Arthrospira platensis accelerates the formation of an endothelial cell monolayer and protects against endothelial cell detachment after bacterial contamination

Abstract

Within the last years a comprehensive number of scientific studies demonstrated beneficial effect of Arthropira platensis (AP) as dietary supplement due to a high content of proteins, minerals and vitamins. Positive effects like promoting the immune system, reducing inflammation and an anti-oxidant capacity are reported. In this study, the effect of an aqueous AP extract on primary human venous endothelial cells (HUVEC) was investigated. In addition, the effect of AP on HUVEC treated with a bacterial toxin (lipopolysaccharide, LPA), inducing an activation of HUVEC and cellular detachment, was analyzed. Depending on the concentration of AP extract a significantly accelerated formation of an endothelial cell monolayer was observed. Furthermore, the detachment of HUVEC after LPA addition was dramatically reduced by AP. In conclusion, the data are promising and indicatory for an application of Arthrospira platensis in the clinical field.

Keywords

Arthrospira platensis endothelial cells xCELLigence system

1 Introduction

Arthrospira platensis (AP) as one representative of this family is a multicellular, photosynthesizing cyanobacterium rich in proteins, vitamins, essential amino acids, minerals and essential fatty acids [1]. Beyond its use as forage with known effects on flesh, egg and plumage color, milk yield and fertility, it has been found to have additional pharmacological properties. Many preclinical and a few clinical studies suggest several therapeutic effects ranging from reduction of cholesterol to enhancement of the immune system, an increase in intestinal lactobacilli, a detoxification of heavy metals and drugs and anti-oxidant, anti-inflammatory properties [1 –7].

The excessive generation of intracellular reactive oxygen species (ROS) has been implicated in the pathogenesis of many cardiovascular diseases, including atherosclerosis, hypertension, heart failure and the associated endothelial dysfunction [8, 9]. Dartsch reported a dose-dependent inactivation of free superoxide radicals (anti-oxidant effect) as well as an anti-inflammatory effect characterized by a dose-dependent reduction of the metabolic activity of neutrophils and a dose-dependent inactivation of superoxide radicals generated during an oxidative burst [10]. In addition, Chu et al. could show that the aqueous extract of AP had a protective effect against apoptotic cell death due to free radicals in fibroblasts [11]. This antioxidant potential has been attributed mainly to phycocyanin prepared from AP by aqueous extraction [12 –15]. A purified peptide from AP inhibited the Angiotensin II induced production of intracellular reactive oxygen species (ROS) in a human endothelial cell line [4]. Moreover, the changes in cell morphology correlated with intracellular ROS production were recovered by treatment with the purified peptide.

These results were confirmed in an animal study in rats, in which the protective effect of AP against 4-nitroquinoline-1-oxide (4NQO) induced hepato- and nephron-toxicity was explored. The 4NQO administration increased the oxidative stress with a concomitant decline in the levels of non-enzymic [reduced glutathione (GSH)] and enzymic antioxidants [Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), and Glutathione-S-transferase (GST)] in both liver and kidney and resulted in increased levels of hepatic and renal markers [Alanine Transaminase (ALT), Aspartate Transaminase (AST), Lactate Dehydrogenase (LDH), urea, creatinine and uric acid] in the serum of experimental animals. The oral pretreatment with aqueous extract of AP prevented those 4NQO-induced changes in the levels of hepatic and kidney enzymes in the serum of experimental rats. It counteracted the 4NQO induced lipid peroxidation and maintained the hepatic and kidney antioxidant defense system at near normal in both liver and kidney [16].

Therefore, the main purpose of this study was to analyze whether an aqueous extract of AP might influence the endothelial cell monolayer formation on tissue culture plates and whether AP is able to attenuate or even prevent the toxic influence of lipopolysaccharides (lipid A, LPA), the toxic component of LPS respectively, on primary human venous endothelial cells (HUVEC [17]).

2 Material and methods

2.1 Study design

The experiments were designed as prospective, controlled in vitro study using human umbilical vein endothelial cells (HUVEC). Optimal HUVEC number was determined in a set of experiments in order to obtain a significant cell index value and a constant cell growth during the entire duration of the experiment. From these experiments the optimum cell density was chosen as 3000 cells/well.

Thereafter, the effect of AP in increasing concentrations (0.125μg/ml, 50μg/ml, 250μg/ml) without and with supplementation of LPA (5μg/ml as toxic component of lipopolysaccharides (LPS) on viability, adhesion and proliferation of human vein endothelial cells was assessed in real-time by the xCELLigence system E-plate 16 (ACEA Biosciences, Inc.) for 80 h at 10 min intervals. The dosages of LPA were based upon previous studies examining the proinflammatory effects of LPS in HUVEC [18, 19] as well as own experience [17].

2.2 Endothelial cells

Human vein endothelial cells (HUVEC) used in this study were purchased from Lonza (Ba-sel, Switzerland). HUVEC were cultured in a standard humidified incubator at 37°C with 5% CO₂ according to optimal media and growth conditions. Cells were used at passage 4 for experiments [20].

2.3 Preparation Arthrospira extract for cell culture experiments

AP powder (Biospirulina, Sanatur GmbH, Singen, Germany) was stirred overnight in sterile 0.9% NaCl solution (B.Braun, Melsungen, Germany) at room temperature (10 mg/ ml). Then the extract was centrifuged at 3400 g for 5 minutes with subsequent filtration using a 0.45 and 0.22μm filter (TPP). The extract was stored at 4°C until further processing.

Phycocyanin – a pigment-binding protein – was described as important ingredient of AP which significantly effects platelet and endothelial cells [21, 22]. Therefore, the concentration of phycocyanin in the aqueous extract was measured. The C-phycocyanin concentration (CPC) in mg/mL was calculated from the optical densities at 652 and 620 nm, using Equation 1 [23]: $CPC = (OD620 0 .474OD652) / 5 .34$ (equation 1)

Extraction yield: the extraction yield was calculated using Equation 2 [24]. $Yield = (CPC)V / DB$ (equation 2)

where Yield is the extraction yield of phycocyanin in mg of C-phycocyanin/dry biomass [g], V is the solvent volume (mL) and DB is the dry biomass [g].

2.4 Cell Index (CI) measurements by RT-CES system

The xCELLigence system consists of a plate station with up to six 96 well E-Plates and a software for automatic and real-time data acquisition and display. The system measures electrical impedance across micro-electrodes integrated on the bottom of tissue culture E-Plates. It allows to monitor changes of ad-herence, spreading and proliferation of HUVEC in real time based on the measured cell-electrode im-pedance [25]. From these data, a parameter termed “Cell Index (CI)” can be calculated, according to $C I = \max_{i = 1, .., N} [(\frac{R_{cell} (f i)}{R_{b} (f i)} - 1)]$ (equation 3)

where R_b(f) and R_cell(f) are the frequency dependent electrode resistances (a component of the impedance) without cells or with cells present, respectively. N is the number of the frequency points at which the impedance is measured.

Thus, CI is a quantitative measure of the status of the cells in an electrode-containing well. Under the same physiological conditions, more cells attached on to the electrodes leads to larger R_cell(f) value, leading to a larger value for CI. Furthermore, for the same number of cells present in the well, a change in the morphology of the cells (spread cells) will also lead to a change in the CI. A “Normalized Cell Index” at a given time point is calculated by dividing the Cell Index at the time point by the Cell Index at a reference time point. Thus, the normalized Cell Index is 1 at the reference time point.

In the absence of living cells (cell culture medium only) or with a suspension of dead cells, the cell index values will be close to zero. After cellular attachment onto the electrode, the measured signal correlates linearly with cell number throughout the experiment with sufficient accuracy, which has been shown in many publications [26 –28].

2.5 Statistics

Arithmetic mean and standard deviation are given for all samples. For two sample comparisons paired, two-sided t-test were used; in the case of multi sample comparisons ANOVA for repeated measures. Differences were considered significant, when p-values were lower 0.05.

3 Results

3.1 Reproducibility of endothelial cell monolayer formation

Both experimental series of HUVEC monolayer formation (E1, E2) without AP or LPA did not differ from each other (p = 0.294). Thus, the experiments – which were performed at intervals of one week – were reproducible (see Fig. 2).

Fig. 1

Sketch of measurements of the Cellular Index under different conditions over time.

Fig. 2

Reproducibility of HUVEC monolayer density. Presented are normalized curves of the mean CI of two subsequent experiments (E1 and E2) with endothelial cell cultures cultivated over 80 h (n = 7 each).

3.2 Influence of AP on HUVEC monolayer formation

Depending on the concentration of AP an increase of the cell index was observed 56 h after adding AP. The cell index (CI) after the addition of 0.125μg/ml AP was CI = 4.53, after adding 50μg/ml CI = 5.25 and CI = 4.97 for the addition of 250μg/ml AP.

Figure 2 shows the HUVEC density after supplementation of the cell culture medium with different concentrations of AP in comparison to untreated HUVEC over cultivation time.

Fig. 2

Course of mean normalized CI of HUVEC during cultivation of untreated cells (control cells), after adding 0.125μg/ml AP, after adding 50μg/ml AP and after adding 250μg/ml AP. Presented are graphs of arithmetic means for n = 7 experi-ments (each treatment).

3.3 Influence of LPA on HUVEC monolayer formation

By adding LPA, a significant and concentration-dependent decrease of the HUVEC monolayer density was observed. The CI was diminished after the addition of 5μg/ml LPA to the cell culture medium to 3.61, see Table 2. It became obvious that most of the HUVEC detached immediately after LPA treatment for a short time period (see CI Fig. 3 below the arrow „treatment“), followed by renewed attachment. This effect was not seen for untreated HUVEC or HUVEC treated with AP (Figs. 1 and 2) although the same volumes (medium or medium with AP extract) were added to the cells. This seemed to be a specific effect of LPA.

Fig. 3

Mean normalized CI of untreated HUVEC in cell culture wells compared to LPA treated HUVEC (5μg/ml LPA) during the cultivation time of 80 hours. Presented are graphs of arithmetic means of n = 7 experiments per treatment.

The samples differed significantly (ANOVA: p < 0.001). Tukey-Kramer test showed that the control culture differed from the HUVEC supplemented with 5μg/ml LPA (p < 0.05 each, see Table 3).

Figure 3 shows the HUVEC density after supplementation of the cell culture medium with 5μg/ml LPA in comparison to untreated HUVEC over cultivation time.

3.4 Effect of AP on endothelial cell impairment by LPA (5μg/ml)

The addition of 5μg/ml LPA to the cell culture medium induced – as already seen in Fig. 3 – a substantial decrease of adherent HUVEC compared to control cells from 4.155 to 3.61 56 hours after adding LPA. This decline was completely repealed by the initial addition of 0.125μg/ml AP (blue curve). A harmful effect of LPA on HUVEC was not observed anymore.

After the supplementation of the cell culture medium with 50μg/ml AP and then adding 5μg/ml LPA even more HUVEC adhered/proliferated compared to untreated control cells (Fig. 4).

Fig. 4

Effect of AP in two concentrations on mean normalized CI of LPA-induced cell impairment/-detachment (5μg/ml) compared to control cells during the cultivation time of 80 hours. Presented are graphs of arithmetic means of n = 7 experiments per treatment.

Figure 4 shows the HUVEC density after supplementation of the cell culture medium with 5μg/ml LPA in comparison to untreated HUVEC over cultivation time.

3.5 Detection of Phycocyanin in AP extract

The amounts of Phycocyanin –as effective ingredient - in the AP extracts were measured. In the extract of 0.125μg/ml AP no Phycocyanin could be detected (values were below the detection limit), in the extract of 50μg/ml AP 1.65μg/ml, in the extract of 250μg/ml AP 9.66μg/ml.

4 Discussion

The study revealed that the aqueous extract of Arthrospira i.) led – in comparison to control cells – to an accelerated formation of an endothelial cell monolayer and ii.) had a protective effect against endothelial cell detachment depending on the AP concentration of the extract.

Different groups described an AP-induced increase of the expression of endothelial nitric oxide synthase (eNOS) [6 , 29–31] which is associated with the amelioration of endothelial NO production [32]. Such an increased NO release would stimulate endothelial cell proliferation [33], what was observed in our study depending on the concentration of the added AP extract.

Also, the anti-inflammatory capacity of AP can support the endothelial proliferation. Several studies have shown that AP lowered TnF-α [34] and TGF-β [35]. A low or even missing TGF-ß and/or TnF-a production would lead to a reduced inhibition and could, therefore, permit increased proliferation of endothelial cells. This effect might support the inhibition of the activation of Adenine Dinucleotide Phosphate (NADPH) oxidase, thereby providing potent anti-oxidative effects which would prevent the overproduction of reactive oxygen species and thereby counteract apoptosis [11 , 37].

Similar to leukocytes, endothelial cells express innate immune receptors, including members of the Toll-like receptor (TLR) family [38, 39]. TLRs are activated following direct recognition of a wide range of PAMPs (pathogen associated molecular patterns) including bacterial lipopolysaccharides (LPS) [40]. Upon sensing such molecules TLRs trigger the expression of inflammatory mediators (cytokines, chemokines and cell adhesion molecules) [41] and, in addition, have been shown to directly regulate cell proliferation [42, 43]. The activation of TLRs is reported to lead to the expression of tissue factor, thereby stimulating endothelial cell proliferation through activation of the extracellular signal regulated kinase 1/2 pathway [44 –46], what was observed in this study.

Exopolysaccharides (PSP) or LPS from AP –both are water soluble [47] –have the potential to induce apoptosis [48, 49] and so to decrease of the number of adherent HUVEC [50]. However, exopolysaccharides dissolve from the outer membrane especially in hot-water AP extracts what was avoided in our study. The destruction of bacteria (the AP powder is mortarized before extraction) leads to the dissemination of lipid A (LPA), the actual endotoxin which anchors the LPS to the outer membrane [51]. However, contrary to LPS of Escherichia coli (E. coli), LPA from Arthrospira was reported to have a very low toxicity and barely induced inflammatory cytokines [52], so that LPA should have a minor influence in our experiments.

Taken together, these effects shed light to the results of our study, that AP can explain –at least in part –the increased proliferation of endothelial cells and in addition the detachment or even apoptosis of endothelial cells after adding LPS. However, we noticed that the effects, observed within this study relied on the concentration of the added AP extract. With increasing concentrations of the AP extract a significant increased CI was detected for HUVEC. In addition, it was recognized that depending on the added AP extract concentration the detachment of HUVEC after LPA addition was significantly antagonized starting with a concentration of 50μg/ml AP.

One must have in mind, that the aqueous extract of AP also contains other substances, such as γ-linolenic acid, α-tocopherol, allophycocyanin, phycobilicyanin, phycoerythrin and other phytochemicals [10, 53] which might also contribute to the effects found in this study. Some studies have attributed the antioxidant capacity [11 , 56], to its content of phenolic compounds, such as γ-linolenic acid, α-tocopherol and phycocyanin [10, 54]. Chu reported that the aqueous extract of AP could reduce apoptotic cell death induced by the free radicals 2,2-diphenyl-1-picrylhydrazyl (DPPH) or 2,2^′-Azino-di(3-ethylbenzthiazolin-6-sulfonsäure) (ABTS) significantly. The antioxidant activity of the extract was higher than isolated phycocyanin based on the DPPH assay, suggesting that a mixture of compounds was more active than a single pure compound.

4.1 Protective potential of AP

There is growing evidence, that AP extracts might have a protective effect against different noxes. Figure 4 shows that the addition of LPA to the cell culture medium (dosage: 5μg/ml) led to a significant decrease of the number of adherent HUVEC. Interestingly, adding an aqueous extract of AP prevented the detachment of HUVEC (induced by LPA) in a concentration-dependent manner. Instead of a LPA-induced decrease of the HUVEC density [17], the density increased after adding 50μg/ml or 250μg/ml significantly (Table 1).

Table 1
Tukey-Kramer significance levels for Arthrospira platensis supplementation

Control 0.125μg/ml AP 50μg/ml AP 250μg/ml AP

Control – – 0.05 0.05

0.125μg/ml AP – 0.05 –

50μg/ml AP – –

250μg/ml AP –

	Control	0.125μg/ml AP	50μg/ml AP	250μg/ml AP
Control	–	–	0.05	0.05
0.125μg/ml AP		–	0.05	–
50μg/ml AP			–	–
250μg/ml AP				–

Table 2

Tukey-Kramer significance levels for LPA supplementation in [μg/ml]

	Control	5μg/ml LPA
Control	–	0.05

Table 3

Tukey-Kramer significance levels for LPA supplementation

	Control	5μg/ml LPA	5μg/ml LPA + 125 ng/ml AP	5μg/ml LPA + 50μg/ml AP
Control	–	0.05	–	–
5μg/ml LPA		–	–	0.05
5μg/ml LPA + 125 ng/ml AP			–	0.05
5μg/ml LPA + 50μg/ml AP				–

This is well in line with a study from Leung et al. [57], in which AP protected against LPS-induced Acute Lung Injury. LPS-induced elevation of protein concentration, nitrite/nitrate level, release of proinflammatory cytokines, the number of total polymorphonuclear cells in bronchoalveolar lavage fluid, and lung edema was observed, accompanied by a remarkable improvement of lung histopathological alterations.

Oral pretreatment with an aqueous extract of AP prevented 4-nitroquinoline-1-oxide (4NQO, a potent carcinogen (it induced papillomas, squamous cell carcinomas, adenocarcinomas, fibrosarcomas and lymphomas in mice, rats, hamsters, guinea pigs and rabbits). AP increased the activity of antioxidant enzymes and counteracted the 4NQO induced lipid peroxidation and maintained the hepatic and kidney antioxidant defense system at near normal in both liver and kidney [58].

5 Conclusion

The study revealed a concentration-dependent positive effect of AP extracted at room temperature on HUVEC proliferation. Furthermore, a protective effect of the AP extract on cellular detachment induced by LPA was observed. Further studies are planned to understand the underlying mole-cular mechanisms.

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Arthrospira platensis accelerates the formation of an endothelial cell monolayer and protects against endothelial cell detachment after bacterial contamination

Abstract

Keywords

1 Introduction

2 Material and methods

2.1 Study design

2.2 Endothelial cells

2.3 Preparation Arthrospira extract for cell culture experiments

3 Results

3.1 Reproducibility of endothelial cell monolayer formation

4 Discussion

4.1 Protective potential of AP

Table 1 Tukey-Kramer significance levels for Arthrospira platensis supplementation Control 0.125μg/ml AP 50μg/ml AP 250μg/ml AP Control – – 0.05 0.05 0.125μg/ml AP – 0.05 – 50μg/ml AP – – 250μg/ml AP –

References

Table 1
Tukey-Kramer significance levels for Arthrospira platensis supplementation

Control 0.125μg/ml AP 50μg/ml AP 250μg/ml AP

Control – – 0.05 0.05

0.125μg/ml AP – 0.05 –

50μg/ml AP – –

250μg/ml AP –