid components in any of the isolates. All the psychrotrophs showed maximum glucose uptake between 15°C and 20°C and the rate decreased rapidly as the temperature was decreased towards 0°C.Abbreviations PS Phosphatidylserine - PE phosphatidylethanolamine - PG phosphatidylglycerol - DPG diphosphatidylglycerol