Nanostructure of Human Aortic Intima in Atherosclerosis ( A Pilot Study )

Материал и методы: Образцы интимы аорты получали из участков с признаками атеросклеротических из менений на различных этапах их формирования. Фрагменты аорт инкубировали в растворе, содержащем цик лодекстрины. В работе использовали раствор препарата НИОДЕКС — пропиленгликолевый эфир бета цикло декстрина. На предметных стеклах с полилизином формировали слой интимы аорты. Образцы помещали в рабочую зону атомного силового микроскопа «Интегра Прима» (NT MDT, РФ) и сканировали поверхности ис следуемых образцов. Число точек сканирования — 512, поля сканирования: 100 100 мкм, 200


Introduction
Endothelial cell damage (in particular, glycoca lyx) plays a key role in the pathogenesis of athero sclerosis [1].The course of aortic atherosclerosis includes several stages.First, a lipid spot appears.Compound complexes consisting of lipids (mainly, cholesterol) and proteins are formed at such sites, and their deposit in intima takes place.Then young connective tissue grows at lipid deposits.An athero sclerotic plaque consisting of lipids and connective tissue is gradually formed.
Researchers have been actively searching for pharmaceutical products that could prevent athero sclerosis.In vitro experiments demonstrated that cyclodextrins can dissolve fibroblast lipids.The greatest portion of cholesterol is eliminated from cells within the first 1-2 hours of incubation [2].A 14-20% decrease in cholesterol and cholesterol ester plasma levels and a decrease in pro atherogenic lipoproteins were registered in mice following oral administration of cyclodextrin [3].Cyclodextrin administration reduced the size of atherosclerotic plaques in mice.Cholesterol outflow from the plaques is activated due to macrophage activation [4].Cyclodextrins were demonstrated to potentiate the anti atherosclerotic of rapamycin as a result of selective mTORC1 inhibition [4].
A new generation, cyclodextrin based antiatherogenic medicinal product, Niodex, has been developed to date.Studies demonstrate its potential to reduce the collagen and polysaccharide content in atherosclerotic plaques.It is obvious from histologi cal and histochemical findings: the intensity of colla gen coloration and the intensity of PAS reaction are decreased.First of all, these changes are registered in the fibrous part of an atherosclerotic plaque and at the sites where cholesterol crystals are located.w w w .r e a n i m a t o l o g y .c o m Все стадии развития атеросклероза широко представлены в литературе изображениями гисто логических срезов при оптической микроскопии [5].
Histological tests permit to analyze macroscop ic changes in the aortic intima (with the resolution of not more than 1 micron), but they do not allow to trace the manifestations of these changes at a molec ular level.
However, this problem may be solved using the atomic force microscopy [6,7].The atomic force microscopy permits to observe not only the early stages of atherosclerotic plaques, but also to trace their suppression by antiatherogenic agents.
The objective of the study is to analyze the nanostructure of human aortic intima in atheroscle rosis and demonstrate the potential effect of Niodex on cholesterol plaques.

Materials and Methods
The structure of aortic intima (24 fragments) taken from 5 patients (who died of atherosclerotic complica tions) was analyzed.Aortic intima specimens were obtained in S. P. Botkin's State Municipal Hospital.
Samples of intima were taken from those parts of aorta, where different stages of atherosclerotic changes were obvious.The intima specimens were treated with cyclodextran dissolved in the saline.The microscopic sec tions were stained with hematoxylin and eosin (Van Gieson staining), and the PAS reaction was performed.The Olympus BX41 microscope (Japan) was used for the histological study.Reference samples of aortic intima were not exposed to the antiatherogenic agent.All studies were performed in accordance with guidelines of the Ethic Committee under the Scientific Research Institute of General Reanimatology.Aortic samples were incubated in a solution containing cyclodextrins.A solution of NIODEX, a propylene glycol ester of beta cyclodextrin, was used in the study.
A layer of aortic intima was separated from other layers and placed onto glass slides to be examined by an atomic force microscope.The intima samples were iso lated from unaltered portions of the internal aortic layer, from lipid spot sites, and from grown atherosclerotic plaques.
A 10 15 mm intima fragment («sample») was isolat ed in each aorta.The samples were fixed on glass slides with polylysine.Then the samples were placed into the working area of an atomic force microscope (Integra Prima, NT MDT, Russian Federation), and their surfaces were scanned.The imaging was performed in the resonant semi contact (tapping) mode using the microscope soft ware [8].The fpN10 cantilevers (NT MDT, Russian Federation) with a 22° apex angle and ~3-5 nm radius.The interaction force during the imaging was 0.1-5 nN.The number of imaging points was 512; and the imaging regions were as follows: 100 100 μm, 2000 2000 nm.First, 100 100 μm aortic intima nanostructures were obtained.Then, the fragments were scanned in a 2000 2000 nm field, if necessary.

Results and Discussion
Unlike the optic microscopy, the atomic force microscopy permits to obtain images of a sample with a resolution of 1 nm in plane and up to 0.1 nm along the Z axis [9].The profile of the aortic intima nanosurface is variable and composite.Therefore, typical objects should be isolated for the further analysis.This paper dwells on nanosurfaces.Therefore, structural differences (hundreds of μm) are not taken into account.A typical intima nanosur face is presented in Fig. 1, a.
In this sample, areas of large and small craters, the ridge, and trabecular fibers are highlighted.These areas have typical shapes and sizes.Let us con sider them in details.. Areas of large and small craters are presented in Fig. 1b, c.These are areas with typical ring shaped objects whose sizes vary within a large range.Large craters may be 1-5 to >100 microns in diameter.At that, their depth may be equal to 200-500 nm.The lipid spots may be several microns deep.The crater surfaces are usually rough.The roughness degree is about 30-150 nm.Small craters are more common in the intima.Their typical diameters are from 120-150 to 900 nm.They are smaller than large craters and may be ring shaped or oblong.Small and large craters overlap other structures and cover almost the whole surface of the intima.
The profile of the intima nanosurface in the area of the ridge and trabecular fibers is presented in Fig. 1, d, e.
Ridges are oblong, high, long structures.The feature of these objects is that their height varies within the range of not more than 40% along their lengths.The height of these objects is 300-700 nm, and there length is dozens of microns.Thread like, curvilinear, trabecule like objects up to 50-150 nm high are called trabecular fibers (Fig. 1, e).
The formation of lipid spots is presented in Fig. 2.
At the same time, lipid spots were identified using standard histological techniques.
Lipid spot outgrowths appear on the typical intima surface (Fig. 1) as a result of abnormal processes described above.Such outgrowths were observed as single (Fig. 2, b) and multiple objects (Fig. 2, a) on the 100 100 μm surface.Since the intima nanosurface is variable and composite, the detection of lipid spots is quite a problem: it is nec essary to differentiate between the plaque out growth and normal surface.The following technique was applied.If the object was near a crater, then the value of 300-350 nm was taken as a midline.This line was equal to 500 nm for the ridge.The spot height was measured against this line.The lipid spots projected above the profile surface by 600-1200 nm.However, this approach was not a guaran w w w .r e a n i m a t o l o g y .c o m По мере развития процесса образования ли пидные пятна увеличивали свои размеры до 4 мкм (как на рис.2, c).Одновременно росла их же сткость [10,11].Она увеличивалась на 10-20% по сравнению с начальной фазой роста (рис.2, c).В tee for identification of required objects.The plaques could be located both in the cavity and on slopes of craters or ridges.Such fragments were con sidered as artifacts and were not taken into account.
At early stages, the lipid spots had similar sizes and shapes.Their stiffness differed from that of the median structure of the intima (both craters and ridge) by 1.2-1.4fold.
Then, with the course of growth, the lipid spots grew up to 4 μm (as seen in the Fig. 2, c).At the same time, their stiffness increased [10,11].It increased by 10-20% as compared to the initial stage (Fig. 2,  c).In this study, for technological reasons, the stiff ness was measured by atomic force spectroscopy using a dry sample.Therefore, stiffness changes are expressed only in relative units.
27 fragments were identified as growing choles terol plaques in 24 samples.16 of them measuring 900-1200 nm were identified near ridges, and 11 near craters (600-1050 nm).The limited number of findings is related, first of all, to diffiulties of identi fication of incipient plaques against the background of a composite intima surface, as well as to labor intensiveness of anlayses of this nanosurface.Differences in shapes and sizes fo the isolated frag ments do not permit to conclude that they are relat ed to craters or ridge, or that this or that object on the nanosurface provokes the growth of atheroscle rotic plaques with a more or less degree of certainty.
Niodex caused destruction of lipid spots and smoothing of the intima surface.More than a half of the 27 identified objects (15) demostrated a 30% and more decrease in size (median 340-400 nm).A 10-15% decrease was registered in 7 fragments; in the remaining 5 fragments, the decrease in the lesion size was less than 10%.The decrease in less than 10% was considered insignificant.

Conclusion
This is a research and experimental study.This study made an attempt to classify nano objects on a composite surface of the human aortic intima.Differences in shapes and sizes of the isolated frag ments do not permit to conclude that that this or that object on the intima nanosurface (ridges, craters) provokes the growth of atherosclerotic plaques with a more or less degree of certainty.Raw data permit to suppose that the effect of Niodex on the aortic intima results in deceleration and decreased intensity of atherosclerotic plaque growth on the intima fragments.Further studies are required to obtain more detailed information about the effica cy of this medicinal product.