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FTIR spectroscopic imaging of endothelial cells response to tumour necrosis factor-#: to follow markers of inflammation using standard and high magnification resolution Ewelina Wiercigroch, Emilia Staniszewska-Slezak, Kinga Szkaradek, Tomasz Wojcik, Yukihiro Ozaki, Malgorzata Baranska, and Kamilla Malek Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03089 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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Analytical Chemistry

FTIR spectroscopic imaging of endothelial cells response to tumour necrosis factor-α α: to follow markers of inflammation using standard and high magnification resolution Ewelina Wiercigrocha, Emilia Staniszewska-Slezaka, Kinga Szkaradekb, Tomasz Wojcikb, Yukihiro Ozakic, Malgorzata Baranskaa,b*, Kamilla Maleka* a

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland.

b

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University

Bobrzynskiego 14, 30-348 Krakow, Poland. c

Department of Chemistry, School of Science and Technology, Kwansei Gakuin University,

Gakuen 2-1, Sanda, Hyogo 669-1337, Japan.

*CORRESPONDING AUTHOR: K. Malek, e-mail: [email protected], M. Baranska, email: [email protected] phone/fax: +48 12 686 2394/+48 12 686 2750

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Two endothelial cell lines were selected as models to investigate an effect of incubation with cytokine tumour necrosis factor type α (TNF-α) using FT-IR imaging spectroscopy. Both cell lines are often used in laboratories, and are typical lung vascular endothelial cells (HMLVEC) and derived from the fusion of umbilical vein endothelial cells with lung adenocarcinoma cells (EA.hy926). This study was focused on alteration of spectral changes accompanying inflammation at the cellular level by applying two resolution systems of FT-IR microscopy. The standard approach, with a pixel size of ca. 5.5 µm2, determined the inflammatory state of the whole cell, while a high magnification resolution (pixel size of ca. 1.1 µm2) provided information at the sub-cellular level. Importantly, the analysis of IR spectra recorded with different modes produced similar results in overall and yielded to unambiguous classification of inflamed cells. Generally, the most significant changes in the cells under the influence of TNF-α are related with lipids - their composition and concentration; however segregation of cells into sub-cellular compartments provided an additional insight into proteins and nucleic acids related events. The observed spectral alterations are specific for the type of endothelial cell line.

Keywords: FTIR imaging microscopy, single cells, sub-cellular compartments, inflammation, in vitro, TNF-α, endothelium


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Introduction Tumour necrosis factor α (TNFα) belongs to the group of pro-inflammatory cytokines and chemically is a 182-amino acid glycoprotein 1–3. TNFα affects pathological processes by regulating the immune response in acute and chronic inflammatory diseases such as septic shock, AIDS and cancer

1,2

. It plays an important role in cardiovascular diseases and

contributes to the disruption of macrovascular and microvascular circulation throughout the production of reactive oxygen species (ROS) inducing changes in vascular endothelial cells. In particular, inflammatory state of endothelial cells is strongly related with vascular injury and is one of the primary factors in pathogenesis of many cardiovascular diseases. However, exact mechanisms underlying this dysfunction of endothelium have still remained unclear 1–5. Many features of TNFα were found to be specific for a given cell type, and the specificity of TNF-induced cellular response is determined by individual intracellular signaling pathways. For instance, TNFα modifies anticoagulant features of endothelial cells, promotes T cell proliferation and induces the release of different inflammatory cytokines in different cells. The common event of TNFα action is only its binding to surface receptors, TNF-R1 and TNF-R2, present on all types cells in the body 1–3. As well-known in vitro studies play a crucial role in biomedicine. They mostly rely on monitoring of protein and gene expression by using target-designed modalities based on flow cytometry, proteomics, immunohistochemistry and in vitro assays quantifying excretion of specific biomolecules. Mass-spectrometry (IMS), Fourier-transform infrared (FTIR) and Raman spectroscopy (RS) combine microscopic imaging with simultaneous label-free detection of chemical species in biological samples. Although IMS provides detailed information about the concentrations of proteins and lipids, routine chemical images show morphological structures larger than 20 µm only and their construction requires advanced bioinformatics protocols

6,7

. Over the last years, FTIR microscopy has been successfully

employed in in vitro assays studies on several cell lines assess effects of external agents on cells

8–10

to help diagnosis disease and to

11,12

. A primary reason for this application is the fact

that FTIR spectral imaging provides a wealth of chemical information about a biosample without a priori knowledge. Synchrotron-based FTIR microscopy has been the main molecular probe to investigate subcellular biochemical behavior because of brightness advantage of the synchrotron source allowing to achieve a pixel resolution up to 0.54 µm using high magnification objectives13–15. Such detailed cellular probing is comparable with Raman spectral imaging for that lateral resolution up to 0.3 µm (with a laser excitation at 532 3 ACS Paragon Plus Environment


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nm) is routinely applied for intracellular studies 16,17. However, IR spectra collection is much faster than Raman spectra, and the former are not obscured by a fluorescence effect. Recently, the Agilent Technologies introduced to the market technological solution for enhancing spatial resolution (~1.1 µm pixel size with the use of a 15× Cassegrain optics, NA=0.62, spatial resolution of 3.0 µm at 2500 cm-1) in the bench top FTIR FPA microscope allowing collection of IR spectra at the subcellular level

18

. The capabilities of this new system for the

analysis of single eukaryotic cells have been shown by Hughes and co-workers,

18

while Perez-

Guaita et al. have reported automated detection of malaria-infected erythrocytes 19. Highly accurate classification of neoplastic cells has been also achieved by Old et al., who investigated tissue sections of Barrett’s esophagus 20. We employed this instrumentation as well as a standard set up of the Agilent 620 FTIR microscope (~5.5 µm pixel size, spatial resolution of 7.6 µm at 2500 cm-1) to achieve molecular information induced by the pro-inflammatory agent – tumour necrosis factor (TNFα) in representatives of the family of endothelial cells - EA.hy926 and HMLVEC cells. The EA.hy926 cell line is an immortalized human umbilical vein endothelial cell (HUVEC) line, derived from the fusion of HUVECs and lung adenocarcinoma cells. The structural characteristic and functions are similar to lung adenocarcinoma cells but they also retain many endothelial cell features. These alveolar endothelial cells have been used to examine leukocyte adhesion to endothelial cells, oxidative and inflammatory stress and protein expression

4,5

. Their TNFα-induced response was compared here with primary

endothelial cell line HLMVEC (human lung microvascular endothelial cells), which has been widely used in studies on pathology and biology of the pulmonary microvasculature in vitro 21

. The aim of this study was to ascertain whether alternation of intracellular chemical

composition upon inflammation is spectrally observable and whether it gives a common/different spectral panel of markers for cells derived from the same family but with different phenotypes. The biological status of the examined cells, by the detection of markers of inflammation, apoptosis, and necrosis, were determined in parallel experiments using fluorescence and flow cytometry. Finally, we examined to which spatial extent inflammation state can be determined in cellular compartments and how IR measurements modes (transmission vs. transflection) affect the spectral profile of cells. We also discussed image quality of single inflamed cells and demonstrated how FTIR spectroscopic imaging can be applied to classify inflamed cells. The inflammatory state appears at an early-stage in most diseases and its detection in clinically applicable timeframe would have an invaluable


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diagnostic potential. Therefore, this work is the first report showing the potential of rapid FTIR spectroscopy screening of inflammatory response.

Experimental Section

Cell culture, fluorescence and flow cytometry EA.hy926 cells and HLMVEC cells were grown in plastic wells in DMEM (Dulbecco's modified eagle medium) supplemented with 10% fetal bovine serum, 2 mM Lglutamine, penicillin, streptomycin, and 2% HAT (hypoxanthine, aminophterin and thymidine). Both cell lines were directly cultured on CaF2 windows in concentration of 6×104 cells/well for transmission IR imaging. EA.hy926 cells were also cultured on Kevley® slides with concentration of 2×106 cell/well for transflection IR imaging. To induce inflammation cells were treated with a medium containing 10 ng⋅mL−1 TNFα for 16 hours. Next, the cells were washed three times with PBS (phosphate buffered saline) and stained with 4% formalin at 20 min. in room temperature with a brief rinse in PBS. Shortly before the measurements, cells were washed gently with doubly deionized water to remove residual PBS from the surface of the cells and then left to air dry. Concentrations of cytokines (interleukin 6 (IL6) and 8 (IL8)) and chemokines (monocyte chemoattractant protein-1 (MCP1) and chemokine β (RANTES)) were determined using ELISA tests and a multiplex technique of Luminex (MAPlex assay). Next, detection of necrosis and apoptosis were performed by using Annexin FITC (Fluorescein) and PI PE (PI, propidium iodide and PE, phycoerytrin) kits on a BD LSR II system.

FTIR imaging IR images were recorded using a Varian 620-IR microscope coupled to a 670-IR spectrometer with a 128×128 pixel focal plane array (FPA) detector. Transmission and transflection measurements were carried out with a 15× Cassegrain objective and condenser collecting 128 and 256 co-scans in standard and high resolution, respectively. High resolution images were recorded immediately after standard resolution IR screening to collect database for the same set of cells. For each experimental group (control and TNF-treated cells of both cell lines), we finally selected 30 images with a pixel size of 5.5 µm, containin7g on average 25 cells each. As well, 30 images of individual cells with a pixel resolution of 1.1 µm were selected for each experimental group. All spectra were recorded in the region of 3600–900



cm–1 with a spectral resolution of 8 cm–1. The total acquisition time for an area of ca. 700×700 µm2 for standard magnification was ca. 5 min, while for an area of ca. 140×140 µm2 for high magnification was ca. 15 min. IR data were collected and initially analyzed with the use of the Varian Resolutions Pro software (ver. 5.0.0.640).

Data pre-processing and analysis Spectral pre-processing and data analysis was performed using a CytoSpec v.2.00.02 software 22. After noise reduction with 7 PCs, second-derivative spectra were calculated using a Savitzky–Golay algorithm (9 points of smoothing). For IR images collected with standard resolution, cluster maps were constructed employing unsupervised hierarchical cluster analysis (UHCA) in the spectral regions of 900–1800 and 2800–3600 cm−1. A Ward’s algorithm was applied while spectral distances were computed as D-values. Two classes were adjusted to extract cells and background. For images recorded with enhanced resolution, cluster maps were constructed with a help of KMC (k-means cluster) analysis in the same spectral regions as for UHCA. We adjusted a number of classes to discriminate central and peripheral parts of a single cell. In addition, we developed KMC maps with a larger number of classes to discriminate subcellular compartments. For KMC, a Euclidian algorithm was used and the number of iteration was 20. Our approach of data analysis is summarized in Figure S1. Principal Component Analysis (PCA) was performed using a Unscrambler X software (v. 10.3, Camo). IR spectra extracted from cluster maps were pre-processed by calculating second derivative spectra (as above) and correcting them by Extended Multiplicative Signal Correction (EMSC) in the entire spectral region. PCA was computed for the “bio-region” (3100–2800 cm–1 and 1750–900 cm–1) by the use of the leave-out-one cross-validation approach and Nipal’s algorithm for PCA decomposition. Seven PCs were chosen for the initial decomposition and 20 iterations were performed for each PC. Two-dimensional (2D) score plots and the corresponding PC loading plots were graphed. Mean second derivative spectra were used for calculation of integral intensity of various bands and their ratios using OPUS 7.0 program (Bruker Optics). For this purpose, a linear baseline was drawn through the peak edges, and the spectrum below this line was integrated over the wavenumber range of the band. Band ratios are expressed as their mean ± standard error (SE). An analysis of variance was performed using a statistical model Anova in a Origin Pro 9.1 software. Tukey’s test was employed to compute significance values p.


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Results and discussion 

Markers of inflammation induced by tumour necrosis factor Inflammation of endothelial cells induced due to exposure to TNFα was confirmed by quantitative determination of pro-inflammatory cytokines and chemokines (Figure S2 in ESI). This agent in concentration of 10 ng/mL stimulated after 16 h a significant release of the basal inflammatory markers, i.e. IL 6 (108 vs. 2 pg/mL in control), IL 8 (1320 pg/mL vs. 41 pg/mL in control), as well as the over 6-hold increase of chemokines level, MCP1 and chemokine β, was observed. The generation of ROS inside cells and in mitochondria and the overexpression of vascular cell adhesion molecules (ICAM1, VCAM1, selectins E and P) confirmed oxidative stress and endothelial dysfunction (data not shown). The characteristics of the inflammatory state were similar for both cell lines. To exclude the induction of apoptotic and necrotic processes in cells upon TNFα action we performed viability flow cytometric analysis (Figure S3 in ESI). It showed that the necrotic process must be excluded as response to TNFα. In turn, the incubation of HLMVEC cells with TNFα resulted in an increase of the number of apoptotic cells to 4.3% of gated cells population from 2.7% in untreated control. In the case of EA.hy926 cells, 4.8 and 10.1% untreated and TNFα-treated cells were apoptotic, respectively. A small population of cells in late apoptosis was also identified; 0.7 (control) vs. 2.0% (TNFα-treated EA.hy926) and 1.5 (control) vs. 1.8% (TNFα-treated HMLVEC). This very likely occurred due to spontaneous cell death as a result of the staining protocol. A major population of cells was observed to be FITC Annexin V and PI negative, indicating that EA.hy926 and HMLVEC cells were viable after 16h incubation with TNFα.

FTIR spectroscopic imaging in standard resolution - transmission mode Figure 1A and B shows our approach to extract rapidly spectral information from IR image of an area of 700×700 µm2 which records IR signal from ca. 20-30 cells. Instead of selecting one spectrum from the center of a cell, we employed a 2-class UHCA analysis to separate the IR spectrum of a whole cell from background. After rejecting images with poor S/N, artefacts, etc., we finally obtained a set of 30 images per experimental group. The average IR spectra with their second derivatives are displayed in Figure 1 C and D. Based on the number of cells in UHCA false-color maps we estimated that we recorded IR signal from ca. 1200 EA.hy926 cells and ca. 700 HMLVEC cells. FTIR spectra of EA.hy926 and HMLVEC cells in Fig. 1C indicate variations in bands shapes which could result from 7 ACS Paragon Plus Environment


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different cell thickness and consequently from different S/N ratio of IR signal. Despite the fact that the same number of cells were seeded on each CaF2 slide (6×104 cells per well), visible microphotographs and IR images constructed for amide I band of proteins show that the number of EA.hy926 cells within an area of 700×700 µm2 and the average absorbance of amide I band are roughly 30% higher than for HMLVEC (Figure S4A and C in ESI). Indeed confluency of immortalized EA.hy926 cells on a CaF2 window is larger than for regular endothelial cells and the former creates a monolayer of cells that is locally thicker than wellseparated cells of the HMLVEC cell line. The overall spectral features of whole cells can be deduced from their second derivative IR spectra (Fig. 1D and Table S1 in ESI). The amide I bands of EA.hy926 and HMLVEC cells were located at around 1655 cm-1 (α-helix 23) whereas a shoulder (unordered random coils and turns

23

) appears at 1686 cm-1 in the spectrum of the EA.hy926 cell line

only. No shift of these bands was found in IR spectra of TNFα-treated cells indicating the lack of apoptosis and necrosis

11,12

. In turn, lipid bands at 2925 [νas(CH2)] and 2853 cm-1

[νs(CH2)] and the bands due to the ester groups of phospholipids and triacylglicerols at ca. 1740 cm-1 [ν(C=O)] were found at positions similar for other FTIR studies on cells

14,24

.

According to Whelan and co-workers 10, the 1238 cm-1 band of the νas(PO2-) mode is the main marker of A-DNA conformation formed due to cell dehydration (Fig. 1D), however, the contribution to this band from RNA, phospholipids and phosphorylated proteins cannot be excluded Minima in the spectrum of EA.hy926 cells revealed the presence of additional bands of nucleic acids at 1119 and 1059 cm-1, which were assigned to the stretching mode of the ribose C-O group in RNA

13

and the backbone of A-DNA, respectively

10

. This region

differed from the corresponding one in the spectrum of HMLVEC; here, the RNA band is present at 1109 cm-1 while a single band at 1045 cm-1 appears instead of well-separated 1085 band accompanied by a shoulder at 1059 cm-1 (Fig. 1D and Table S1 in ESI). The 1045 cm-1 band was also observed in IR spectrum of human Jurkat cells, and its assignment is not straightforward since it can be attributed to the C-O and C-C vibrations of the phosphate moieties, oligo- and polysaccharides and cholesterol esters. The presence of glycogen was detected by a band at 1165 cm-1 in the spectra of both cell lines 8. Alternations in metabolic processes can be also evaluated by the numerical variations of the bands intensities and their ratios (Table 1). Modifications in lipid synthesis and/or plasma membrane structure were expressed by the sum of intensities of the 2923 and 2850 cm-1 maxima. The total content of lipids decreased in both cell lines due to the exposure to 8 ACS Paragon Plus Environment

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TNFα. This process rather results from higher fluidity and lower order of lipid membranes in inflamed cells as observed in other FTIR studies on cells under stress

11,12,24

than from

intracellular lipid consumption. The latter was excluded from our Raman studies that showed the production of lipid droplets composed of fatty acids in dysfunctional endothelial cells 16,17. In the IR spectra of EAhy.926 we also observed a higher percentage of branched lipid alkyl chains which were associated with changes in cell membrane (ratio of νas(CH2):νas(CH3) in Table 1)

13 25

, . Next, variation of a total protein level suggested an enhanced synthesis of

proteins in HMLVEC and proteins downregulation in EAhy.926 (the sum of amide I and II bands, Table 1). This funding was congruent with reports in 26 showing a significant decrease in the protein synthesis in malignant tumour and the production of new proteins (e.g. COX-2, GTP cyclohydrolase I, E-selectin, etc.) regulating cellular TNF signaling in the inflammatory response of vascular endothelial cells

27

. Another marker for endothelial inflammation is

biosynthesis of glycogen activated by growth factors, and it is a common feature for dysfunctional endothelium and cancer cells

28

. This fact was reflected here by intensity

-1

changes of the glycogen IR band (the 1165 cm band, Table 1). Next, lipid to protein ratio was almost unchanged in inflamed EAhy.926 cells whereas a pronounce decrease by 27% was found in HMLVEC, suggesting that processes associated with lipid down-regulation overcame biochemical events of protein synthesis (Table 1). The use of PCA enabled us to explore in an efficient way the spectra of healthy and inflamed cells and to identify additionally the significant spectral variations of this process. PC-1 vs. PC-2 score plots for FTIR spectra of whole cells are displayed in Figure S5A and C in ESI. Excellent discrimination of spectral data into separated clusters were obtained according PC-1 with variance of 81 and 56% for EA.hy926 and HMLVEC, respectively. Loadings plots and their most significant discriminant functions are presented in Figure S5B and D, Table S1 in ESI. The highest loadings were found in the region of 1700 – 1400 cm-1, however, the regions below 1400 and above 2800 cm-1 also contributed to the segregation. It is important to highlight here that PC loadings indicating up- and down regulation of proteins and lipids in inflammatory state varied between investigated endothelial cell lines. This demonstrated that FTIR microscopy combined with multivariate analysis of whole cells without differentiation of sub-cellular constituents provides the identification of inflamed cells, and the “TNFα IR signature” is cell-type dependent as exhibited by the discriminators found in the PCA analysis. Up- and down-regulation of macromolecules found in the PC-1



loading plots (Table S1 in ESI) was congruent with variation of bands intensities discussed above (Table 1).

FTIR spectroscopic imaging in enhanced resolution - transmission mode To assess variations of spectral features of inflamed cells in intracellular domain, we performed segmentation of cells into nuclear and cytoplasmic functionality with the support of k-means cluster analysis (KMC) (Figure S1). Average IR spectra of 30 cells for each group are displayed in Figure 2 and their inspection indicated that FTIR spectroscopic imaging was able to detect the inflammation response at this level of cellular structure. Spectral features were clearly differentiated by PCA score plots (Figure S6 in ESI). Healthy nuclei and cytoplasm of EAhy.926 cells were differentiated from the TNFα-treated compartments along PC-2 with variation of 10 and 23 %, respectively, whereas their counterparts for HLMVEC were segregated along PC-1 with variation of 53 and 77 % for nuclei and cytoplasm, respectively. These results indicated that the inflammation process was dominant for cytoplasm, and the lung microvascular endothelial cells are more sensitive to the TNFα action than the fused adenocarcinoma and endothelial cells. The high variation values demonstrated the power of enhanced-resolution FTIR imaging spectroscopy for the successful discrimination of healthy and inflamed endothelial cells. The corresponding loadings plots and positions of all PCA discriminators indicated the localization of biochemical changes (Figure S6 and Table S2 in ESI). We found that a decrease of the total lipid level appeared in nucleus and cytoplasm confirming our hypothesis from the analysis of whole cells about degradation and permeability of cell membrane due to inflammation. We also observed this process by changing intracellular pH of EA.hy926 incubated with TNFα 29. A PC-2 loadings plot for nuclei and cytoplasm of EA.hy926 indicated a higher content of triacylglycerols in the peripheral part of cells than in the central one. This spectral discriminator was not highlighted by PCA for HMLVEC; however, we noticed the presence of strong negative signals at ca. 1700 cm-1 in the both loading plots of this cell line, suggesting the production of fatty acids (Figure S6 C,D and Table S2 in ESI). This fact was found in agreement with accumulation of unsaturated lipid droplets in endothelial HMEC-1 cells found in our Raman images 16,17. They were randomly distributed in cytoplasm with the size of 0.5 – 2 µm. Next, the profile of the amide I region confirmed our observation from FTIR imaging with standard resolution, and also indicated that cellular proteomics is specific for subcellular compartments in each cell line (Table S2 in ESI).


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Exploiting the enhanced magnification system of the Agilent FTIR microscope that provides the spatial resolution of ca. 3 µm at 2500 cm-1, selected IR images of EA.hy926 and HMLVEC cells were examined by k-means cluster analysis (KMC) to elucidate chemical composition at the sub-cellular level. The use of the KMC analysis was proposed by Hughes and co-workers in their work on IR spectra of single cells recorded by enhanced-resolution of the Agilent microscope

18

. Figures 3A and 4A display false-color KMC maps for EA.hy926

and HMLVEC cells, respectively. We see clustering of the cells into nucleus (red class), perinuclear cellular compartment such as endoplasmic reticulum and Golgi apparatus (green class), cytoplasm (pink and blues classes) along with cytoplasmic periphery (grey class). Similar discrimination and distribution of cellular compartments were obtained by synchrotron IR imaging of monkey fibroblast cell line cells

25

, mouse hypothalamic and scrapie

30

. Figures 3B-D and 4B-D illustrate mean second derivative IR spectra normalized in

the 1760-900 cm-1 region to exclude an effect of thickness differences in cells according to 18. FTIR spectra of each class of the KMC maps before normalization are displayed in Figure S7 (in ESI). The main factors of classification were associated with specific constituents, which content related to individual parts of the analyzed cells. The major spectral differences between classes both for the control and TNF-treated EA.hy926 cells were observed in the region of bands specific for DNA and RNA (Figure 3D). The A-DNA markers were found at 964 and 1235 cm-1 in the spectra of both cells 10. On contrary, RNA bands were shifted in the TNFα-treated cells comparing to control, i.e. from 1114 and 1059 cm-1 to 1111 and 1050 cm1

, respectively

31

. This may indicate changes in RNA conformation due to interactions with

proteins and/or ion binding and this effect was well recognized in cellular biochemistry including TNFα action

32–35

. We also noticed an alternation of the ratio of nucleic acids and

RNA peaks at 1081 and ca. 1050 cm-1, respectively. Note that this ratio significantly varied in the control cell. The intensity of the RNA band became less pronounced than the nucleic acids counterpart in the peripheral part of the cell (pink and grey classes) with respect to the nucleus and its surrounding (red and green classes). A different trend was observed in the inflamed EA.hy926 cell; the 1081 cm-1 band was more intense than the 1050 cm-1 maximum in the nuclear red class and this ratio decreased in the external periphery of the cell. These spectral changes of nucleic acids/RNA bands could indicate adaptive gene regulation due to stress conditions since transduction mechanisms are activated within minutes and are mainly associated with modifications of selective mRNA and protein production 36.



Noticeable differences between classes from the central and peripheral parts of EA.hy926 cells were also detected in the spectral regions specific for lipids and proteins (Figure 3B and C). A lower intensity of bands assigned to the stretching vibrations of the CH2 and ester C=O groups assigned to lipids in general was found in the nuclei of the control and TNFα-treated cells than in their cytoplasm. Triacylglicerols were accumulated in the outer cytoplasmic periphery of the healthy cell (grey and pink classes) and in the blue class of cytosol in the inflamed cell (Fig. 3C). This may correspond to the formation of lipid bodies in endothelial cells upon stress 17. A shape of amide I bands suggested conformational changes in protein structures as indicated by PCA analysis discussed above (Figure S6 and Table S2 in ESI). The main amide I band was centered at 1658 cm-1 in control (α-helices) and shifted to 1662 cm-1 in the TNFα-treated cell (turns and coils). In addition, a shoulder of the amide I band at 1639 cm-1 (β-sheets) was only slightly altered in the non-treated cell on contrary to the inflamed cell (Fig. 3C). Here, intensity of this band increased considerably in all classes, except the red cluster. In the case of HMLVEC cells, the KMC analysis segmented the cells into centroid classes (Figure 4A). However, the comparison of bands intensities and their ratios in the entire range of mean IR spectra excluded clustering according to the thickness of the cells (Figure 4B-D). Probably, rehydration process of these thin and sensitive cells affected the preservation of intracellular compartments. Similarly to EA.hy926 cells, the most significant spectral variations between individual parts of the HMLVEC cells were found in the DNA and RNA region as above (Fig. 4D). We observed shifts of some bands at 1094/1088 (nucleic acids), 1057 (RNA) and 964 cm-1 (DNA) in control to 1082, 1050 and 970 cm-1 in treated cells

10,31

. Changes in the ratio of the 1080/1050 cm-1 bands were similar to EA.hy926. In

addition, a band at 980 cm-1 exclusively appeared in the blue and grey classes of both HMLVEC cells and its intensity decreased due to inflammation. We proposed to assign this signal to protein phosphorylation process as observed in synchrotron IR spectra of PC12 cells 8

. On contrary to EA.hy926 cells, no changes were visibly found in the protein conformations

(Fig. 4C) whereas the distribution of lipids, including triglycerides, changed from a high content in the periphery of the healthy cell toward the central classes in the TNFα-treated cell (Fig. 4B,C). We probably observed here migration of lipid structures into the central part of the HMLVEC cell, which then surrounded the nucleus and contributed to the IR spectrum of the nucleus.


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FTIR spectroscopic imaging in the enhanced resolution of inflamed EA.hy926 cells transflection mode The transflection geometry with the use of Ag/SnO2-coated microscope slides (low-e slides from Kevley Technologies) has the advantages of a stronger absorption and costeffective substrates. On one hand, several reports have showed that the electric field standing wave (EFSW) leads to significant distortion of transflection FTIR spectra of biological samples, giving false indicators what spectral features can be used as potential biomarkers 37,38

. However, Wood and co-workers have demonstrated that transflection measurements

were capable to identify cancerous cells in tissues despite the presence of EFSW artefacts 39. Therefore, we tested this capability of transflection IR imaging to detect inflammatory stress of EA.hy926 cells. From our best knowledge it is the first report that shows transflection IR spectra of cells images recorded in high resolution system of the Agilent IR microscope. Similarly to IR imaging dataset recorded in transmission mode, we examined whole-cell spectra and spectra of nuclear and cytoplasm regions by employing cluster analysis and PCA (Figure S1 in ESI). It is worthy to note that we did not observe an effect of silver-coated low-e slides on viability of mammalian cells as reported by Wehbe et. al. (Fig. S4A,B in ESI) 37. We noticed that IR spectra registered in transflection mode were more distorted in the region of 1800-2800 cm-1 than transmission spectra due to dispersion and resonant Mie scattering (Figure S8 in ESI)

37,38

. Additionally, the position of amide I band was shifted in

spectra of nucleus and cytoplasm from 1653 cm-1 in transmission spectra to 1642 cm-1 in the transflection mode. But no shift of amide bands appeared in transflection spectra of the whole cells. Wehbe and others reported the 6 cm-1 shift only 37. Similarly to Bassan et al. 40 one can also observe disturbed ratio between high-wavenumber and fingerprint regions due to EFSW. The results of PCA for transflection spectra are presented in Figure S9 (in ESI). Taking into account discrimination of control from inflamed cells, the type of measurement technique did not affect significantly the ability of FTIR spectroscopic imaging of whole cells for the identification of TNFα-treated cells (Figs. S5 and S9A in ESI). Variation of 54% along PC-1 was achieved for the transflection spectra (vs 81% for transmission) and separation of two groups were more pronounced in transflection mode than in transmission. However, the loadings plots revealed completely different spectral responses due to the TNFα action (Figs. S5 and S9A in ESI). Most of PCA discriminators showed opposite spectral features, e.g. in the high-wavenumber and amide I regions. Next, PCA segregation of transflection IR spectra of nuclei and cytoplasm was not pronounced like their counterpart in transmission. Discrimination of these cellular compartments due to inflammation was achieved according to 13 ACS Paragon Plus Environment


PC-5 with variation of 3-4 % only versus to 10-23 % variation gathered for PC-2 for transmission (Figs. S6A,B and S9B,C in ESI).

Conclusion This is the first FPA FTIR imaging study of endothelial cells using two different approaches, with standard and high magnification, and discovering meaningful biochemical changes due to inflammation induced by TNF-α. Generally, for both cell lines the total content of lipids decreased due to exposure to TNFα and processes associated with protein synthesis overcame biochemical events of lipid down-regulation. These results demonstrate that FTIR microscopy combined with multivariate analysis on whole cells without differentiation of sub-cellular constituents provides the identification of inflamed cells and the “TNFα signature” is cell-type dependent. The major advantage of FTIR microscopy is the possibility to sort rapidly the inflammatory status of the cells with various magnification. With a high magnification approach it was easily possible to perform segmentation of cells into nuclear and cytoplasmic compartments and even into smaller cellular structures. It was shown, that the spectral signature of the inflammation process is dominant for cytoplasm and HMLVECs are more sensitive to TNFα action than EA.hy926 cells. For lipid metabolism, the decrease in total lipid level appeared in nucleus and cytoplasm as previously observed for whole cells, confirming the hypothesis about degradation and permeability of cell membrane due to inflammation. For the first time, IR spectroscopic imaging of cells with high magnification was performed in transflection mode. It was shown that IR detection of inflamed cells after segregation of nuclei and cytoplasm is less pronounced than from transmission spectra. Further work is needed to examine reliability of the low-cost e-low slides for IR imaging of cells with enhanced spatial resolution. Taking into account a possible FTIR-based classification of the inflammatory response in cells we demonstrated that rapid and sensitive screening applications are easily achievable at various levels of spatial resolution although further studies involving different cells lines and the determination of optimum processing methods are clearly required. Overall, potential end-users of such investigations can monitor inflammation events by FTIR spectroscopic imaging with maximum flexibility in selection of the cellular organelles without cost- and time consuming procedures.


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Acknowledgements This work was supported by the National Science Centre in Poland (DEC2016/22/M/ST4/00150).

Supporting Information Available: A schematic of spectral data processing, biochemical parameters of inflamed cells, results of principal component analysis (PCA), and IR spectra recorded with the use of high spatial resolution and transflection technique.

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Figure captions Figure 1. (A) An exemplary 3D IR images of the whole endothelial cells with an applied 2class UHC analysis (B), average FTIR spectra with standard deviation (SD) (C) and their second derivative spectra (D) (N=30 for each group).

Figure 2. (A) An exemplary 3D IR image of whole endothelial cells with a KMC segregation into nucleus and cytoplasm (B), average FTIR spectra and their second derivative for nucleus (C,D) and cytoplasm (E,F) (N=30 cells for each group).

Figure 3. (A) KMC false-color maps of control (left) and TNFα-treated (right) EA.hy926 cells with mean second derivative IR spectra in the regions of 3050-2800 (B), 1760-1420 (C), and 1420-940 cm-1 (D). The colors of spectra correspond to the colors of classes in the maps.

Figure 4. (A) KMC false-color maps of control (left) and TNFα-treated (right) HMLVEC cells with mean second derivative IR spectra in the regions of 3050-2800 (B), 1760-1420 (C), and 1420-940 cm-1 (D). The colors of spectra correspond to the colors of classes in maps.


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Table of Content


Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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Table 1. Changes in the integral intensity values of the spectral markers for non-treated control and TNFα-treated EAhy.926 and HLMVEC cells (means ± SE). EAhy.926 Spectral markers

Total lipids (TL)

HMLVEC

Spectral metrics

νas(CH2)+νs(CH2)

control

TNFα-treated

control

TNFα-treated

9.10×10-3±0.11×10-3

8.26×10-3±0.15×10-3**

5.50×10-3±0.22×10-3

4.35×10-3±0.19×10-3***

(2924+2853 cm-1) Branched chain

νasCH2/νasCH3

fatty acids

(2924 cm-1/2963 cm-1)

Total proteins

amide I + amide II

(TP)

(1655+~1540 cm-1)

(↓ ↓9%) 1.74±0.04

1.58±0.04*

--

--

2.11×10-2±0.07×10-2

2.29×10-2±0.08×10-2

(↓ ↓9%) 3.62×10-2±0.03×10-2

3.36×10-2±0.02×10-2** (↓ ↓7%)

0.251±0.004

TL/TP

(↓ ↓21%)

0.246±0.004

(↑ ↑9%) 0.261±0.010

(↓ ↓2%) Glycogen

νas(CO-O-C) (1165 cm-1)

1.22×10-3±0.06×10-3

1.86×10-3±0.11×10-3*** (↑ ↑52%)

20

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0.190±0.009*** (↓ ↓27%)

4.25×10-4±0.31×10-4

1.32×10-3±0.13×10-3*** (↑ ↑69%)

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*p

FT-IR Spectroscopic Imaging of Endothelial Cells ... - ACS Publications

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