2-Chlorofatty acids induce Weibel-Palade body mobilization[S]

Endothelial dysfunction is a hallmark of multiple inflammatory diseases. Leukocyte interactions with the endothelium have significant effects on vascular wall biology and pathophysiology. Myeloperoxidase (MPO)-derived oxidant products released from leukocytes are potential mediators of inflammation and endothelial dysfunction. 2-Chlorofatty acids (2-ClFAs) are produced as a result of MPO-derived HOCl targeting plasmalogen phospholipids. Chlorinated lipids have been shown to be associated with multiple inflammatory diseases, but their impact on surrounding endothelial cells has not been examined. This study tested the biological properties of the 2-ClFA molecular species 2-chlorohexadecanoic acid (2-ClHA) on endothelial cells. A synthetic alkyne analog of 2-ClHA, 2-chlorohexadec-15-ynoic acid (2-ClHyA), was used to examine the subcellular localization of 2-ClFA in human coronary artery endothelial cells. Click chemistry experiments revealed that 2-ClHyA localizes to Weibel-Palade bodies. 2-ClHA and 2-ClHyA promote the release of P-selectin, von Willebrand factor, and angiopoietin-2 from endothelial cells. Functionally, 2-ClHA and 2-ClHyA cause neutrophils to adhere to and platelets to aggregate on the endothelium, as well as increase permeability of the endothelial barrier which has been tied to the release of angiopoietin-2. These findings suggest that 2-ClFAs promote endothelial cell dysfunction, which may lead to broad implications in inflammation, thrombosis, and blood vessel stability.

out of neutrophils, but much remains unknown as to the role of 2-ClFAs in circulation and in microenvironments, such as the blood-endothelial interface (29,30). During inflammation, 2-ClFAs are produced at the site of neutrophil infiltration, and, once released, 2-ClFAs could alter nearby cell function (31).
Chlorinated lipids, including 2-ClFALD and 2-ClFA, have been linked to inflammatory diseases, including endotoxemia and atherosclerosis (27,(32)(33)(34). Activated neutrophils and monocytes isolated from human blood have elevated levels of 2-ClFA, approaching 20 µM, which may be suggestive of the concentration at the site of production near the leukocyte-endothelial interface (28,35). The concentration of 2-ClFA in the systemic circulation is lower, ranging from 1 to 100 nM, due to dilution in the volume of blood. As demonstrated in rats, lipopolysaccharide-mediated endotoxemia results in tripling the levels of plasma 2-ClFA compared with naïve rats (27). Furthermore, mice exposed to a sublethal dose of chlorine gas were found to have plasma levels of 2-ClFA greater than 100 nM (32). Chlorinated lipids have been shown to elicit recruitment of macrophages to the lung, disrupt the blood-brain barrier, and induce apoptosis (32,35,36). The underlying mechanisms of endothelial dysfunction, seen in multiple inflammatory diseases, remain to be completely understood.
The present study was designed to elucidate a common mechanism by which 2-ClFAs alter the blood-endothelial cell interface. By using a novel click chemistry approach, results demonstrate that the 2-ClFA species 2-chlorohexadecanoic acid (2-ClHA) localizes to Weibel-Palade bodies in human coronary artery endothelial cells (HCAECs). Weibel-Palade bodies, unique to endothelial cells, are exocytic storage granules that release crucial bioactive mediators after exposure to an external stimulus (37,38). Accordingly, further studies show that 2-ClHA induces surface expression of P-selectin and E-selectin, as well as the release of von Willebrand factor (VWF) from Weibel-Palade bodies. Consequently, 2-ClHA increases adherence of platelets and neutrophils to HCAECs. Furthermore, 2-ClHA stimulates the release of angiopoietin-2 release from the Weibel-Palade bodies and the loss of HCAEC barrier function.

Instrumentation
NMR spectroscopy was performed with a JEOL ECS-400 NMR spectrometer. Mass spectrometry (MS) was performed with a Thermo Fisher triple quadrupole Quantum Ultra mass spectrometer. For experiments applying LC/MS, a Thermo Fisher Surveyor LC system was coupled to the Quantum Ultra. LC/MS data analysis was completed by using XCalibur software (Thermo Fisher). GC/MS analysis was performed by using a Hewlett Packard GC 6890-5973. GC/MS data analysis was completed by using MSD ChemStation GC/MS Data Analysis.
Column fractions were loaded onto 60 Å silica gel TLC plates (Millipore Sigma; catalog no. 105721). Mobile phase for TLC was composed of ethyl acetate/hexane/toluene/acetic acid (30/ 10/60/5, v/v/v/v). TLC plates were visualized with phosphomolybdic acid (TCI America; catalog no. P1910). TLC lanes used for purification were not stained but scraped and extracted with 8 ml of chloroform/methanol (1/1, v/v) supplemented with 0.1% acetic acid. The solution was centrifuged, and the supernatant was removed. After a second extraction with chloroform/methanol (1/1, v/v), the combined extracts from the silica were dried under nitrogen and suspended in methanol.
Lipids were then conjugated to fatty acid-free BSA, which was used for all cell treatments. Briefly, 5 mg of 2-ClHyA or HyA was dried down and added to sterile-filtered PBS. A BSA blank was made with PBS alone. Each solution was heated to 70°C for 10 min, and 1 M NaOH was added dropwise until the solution was clear. After heating to 70°C for 30 min, a sterile-filtered 6% BSA (w/v; MP Biomedicals; catalog no. 152401) in PBS solution was added while shaking. The resulting solutions were aliquoted and stored at 20°C. Final solutions were quantified by pentafluorobenzyl bromide (PFB-Br) derivatization. The 2-ClHA and 2-ClHyA were converted to their respective PFB ester derivative as previously described (28). The 2,3,4,5,6-pentafluorobenzyl bromide (Sigma-Aldrich; catalog no. 101052) and N′,N′-diisopropylethylamine (Sigma-Aldrich; catalog no. D125806) in a solution containing acetonitrile was added to dried lipid. After reaction for 1 h at 45°C, PFB ester derivatives were suspended in ethyl acetate and subsequently subjected to GC/MS analysis by using negative ion chemical ionization.

NMR analyses
To confirm the structures of 2-ClHyA and HyA, samples were dried down and suspended in CDCl 3 to be analyzed with NMR. NMR spectra were collected on a JEOL ECS-400 NMR spectrometer. 1 H NMR spectra were reported in ppm from tetramethylsilane on the d scale. Data are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet, etc.; m, multiplet; b, broadened; obs, obscured; ABq, AB quartet), coupling constants, and assignments or relative integration where appropriate. 13 C NMR spectra were reported in ppm from the central deuterated solvent peak (multiplicities indicated when determined). Grouped shifts are provided where an ambiguity has not been resolved.

MTT assay
The metabolic activity of HCAECs was examined by using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells were plated in a clear 96-well plate and grown to confluence. Cells were treated with the indicated concentrations of BSA-conjugated lipids in phenol red-free growth medium with 5% FBS for the indicated time points up to 18 h. MTT (Sigma-Aldrich; catalog no. M2128; 1.2 mM, in 100 µl of serumfree, phenol red-free medium) was added to cells for 4 h. Seventyfive microliters of medium was removed, and 50 µl of DMSO was added for 10 min to lyse the cells. Absorbance was read at 540 nm on an Enspire multimode plate reader and corrected for background absorbance. Triton at a concentration of 0.1% in PBS was used as a positive control. MTT reduction is expressed as percent MTT reduction (BSA blank is designated as 100%).

Detection of HyA and 2-ClHyA in HCAEC by immunofluorescence
Cells were plated on sterile coverslips in 6-well plates and grown to confluence. Cells were treated with 10 µM HyA or 2-ClHyA for 30 min as described above. After washing with PBS, cells were fixed with formalin for 10 min. Cells were permeabilized with 0.25% Triton X-100 for 10 min. Cells were subsequently washed with 3% (w/v) BSA in PBS and labeled with 5 M azide-carboxytetramethylrhodamine (azide-TAMRA) (Sigma-Aldrich; catalog no. 760757) by using the Click-It Cell Reaction Buffer Kit (Thermo Fisher; catalog no. C10269) following manufacturer's protocols.

Confocal microscopy
A Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany) with a 63 x 1.4 oil immersion objective was used to acquire images. Cy2 fluorescence was excited at 488 nm and detected between 500 and 540 nm. Azide-TAMRA fluorescence was excited at 543 nm and detected between 570 and 650 nm. DAPI fluorescence was detected between 440 and 470 nm. All fluorescence signals were acquired simultaneously. Total corrected cell fluorescence was calculated by using ImageJ. The fluorescence was measured in at least 30 cells in five fields of view per condition.

Selectin surface expression
HCAECs were plated in a 24-well plate and grown to confluence. Cells were treated with 10 M BSA-conjugated lipids in growth medium with 5% FBS for 30 min (P-selectin) or 1 h (E-selectin). Thrombin (0.05 IU/ml; Sigma-Aldrich; catalog no. T6884) was used as a positive control. Cells were fixed with 1% paraformaldehyde overnight. Cells were washed with PBS and blocked with PBS containing BSA and fish gelatin for 1 h. Cells were incubated with primary antibodies (1:50) against P-selectin and E-selectin for 1 h at 37°C. Cells were washed with PBS and incubated with HRP-conjugated goat anti-mouse secondary antibody (1:5000) for 30 min at 37°C. Cells were washed with PBS and incubated with 3,3,5,5,tetramethylbenzidine (TMB; Sigma-Aldrich; catalog no. T0440) substrate system for 30 min in the dark. The color reaction was stopped with 8 N H 2 SO 4 , and absorbance was read at 450 nm. Values were normalized to the secondary antibody alone.

Platelet and neutrophil adherence to HCAECs
Adherence of platelets to HCAECs was determined as previously described (40). Briefly, HCAECs were grown to confluence in a 24-well plate and treated with 10 M BSA-conjugated lipids in growth medium for 30 min. PMA and thrombin were used as positive controls. Meanwhile, platelets were isolated from the whole blood of healthy volunteers as previously described (41) and as authorized by Saint Louis University Institutional Review Board Protocol 12369. Platelet-rich plasma was collected by centrifuging whole blood for 15 min at 100 g. The plasma was gently removed and centrifuged for 20 min at 2,000 g. Platelets were washed and stained with Calcein-AM (2.5 mol/liter; Thermo Fisher; catalog no. C3100MP) for 15 min at 37°C in the dark. Fluorescencelabeled platelets (50 × 10 6 cells in 500 µl) were subsequently added to each well containing HCAECs and incubated for 20 min at 37°C. Following multiple washes with PBS, bound platelets were lysed in lysis buffer (1% NP40, 150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 20 µg/ml soybean trypsin inhibitor) for 10 min, and fluorescence was measured with a plate reader (excitation at 492 nm, emission at 535 nm).
For neutrophil adherence assays, HCAECs were grown to confluence in a 12-well plate and treated with 10 M BSA-conjugated lipids in growth medium for 30 min. PMA and thrombin were used as positive controls. Neutrophils were prepared from whole blood of healthy volunteers as previously described (42,43) and as authorized by Saint Louis University Institutional Review Board Protocol 9952. Whole blood was anticoagulated with EDTA (final concentration 5.4 mM) prior to the isolation of neutrophils by using a Ficoll-Hypaque gradient. Five hundred microliters (4 × 10 6 cells/ml) was subsequently added to each well containing HCAECs and incubated for 20 min. Following multiple washes, bound neutrophils were lysed with 0.2% Triton X-100, and MPO activity was measured in HBSS containing BSA, 1,9-dimethylmethylene blue, and 0.5% hydrogen peroxide. Samples were incubated for 15 min at room temperature. Sodium azide (1%) was added to stop the reaction. Absorbance was measured at 460 nm.

Endothelial cell permeability
HCAECs were grown to confluence on Transwell polycarbonate filters (Corning Inc., Corning, NY) mounted in a chamber insert. Resistance across cells was monitored daily by using an EVOM volt-ohmmeter (World Precision Instruments). Once the resistance remained consistent for 3 consecutive days, experiments were performed. BSA-conjugated lipids (10 M) were added to each well, and then the resistance across each well was monitored at 15, 30, and 60 min, followed by 2, 4, 8, and 24 h.

Statistical analyses
Student's t-test was used for comparisons between two groups. ANOVA with the Dunnett post hoc test was used for comparisons between three or more groups to the control condition. All data are presented as mean ± SEM unless otherwise noted.

Characterization of 2-ClHyA
The synthetic scheme of 2-ClHyA is shown in supplemental Fig. S1A. These reactions yielded adequately pure (>98%) HyA and 2-ClHyA. To confirm 2-ClHyA structure, 1 H and 13 C NMR were performed (see complete NMR assignment in supplemental Table S1). The terminal alkyne proton absorption (labeled "a" in supplemental Fig. S1B; see inset) is clearly seen at 1.92 ppm as a triplet (J = 2.80 Hz). To further characterize 2-ClHyA, both 2-ClHA and 2-ClHyA were converted to their respective PFB ester derivatives and analyzed by GC/MS. The respective ion intensity ratios and proposed fragmentation patterns are shown in supplemental Fig. S1C, D. The intensity ratio of fragment

Effect of 2-ClHA and 2-ClHyA on the metabolic activity of HCAECs
To examine the effects of 2-ClFA on the endothelium, HCAECs were used for these studies. To ensure that there were no differential effects of 2-ClHyA compared with 2-ClHA on the metabolic activity in HCAECs, an MTT assay was performed (supplemental Fig. S2). There were no significant differences in metabolic activity in HCAECs after 2-ClHyA and 2-ClHA treatment at all time points and concentrations assessed. Similarly, MTT reduction was not significantly decreased in HCAECs treated with hexadecanoic acid (HA).

Visualization of 2-ClHyA in HCAECs
To visualize 2-ClHyA intracellularly, HCAECs were incubated with BSA-conjugated 2-ClHyA and then fixed, permeabilized, and subjected to a click chemistry reaction with azide-TAMRA. Fluorescence, as an indicator of the subcellular fate of 2-ClHyA, was detected as red punctate foci in the perinuclear region (Fig. 1A) in cells treated with concentrations ranging from 500 nM to 10 µM. Total cell fluorescence, corrected for background, was calculated in cells from multiple fields of view. Total corrected cell fluorescence increased with increasing concentrations of 2-ClHyA (Fig. 1B). To demonstrate the specificity of 2-ClHyA for localization shared with 2-ClHA, 2-ClHyA was outcompeted with simultaneous 2-ClHA treatment (either 10-or 20-fold molar excesses of 2-ClHA) (Fig. 1A, C). Simultaneous treatment with either 10-or 20-fold molar excesses of the nonchlorinated fatty acid, HA, did not alter 2-ClHyA subcellular localization or total cell fluorescence, highlighting the specificity of 2-ClHyA for 2-ClHA localization to the punctate foci. Alternatively, these results may indicate that 2-ClHA uptake by HCAECs is mediated by a unique mechanism compared with HA.
The 2-ClHyA accumulation in HCAECs increased over time, until reaching a plateau at 2 h. Representative images are shown in Fig. 2A. After images containing multiple fields of view were collected, the total corrected cell fluorescence and the percent of cells containing foci (number of cells containing foci/total number of cells × 100) were calculated, as shown in Fig. 2B, C.

Subcellular localization of 2-ClHyA
Colocalization analyses with known organelle markers were performed to identify 2-ClHyA subcellular localization in HCAECs. Immunofluorescence studies revealed that 2-ClHyA colocalized with the Weibel-Palade bodies (as indicated by P-selectin and VWF) and the mitochondria (as indicated by COX IV) (Fig. 3). Interestingly, after 2-ClHyA treatment, the density of VWF staining of Weibel-Palade bodies was decreased, and the Weibel-Palade bodies appeared rounded when compared with control treatment (supplemental Fig. S3). In contrast to 2-ClHyA, the localization of the nonchlorinated control HyA was most apparent with the Golgi and endoplasmic reticulum (ER; supplemental Fig. S4). HyA localization was not as striking as 2-ClHyA localization to the Weibel-Palade bodies.

Effect of 2-ClHA and 2-ClHyA on selectin surface expression and neutrophil adherence
Based on colocalization with known markers of the Weibel-Palade bodies, 2-ClHA and 2-ClHyA were examined for functional properties related to the Weibel-Palade bodies. Both 2-ClHA and 2-ClHyA significantly increased P-selectin and E-selectin surface expression compared with treatments with either BSA vehicle (control) or HA (Fig. 4A, B). The increase in P-selectin surface expression elicited by 2-ClHA was comparable to that after thrombin treatment (supplemental Fig. S5A). The relative response of for E-selectin surface expression was more robust for thrombin (supplemental Fig. S5B). A modest increase in selectin surface expression was also observed in HA-treated HCAECs compared with BSA vehicle-treated HCAECs.
The selectin adhesion molecules are primarily responsible for the adherence of leukocytes to the endothelium (44,45). Because these molecules are increased after 2-ClFA treatment, the adherence of neutrophils to HCAECs after 2-ClFA treatment was examined. Both 2-ClHA and 2-ClHyA treatments of HCAECs significantly increased the amount of neutrophils adhered to HCAECs compared with treatments with either BSA vehicle (control) or HA (Fig. 4C). Again, this increase was comparable to the increase in neutrophil adherence using thrombin and PMA as positive controls (supplemental Fig. S5C). A modest increase in neutrophil adherence was also observed in HAtreated HCAECs compared with BSA vehicle-treated HCAECs.

Effect of 2-ClHA and 2-ClHyA on VWF release and platelet adherence
The major component of Weibel-Palade bodies, acting as a backbone for formation, is VWF (38). In HCAECs, both 2-ClHA and 2-ClHyA significantly increased the release of VWF compared with treatments with BSA vehicle or HA (Fig. 5A). Pretreatment with Bis I (protein kinase C inhibitor) or BAPTA-AM (intracellular calcium chelator) reduced 2-ClHA-elicited or 2-ClHyA-elicited VWF release to levels similar to control treatments. This demonstrated that VWF release induced by both 2-ClHA and 2-ClHyA is dependent on protein kinase C and calcium. As a positive control in these studies, Bis I-sensitive or BAPTA-AM-sensitive VWF release was observed in response to PMA (supplemental Fig. S5E). Because VWF mediates platelet adherence to the endothelium (44,45), we further demonstrated that Cells were fixed with formalin, permeabilized with Triton X-100, and subjected to click chemistry with azide-TAMRA (red). Cells were incubated with primary antibodies against Golgin subfamily A member 2 (Golgi; GM130), P-selectin, VWF, calnexin (ER), and COX IV (mitochondria), then labeled with a Cy2conjugated secondary antibody (green). Cells were mounted in DAPI-containing solution (blue) and imaged with a Leica SP5 microscope. All images were taken by using the same settings. both 2-ClHA and 2-ClHyA significantly increased the amount of platelets adhered to HCAECs compared with BSA vehicle or HA treatments (Fig. 5B). The increases in platelet adherence by using thrombin and PMA as positive controls were greater than that elicited by 2-ClHA (supplemental Fig. S5F).

Angiopoietin-2 release as a mechanism of HCAECs permeability
Another key protein stored in Weibel-Palade bodies is angiopoietin-2, which contributes to endothelial cell permeability as an antagonist to Tie2 signaling pathways (46). 2-ClFA was examined for both release of angiopoietin-2 from HCAECs and alterations in endothelial cell electrical resistance. 2-ClHA and 2-ClHyA significantly increased angiopoietin-2 release from HCAECs compared with controls, in a concentration-dependent manner (Fig. 6A). The levels of angiopoietin-2 release by 2-ClHA and 2-ClHyA approached the levels stimulated by the positive control, PMA (supplemental Fig. S5D). Furthermore, 2-ClHA and 2-ClHyA significantly decreased resistance across HCAECs, an indicator of endothelial cell permeability (Fig. 6B). Additionally, HA treatment elicited a significant release of angiopoietin-2 from HCAECs compared with control conditions, which was accompanied by a slight decrease in resistance across HCAECs. It should, however, be appreciated that 2-ClHA-stimulated and 2-ClHyA-stimulated angiopoietin-2 release from HCAECs, as well as decreased resistance across HCAECs, were significantly greater than those HA-elicited changes.
To determine 2-ClFA localization in endothelial cells, we synthesized a "clickable" analog of 2-ClHA, termed 2-ClHyA, containing an alkyne bond at the omega end of the fatty acid. HCAECs were treated with this analog, and then the analog was clicked to the fluorescent probe, azide-TAMRA, to determine subcellular distribution. The click and then incubated with HRP-conjugated secondary antibodies. Surface expression of selectins was measured spectrophotometrically after addition of TMB substrate. Values are normalized to secondary antibody alone. To measure neutrophil adherence, freshly isolated neutrophils were added to the HCAECs for 20 min. HCAECs were washed thoroughly, and MPO activity was quantified (C). n = 6 for each treatment, mean ± SEM. * P < 0.05, *** P < 0.001 for comparisons with control treatment. ### P < 0.001 for comparisons with HA treatment. reaction used in these studies was the copper-catalyzed Huisgen 1,3-dipolar cycloaddition reaction, which is ideal, as the reaction is selective, quick, and minimizes unwanted byproducts (48,49). This approach revealed the selective, uniform distribution of 2-ClHyA-containing foci throughout HCAECs, which increased over time and then plateaued after 2 h of treatment. The plateau of 2-ClHyA uptake may reflect the complete saturation of the binding sites for 2-ClHyA in Weibel-Palade bodies or the intracellular metabolism of the lipid. These foci were associated with Weibel-Palade bodies, as determined by colocalization with VWF and P-selectin. 2-ClHyA treatment altered the shape (cigar-like to round) and abundance of Weibel-Palade bodies detected by VWF staining. Changes in the shape of VWF-detected Weibel-Palade bodies has been attributed to packaging of VWF and maturation of Weibel-Palade bodies (50), acidification of Weibel-Palade bodies (50), and the process of Weibel-Palade body exocytosis and release of body contents (51).
Interestingly, 2-ClHyA also localizes to the mitochondria, a key player in calcium homeostasis and cell respiration. Based on the well-established role of calcium in Weibel-Palade body mobilization, 2-ClFAs may alter intracellular calcium levels and further contribute to endothelial dysfunction and inflammation in a mitochondria-mediated pathway. The importance of 2-ClHyA localization to the mitochondria and the impact on mitochondrial respiration needs to be examined in future studies.
Multiple lines of evidence support that the localization of the alkyne analog, 2-ClHyA, is analogous with the localization of 2-ClHA. First, 2-ClHyA localization to the Weibel-Palade bodies was reduced in competition studies with 10-to 20-fold molar excesses of 2-ClHA. In contrast, 2-ClHyA localization to the Weibel-Palade bodies was not reduced in competition studies with 10-to 20-fold molar excesses of the nonchlorinated fatty acid, HA. These studies demonstrate the shared properties of 2-ClHA and 2-ClHyA while ruling out nonspecific lipid effects. Second, 2-ClHA and 2-ClHyA were shown to elicit similar metabolic responses from HCAECs, as assessed by the MTT assay. Finally, both 2-ClHA and 2-ClHyA, compared with HA, elicited Cells were pretreated with vehicle, Bisindolylmaleimide I, or BAPTA-AM (A). Cells were treated with 10 M BSA-conjugated lipids for 30 min or PMA as a positive control for 20 min. Medium was removed and centrifuged to remove cell debris. Fifty microliters of medium was assayed for VWF by using an ELISA following manufacturer's protocol. To quantify platelet adherence, HCAECs were grown to confluence and treated with 10 M BSA-conjugated lipids for 30 min. Calcein-AM-labeled platelets were added to the HCAECs for 20 min. HCAECs were washed thoroughly, and fluorescence was immediately measured (B). n = 4 or 6 for each treatment, mean ± SEM. *** P < 0.001 for comparisons with control treatment. ### P < 0.001 for comparisons with HA treatment. Fig. 6. Angiopoietin-2 release from HCAECs and permeability of HCAECs. Cells were treated with the indicated concentrations of BSA-conjugated lipids for 30 min or PMA as a positive control for 20 min. Medium was removed and centrifuged to remove cell debris. Fifty microliters of medium was assayed for angiopoietin-2 by using an ELISA following manufacturer's protocol (A). To measure resistance (B), cells were grown to confluence on Transwell polycarbonate filters mounted in a chamber insert. Resistance across the cells was monitored daily by using an EVOM volt-ohmmeter. Once the resistance remained constant for 3 consecutive days, experiments were performed. A concentration of 10 M BSA-conjugated lipids were added to each well, and then the resistance across each well was monitored at 15, 30, and 60 min followed by 2, 4, 8, and 24 h. n = 6 for each treatment, mean ± SEM. * P < 0.05, *** P < 0.001 for comparisons with control treatment. # P < 0.05, ### P < 0.001 for comparisons with HA treatment. nearly identical changes in multiple endothelial cell responses including: 1) increased plasma membrane surface expression of P-selectin and E-selectin; 2) increased neutrophil adherence to endothelial cells; 3) release of VWF; 4) increased platelet adherence to endothelial cells; 5) release of angiopoietin-2; and 6) decreased endothelial cell permeability barrier. Collectively, both the confocal microscopy studies and the functional studies support that 2-ClHyA is a 2-ClHA analog sharing the same subcellular localization and biological properties in HCAECs.
Additional studies investigated the properties of the nonchlorinated fatty acid, HA, on endothelial function. By using the click analog of HA to assess subcellular distribution, it was clear that the intracellular distribution of HyA in HCAECs was different than that of 2-ClHyA. HyA predominantly localized to the Golgi with some localization also observed in the mitochondria, ER, and Weibel-Palade bodies. In contrast to the robust increases in P-selectin and E-selectin in response to ClFA, there were minor, yet significant, increases in membrane surface expression of Pselectin and E-selectin in response to HA. Minor changes elicited by HA were accompanied with corresponding levels of functional outcomes, including modest neutrophil adherence to and reduced resistance across HCAECs. The changes in endothelial barrier function elicited by HA have been reported by others, demonstrating that the permeability of the endothelium is impacted by HA at high concentrations (>20 µM) for long time periods (24 h) (52).
Endothelial cell biology is modulated by the status of Weibel-Palade bodies, which store multiple bioactive proteins poised to be released from the endothelial cell (38,53,54). The present findings suggest an intimate relationship between neutrophils and the endothelium mediated by neutrophil-derived lipid products that potentially enhance blood-endothelial interaction due to 2-ClFA targeting Weibel-Palade bodies. A key research direction that remains is revealing the underlying mechanism responsible for 2-ClFA selectively associating with Weibel-Palade bodies. We speculate that there are specific proteins associated with Weibel-Palade bodies that bind 2-ClFA. In addition to determining the mechanism responsible for 2-ClFA association with Weibel-Palade bodies, it will also be important to determine the mechanism by which 2-ClFA causes Weibel-Palade body mobilization. The changes in shape of VWF-stained Weibel-Palade bodies may indicate that 2-ClFA leads to acidification. Both actomyosin-dependent and -independent mechanisms have been attributed to Weibel-Palade body unpacking (51,55), and our observations in morphological changes in VWF staining are consistent with both of these mechanisms.
These studies used a click analog of the novel neutrophil-derived oxidation product, 2-ClFA, to gain insights into the subcellular localization in neighboring endothelial cells. Surprisingly, these experiments revealed that 2-ClFA specifically localizes to the Weibel-Palade bodies in HCAECs. This revelation led to the discovery that 2-ClFA leads to profound changes in endothelial biology through the mobilization of the contents of Weibel-Palade bodies. Thus, 2-ClFA targeting the Weibel-Palade bodies causes endothelial cell dysfunction, which may alter multiple processes, such as leukocyte extravasation, thrombosis, and endothelial barrier function.