Cellular models of atherosclerosis and their implication for testing natural substances with anti-atherosclerotic potential.
Background: Atherosclerosis remains a major problem in the modern society being a cause of life-threatening cardiovascular diseases. Subclinical atherosclerosis can be present for years before the symptoms become obvious, and first manifestations of the disease in a form of acute ischemia of organs are often fatal. The development of atherosclerosis is characterized by lipid accumulation in the aortic wall and formation of foam cells overloaded with large amounts of lipid inclusions in the cytoplasm. Current therapy of atherosclerosis is aimed mostly at the normalization of the blood lipid profile, and has no direct activity on the atherosclerotic plaque development. It is therefore necessary to continue the search for substances that possess a direct anti-atherosclerotic effect, preventing the cholesterol deposition in the arterial wall cells and reducing the existing plaques.
Purpose: Medicinal plants with potential anti-atherosclerotic activity are especially interesting in that regard, as plant-based medications are often characterized by good tolerability and are suitable for long-term therapy. The evaluation of novel active substances requires the establishment of reliable models of atherogenesis. In this review we discuss cellular models based on cultured human aortic cells. We also discuss several examples of successful application of these models for evaluation of anti-atherosclerotic activity of natural products of botanical origin based on measurable parameters, such as intracellular cholesterol accumulation.
Chapters: We describe several examples of successful screening and clinical studies evaluating natural products that can be beneficial for prevention and treatment of atherosclerosis, including the subclinical (asymptomatic) forms.
Conclusion: Several substances of botanical origin have been demonstrated to be active for treatment and prevention of atherosclerosis. The obtained results encourage future studies of naturally occurring anti-atherosclerotic agents.
Atherosclerosis remains one of the major medical and social problems in the developed countries, since associated with it cardiovascular disorders account for a great part of overall morbidity and mortality (Simmons et al. 2012). Atherosclerosis development is a multifactorial process, which is characterized by degenerative changes in the wail of large arteries with subsequent blood vessel occlusion and ischemia of organs and tissues. Subclinical (asymptomatic) atherosclerosis is a wide spread condition in modern society. Atherosclerotic lesions exist already in relatively young people and can progress for decades until first clinical manifestations become evident (Simmons et al. 2012; Berenson et al. 1998; McGill et al. 2002; Tuzcu et al. 2001). It has been shown that middle-aged people do not present with symptoms of atherosclerosis while having atherosclerotic lesions at various stages of progression in nearly 100% of cases (Berenson et al. 1998; McGill et al. 2002; Tuzcu et al. 2001). At the same time, no specific therapy is developed so far for direct prevention and treatment of subclinical atherosclerosis, partly because current understanding of the exact mechanisms and hence relevant therapeutic targets is not sufficient.
The results of epidemiological studies define a number of factors associated with the increased risk of vascular occlusion, including several clinical and biochemical syndromes (Anderson et al. 1991). Elimination of such risk factors is regarded as the most promising approach for primary prevention of atherosclerosis (Fowkes et al. 2013). These preventive measures, however, are indirect, as they are aimed to alter certain conditions, which are not immediately related to the molecular and cellular mechanisms of atherogenesis. Another therapeutic strategy implicates a "direct" therapy, which is aimed to prevent the onset and progression of atherosclerotic lesions by inhibiting the molecular and cellular mechanisms of atherogenesis in human arteries (Orekhov and Tertov 1997; Orekhov et al. 1986a; Sazonova et al. 2009). This approach to atherosclerosis prevention and treatment is currently under development.
Statins are currently regarded as most promising therapeutic agents for prevention and treatment of atherosclerosis (Stein and Raal 2014). Regular and long-term statin therapy helps preventing the development of novel and promoting the regression of existing atherosclerotic lesions. However, recent opinion is that, although statins are effective at reducing cholesterol levels, they fail to substantially improve cardiovascular outcomes. It became known that the directors of the clinical trials have succeeded in minimizing the significance of the numerous adverse effects of statin treatment (Diamond and Ravnskov 2015). However, the target level of LDL cholesterol cannot be achieved in many patients because of statin intolerance (Banach et al. 2015). Noteworthy, therapy aimed at prevention and treatment of early stages of atherosclerosis should necessarily be long-term, making the good tolerability and low cost essential. Therefore, anti-atherosclerotic drugs based on natural products can be a preferred alternative, as they have virtually no adverse effects and are suitable for long-term or even life-long treatment (Orekhov et al. 2013).
Natural products with anti-atherosclerotic potential
Natural products with potential anti-atherosclerotic properties are currently gaining attention of the researchers (Orekhov et al. 2013, Badimon et al. 2010). Many of the well-known dietary products have strong positive effects on the blood lipid profile, reducing the risk of atherosclerosis development, others can be used to reduce inflammation and modulate platelet aggregation. Nutraceuticals that can reduce cIMT can be considered as direct anti-atherosclerotic agents. The protective effect of Mediterranean diet, which includes high amounts of mono- and polyunsaturated fatty acid sources, such as olive oil, has been evaluated in several controlled studies (Mensink et al. 2003). Garlic (Allium sativum) has been demonstrated to contain active substances that can possess not only protective, but also curative effects on atherosclerosis (Koscielny et al. 1999). Vegetables, fruits, cereals and other dietary substances contain such biologically active molecules as polyphenols, phytosterols and phytostanols, vitamins and antioxidants and dietary fiber, all of them being considered to have more or less prominent anti-atherosclerotic activity (Badimon et al. 2010).
Several recent studies evaluated certain nutraceuticals as potential agents for development of dietary supplements for lowering the risk of cardiovascular disease development. Chitosan, a dietary fiber derived from fungal mycelium, has been demonstrated to reduce plasma and LDL cholesterol and triglycerides and increase HDL cholesterol in patients with elevated plasma triglyceride levels, not taking lipid-lowering medications (Rizzo et al. 2014, Patti et al. 2015b). It could be interesting to study the beneficial activity of chitosan on plasma lipid profile in a larger study. Another recent study has evaluated the beneficial effects of a natural supplement containing several biologically active substances: curcumin (Curcuma longa), silymarin (Silybum marianum), guggul (Commiphora wightii), chlorogenic acid and inulin (Patti et al. 2015a). The supplement was tested on 78 patients with metabolic syndrome as add-on therapy for 4 months. Treatment with the product resulted in significant reductions of body weight and body mass index (BMI), total cholesterol levels (p = 0.03) and fasting glucose levels (p = 0.14). Levels of LDL cholesterol were decreased as well, although statistical significance has not been achieved. Here again, a larger study is required to evaluated the protective effects of the supplement for prevention and treatment of atherosclerosis.
Despite the considerable progress made during the recent years, our understanding of possible cardioprotective effects of nutraceuticals, such as lowering of blood cholesterol and blood pressure regulation, and their impact on cardiovascular disease risk factors is still limited (Rai et al. 2013; Al-Waili et al. 2013; Ried et al. 2013; Hopkins et al. 2013; Sobenin et al. 2008a; Sobenin et al. 2010; Sobenin et al. 2009). This can partly be explained by the limited methodology for studying anti-atherosclerotic activity of drug substances, especially regarding the early stages of the pathogenesis, and the lack of adequate pathophysiological models. Therefore, efforts should be taken to develop reliable new methods for assessment of the therapeutic potential of natural products.
Mechanisms of atherogenesis in humans
Current understanding of cellular and molecular mechanisms of atherogenesis is based on the classical lipid theory of atherosclerosis, postulating that the most important event in the disease development is the accumulation of extracellular and intracellular lipids in the arterial intima (Schonfelder 1969; Konstantinov et al. 2006). Low-density lipoprotein (LDL) serves as the major source of lipid deposits in the arterial wall. During the last decades, it became obvious that atherogenesis is caused not by native LDL, but rather by its modifications, including desialylation, the change of the total surface charge (electronegativity), change of hydrated density of lipoprotein particles and oxidation Qaakkola et al. 1993; Sobenin et al. 1998; Tertov et al. 1992a; Tertov et al. 1995a). It is likely that we actually deal with the same type of multiple atherogenic modifications, but differently evaluated by different methods of laboratory diagnostics (Tertov et al. 1995a; Tertov et al. 1995b; Tertov et al. 1996; Tertov et al. 1992b). The elevated levels of modified LDL in the bloodstream trigger additional mechanisms that enhance its atherogenic potential, most importantly, the formation of large complexes. The modified LDL species acquire the ability to spontaneous self-association because of altered surface charge. Additionally, modified LDL possesses antigenic properties, inducing the production of anti-LDL autoantibodies, which ultimately leads to the formation of LDL-containing circulating immune complexes (Sobenin et al. 2014a). Modified LDL also has high avidity for connective tissue matrix components. The resulting large LDL-containing associates are characterized by altered cellular metabolism. Rather than being taken up by the receptor-mediated internalization, these complexes are internalized by vascular cells in process of uncontrolled phagocytosis and processed differently than native LDL particles (Goldstein and Brown 1987). As a result, larger amounts of LDL-containing phagocytized particles accumulate in cellular cytoplasm mainly in the form of lipid droplets, leading to the lipid retention in the arterial wall. Such lipid-loaded cells are defined as foam cells and are a common feature of atherosclerotic lesions.
The key steps of atherosclerosis development are represented on Fig. 1. The initiation of atherosclerotic lesion depends on the atherogenic modification of circulating LDL and on local change in the endothelial permeability (Vanhoutte 2009). Modification of LDL can occur both in the bloodstream and directly in the intima, after penetration of LDL particles via the luminal endothelium. One of the earliest events of multiple modification of LDL is desialylation of lipoprotein glycoconjugates caused by the transsialidase circulating in the blood (Tertov et al. 1998). Endothelial heterogeneity underlying the local changes in permeability includes the presence of clusters of multinudeated giant endothelial cells and mosaically located small endothelial cells (Antonov et al. 1986). Different structural types of endothelium are present along the luminal surface of blood vessels and can possibly determine the mosaicism and "focality" of the atherosclerotic lesion development. Penetration of modified LDL into the subendothelial space in areas with increased endothelial permeability leads to the formation of focal lipid infiltrations into the intima. Intimal cells comprise resident cells of mesenchymal origin and infiltrated hematogenous cells and internalize the modified LDL particles leading to the lipid deposition. The LDL particles modified in the bloodstream or in the intima associate with each other or with autoantibodies against modified LDL. In the subendothelial space, modified LDL particles can associate with extracellular matrix components, complicating the characteristics of modified LDL (Orekhov et al. 1987a). Altered conformation of modified LDL particles results in decreased interaction with LDL receptors on the cell surface and preferential uptake of the particles via a non-receptor pathway resulting in foam cell formation (Goldstein and Brown 1987).
Uptake of LDL associates by phagocytosis may be regarded as a part of the innate immunity response. Indeed, such large complexes can be perceived by intimal cells as pathogens and destined to the destruction by phagocytosis. This destruction is performed by monocyte-derived macrophages and resident mesenchymal cells (smooth muscle cells and pluripotent pericyte-like cells) that, upon the uptake of the LDL associates, release innate immunity signals in a form of soluble signaling factors that attract the adjacent arterial cells and circulating inflammatory cells into the inflammation site (Gratchev et al. 2012). These signals promote further lipid accumulation by resident and inflammatory cells gathered in the intima, leading to the retention of lipids and foam cell formation. Another process induced by the lipid accumulation is proliferation and fibrosis (extracellular matrix synthesis), which is typical for the reparative phase of inflammation, accompanied by the decrease of cell-to-cell contacts between the resident cells (Andreeva et al. 1995; Andreeva et al. 1997).
The reparation can develop rapidly in favorable conditions. Former lesion sites are characterized by the increased cell numbers and excessive production of extracellular matrix components. These processes can continue for years resulting in a diffused fibrotic thickening of the arterial wall frequently observed in adults. However, such successful reparation is not always the case. Chronic lipid infiltration of the intima can prompt the immune cells to the development of the enhanced inflammatory response. The increasing local accumulation of cells, proliferation and fibrosis will intensify resulting in delayed and inefficient reparation. Local chronic inflammation can be associated with increased lipidosis caused by constant lipid infiltration, loss of intracellular contacts, increased proliferation and intensification of fibrosis (Andreeva et al. 1995; Orekhov et al. 1990a). These processes lead to the formation of atherosclerotic lesion: a fatty streak and a fibrolipid plaque. Tissue reaction, caused by a prolonged local inflammation in the subendothelial intima, is accompanied by the formation of a fibrous cap to isolate the center of the inflammation site and create a barrier against further penetration of lipoproteins and immune cells from the bloodstream. A favorable outcome of this process would be a complete separation of the inflammation site with the suppression of the inflammatory response and the gradual and partial restoration of tissue functions. Alternatively, unfavorable outcome would be the development of a fibrolipid plaque susceptible to rupture and thrombus formation, which can have fatal consequences (Badimon and Vilahur 2014). Fibrolipid plaque formation is controlled by two opposing processes, namely by infiltration and reparation (Fig. 2). Shifting of balance toward reparation, results in the formation of a fibrous plaque, representing a favorable clinical outcome. Inefficient reparation and prevailing lipid infiltration can lead to the plaque rupture, resulting in lethal consequences in almost 50% of cases. Therefore, lipidosis (lipid infiltration of arterial intima and accumulation in intimal cells) is a crucial factor for the initiation and development of atherosclerotic lesions. This underscores the importance of the development of therapeutic approaches aiming to prevent lipidosis.
Cellular models for development of anti-atherosclerotic drugs
No contemporary drugs can be classified as direct anti-atherosclerotic agents so far. Some therapeutic agents can help to reduce atherogenic potential, or atherogenicity of blood serum in patients with atherosclerosis (Orekhov et al. 1986a; Orekhov 2013; Sobenin et al. 2013; Orekhov et al. 1991). The phenomenon of serum atherogenicity was first detected in patients with coronary atherosclerosis, and is defined as the ability of serum to induce cholesterol accumulation in cells (Chazov et al. 1986). Therapies lowering this parameter may help prevent the lipid accumulation in the arterial wall and inhibit the development of atherosclerosis at the initial stages (Orekhov 2013). Cellular tests could be an appropriate model to study the early stages of atherogenesis and blood atherogenicity (Orekhov et al. 1986a; Orekhov et al. 1990b; Orekhov et al. 1991).
In vitro model
A cellular model based on primary culture of human aortic cells has been developed for screening of substances with anti-atherosclerotic potential. Cells were isolated from the subendothelial part of normal (unaffected by atherosclerosis) human aortic intima, an area, which is localized between the endothelial lining and the tunica media (Chazov et al. 1986). Samples were collected shortly (1.5-3 h) after sudden death of male and female subjects between 40 and 65 years old. Living cells were isolated by treatment of the tissue sample with collagenase and elastase and brought to primary culture at 37[degrees]C for 7-10 days in daily refreshed medium (Orekhov et al. 1986a; Orekhov et al. 1984a; Orekhov et al. 1985; Orekhov et al. 1986b). Immunocytochemical analysis revealed that the obtained primary cultures contained a mixture of different cell types, including smooth muscle cells (20-50%), pericyte-like cells (30-70%), inflammatory hematogenous cells and tissue macrophages (10%) (Orekhov et al. 1986a; Orekhov et al. 1985; Orekhov et al. 1986b). Smooth muscle cells and pericytes were identified with antibodies against smooth muscle a-actin. Smooth muscle cells and stellate-shaped pericyte-like cells represented the major portion of the in cell culture. Cells of hematogenous origin were detected by antibodies against leukocytes (CD45+), monocytes (CD14+) and tissue macrophages (CD68+) represented only a minor portion of the cultured cells.
Cellular lipidosis was stimulated by incubation of cultured primary aortic cells with atherogenic serum obtained from patients with assessed atherosclerosis. Such serum has been shown to induce a two-fold increase of intracellular cholesterol after 24 hours of incubation (Chazov et al. 1986). Tested substances were added in a form of aqueous solutions to the cells, together with the atherogenic serum. Substances were defined as potential anti-atherogenic agents if they caused a reduction of intracellular cholesterol accumulation induced by the atherogenic serum. The anti-atherosclerotic effect was expressed as percentage of suppression of this accumulation. The described model was used for testing of various drugs and chemicals (reviewed in: Orekhov 2013). Some of the tested substances were found to have anti-atherosclerotic activity; others had no effect or even pro-atherogenic effect, intensifying the intracellular cholesterol accumulation. The in vitro cellular models have been used for studying several natural products, as well as drug substances, such as calcium antagonists, beta blockers, antioxidants and others (see Table 1 for references) (Table 2).
Ex vivo model
Ex vivo model was developed on the basis of primary culture deriving from normal (unaffected by atherosclerosis) human aortic intima (Orekhov 1990). This model can be used for testing the atherogenic potential of the blood serum of patients with confirmed atherosclerosis after administration of different medicinal substances with potential anti-atherogenic activity. In this experimental setup, blood is taken before and at certain time points after a single dose drug administration. Samples of blood serum are added to the primary culture of normal aortic cells, and the accumulation of cholesterol esters and/or free cholesterol is measured 24 h after the addition. The anti-atherosclerotic effect of the medication is evaluated here as the decrease of blood serum atherogenicity and expressed as % of the baseline. The described model was used to test the anti-atherosclerotic activity of various natural products, mainly botanicals. The study included volunteers (groups of 4-8 people; men and women aged 45-60 years) with atherosclerosis and evaluated the effect of a single dose (300 mg) of tested substance on blood atherogenicity (Orekhov 2013).
One of the tested substances was onion in a form of capsulated bulb powder (300 mg), which was found to have anti-atherogenic effects manifested in a moderate reduction of serum atherogenicity by 12%, 28%, and 24% from baseline after 2, 4, and 6h after a single dose administration, respectively. Among the tested natural products, wheat (Triticum vulgaris) seedlings and garlic (Allium sativum) powder were demonstrated to possess the most pronounced and long-lasting anti-atherosclerotic activity, reducing the serum atherogenicity by 3 folds, with biological effect lasting for 6 h. Dry beet (Beta vulgaris) juice had a moderate but prolonged anti-atherosclerotic effect (Orekhov 2013). The ex vivo model was also used to determine the effective dose and adequate posology of the natural products with revealed anti-atherosclerotic activity. For this purpose, 2 blood collections (after 2 and 4h) were performed from atherosclerosis patients, received a single dose of the product. Dose-dependence of the effect was evaluated by comparing the efficacy of 2 doses, each of them assessed in at least 6 different serum samples obtained from patients. It was found that the garlic powder had anti-atherosclerotic effect in a dose range of 50-300 mg, with half-maximal effect achieved at a dose of 100 mg and the maximal effect achieved at a dose of 150 mg (Orekhov 1992). Taken together, these results demonstrate that certain natural products can be regarded as potential drug substances for the development of direct anti-atherosclerotic therapy. As a result, several herbal preparations were developed and clinical studies were conducted to evaluate their anti-atherosclerotic efficacy.
The activity of the garlic-based herbal preparation (Allicor, INAT-Farma, Russia) was evaluated in an open-label prospective pilot study performed in 28 apparently healthy men aged 46-58 (mean age 52.0, SD = 9.0). Anti-atherosclerotic effect of the herbal preparation was measured by the carotid intima-media thickness (cIMT). The study participants were normolipidemic or mildly hyperlipidemic and had no clinical signs of coronary heart disease (CHD) and no chronic diseases requiring continuous use of vasoactive drugs, diuretics, lipid-lowering or anti-diabetic drugs. The diagnostics of diffused intimal thickening was performed by the cut-off cIMT value of 0.7 mm in the distal segment of at least one common carotid artery using the ultrasound B-mode examination described elsewhere (Salonen et al. 1995). The mean cIMT value at the baseline was 0.832 [+ or -] 0.024 mm. One group of study participants (n = 16) received 600 mg Allicor daily, the other (n = 12) was the control group. Interview and ultrasound examination of the carotid arteries were held every 3 months, and the total duration of follow-up was 12 months. No adverse effects were observed during the follow-up period, indicative of the good tolerability of the product. Regression analysis revealed a significant difference between the trends in cIMT dynamics (p<0.05). A tendency to dMT increase was detected in the control group, which was significantly different from that of null hypothesis of no change (F-test, 31.72; p = 0.011). In the Allicor-treated group, a tendency to cIMT decrease was revealed, which was also significantly different from that of null hypothesis (F-test, 28.81; p = 0.013).
The obtained results indicated that the therapy with Allicor might potentially inhibit the development and induce the regression of subclinical atherosclerosis. The statistical power of this pilot study was insufficient to avoid type 2 error. These results allowed designing a larger prospective clinical study with the assessment of a number of clinical and biochemical parameters associated with atherogenesis and the risk of atherosclerosis, including the assessment of serum atherogenicity.
This double-blind placebo-controlled clinical study was designed to estimate the effect of Allicor on the progression of cIMT in 211 asymptomatic men aged 40-74 (ClinicalTrials.gov identifier, NCT01734707). The progression rate of subclinical atherosclerosis estimated by B-mode ultrasonography as the increase of cIMT was set as the primary outcome. By the end of the first 12 month follow-up period, a decrease of cIMT by 0.028 [+ or -] 0.008 mm was observed in Allicor-treated group, whereas in placebo group there was a moderate progression at the rate of 0.014 [+ or -] 0.009 mm (p = 0.002). Serum atherogenicity was lowered in Allicor-treated patients by 45% from the baseline and remained constant in the placebo group (Sobenin et al. 2014b). By the end of the 24-months follow-up period, the mean rate of cIMT decrease was 0.022 [+ or -] 0.007 mm per year in the Allicor-treated group, which was significantly different (p = 0.002) from the placebo group, where a moderate progression of 0.015 [+ or -]0.008 mm was observed (Orekhov et al. 2013; Sobenin et al. 2013). Within Allicor-treated group, significant reduction of cIMT was registered in 47.3% study participants versus 30.1% in placebo group (p<0.05). Further significant cIMT increase was registered in 32.2% study participants in Allicor-treated group versus 47.3% in the placebo group (p<0.05). Serum taken from study participants at the baseline induced a 1.56-fold increase in intracellular cholesterol content in cell culture test. Serum atherogenicity was lowered in Allicor-treated study participants by 30% in average. In the placebo group, serum atherogenic potential did not change during the study. A significant correlation has been revealed between the changes in blood serum atherogenicity during the study and the changes in intima-media thickness of common carotid arteries (r = 0.144, p = 0.045). Therefore, it was demonstrated that long-term treatment with Allicor had a direct anti-atherosclerotic effect on subdinical carotid atherosclerosis, and this effect could be explained by the improvement of serum atherogenicity (Orekhov et al. 2013; Sobenin et al. 2013).
Inflammation plays a key role at all the stages of the atherosclerosis development (Libby 2012; Wolf et al. 2014). Medications with systemic anti-inflammatory activity may therefore be beneficial for the prevention of atherosclerosis. It has been demonstrated that several natural compounds possess not only anti-inflammatory, but also anti-atherogenic effect, among them are calendula (Calendula officinalis), elder (Sambucus nigra) and violet (Viola tricolor) (Gorchakova et al. 2005; Gorchakova et al. 2007a; Gorchakova et al. 2007b). The combination of these three herbs was used for the development of a herbal preparation Inflaminat (INAT-Farma, Russia) (Gorchakova et al. 2007c). Its effect on clMT dynamics was evaluated in a pilot phase of placebo-controlled double-blind study performed on 67 asymptomatic men (ClinicalTrials.gov Identifier, NCT01743404) (Orekhov et al. 2013; Gorchakova et al. 2009). The protocol of the study was similar to that described above, with the follow-up period of 12 months. Inflaminat induced clMT regression in subdinical atherosclerosis, and clMT changes were statistically significant compared to the baseline as placebo group. Thus, Inflaminat had anti-inflammatory and anti-atherosclerotic effects on cellular level revealed in cell culture, and induced regression of subdinical atherosclerosis in asymptomatic men.
Several natural phytoestrogen-rich components were screened for possible anti-atherogenic activity using in vitro and an ex vivo test systems (Sobenin et al. 2003; Korennaya et al. 2006; Nikitina et al. 2006). The most promising of these compounds were garlic powder, extract of grape (Vitis vinifera) seeds, green tea (Camellia sinensis) leaf and hop (Humulus lupulus) cones. In all cases, a significant anti-atherogenic effect was registered. Based on these results, an isoflavonoid-rich herbal preparation containing a combination of the abovementioned natural components was developed (Karinat, INAT-Farma, Russia), which was characterized by a potent anti-atherogenic effect in cell culture models and improved phytoestrogen profile, providing additional amounts of biologically active polyphenols including resveratrol, genistein and daidzein. The atherosclerotic effect of Karinat was evaluated in a pilot phase of randomized double-blind placebo-controlled pilot clinical study in 157 asymptomatic postmenopausal women (ClinicalTrials.gov Identifier, NCT01742000) (Sobenin et al. 2007; Myasoedova and Sobenin 2008). The primary endpoint was the annual rate of clMT changes. The protocol of the study was similar to that reported above, with a follow-up period of 12 months. An annual increase in the mean clMT of > 100 jam was observed in the placebo group, indicative of a high yearly rate of clMT progression (13%) and atherosclerotic plaque growth (40%) in postmenopausal women. In the Karinat-treated group, mean clMT remained stable, with a statistically insignificant increase of 6/rm per year (<1%). The tested phytoestrogen complex is therefore demonstrated to suppress the formation of new atherosclerotic lesions in postmenopausal women (Orekhov et al. 2013; Sobenin et al. 2008b).
Direct anti-atherosclerotic therapy would allow a better control over the pathological process, including the early predinical stages. Such therapy should inhibit the lipid accumulation in the aortic cells, which causes the atherosclerotic plaque growth. Cellular models appear to be a useful tool for studying the anti-atherogenic potential of various drugs and substances both by direct effect in cultured cells (in vivo model) and by lowering the blood serum atherogenicity (ex vivo model). Several examples of successful application of such models have been discussed, resulting in the development of a number of herbal preparations based of natural products with anti-atherogenic potential. These preparations have advantages of good tolerability and direct anti-atherosclerotic effect that make them suitable candidates for a long-term therapy for prevention and treatment of atherosclerosis.
Received 14 September 2015
Revised 7 January 2016
Accepted 8 January 2016
Conflict of interest
The authors report no conflict of interest.
This work was supported by Russian Ministry of Education and Science (Project # RFMEFI61614x0010).
Akopov, S.E., Orekhov, A.N., Tertov, V., et al., 1988. Stable analogues of prostacyclin and thromboxane A2 display contradictory influences on atherosclerotic properties of cells cultured from human aorta. The effect of calcium antagonists. Atherosclerosis 72, 245-248.
Al-Waili, N., Salom, K., Al-Chamdi, A., et al., 2013. Honey and cardiovascular risk factors, in normal individuals and in patients with diabetes mellitus or dyslipidemia. J Med Food 16, 1063-1078.
Anderson, K.M., Wilson, P.W., Odell, P.M., et al., 1991. An updated coronary risk profile. A statement for health professionals. Circulation 83, 356-362.
Andreeva, E.R., Pugach, I.M., Orekhov, A.N., 1997. Collagen-synthesizing cells in initial and advanced atherosclerotic lesions of human aorta. Atherosclerosis 130, 133-142.
Andreeva, E.R., Serebryakov, V.N., Orekhov, A.N., 1995. Gap junctional communication in primary culture of cells derived from human aortic intima. Tissue Cell 27, 591-597.
Antonov, A.S., Nikolaeva, M.A., Klueva, T.S., et al., 1986. Primary culture of endothelial cells from atherosclerotic human aorta. Part 1. Identification, morphological and ultrastructural characteristics of two endothelial cell subpopulations. Atherosclerosis 59, 1-19.
Badimon, L., Vilahur, G., Padro, T., 2010. Nutraceuticals and atheroslcerosis: human trials. Cardiovasc. Ther. 28. 202-215.
Badimon, L., Vilahur, C., 2014. Thrombosis formation on atherosclerotic lesions and plaque rupture. J. Intern. Med. 276, 618-632.
Baldenkov, G.N., Akopov, S.E., Ryong, L.H., et al., 1988. Prostacyclin, thromboxane A2 and calcium antagonists: effects on atherosclerotic characteristics of vascular cells. Biomed. Biochim. Acta. 47, S324-S327.
Banach, M., Rizzo, M., Toth, P.P., et al., 2015. Statin intolerance--an attempt at a unified definition. Position paper from an International Lipid Expert Panel. Expert Opin. Drug Safety 14, 935-955.
Berenson, G.S., Srinivasan, S.R., Bao, W., et al., 1998. Association between multiple cardiovascular risk factors and atherosclerosis in children and young adults. The Bogalusa Heart Study. N. Engl. J. Med. 338, 1650-1656.
Chazov, E.I., Tertov, V.V., Orekhov, A.N., et al., 1986. Atherogenicity of blood serum from patients with coronary heart disease. Lancet 2, 595-598.
Diamond, D.M., Ravnskov, U., 2015. How statistical deception created the appearance that statins are safe and effective in primary and secondary prevention of cardiovascular disease. Expert Rev. Clin. Pharmacol. 8, 201-210.
Fowkes, F.G., Rudan, D., Rudan, I., et al., 2013. Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010: a systematic review and analysis. Lancet 382, 1329-1340.
Giessler, C., Fahr, A., Tertov, V.V., et al., 1987. Trapidil derivatives as potential anti-atherosclerotic drugs. Arzneimittelforschung 37-1, 538-541.
Goldstein, J.L., Brown, M.S., 1987. Regulation of low-density lipoprotein receptors: implications for pathogenesis and therapy of hypercholesterolemia and atherosclerosis. Circulation 76, 504-507.
Gorchakova, T., Suprun, L, Sobenin, I., et al., 2005. The suppression of the inflammatory cytokines expression by natural substances. Atherosclerosis 6, 66-67.
Gorchakova, T.V., Suprun, I.V., Sobenin, LA., et al., 2007a. Use of natural products in anticytokine therapy. Bull. Exp. Biol. Med. 143, 316-319.
Gorchakova, T.V., Sobenin, LA., Orekhov, A.N., 2007b. The reduction of proinflammatory cytokine expression by natural components: a new approach to the prevention and treatment of atherosclerosis at the cellular level, J. Clin. Lipidol. 1, 492.
Gorchakova, T.V., Suprun, I.V., Sobenin, LA., et al., 2007c. Combined antiinflammatory and anti-atherogenic activity of natural drug Inflaminat--a perspective for long-term atherosclerosis prevention and treatment. Atheroscler. Suppl. 8, 224.
Gorchakova, T., Myasoedova, V., Sobenin, L, et al.. 2009. Atherosclerosis prevention with the anti-inflammatory dietary supplement Inflaminat. Atheroscler. Suppl. 10. 387.
Gratchev, A., Sobenin, I., Orekhov, A., et al., 2012. Monocytes as a diagnostic marker of cardiovascular diseases. Immunobiology 217, 476-482.
Heinroth-Hoffmann, L, Kruger, J., Tertov, V.V., et al., 1990. Influence of trapidil and trapidil derivatives on the content of cyclic nucleotides in human intima cells cultured from atherosclerotic plaques. Drug Dev. Res. 19, 321-327.
Hopkins, A.L., Lamm, M.G., Funk, J.L., et al., 2013. Hibiscus sabdariffa L in the treatment of hypertension and hyperlipidemia: a comprehensive review of animal and human studies. Fitoterapia 85, 84-94.
Jaakkola, O., Solakivi, T., Tertov, V.V., et al., 1993. Characteristics of low-density lipoprotein subfractions from patients with coronary artery disease. Coron. Artery Dis. 4, 379-385.
Konstantinov, I.E., Mejevoi, N., Anichkov, N.M., 2006. Nikolai N. Anichkov and his theory of atherosclerosis. Tex Heart Inst. J. 33, 417-423.
Korennaya, V.V., Myasoedova, V.A., Nikitina, N.A., et al., 2006. Bioflavonoid-rich botanicals reduce blood serum atherogenicity in perimenopausal women. Atheroscler. Suppl. 7, 444.
Koscielny, J., Klussendorf, D., Latza, R., et al., 1999. The antiatherosclerotic effect of Allium sativum. Atherosclerosis 144. 237-249.
Kudryashov, S.A., Tertov, V.V., Orekhov, A.N., et al., 1984. Regression of atherosclerotic manifestations in primary culture of human aortic cells: effects of prostaglandins. Biomed. Biochim. Acta 43, S284-S286.
Li, H.R., Tertov, V.V., Vasil'ev, A.V., et al., 1989. Anti-atherogenic and antiatherosclerotic effects of mushroom extracts revealed in human aortic intima cell culture. Drug Dev. Res. 17, 109-117.
Libby, P., 2012. Inflammation in atherosclerosis. Arterioscler. Thromb. Vase. Biol. 32, 2045-2051.
McGill Jr., H.C., Herderick, E.E., McMahan, CA, et al., 2002. Atherosclerosis in youth. Minerva Pediatr. 54, 437-447.
Mensink, R.P., Zock, P.L., Kester, A.D., Katan, M.B., 2003. Effects of dietary fatty acids and carbohydrates on the ratio of serum total to HDL cholesterol and on serum lipids and apolipoproteins: a meta-analysis of 60 controlled trials. Am. J. Clin. Nutr 77, 1146-1155.
Myasoedova, V.A., Sobenin, I.A., 2008. Background, rationale and design of clinical study of the effect of isoflavonoid-rich botanicals on natural history of atherosclerosis in women. Atheroscler. Suppl. 9, 171.
Nikitina, N.A., Sobenin, 1A, Myasoedova, V.A., et al., 2006. Antiatherogenic effect of grape flavonoids in an ex vivo model. Bull. Exp. Biol. Med. 141, 712-715.
Orekhov, A.N., Tertov, V.V., Smirnov, V.N., 1983. Prostacyclin analogues as antiatherosclerotic drugs. Lancet 2, 521.
Orekhov, A.N., Andreeva, E.R., Krushinsky, A.V., et al., 1984a. Primaiy cultures of enzyme-isolated cells from normal and atherosclerotic human aorta. Med. Biol. 62, 255-259.
Orekhov, A.N., Misharin, A.Yu., Tertov, V.V., et al., 1984b. Artificial HDL as an antiatherosclerotic drug. Lancet 2, 1149-1150.
Orekhov, A.N., Tertov, V.V., Novikov, I.D., et al., 1985. Lipids in cells of atherosclerotic and uninvolved human aorta. I. Lipid composition of aortic tissue and enzymeisolated and cultured cells. Exp. Mol. Pathol. 42,117-137.
Orekhov, A.N., Tertov, V.V., Kudryashov, S.A., et al., 1986a. Primaiy culture of human aortic intima cells as a model for testing antiatherosclerotic drugs. Effects of cyclic AMP, prostaglandins, calcium antagonists, antioxidants, and lipid-lowering agents. Atherosclerosis 60,101-110.
Orekhov, A.N., Krushinsky, A.V., Andreeva, E.R., et al., 1986b. Adult human aortic cells in primary culture: heterogeneity in shape. Heart Vessels 2, 193-201.
Orekhov, A.N., Tertov, V.V., Mazurov, A.V., et al., 1986c. Regression" of atherosclerosis in cell culture: effects of stable prostacyclin analogues. Drug Dev. Res. 9, 189201.
Orekhov, A.N., Tertov, V.V., Mukhin, D.N., et al., 1987a. Association of low-density lipoprotein with particulate connective tissue matrix components enhances cholesterol accumulation in cultured subendothelial cells of human aorta. Biochim. Biophys. Acta 928, 251-258.
Orekhov, A.N., Tertov, V.V., Khashimov, KA, et al., 1987b. Evidence of antiatherosclerotic action of verapamil from direct effects on arterial cells. Am. J. Cardiol. 59, 495-496.
Orekhov, A.N., Baldenkov, G.N., Tertov, V.V., et al., 1988a. Cardiovascular drugs and atherosclerosis: effects of calcium antagonists, beta-blockers, and nitrates on atherosclerotic characteristics of human aortic cells. J. Cardiovasc. Pharmacol. 12 (Suppl 6), S66-S68.
Orekhov, A.N., Ruda, M.Ya., Baldenkov, G.N., et a!., 1988b. Atherogenic effects of beta blockers on cells cultured from normal and atherosclerotic aorta. Am. J. Cardiol. 61, 1116-1117.
Orekhov, A.N., 1990. In vitro models of antiatherosclerotic effects of cardiovascular drugs. Am. J. Cardiol. 66, 231-281.
Orekhov, A.N., Tertov, V.V., Kudryashov, SA, et al., 1990a. Trigger-like stimulation of cholesterol accumulation and DNA and extracellular matrix synthesis induced by atherogenic serum or low density lipoprotein in cultured cells. Circ. Res. 66, 311-320.
Orekhov, A.N., Baldenkov, G.N., Tertov, V.V., et al., 1990b. Antiatherosclerotic effects of calcium antagonists. Study in human aortic cell culture. Herz 15,139-145.
Orekhov, A.N., Tertov, V.V., Lyakishev, A.A., et al., 1991. Use of cultured atherosclerotic cells for investigation of antiatherosclerotic effects of anipamil and other calcium antagonists. J. Hum. Hypertens. 5, 425-430.
Orekhov, A.N., 1992. Direct anti-atherosclerotic and anti-atherogenic effects of garlic. Eur. J. Clin. Res. 3A, 1-12.
Orekhov, A.N., Tertov, V.V., 1997. In vitro effect of garlic powder extract on lipid content in normal and atherosclerotic human aortic cells. Lipids 32, 1055-1060.
Orekhov, A.N., 2013. Direct anti-atherosclerotic therapy; development of natural anti-atherosclerotic drugs preventing cellular cholesterol retention. Curr. Pharm. Des. 19, 5909-5928.
Orekhov, A.N., Sobenin, IA, Korneev, N.V., et al., 2013. Anti-atherosclerotic therapy based on botanicals. Recent Pat. Cardiovasc. Drug Discov. 8, 56-66.
Patti, A.M., Al-Rasadi, K., Katsiki, N., et al., 2015a. Effect of natural supplement containing curcuma longa, guggul, and chlorogenic acid in patients with metabolic syndrome. Angiology 66, 856-861.
Patti, A.M., Katsiki, N., Nicolic, D., et al., 2015b. Nutraceuticals in lipid-lowering treatment: a narrative review on the role of chitosan. Angiology 66, 416-421.
Rai, A.K., Debetto, P., Sala, F.D., 2013. Molecular regulation of cholesterol metabolism: HDL-based intervention through drugs and diet. Indian J. Exp. Biol. 51, 885-894.
Ried, K., Toben, C., Fakler, P., 2013. Effect of garlic on serum lipids: an updated meta- analysis. Nutr. Rev. 71, 282-299.
Rizzo, M., Giglio, R.V., Nicolic, D., et al., 2014. Effects of chitosan on plasma lipids and lipoporoteins: a 4-month prospective pilot study. Angiology 65, 538542.
Salonen, R., Nyyssonen, K., Porkkala, E., et al., 1995. Atherosclerosis Prevention Study (KAPS). A population-based primary preventive trial of the effect of LDL lowering on atherosclerotic progression in carotid and femoral arteries. Circulation 92, 1758-1764.
Sazonova, M., Budnikov, E., Khasanova, Z., et al., 2009. Studies of the human aortic intima by a direct quantitative assay of mutant alleles in the mitochondrial genome. Atherosclerosis 204, 184-190.
Schfinfelder, M., 1969. Ortologie und patologie der Langhans-zellen der aortenintima des menschen. Pathol. Microbiol. (Basel) 33, 129-145.
Simmons, A., Steffen, K., Sanders, S., 2012. Medical therapy for peripheral arterial disease. Curr. Opin. Cardiol. 27, 592-597.
Sobenin, LA., Maksumova, M.A., Slavina, E.S., et al., 1994. Sulfonylureas induce cholesterol accumulation in cultured human intimal cells and macrophages. Atherosclerosis 105, 159-163.
Sobenin, I.A., Tertov, V.V., Orekhov, A.N., 1998. Optimization of the assay for sialic acid determination in low density lipoprotein. J. Lipid Res. 39, 2293-2299.
Sobenin, 1A, Nikitina, N.A., Myasoedova, V.A., et al, 2003. Antiatherogenic properties of isoflavones from phytoestrogen-rich botanicals. Atheroscler. Suppl. 4, 339.
Sobenin, IA, Myasoedova, V.A., Orekhov, A.N., 2007. Antiatherogenic action of isoflavonoid-rich botanicals: an implementation for atherosclerosis prevention in postmenopausal women. J. Clin. Lipidol. 7, 491.
Sobenin, IA, Nedosugova, L.V, Filatova, L.V, et al, 2008a. Metabolic effects of time- released garlic powder tablets in type 2 diabetes mellitus: the results of double-blinded placebo-controlled study. Acta Diabetol. 45, 1-6.
Sobenin, I., Myasoedova, V., Orekhov, A., 2008b. Atherosclerosis prevention in postmenopausal women with the isoflavonoid-rich dietary supplement Karinat. J. Clin. Lipidol. 2, S26-S27.
Sobenin, I.A., Andrianova, I.V., Fomchenkov, I.V., et al, 2009. Time-released garlic powder tablets lower systolic and diastolic blood pressure in men with mild and moderate arterial hypertension. Hypertens. Res. 32, 433-437.
Sobenin, I.A., Pryanishnikov, V.V., Kunnova, LM., et al, 2010. The effects of time- released garlic powder tablets on multifunctional cardiovascular risk in patients with coronary artery disease. Lipids Health Dis. 9, 119.
Sobenin, I.A., Chistiakov, D.A., Bobryshev, Y.V., et al, 2013. Blood atherogenicity as a target for anti-atherosclerotic therapy. Curr. Pharm. Des. 19, 5954-5962.
Sobenin, I.A., Korneev, N.V., Romanov, I.V., et al, 2014a. The effects of garlic powder tablets in subclinical carotid atherosclerosis. Exp. Clin. Cardiol. 20, 629-638.
Sobenin, I.A., Salonen, J.T., Zhelankin, A.V., et al, 2014b. Low density lipoprotein- containing circulating immune complexes: role in atherosclerosis and diagnostic value. Biomed. Res. Int. 2014, 205697. doi:10.1155/2014/205697.
Stein, E.A., Raal, F.J., 2014. New therapies for reducing low-density lipoprotein cholesterol. Endocrinol. Metab. Clin. North Am. 43, 1007-1033.
Tertov, V.V., Orekhov, A.N., Repin, V.S., et al, 1982. Dibutyryl cyclic AMP decrease proliferative activity and the cholesteryl ester content in cultured cells of atherosclerotic human aorta. Biochem. Biophys. Res. Commun. 109, 1228-1233.
Tertov, V.V., Khashimov, KhA, Orekhov, A.N., et al, 1985. LDL apheresis and regression of atherosclerosis. Lancet 1,1108-1109.
Tertov, V.V., Orekhov, A.N., Smirnov, V.N., 1986. Effect of cyclic AMP on lipid accumulation and metabolism in human atherosclerotic aortic cells. Atherosclerosis 62, 55-64.
Tertov, V.V., Orekhov, A.N., Grigorian, G.Yu, et al, 1987. Disorders in the system of cyclic nucleotides in atherosclerosis: cyclic AMP and cyclic GMP content and activity of related enzymes in human aorta. Tissue Cell 19, 21-28.
Tertov, V.V., Panosyan, A.G., Akopov, S.E., et al, 1988. The effects of eicozanoids and lipoxygenase inhibitors on the lipid metabolism of aortic cells. Biomed. Biochim. Acta 47. S286-S288.
Tertov, V.V., Sobenin, I.A., Gabbasov, Z.A., et al, 1992a. Multiple-modified desialylated low density lipoproteins that cause intracellular lipid accumulation. Isolation, fractionation and characterization. Lab. Invest. 67, 665-675.
Tertov. V.V., Sobenin, I.A., Gabbasov, Z.A., et al, 1992b. Three types of naturally occurring modified lipoproteins induce intracellular lipid accumulation in human aortic intimal cells--the role of lipoprotein aggregation. Eur. J. Clin. Chem. Clin. Biochem. 30, 171-178.
Tertov, V.V., Sobenin, I.A, Orekhov, A.N., 1995a. Modified (desiaiylated) low-density lipoprotein measured in serum by lectin-sorbent assay. Clin. Chem. 41, 1018-1021.
Tertov, V.V., Bittolo-Bon, G., Sobenin, I.A., et al, 1995b. Naturally occurring modified low density lipoproteins are similar if not identical: more electronegative and desiaiylated lipoprotein subfractions. Exp. Mol. Pathol. 62, 166-172.
Tertov, V.V., Sobenin, I.A., Orekhov, A.N, 1996. Similarity between naturally occurring modified desiaiylated, electronegative and aortic low density lipoprotein. Free Radic. Res. 25, 313-319.
Tertov, V.V., Kaplun, V.V., Sobenin, I.A., et al., 1998. Low-density lipoprotein modification occurring in human plasma. Possible mechanism of in vivo lipoprotein desialylation as a primary step of atherogenic modification. Atherosclerosis 138, 183-195.
Tuzcu, E.M., Kapadia, S.R., Tutar, E., et al., 2001. High prevalence of coronary atherosclerosis in asymptomatic teenagers and young adults: evidence from intravascular ultrasound. Circulation 103, 2705-2710.
Vanhoutte, P.M., 2009. Endothelial dysfunction: the first step toward coronary atherosclerosis. Circ. J. 73, 595-601.
Wolf, D., Stachon, P., Bode, C, et al., 2014. Inflammatory mechanisms in atherosclerosis. Hamostaseologie 34, 63-71.
Alexander N. Orekhov (a,b,c), Ekaterina A. Ivanova (d), *
(a) Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology, Russian Academy of Sciences, Moscow 125315, Russia
(b) Institute for Atherosclerosis Research, Skolkovo Innovative Center, Moscow 121609, Russia
(c) Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, Moscow 119991, Russia
(d) Department of Development and Regeneration, Katholieke Universiteit, Leuven 3000 Belgium
Abbreviations: CHD, coronary heart disease; clMT, carotid intima-media thickness; HDL, high density lipoprotein; LDL, low density lipoprotein.
* Corresponding author. Tel.: +32 488 46 16 92.
E-mail addresses: firstname.lastname@example.org (A.N. Orekhov), email@example.com (E.A. Ivanova).
Table 1 Substances tested with in vitro cellular model. Substance References Anti-atherosclerotic Cyclic AMP Orekhov et al. 1986a; Tertov et al. 1982; Tertov et al. 1985; Tertov et al. 1986; Tertov et al. 1987 Prostacyclin Orekhov et al. 1986a; Akopov et al. 1988; Baldenkov et al. 1988; Kudryashov et al. 1984; Orekhov et al. 1983; Orekhov et al. 1986c Prostaglandin [E.sub.2] Orekhov et al. 1986a; Kudryashov et al. 1984, Tertov et al. 1988 Artificial HDL * Orekhov et al. 1984b Antioxidants Orekhov et al. 1986a Calcium antagonists Orekhov et al. 1986a; Baldenkov et al. 1988; Orekhov et al. 1987b; Orekhov et al. 1988a; Orekhov et al. 1990a Trapidil and its derivatives Giessler et al. 1987; Heinroth- Hoffmann et al. 1990 Lipoxygenase inhibitors Tertov et al. 1988 Lipostabil Orekhov et al. 1986a Mushroom extracts Li et al. 1989 Pro-atherogenic Beta-blockers Orekhov et al. 1988a; Orekhov et al. 1988b Thromboxane [A.sub.2] Baldenkov et al. 1988; Tertov et al. 1988 Phenothiazine Orekhov et al. 1988a Indifferent Nitrates Orekhov et al. 1988a Cholestyramine Orekhov et al. 1988a Sulfonylureas Sobenin et al. 1994 * HDL, high density lipoprotein. Table 2 Integral estimation of anti-atherogenic actions of natural products *. Botanical and its source The mean efficiency Maximum of atherogenic effect, % reduction, % Spirulina platensis powder 50.7% 61 Onion (Allium cepa) bulb powder 21.4% 28 Beet (Beta vulgaris) juice powder 30.7% 40 Wheat (Triticum vulgaris) 70.0% 100 seedlings powder Licorice (Glycyrrhiza glabra) 54.6% 32 root powder Salsola collina leaf powder 10.9% 28 Garlic (Allium sativum) bulbs 76.6% 100 powder Pine (Pinus sylvestris) needles 52.1% 62 extract * The integrated effect was calculated as a mean reduction in serum atherogenicity for 6 h after a single oral dose
Please note: Some tables or figures were omitted from this article.
|Printer friendly Cite/link Email Feedback|
|Author:||Orekhov, Alexander N.; Ivanova, Ekaterina A.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Oct 15, 2016|
|Previous Article:||Cardiovascular aspects of geriatric medicines in traditional Persian medicine; a review of phytochemistry and pharmacology.|
|Next Article:||Anti-cytokine therapy for prevention of atherosclerosis.|