THE EFFECT OF OMEGA-3 FATTY ACIDS ON EXPRESSION OF CONNEXIN-40
IN WISTAR RAT AORTA AFTER LIPOPOLYSACCHARIDE ADMINISTRATION

Vascular gap junctions (GJs) are low-resistant transmembrane channels playing an important role in coordination and synchronization of physiological events of vascular wall cells to maintain the tissue homeostasis and vasomotion (1). GJs allow a direct exchange of ions, small metabolites and second messengers (<1 kDa) between cytoplasm of adjacent cells of endothelium and smooth muscle (EC-EC, SMC-SMC, EC-SMC) and ensure their mechanic and electrical coupling. Vascular GJs are composed of four Cx isoforms - Cx37, Cx40, Cx43 and Cx45 (1). Their expression is not uniform. It depends on a type of cells, size and type of blood vessels, age and species (2-4) as well as on pathophysiological conditions (1, 5, 6). Abnormal expression of Cxs has been shown to contribute to disturbances in endothelial homeostasis and permeability and contractility of smooth muscle cells as well, supporting GJs as an attractive therapeutic target for the treatment of vascular disorders.

Increasing evidence suggests relationship between Gram-negative bacterial lipopolysaccharide (LPS)-induced chronic low-grade inflammatory and development of cardiovascular diseases including atherosclerosis (7-10). LPS triggers a variety of proinflammatory responses including increased production of cytokines, chemokines, growth factors and reactive oxygen species. These via paracrine pathways can locally induce endothelial activation and/or dysfunction respectively, to increase vascular leak and subsequently the impairment of vascular wall function and its structural remodeling.

LPS-induced abnormal expression of Cx isoforms and GJ communication in the vascular wall cells cultured endothelial cells, leukocytes between EC and leukocytes (11-15) as well as in the cells of immune system (16) indicate that GJs could be involved in the development of inflammation-induced vascular wall injury. Mentioned changes were detected a few hours after LPS administration mimicking the acute phase of inflammatory process on Cxs expression. According to our knowledge, data concerning the effect of a systemic LPS-induced inflammation on Cxs expression in cardiovascular system are missing.

Regular chronic consumption of fish oil and/or isolated omega-3 polyunsaturated fatty acids (n-3 PUFA) respectively is well-known for improving of health. N-3 PUFA are considered to be powerful intracellular and intercellular mediators (17). They possess multifunctional beneficial effects (18, 19). They reduce lipid levels and blood pressure, attenuate thrombosis, improve NO production and suppress reactive species production. N-3 PUFA are also precursors for the synthesis of eicosanoids and modulators of the cell signaling network and cellular membrane fluidity that can lead to various effects on inflammatory processes (20). N-3 PUFA protected the renal Na,K-ATPase activity in endotoxemic rats (21) and HTG rats (22). Moreover, modulation of Cx43 expression in the rat heart and the aorta with chronic n-3 PUFA intake demonstrated in our previous studies (23-26) suggested a link of cardioprotective effects of n-3 PUFA with GJ communication. Whether shorter n-3 PUFA supplementation can have protective effects on vascular Cx isoforms expression during LPS-induced inflammation has not been yet studied.

From this perspective, the aim of the present work was to study the potential anti-inflammatory effect of 10-day n-3 PUFA diet on Cx40 expression in the rat aorta after a single dose of LPS-induced inflammatory process.

MATERIAL AND METHODS

Animal model

The procedures of animal experiments in this study were performed in the accordance with institutional guidelines and the protocols approved by the State Veterinary and Food Administration of the Slovak Republic.

The experiments were performed on adult normotensive male Wistar (W) rats (220–240 g of body weight). Prior to the experiments, the rats were acclimatized and housed in a temperature- and light-controlled room (23±1°C, 12 h light: 12 h dark cycle). All rats had ad libitum access to a standard food and drinking water before and after the beginning of the experiments. At the beginning of experiments, the rats were divided into four groups (n=6 per each group): 1) C - control healthy animals,

2) PUFA - healthy rats were daily treated with n-3 PUFA in food supplementation, 30 mg/kg/day for 10 days (57% eicosapentaenoic acid and 43% docosahexaenoic acid, commercial nutritional supplement MaxiCor, SVUS Pharma, Czech Republic), 3) LPS - animals injected with a single dose of LPS, i.p., 1 mg/kg of body weight, 4) PUFA+LPS - rats injected with LPS were supplemented with n-3 PUFA for 10 days, first dose of n-3 PUFA was applied on the day of vaccination. LPS (E. coli serotype 055:B5, Sigma Chemicals, Germany) was dissolved in sterile 0.9% NaCl solution. The groups 1) and 2) were injected with the same volume of sterile 0.9% NaCl solution. At the end of the experiments, the thoracic aorta was excised from anaesthetized animals, cleaned of adherent tissue and processed for further analysis.

Basic characteristics of experimental animals

Blood pressure was measured at the beginning and at the end of the experiments according to Dlugosova et al. (23) using noninvasive plethysmography (Statham Pressure Transducer, AD Instruments, Germany). The body weight of the rats was measured at the same periods of the experiment. The weight of the whole heart and the left ventricle weight were measured at the end of the experiment only.

Blood samples were collected for plasma separation. After this process the aorta was isolated. Plasma was stored at –82°C until analysis of levels of high sensitive C-reactive protein (CRP) as a marker of acute inflammation and malondialdehyde (MDA) as an index of lipid peroxidation (27). The specific activity of N-acetyl-β-D-glucosaminidase (NAGA) as a marker of cellular injury and chronic phase of inflammation (28) was measured in plasma.

Hs-CRP levels were measured using commercial clinical Cholestech LDX System (California, USA). MDA concentration was measured spectrophotometrically with measurement of the color produced during the reaction of MDA with thiobarbituric acid according to Draper and Hadley (29). The activity of lysosomal NAGA was assayed according to standard methods as described previously in Navarova et al. (30).

Transmission electron microscopy

The aorta of the control rats and the LPS rats (n=4 for each groups) was cut into 3 mm rings. The specimens were immersion fixed in 2.5 % glutaraldehyde in 0.1 mol/l cacodylate buffer (pH 7.4) for 3 hour, then rinsed in buffer, postfixed in 1% OsO4, dehydrated in ethanol and embedded in Epon 812 (31). Thin sections after staining with uranyl acetate and lead citrate were examined with TESLA 500 electron microscope (Brno, Czech Republic).

Cx40 immunofluorescence

Indirect immunofluorescent detection of Cx40 was performed on frozen cross-sections of the non-fixed aortic rings of the rats of all experimental groups. Series of the sections were fixed in 4% paraformaldehyde, incubated in 0.3% Triton X-100, blocked by 10% goat serum and incubated with primary monoclonal antibody rabbit anti-Cx40 (1:500, Santa Cruz Biotechnology, Inc.) over night at 4°C. The sections were subsequently washed with phosphate-buffered saline (PBS) and then followed the application of secondary goat anti-rabbit conjugated with FITC-fluorescein isothiocyanate (1:500, Jackson ImmunoResearch Laboratory, Inc.). The primary antibody was omitted in negative controls to check the specificity of immunolabeling. After washing, the aortic sections were mounted into Vectashield mounting medium and viewed by confocal microscope Olympus FV1000 (Olympus, Japan).

Image densitometric analysis of Cx40

Both the density and the area of immunofluorescent Cx40 clusters were evaluated in endothelium and media using morphometric analysis system (Soft Imaging System, Germany) according to Dlugosova et al. (24) with a minor modification: after set of magnification, color of immunofluorescent signal of Cx40 was changed into gray scales. Endothelial layer and media were defined as a region of interest (ROI). Setting of threshold was used to determine minimal and maximal borders of a gray scale for immunoreaction. After the processes, we defined data for measurement of the density of Cx40 spots, their total area/mm2 of endothelium and media and the average size of one Cx40 spot. Signal was analyzed for each animal. The aorta from 6 animals in each group was cut to 6 rings, 3 to 8 sections per aortic ring were analyzed for each animal and the results were expressed in percentages.

Western blot of Cx40 and CD68

Frozen aortic tissue of the rats of all experimental groups was homogenized in SB20 solution (20% SDS, 10 mmol/l EDTA, 0.1 mol/l TRIS, pH 6.8) by sonificator UP 100H (Dr. Hielscher, Germany). An equal amount of the total protein in each sample was separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane.

To analyze Cx40 expression the membrane was incubated with rabbit monoclonal antibody (Santa Cruz Biotechnology, Inc., 1:500) at a room temperature for 2 hours and followed by the incubation with secondary donkey anti-rabbit antibody coupled with horseradish peroxidase diluted 1:2000 (GE Healthcare).

To detect monocytes/macrophages in the aortic tissue, the membrane was incubated with primary mouse monoclonal antibody anti-CD68 diluted 1:1000 (Millipore) and followed by further incubation with secondary sheep anti-mouse antibody coupled with horseradish peroxidase 1:2000 (GE Healthcare).

The optical density of specific bands was detected by Kodak In-vivo Imaging System Fx Pro (USA) using “Carestream MI SE“software. The optical density of each band was verified in relation to the optical density of GAPDH.

Measurement of nitric oxide synthase activity and nitric oxide-dependent relaxation of aorta

The activity of NO synthase (NOS) was assayed in a homogenate of the aorta by determination of [3H]-L-citruline formation from [3H]-L-arginine (MP, Biomedicals) as described previously in Bernatova et al. (32).

The endothelium-dependent relaxation of the thoracic aorta was studied according to Sotnikova et al. (33). The rings of the aorta were mounted between two hooks in water-jacketed (37°C) chambers containing physiological saline solution (PSS) bubbled with a mixture of 95% O2 and 5% CO2 at pH 7.4. Special care was taken not to damage the endothelium. The composition of PSS was (in mmol.l–1): NaCl (118.0), KCl (4.7), KH2PO4 (1:2), MgSO4 (1.2), CaCl2 (2.5), NaHCO3 (25.0) and glucose (11.0). The preparations were connected to an isometric force transducer (Experimetria Hungary). After the equilibration period, vascular rings were precontracted with submaximal concentration of phenylephrine (10–6 mol.l–1). At the plateau of the contraction, the effect of acetylcholine in the cumulative concentrations of

10–8–10–5 mol.l–1 was tested to measure the endothelial function. The relaxation responses to acetylcholine are expressed in percentages of phenylephrine-induced contraction. Concentration-response curves of sodium nitropruside (10–10–10–7 mol/l) were performed in phenylephrine (10–6 mol/l)-precontracted preparations to measure endothelium-independent vasodilation of the aorta.

Statistical analysis

Obtained biochemical and functional data were statistically evaluated using ANOVA with Bonferroni multiple comparison test. Morphometric image analysis of Cx40 and Western blotting were evaluated using Student-Newman-Keuls method. The results were considered to be statistically significant when p values were less 0.05.

RESULTS

Basic characteristics

LPS significantly increased the levels of CRP in plasma (Table 1) when compared to controls. The 10 day n-3 PUFA supplementation markedly suppressed CRP concentration in the endotoxemic rats. Although CRP level was significantly lower in the LPS+PUFA group than in the LPS group, it is still significantly higher than in control rats. Treatment of healthy rats with n-3 PUFA had no effect on CRP levels.

Table 1. Biomarkers in plasma: high sensitive C-reactive protein (Hs-CRP), malondialdehyde (MDA), N-acetyl-β-D-glucosaminidase (NAGA). (n=6 animals per group) Results are expressed as mean ± S.E.M. *p<0.05 versus Controls, #p<0.05 versus LPS, **p<0.05 versus LPS + PUFA.

The plasma concentration of MDA (Table 1) was significantly higher in LPS group than in controls. N-3 PUFA administration to LPS rats suppressed MDA levels on the control level. In the healthy animals, n-3 PUFA supplementation had no effect on MDA production.

Measurement of the activity of NAGA in plasma (Table 1) demonstrated that LPS alone caused its significant increase. The 10-day n-3 PUFA treatment of the LPS rats did not significantly reduce the enzyme activity when compared to the LPS group, but it was still significantly higher than in the control group. Supplementation of the healthy rats with n-3 PUFA did not affect the activity of NAGA.

No significant reduction in the body weight gain was observed in the LPS group when compared to the controls (Table 2). N-3 PUFA feeding the LPS rats for 10 days did not affect the body weight gain comparing to the LPS group. N-3 PUFA had no effect on the body weight of the control rats. The evaluation of the body weight gain indicates a detail evidence for the LPS-induced changes independently of n-3 PUFA treatment. No relevant differences concerning systolic blood pressure, the heart rate and the relative heart weight were observed among the experimental groups (Table 2).

Table 2. Biometric parameters of rats: body weight gain (BWG), systolic blood pressure (SPB), heart rate (HR), relative heart weight (HW/BW, HW - heart weight, BW - body weight). (n=6 animals per group). Results are expressed as mean ± S.E.M.

Transmission electronmicroscopy

Electronmicroscopy revealed a standard architecture of the endothelial cells of the aorta of the control animals (Fig. 1a). Focal irregularly extended tight junctions were rarely seen between adjacent endothelial cells. In the aorta of LPS rats (Fig. 1b-1d), the endothelium besides the healthy cells locally displayed slight to moderate ultrastructural abnormalities. They were manifested by edematous mitochondria (Fig. 1b), clumping of chromatin, increased presence of lysosomes and Weibel-Palade bodies (Fig. 1c) and increased amount of pinocytic vesicles (Fig. 1d). Cells with electronlucent cytoplasm (Figs. 1b, 1c) contained decreased amount of subcellular organelles (Fig. 1b) while increased amount of vacuoles. The inter-endothelial connections of the aorta of LPS rats, in contrast to the controls, frequently consisted of irregularly extended tight junctions (Fig. 1d). Overlapping of endothelial cells was frequently seen (Fig. 1b, 1c). Locally, monocyte contacting endothelial cell through gap junction was observed in subendothelial area (Fig. 1b). The subcellular alterations of endothelium represent the structural substrate of the endothelial activation/dysfunction.

Fig. 1.Ultrastructure of aortic endothelial cells a) of control Wistar rats (n=4 per group), b-d) after application of lipopolysaccharide (n=4).
L - lumen, EC - endothelial cells, N - nucleus, r - ribosomes, er - rough endoplasmic reticulum, m - mitochondria, G - Golgi complex, pv - pinocytic vesicles, ly - lysosomes, M - monocyte, thick arrow - gap junction, arrow - tight junction, arrowhead - overlapping of endothelial cells, asterisk - extended tight junctions. Original magnification: a) 14,000×, b) 10,000×, c) 8,000×, d) 14,000×.

Cx40 distribution and spatial expression in the aortic wall

The immunolabeling of Cx40 was found in the sections of the rat aortic wall of all experimental groups (Fig. 2a): it was relatively regularly distributed within the endothelium, while heterogeneously scattered Cx40 punctuated pattern was observed throughout media.

Fig. 2. a) Representative figures of Cx40 immunolabeling in endothelium (E) and media (M) of the aorta of the control group (C), the inflammatory group (LPS), the control group treated with PUFA (PUFA) and the treated inflammatory group (LPS + PUFA). (n=6 animals per group.) Original magnification 40×.

Morphometric analysis quantifies the total immunofluorescent area of Cx40 clusters per unit area of the individual aortic layers (endothelium and media) indicating Cx40 pattern expression. Considerably higher Cx40 expression pattern was demonstrated in the endothelium than in media of the aorta of all groups (Fig. 2b). Endothelial Cx40 expression was significantly higher in LPS group than in the control one. The administration of n-3 PUFA to the endotoxemic rats markedly reduced the Cx40 immunolabeling in the endothelium only when compared to LPS rats. In media, there were no differences among the groups.

Fig. 2. b) Densitometric analysis of Cx40 spatial pattern in endothelium (E) and media (M) of the aorta of the control group (C), the treated control group (PUFA), the inflammatory group (LPS) and the treated inflammatory group (LPS + PUFA). (n=6 animals per group). Results are mean ± S.E.M. * p<0.05 versus media, + p<0.05 versus Controls, # p<0.05 versus LPS group.

LPS injection resulted in the significant increase in Cx40 density clusters (Fig. 2c) in endothelium, while not in media comparing with the healthy animals. In media, there was a tendency of Cx40 cluster density to be increase. Daily n-3 PUFA supplementation of LPS rats for 10 days suppressed Cx40 density in endothelium when compared to LPS animals. No significant differences were observed between media of the treated and the untreated endotoxemic rats. N-3 PUFA diet did not change Cx40 density in endothelium and media of the control rats.

Fig. 2. c) Morphometric analysis of Cx40 density in endothelium (E) and media (M) of the rat aorta of the control group (C), the treated control group (PUFA), the inflammatory group (LPS) and the treated inflammatory group (LPS + PUFA). (n=6 animals per group). Results are mean ± S.E.M. *p<0.05 versus media, +p<0.05 versus Controls, #p<0.05 versus LPS group.

Quantification of the average area of Cx40 clusters (Fig. 2d) demonstrated that LPS injection caused their enlargement in endothelium when compared to the controls, while no changes were seen in media between both groups. N-3 PUFA treatment of LPS rats significantly decreased the cluster area in the endothelium only when compared to LPS rats. N-3 PUFA supplementation of the healthy rats augmented the cluster area in the endothelium only when compared to healthy rats.

Fig. 2. d) Average size of single Cx40 clusters in endothelium (E) and media (M) of the rat aorta of the control group (C), the treated control group (PUFA), the inflammatory group (LPS) and the treated inflammatory group (LPS + PUFA). (n=6 animals per group). Results are mean ± S.E.M. *p<0.05 versus media, +p<0.05 versus controls, #p<0.05 versus LPS group.

Western blotting of Cx40 and CD68

Western blotting analysis of Cx40 expression in the tissue of aorta showed significant differences among the experimental groups (Fig. 3a, 3b): alone, LPS significantly up-regulated Cx40 expression when compared to controls. PUFA diet of the endotoxemic rats induced marked Cx40 down-regulation when compared to the LPS group and the control group. PUFA supplementation of the control rats did not significantly affect Cx40 expression.

Fig. 3. a) Representative Western blot of CD68, Cx40 and GAPDH expression in the rat aorta of the control group (C), the inflammatory group (LPS), the treated inflammatory group (LPS + PUFA) and the treated control group (PUFA). (n=6 animals per group).

Fig. 3. b) Expression of Cx40 in the rat aorta of the control group (C), the inflammatory group (LPS), the treated inflammatory group (LPS + PUFA) and the treated control group (PUFA) normalized to GAPDH. (n=6 animals per group) Results are mean ± S E M. *p<0.05 versus Controls, #p<0.05 versus LPS group.

Western blotting of CD68 as the macrophage/monocyte receptor marker in the aortic tissue revealed its expression in the aorta of the control rats (Fig. 3a). Up-regulation of CD68 was found in the aorta of endotoxemic rats when compared to the controls (Fig. 3c). N-3 PUFA treatment of LPS rats significantly suppressed Cx40 expression when compared to LPS group. Treatment of the healthy rats with n-3 PUFA did not significantly change CD68 expression.

Fig. 3. c) Expression of CD68 in the rat aorta of the control group (C), the inflammatory group (LPS), the treated inflammatory group (LPS + PUFA) and the treated control group (PUFA) normalized to GAPDH. (n=6 animals per group) Results are mean ± S.E.M. *p<0.05 versus Controls, #p<0.05 versus LPS group.

The nitric oxide-dependent relaxation of the aorta and the nitric oxide synthase activity

As is demonstrated on Fig. 4, LPS injection impaired the NO-dependent relaxation of the aorta when compared to the healthy animals. N-3 PUFA administration to the LPS group did not protect the relaxation neither had influence on the aortic relaxation in the control group. Endothelium-independent relaxation of the aorta induced by sodium nitroprusside did not differ among the experimental groups (not shown).

We further measured the NOS activ

Fig. 4. NO-dependent relaxation of the rat aorta of the control group (C), the inflammatory group (LPS), the treated inflammatory group (LPS + PUFA) and the treated control group (PUFA). (n=6 animals per group) Results are mean ± S.E.M. *p<0.05 versus controls.

ity in the aorta to correlate functional data with biochemical one (Fig. 5). LPS caused marked increase in the NOS activity when compared to the controls. Interestingly, n-3 PUFA supplementation of the endotoxemic rats did not reduce the enzyme activity it even increased more than twice when compared to the LPS group. N-3 PUFA significantly stimulated the NOS activity in the aorta of the healthy rats as well.

Fig. 5. NO-synthase activity measured in the rat aorta of the control group (C), the inflammatory group (LPS), the treated inflammatory group (LPS + PUFA) and the treated control group (PUFA). (n=6 animals per group) Results are mean ± S.E.M. Results are mean ± S.E.M. *p<0.05 versus Controls, #p<0.05 versus LPS.

DISCUSSION

Experimental inflammation induced by various bacterial LPS has been used as a common model of infection and chronic inflammation which as it is mentioned above is recognized as one of risk factors for cardiovascular diseases. In 10-day experiment, we injected the adult normotensive healthy rats with a single dose of LPS (1 mg/kg b.w.). The used concentration when compared to higher one (4–10 mg/kg) (34) did not affect the body weight of the rats, neither heart rate nor systolic blood pressure. The dose was low enough not to induce sepsis and the mortality, as rodents are much less sensitive to LPS when compared to human (35). However, it was high enough to elicit more than six times increased level of CRP the circulating marker of acute inflammation and more that 100% elevation of the NAGA activity the marker of chronic inflammation and lysosomal injury of cells when compared to controls. This was associated with significantly increased levels of MDA indicating elevated generation of oxygen free radicals playing an important role in the progression of inflammatory disorders (36). Under inflammation, the endothelium is a major target of proinflammatory mediators and oxygen free radicals which are cause the endothelial dysfunction, affect intercellular connections and increase endothelial permeability (36, 37), resulting in changes in vascular tone. Ten days after LPS administration we demonstrated reduced the NO-dependent relaxation of the aorta while no changes in NO-independent vasodilation associated with the increased NOS activity suggesting changes in endothelial function. Our results are consistent with numerous literature data (38-40) demonstrating that the LPS-related increase in the NOS activity can be attributed to iNOS isoform. The subcellular aortic endothelial abnormalities including the intercellular connections which we observed in LPS rats correlate with the increased NAGA activity and can contribute to the further progression of the endothelial dysfunction and impaired endothelial barrier integrity. The latter allows increased passage of circulating inflammatory cells into the aorta the presence of which we demonstrated by the upregulation of CD68. Inflammation-induced production of superoxide by adhered neutrophils in the vicinity of endothelial and smooth muscle cells and its interaction with NO result in endothelial damage, decrease of the endothelium-dependent relaxation and can induce also vascular contraction (41). Detrimental effects of a single dose of LPS on the Na,K-ATPase activity regulating Na+ homeostasis in the body was demonstrated in rat kidney in our recent study (21). The results together demonstrate that a single dose of LPS injected to the healthy rats resulted in the moderate inflammatory response associated with endothelium-dependent aortic wall disease. However, it was reported that the progression of a disease can depend on the individual sensitivity to LPS and various genetic predispositions (42-44).

Vascular cell-to-cell coupling plays an important role in the propagation of electrical signals along blood vessels that contribute to regulation of vasomotor responses. We observed that LPS administration resulted in upregulation of Cx40 expression in the aortic tissue associated with the significant increase in endothelial Cx40 spatial expression only. The latter could represent interendothelial Cx40-channels as well as LPS-induced activation and stimulation of Cx40 hemmichannels (45, 46). Both, channels and hemmichannels are known to optimize cellular responses to actual (patho)physiological conditions and/or they to cause progress of disturbances in cellular function respectively. In media we observed a tendency of Cx40 expression to be increased. It seems to be important in relationship to upregulation of CD68 demonstrating increased infiltration of inflammatory cells into aortic tissue.

Surprisingly, we observed in contrast to others (2) Cx40 distribution in media of the aorta of healthy rats, too. This might indicate rat strain differences (47) or inflammatory cells such as macrophages and monocytes demonstrated by CD68. The latter are also known to express Cx40 (14, 15). The suggestion needs to be clarified in future studies. The local infiltration of inflammatory cells into the aortic wall of control group can occur due to the focal age-dependent abnormalities of endothelial monolayer structure. Because the aorta is an electrically quiescent tissue and the coordination of the responses of smooth muscle cells to diverse neural and endothelial signals particularly depends on gap junctional communications (48), the Cx40 immunolabeling in both aortic layers supports the role of Cx40-gap junction communication in maintaining of the aortic function. However, the function of Cx40 in media is not well-known. Therefore, we suggest that abnormal endothelial Cx40 expression caused by LPS might contribute to endothelial dysfunction and impaired aortic function.

Modulation of Cx isoforms during LPS-mediated response was reported in several studies. However, the results are diverse. LPS intake induced the decrease in Cx40 and Cx37 expression in endothelium of mouse aorta (49), microvascular endothelium (50) and HUVEC cells (51) as well. LPS-induced downregulation of Cx40 was also demonstrated in mouse lung (52). On the other hand, upregulation of Cx40 expression was found in the endothelium of Wistar rat aorta during sepsis (12). In all above mentioned studies LPS was administered only for a few hours to mimic acute-phase of inflammatory response. In our work, we demonstrated upregulation of Cx40 at the day 10 after LPS administration that in view of the life span of rat mimicked rather subchronic and/or chronic phase of inflammation respectively and could be relatively sufficient to cause/progress the vascular wall disease. We correlated our results with the works of Wang et al. (53) and Kwak et al. (54) who studied Cx40 expression in the atherosclerotic aorta of rabbit and mouse two weeks after high cholesterol diet which is associated with inflammation as well. Wang et al. observed Cx40 upregulation, while Kwak et al. found no changes. The effect of chronic LPS application alone induced upregulation of Cx32 and Cx30, while downregulation of Cx43 in rat hippocampus (55, 56). Data together suggest selective modulation of Cxs due to species-dependent variability, duration of inflammation as well as a dose and a route of LPS application (35, 57).

Cx40 expression in the aorta of endotoxemic rats might be modulated with high concentration of oxygen and nitrogen free radical (38, 58) as well as by proinflammatory cytokines (59). The NO pathway represents further potent modulator of Cx expression and their specific interactions are considered as key mechanisms in the control of a vascular function (60, 61). Alonso et al. (62) observed association of reduced eNOS activity with downregulation of Cx40 in the aorta of Cx40–/– mice. Modulation of Cx43 expression with NOS activity in the rat hearts was observed in our previous study (25). The present study demonstrated LPS-induced the increased NOS activity associated with upregulation of Cx40 expression in the rat aorta. The impaired NO-dependent relaxation of the aorta in correlation with literature data indicating the decreased eNOS activity and LPS-related elevation of the NOS activity suggests the increased activity of iNOS isoform and NO overproduction. Our results correlate with data showing that de novo formation of Cx40 was associated with the overproduction of NO in cultured endothelial cell (63). Our results thus indicate that LPS-induced Cx40 upregulation might be related to increased iNOS activity. The issue needs more detail study.

The chronic consumption of n-3 PUFA represents a known powerful tool aimed on the reduction of cardiovascular disease and supporting good health. Despite the extensive studies, the exact mechanisms involved in their protective effects are still not completely known. The improved Cx43 expression in the aorta and the heart of hypertensive and HTG rats after chronic supplementation with n-3 PUFA (23-26) indicated the involvement of GJs as one of the mechanisms through which n-3 PUFA can protect the function of cardiovascular system. In the present study we demonstrated that 10-day n-3 PUFA supplementation of LPS rats reduced Cx40 expression of the aorta supporting the involvement of Cx40-GJ as the target for n-3 PUFA. Cx40 downregulation could result from n-3 PUFA-related reduction of inflammation and oxidative stress as the levels of inflammatory mediators CRP, MDA, CD68 expression and the NAGA activity were decreased. The anti-inflammatory and anti-oxidative effects of n-3 PUFA are consistent with further studies (64). The results also indicate NO-independent modulation of Cx40 expression as well. N-3 PUFA are a part of cellular membrane and affect its absorption and transport properties. Moreover, they are known to modulate lipid metabolism (65). During the n-3 PUFA diet, these can be incorporated into membrane to influence lipid microdomains which are rich in Cx proteins (66). Cxs are dynamic transmembrane proteins therefore the modulation of Cx40 with n-3 PUFA incorporation seems very likely.

Our results indicated that 10-day n-3 PUFA intake could protect the function of the aorta. Unawares, n-3 PUFA administration to the LPS rats did not improve the NO-dependent relaxation response of the aorta, nor protected the NOS activity, which even enhanced. The results do not allow the elucidation of this situation. However, n-3 PUFA are known with their diverse (healthy and toxic) effects and their co-existence could occur (67, 68). It was reported that the administration of n-3 PUFA prior a pathological insult for chronic period ameliorated the symptoms of inflammation and protected against the effects of endotoxin in some animal models (69). We applied n-3 PUFA at the same day as a pathological insult, LPS in our case. At the end of experiment we observed reduced inflammation, but the levels of CRP and the activity of NAGA in LPS+PUFA group were still higher than in controls suggesting the persistence of a lower inflammation. Because n-3 PUFA are highly unsaturated and highly susceptible to the oxidation to form toxic oxidation products (70-72), we suggest that the suppression of inflammatory process could relatively be low enough to decrease the expression of Cx40 but it might still affect n-3 PUFA to produce oxidative form products and contribute to the deterioration of endothelium-dependent vasomotor response. Our work thus indicates the importance of the timing of n-3 PUFA administration. The results could also be dependent on the dose and the period of n-3 PUFA supplementation (73, 74) as well as on the type of the used artery and the age of experimental animals (23, 75, 76). Moreover, the farnesoid X receptors due to their anti-inflammatory properties in atherosclerosis and lipid homeostasis may represent further potential mechanism of n-3 PUFA involved in the modulation of NO-dependent vascular reactivity observed in the thoracic aorta (77). However, the exact mechanisms underlying the effects of n-3 PUFA on the aortic function and Cx40 expression remain to be elucidated.

Our results demonstrated that a single dose of LPS resulted in upregulation of Cx40 expression in the aorta associated with the moderate inflammation. We also observed that 10-day n-3 PUFA diet in vivo normalized Cx40 expression in the aorta of LPS rat and had a partial antiinflamatory effects. The results indicate Cx40 as a potential target of n-3 PUFA diet to protect the aortic function against LPS-induced inflammation.

Acknowledgements: This study was supported by VEGA grants No. 2/0108/10 and 2/0065/13. We thank V. Mocsonokyova and V. Dytrichova for their technical help.

Conflict of interests: None declared.

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