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Review article

Vascular diseases investigated ex vivo by using Raman, FT-IR andcomplementary methods

Katarzyna M. Marzec a, Anna Rygula a, Marlena Gasior-Glogowska a, Kamila Kochan a,b,Krzysztof Czamara a,b, Katarzyna Bulat a,b, Kamilla Malek a,b, Agnieszka Kaczor a,b,Malgorzata Baranska a,b,*a Jagiellonian Center for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Polandb Faculty of Chemistry, Jagiellonian University, Krakow, Poland

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

General chemical composition of atherosclerotic plaque. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

High-resolution images (Raman and AFM) of atherosclerotic plaque and vessel wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Spectroscopic markers of endothelium dysfunction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Monitoring of tissue calcification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Role of the funding source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

A R T I C L E I N F O

Article history:

Received 14 February 2015

Received in revised form 30 April 2015

Accepted 5 May 2015

Available online xxx

Keywords:

Raman spectroscopy

Infrared spectroscopy

Atomic force microscopy

Vascular diseases

Atherosclerosis

A B S T R A C T

This work shows the application of vibrational spectroscopy supported by other complementary

techniques in analysis of tissues altered by vascular diseases, in particular atherosclerosis. The analysis of

atherosclerotic plaque components, as well as label-free imaging of vessels and identification of

biochemical markers of endothelial dysfunction are reported. Additionally, the potential of vibrational

spectroscopy imaging in following the disease progression (including calcification) and pathological

changes in heart valves is described. The presented research shows the effectiveness of techniques used

in the biochemical studies of altered tissues and summarizes their capabilities in research on vascular

diseases.

The scope of the paper is to collect previously published work connected with the application of

Raman spectroscopy, FT-IR spectroscopy and complementary methods for the investigation of vascular

diseases ex vivo and presenting it in a comprehensive overview.

� 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights

reserved.

Contents lists available at ScienceDirect

Pharmacological Reports

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* Corresponding author.

E-mail address: baranska@chemia.uj.edu.pl (M. Baranska).

Please cite this article in press as: Marzec KM, et al. Vascular diseasemethods. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.

http://dx.doi.org/10.1016/j.pharep.2015.05.001

1734-1140/� 2015 Institute of Pharmacology, Polish Academy of Sciences. Published b

Introduction

QThe concentration of blood lipids, total cholesterol, low-densitylipoprotein (LDL) cholesterol, high-density lipoprotein (HDL)cholesterol, and triglycerides, plays a major role in atherosclerosisdevelopment [1]. During the hypercholesterolemic state, the LDLconcentration in blood plasma is elevated causing the infiltration

s investigated ex vivo by using Raman, FT-IR and complementary2015.05.001

y Elsevier Sp. z o.o. All rights reserved.

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the vessel wall by LDL particles [2]. Oxidized LDL particlesxLDL) trigger the activation of endothelial cells and, as ansequence, a chemotactic recruitment of adherent monocytesd lymphocytes to the forming lesion is mediated [3–5]. Suchdothelial dysfunction leads to the different vascular diseases, inrticular to the chronic inflammatory disease of the blood vesselsown as atherosclerosis, a leading cause of death in westernizeduntries [6]. Atherosclerosis affects the arteries of all vital organsd its development is closely associated with the progress ofher disorders. The risk factors typical for atherosclerosis, i.e. age,nder, smoking, elevated concentration of lipids in the bloodrum, also have a pernicious impact on the proper functioning ofart valves [7,8]. According to one survey, atherosclerotic lesions

the coronary arteries are also observed in approximately 40% oftients with aortic stenosis [9]. In both cases, the injury ofdothelium is considered a trigger for further disease progression0]. In the advanced stage of both diseases calcified lesionsntaining inflammatory cells and lipids are formed [11].Among the various imaging techniques applied to investigate

rtain tissues changed due to atherosclerosis, label-free techni-es hold a special place. Vibrational spectroscopy combined with

her supplementary techniques, such as confocal microscopy andomic force microscopy (AFM), gives a unique approach tocognizing various changes occurring in tissues. These experi-ental techniques, with proper data classification algorithms,ovide a powerful approach to be used besides other classicalethods (e.g. histochemical staining). They were found to be awerful tool for analysis of the biochemical alterations of tissues

different animal models, mostly based on diet–induced changes on a combination of genetic modification and nutrition effects2–18]. Most of the spectroscopic studies carried out have beenccessfully applied ex vivo, however, there are also reports whichow good prospects for in vivo spectroscopy [19–21].A detailed description of the extensive history of research on

herosclerotic plaque and vessels with the use of vibrationalectroscopy was previously described [22]. This work reportedamples of vibrational spectroscopy analysis combined withM applied to the biochemical analysis and visualization of theost important plaque features, components of the vascularall, markers of endothelium dysfunction and heart valveenosis. The presented results prove that vibrational spectro-opies offer high content images of the morphological structure

the tissue, that is of diagnostic, prognostic and therapeuticgnificance.

neral chemical composition of atherosclerotic plaque

Fourier transform infrared spectroscopy (FT-IR) and Ramanectroscopy have been widely applied for identification andaluation of atherosclerotic lesions, aiming mostly at a generalerview of the composition changes occurring in a disease state1–29].FT-IR allows the investigation of the distribution of various

mpounds in the tissue, e.g. lipids occurring in the vascular wallith developed atherosclerosis [28,30,31]. The ability of FT-IRectroscopy to detect and characterize directly various lipid

asses of the lesions is a major advantage of this technique overstological staining methods [22,25,26]. A comparative analysis of-IR spectra of atherosclerotic plaques and the reference lipidsnfirmed the presence of cholesterol (10–30%) and cholesterylters (45–78%) [30,32,33]. The stretching vibration of the C55Orbonyl band in the ester group (1710–1750 cm�1) is used as aarker of lipids in general [27,30,31]. Cholesterol alone shows aecific band at 1058 cm�1 that originates from the C–O bendingbration, while the band at 3005 cm�1 is assigned to cholesteryleate and cholesteryl linoleate [22,23,28,30–34]. This high

Please cite this article in press as: Marzec KM, et al. Vascular diseasmethods. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep

wavenumber band is generally characteristic of unsaturatedaliphatic compounds, which are distinctive of foam cells[30]. FT-IR imaging of the artery enables differentiation of normalvessel tissue from the atherosclerotic lesion [22,28,30,34]. The IRclass of proteins of the vessel wall can be easily distinguished bythe integration of the CH2/CH3 stretching band (2800–3000 cm�1).Although this band is not specific only to lipids, its absorbance ismuch bigger for lipids than for proteins. The carbonyl stretchingmode (1730 cm�1) used as a marker of lipids can be assigned morespecifically to cholesteryl esters and triglycerides [30]. To visualizethe structure of the brachiocephalic artery (BCA) and identifyatherosclerotic lesions, a multivariate analysis, i.e. HierarchicalCluster Analysis (HCA) and Fuzzy C-means Clustering (FCM) can beused to interpret the FT-IR imaging results [23,30,31] (Fig. 1).

The vascular wall is composed mainly of proteins representedin the spectra by the amide I band (�1652 cm�1) and amide II(1539 cm�1) as well as by other amide bands (amide III at1236 cm�1, amide A and B at around 3500 and 3100 cm�1,respectively). A region of atherosclerotic plaque can be clearlyidentified by HCA as well as its two sub-regions characterized bythe high content of cholesterol or cholesteryl esters, respectively[31]. By using FT-IR imaging, an estimation of the lesion size andquantification of its lipids components is also possible [13].

FT-IR spectroscopy may also provide information about thechanges of proteins in the vascular wall during atherosclerosisprogression. The analysis, based on the amide I and II spectralrange (1718–1487 cm�1), sensitive for alterations of the secondarystructure of proteins, showed an increase of structures related tob-sheet and a decrease of helical and unassigned arrangements inatherosclerotic murine tissue in comparison to the healthy murinetissue [33]. FT-IR spectroscopy allows also for the detection ofcalcification described further on.

High-resolution images (Raman and AFM) of atheroscleroticplaque and vessel wall

Raman microscopy enables chemical images to be obtainedwith better spatial resolution than FT-IR spectroscopy, but themethod is usually more time-consuming [23]. Raman mapping ofthe atherosclerotic plaque results in images of micro and sub-micro resolution and also can provide information about the earlystages of atherosclerosis in the endothelium.

Previously reported Raman spectroscopy studies of theatherosclerosis have focused mainly on the identification of theatherosclerotic plaque, the analysis of its biochemical composition,and detection of the so-called vulnerable plaque [35–39]. Thismethod was successfully used to study plaque features such as:fibrous cap, internal elastic lamina, collagen fibers, smooth musclecells, adventitial fat, necrotic core/foam cells, ceroid, thrombus onthe surface of plaque, intraplaque hemorrhage, carotene crystals,and calcification [22,23,35–39]. The comparison of the distributionof the major plaque components obtained using Raman mappingand histopathological staining is presented in Fig. 2 [23].

The Raman spectra of the lipid fraction contain bands connectedmainly to cholesterol esters and cholesterol, observed at 1674 cm�1

(C55C stretching mode), 1443 cm�1 (C–H bending mode), 1740 cm�1

(C55O stretching mode), 2885 cm�1 at (C–H stretching mode), and704 cm�1 (vibration modes of steroid rings) [40,41]. Raman confocalmapping additionally provides a unique advantage in obtaininginformation of the tissue composition directly from a chosenlocation (i.e. lipid droplets, cholesterol crystals) as well as the abilityto study significant changes in the degree of unsaturation of thelipids with submicron resolution [23,42,43].

The internal and external elastic laminas, which contain mainlyelastin and collagen, demonstrate a weak Raman signal and strongautofluorescence when the 532 nm laser excitation wavelength is

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Fig. 1. (a) An exemplary microphotograph of the cross section of mice brachiocephalic artery; (b) its spectroscopic image in the range 3025–2800 cm�1 of the IR spectra

showing distribution of lipids in general; (c) FCM visualization showing distribution of lipids (red color denotes high concentration of lipids and blue represents a vascular

wall); and (d) corresponding average spectra of two classes obtained with FCM.

K.M. Marzec et al. / Pharmacological Reports xxx (2015) xxx–xxx 3

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used [23]. The Raman spectrum of remodeled tunica media(pathologically altered muscle cells tissue) originates mainly fromthe protein features with the main bands at 1660 cm�1 (amide I),1244 cm�1 (amide III), and at 1004 cm�1 (phenylalanine). Plaquecontaining hemoglobin (Hb) fraction (intraplaque hemorrhage,thrombotic plaques) can be easily detected and visualized with theuse of resonance Raman imaging. The excitation with 532 nm laserresults in high intensity bands in the spectra observed at 750,1130 and 1580 cm�1, assigned to heme vibrations. The band at964 cm�1 is characteristic of the calcium deposits and is describedfurther on. Presented Raman results were recorded using a WITecconfocal CRM alpha 300 Raman microscope which was equippedwith an air-cooled solid-state laser operating at 532 nm and a CCDdetector. The integration time for a single spectrum was 0.3 s, andthe spectral resolution was equal to 3 cm�1.

The detailed description of Raman spectra of plaque compo-nents such as collagen, elastin, cholesteryl esters, cholesterolcrystals, Hb, calcium salts, ascorbic acid, a-tocopherol, andb-carotene also can be found elsewhere [22,23,30–39,44].

A study on biochemical markers of atherosclerotic plaque wasreported [23]. The AFM imaging, having substantially better spatialresolution compared to spectroscopic techniques, reveals impor-tant and valuable surface details, e.g. the thickness of the fibrouscap, the morphology of which is a key marker of plaque stability.Both fibrous cap and elastic lamina have similar physicalproperties, as they are the stiffest parts of the tissue or plaque

Please cite this article in press as: Marzec KM, et al. Vascular diseasemethods. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.

and serve as their framework. It should be stressed that the fibrouscap thickness is one of the main criteria in the detection of unstableplaque, and when it is very thin or distorted, the staining methodsfail in its recognition. This applies mainly to animal model studieswhere such structures are much smaller compared to humans. It isworth mentioning that AFM could also provide information aboutmechanical properties of the fibrous cap.

Spectroscopic markers of endothelium dysfunction

The spectroscopic approach was applied, aimed at identifyingbiochemical alterations in atherosclerotic endothelial dysfunctionwith the use of Raman spectroscopy [45]. The method was basedon the quantification of the ratio of tyrosine (Tyr) to phenylalanine(Phe) contents in the endothelium. It is known [46] that thesynthesis of Tyr from Phe requires the presence of tetrahydro-biopterin (BH4) as a co-factor of phenylalanine hydroxylase (PAH).Moreover, a limitation of BH4 availability in the endothelium,which is also required for enzymatic synthesis of nitric oxide (NO),is a hallmark of endothelium dysfunction.

It was shown that using Raman spectra, the ratio of the markerbands of Tyr to Phe can be calculated and consequently, thepathological state of endothelium could be detected (Fig. 3).The evidence that the Tyr/Phe intensity ratio can discriminateendothelial dysfunction in ApoE/LDLR�/� mice was based onthe comparison with control mice (C57). It is clearly seen that the

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Fig. 2. (a) A microphotograph of the part of the atherosclerotic plaque from the interior side of the cross section of a brachiocephalic artery with the labeled investigated area

(red); (b) investigated area of the plaque stained with hematoxylin and eosin (pink) and Van Koss (calcium depositions denoted in black); (c) the K-means Clustering (KMC)

results with the five main classes including remodeled media, heme, internal elastic lamina/fibrous cap, calcification and lipids and (d) average Raman spectra of each KMC

class; Raman images based on integration of: (e) the CH stretching band (2800–3020 cm�1) and (f) hydroxyapatite band (964 cm�1); (g) Autofluorescence of the sample

connected with the presence of the internal elastic lamina and fibrous cap [23].

Fig. 3. The integration ratio of the bands of Tyr vs. Phe, for ApoE/LDLR�/�, ApoE/

LDLR�/�-MNA and C57 models. The intensity ratios were calculated for Phe and Tyr

bands in the 1014–991 cm�1 and 872–823 cm�1 regions, respectively.

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lue of Tyr/Phe ratio is significantly lower for ApoE/LDR tissuesan for the control samples (Fig. 3). It was also demonstrated thatNA treatment tended to increase the Tyr/Phe bands ratio in thedothelium, consistent with the improvement of endothelialnction in MNA-treated ApoE/LDLR�/� mice. More details ofalysis can be found in Ref. [45].

Please cite this article in press as: Marzec KM, et al. Vascular diseasmethods. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep

Monitoring of tissue calcification

There are many similarities between atherosclerotic plaqueformation and heart valves disorder. The outbreak of the pathologyof both alterations develops from the damage of the layer ofendothelial cells covering the inner wall of the vessel and valvecusps [10]. The experimental and clinical data reveal moresimilarities, including for example, increased production of tissuegrowth factor beta (TGF-b) [47] and enhanced activity ofmetalloproteinases [48,49]. Additionally, in both atheroscleroticplaque formation and aortic stenosis, calcified lesions are observedin the advanced stage of pathology.

As already presented in Fig. 2b, calcium content is commonlyvisualized in the atherosclerotic plaques with Von Kossa staining,while vibrational techniques enable label-free imaging of the maincalcification component, i.e. hydroxyapatite (Fig. 2f). This mineralgives a strong Raman signal in the spectra and the propervisualization can be obtained even at low concentrations ofcalcification inside the plaque. The bands around 964 cm�1 and1070–1080 cm�1, observed in the Raman spectra of hydroxyapa-tite, are assigned to the stretching vibrations of the phosphate andcarbonate groups, respectively [22,23]. Raman spectra reveal thatthe calcification class inside atherosclerotic plaques may alsocontain the lipids fraction (as this is located inside the lipid core) aswell as proteins (as these may be mixed with collagen) [22,23]. Itwas also previously reported that some calcified lesions of theplaque may contain nano-sized particles of titanium dioxide (TiO2)[23,50]. Although hydroxyapatite detection is more pronounced inRaman spectra, it is also possible to study plaque calcification withthe use of FT-IR [22]. The presence of calcification in the plaque is

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Fig. 4. Reflected light image of aortic valve tissue (A) and distribution images of calcium phosphate salts: tricalcium phosphate, mixed with the salt containing the acidic

phosphate groups (HPO42�) (B) and B-type carbonated hydroxyapatite (C) obtained by integration of the marker band (n1 PO4

3�) for different stages of calcification process

along with the respective clusters on image (D; K-means, Manhattan method). The averaged Raman spectra of the most important clusters (E) correspond to the structure of

big deposits (red) and medium-size grains (blue), respectively.

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evidenced by IR bands at ca. 3400, 1080–1100 and 600 cm�1. However,these spectral regions are common to many other compoundsoccurring in the tissue, therefore FT-IR analysis of calcification islimited [22,23].

Raman spectroscopy also has great potential to investigatecalcification in heart valve leaflets [51–53]. The analysis of Ramanimages of human aortic valves with aortic stenosis combined withchemometric methods (CA) enabled monitoring of the progress ofvalve mineralization and the distinguishing of three differentstages of calcification according to the structure of phosphate saltsand the size of deposits (Fig. 4) [54].

Fig. 4 presents the distribution of inorganic salts in aortic valvetissue upon mineralization process. The medium-size grains(Fig. 4C) of diameter 0.7–1.6 mm are built of tricalcium phosphate,mixed with the salt containing the acidic phosphate groups(HPO4

2�). After subsequent disease progression they transforminto final stable form in big deposits (Fig. 4B) composed of B-typecarbonated hydroxyapatite [54]. It was also reported thatcalcification is partially co-localized with lipids, mostly cholesteroland its esters [55]. Despite the local increase of the cholesterollevel in the proximity of calcium salt deposits, globally a decreaseof the relative lipid to protein content was observed [55].

Conclusions and perspectives

In this paper we have shown the usefulness of vibrationalspectroscopy for ex vivo analysis of various tissues affected by

Please cite this article in press as: Marzec KM, et al. Vascular diseasemethods. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.

vascular diseases, including atherosclerotic plaque and vessel wall,endothelium and heart valves.

FT-IR spectroscopy is applied to the general identification andevaluation of composition changes occurring in the atheroscleroticplaque. This method provides information about the concentrationof lipids as well as about the content of secondary structures ofproteins. FT-IR imaging combined with multivariate analysisenables the distinguishing of the atherosclerotic lesion from theintact vessel wall. Raman microscopy yields more specificinformation of atherosclerotic plaque with a better spatialresolution and is used successfully in the detection of collagenand elastin, cholesterol and cholesteryl esters, calcium minerali-zation, fibrous cap, necrotic core/foam cells, ceroid, hemoglobin,and intraplaque hemorrhage. Additionally, in both atheroscleroticplaque formation and aortic stenosis, calcified lesions’ progressioncan be studied successfully. Due to submicron resolution of Ramanimages, it is possible to detect the early stages of atheroscleroticchanges in endothelial cells. Moreover, the universal spectroscopicmarker of endothelium dysfunction was proposed, based on thequantification of the ratio of tyrosine to phenylalanine endothelialcontents.

The use of vibrational techniques supports the classical imagingtechniques of tissues and may provide additional informationabout their biochemical composition without prior staining. Now,most research on atherosclerosis with the use of IR and Ramanimaging has been applied successfully to assess different tissuesex vivo. In this type of study, detection and visualization of the

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nsitive biochemical markers of atherosclerotic alterations play ay role. Such markers present a huge potential in following thesease progression and pathological alterations. Optical fiberchnology also makes Raman spectroscopy a potentially impor-nt clinical tool for in vivo diagnosis [19–21]. In situ evaluations ofherosclerotic plaque with the use of Raman/IR techniques offferent animal models [39,56,57] as well as humans6,44,58,59] have been previously reported. Despite manyports, which show the in vivo applications of vibrationalectroscopy techniques, there is still a long way to go in theoper clinical validation and commercial application of theseethods. With the further development of commercial spectro-eters for in vivo studies, the use of vibrational imagingchniques may allow for rapid and non-invasive diagnosis ofsue alterations induced by atherosclerosis [60].

le of the funding source

Publication is funded partly by the European Union under theropean Regional Development Fund (grant coordinated by

ET-UJ, POIG.01.01.02-00-069/09) and by the National Sciencenter (DEC-2013/08/A/ST4/00308).

nflict of interest

None declared.

knowledgements

This work was supported by the European Union from thesources of the European Regional Development Fund under thenovative Economy Programme (grant coordinated by JCET-UJ, NoIG.01.01.02-00-069/09) and by the National Science Center (thecision number DEC-2013/08/A/ST4/00308).

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