Current Pharmaceutical Design - Volume 19, Issue 33, 2013

Atherosclerosis remains one of the main causes of cardiovascular disease, which is the leading cause of death worldwide. It is now accepted that atherosclerosis is an inflammatory, dynamic and complex disease involving multiple cell types, and many antiinflammatory strategies have recently emerged as potential therapeutic approaches for atherosclerotic disease. In this review, we discuss the most recent progress in the development of anti-inflammatory strategies. We highlight the beneficial effects of potent antiinflammatory drugs, including recently developed biologics, and we describe diverse emerging approaches that target inflammatory processes involved in atherosclerosis including tumor necrosis factor antagonists, anti-interleukins, viral-derived serpins, P-selectin inhibition and leukotriene synthesis inhibition.

Cardiovascular diseases (CVDs), largely due to atherosclerosis, are the major causes of death in today’s world. Atherosclerosis is a chronic inflammatory condition initiated by retention and accumulation of cholesterol-containing lipoproteins, in particular lowdensity lipoprotein (LDL), in the artery wall. This initiates pathological responses of immune cells that lead to atherosclerotic plaque formation. T cells are present during all stages of the disease, and play an essential role in the initiation and progression of plaques. Whereas most T effector cell responses have been suggested to aggravate atherosclerosis, regulatory T cells (Tregs) have been shown to limit inflammation and inhibit the formation of lesions. In addition to their effects on the local pathological process, T cells and their released mediators modulate systemic lipid metabolism and can increase risk of CVDs. Such knowledge on the pathological and protective function of these cells has led to significant advances in the field. This review examines experimental and pre-clinical studies approaching the manipulation of cellular immunity in atherosclerosis. Modulation of T cells responses by vaccination, antibody therapies, dendritic cell based-therapies, and using amino acid-derived metabolites have shown benefits against atherosclerotic plaque progression in animal models. The clinical benefit of T cell-based therapies in humans still requires further investigation.

Hidden low grade inflammation underlines various cardio-metabolic diseases. This type of inflammation is triggered by abnormal reaction to unwanted self products or deregulation of cellular response to cytokines. In the case of atherosclerosis hidden inflammation is induced by modified lipoproteins and develops under control of different cytokines including IL-4 and TGFβ. The key innate immune cells reacting on these factors are monocytes and macrophages. It is well established that monocytes represent a heterogeneous cell population that easily reacts to pathologic changes in the organism. The best studied marker which expression on monocytes is changed in inflammatory conditions is CD16. Although this marker was shown to be associated with various pathologic conditions its specificity is highly questionable. There is an urgent need of identification of new monocyte-expressed biomarkers that may help not only to detect hidden inflammation but also to determine its type. Our analysis of type 2 activation of human monocytes resulted in identification of 3 novel biomarkers for hidden type 2 inflammation. These include stabilin-1, FOXQ1 and IL17RB. These markers are expressed by monocytes/macrophages under stimulation with IL-4 or its combination with TGF β – 2 cytokines playing important role in atherogenesis. Stabilin-1 was demonstrated on the monocytes in patients with hyperholisterolemia and in macrophages within atherosclerotic lesions. Association of FoxQ1 and IL17RB with atherosclerosis can be deduced from published data but requires experimental confirmation. Functions of all three proteins suggest that they are not only diagnostic markers, but also involved in atherogenesis and can be used as targets for novel therapeutic approaches.

Circulating low-density lipoprotein (LDL) that enters the blood vessel wall is the main source of cholesterol that accumulates within atherosclerotic plaques. Much of the deposited cholesterol accumulates within plaque macrophages converting these macrophages into cholesterol-rich foamy looking cells. Cholesterol accumulation in macrophages contributes to cholesterol retention within the vessel wall, and promotes vessel wall inflammation and thrombogenicity. Thus, how macrophages accumulate cholesterol and become foam cells has been the subject of intense investigation. It is generally believed that macrophages accumulate cholesterol only through scavenger receptor-mediated uptake of modified LDL. However, an alternative mechanism for macrophage foam cell formation that does not depend on LDL modification or macrophage receptors has been elucidated. By this alternative mechanism, macrophages show receptorindependent uptake of unmodified native LDL that is mediated by fluid-phase pinocytosis. In receptor-independent, fluid-phase pinocytosis, macrophages take up LDL as part of the fluid that they ingest during micropinocytosis within small vesicles called micropinosomes, and by macropinocytosis within larger vacuoles called macropinosomes. This produces cholesterol accumulation in macrophages to levels characteristic of macrophage foam cells in atherosclerotic plaques. Fluid-phase pinocytosis of LDL is a plausible mechanism that can explain how macrophages accumulate cholesterol and become disease-causing foam cells. Fluid-phase pinocytosis of LDL is a relevant pathway to target for modulating macrophage cholesterol accumulation in atherosclerosis. Recent studies show that phosphoinositide 3-kinase (PI3K), liver X receptors (LXRs), the macrophage colony-stimulating factor (M-CSF) receptor, and protein kinase C (PKC) mediate macrophage macropinocytosis of LDL, and thus, these may be relevant targets to inhibit macrophage cholesterol accumulation in atherosclerosis.

The chronic inflammatory nature of atherosclerosis is nowadays widely accepted. Dendritic cells (DCs) are likely to play a crucial role in directing innate and adaptive immunity against altered (self-)antigens, such as oxidized low density lipoproteins (oxLDL). DCs are found in early lesions and their numbers become even higher when the lesion progresses. DCs are most abundant in areas of neovascularization where they are often found near T cells. All stages from precursors to fully mature DCs are present in human plaques. Treatment of atherosclerosis is currently based on reducing risk factors, e.g. by use of statins and beta-blockers. Some of these pharmacological agents also show anti-inflammatory properties and consequently can affect DC function. Yet, many patients remain at risk for acute coronary events, and new therapies to treat atherosclerosis are needed. One therapeutic strategy is based on isolation of patient’s DCs that are then pulsed with appropriate antigen(s) ex vivo, e.g. (immunogenic components of) oxLDL or total extract of atherosclerotic plaque tissue, and returned to the blood stream. Other approaches to ensure immune protection include generation of tolerogenic DCs, or using DCs to deplete detrimental Th1 or Th17 cells. However, the future lies in direct targeting of DCs by manipulating functions of different DC subsets. Therefore, it would be useful to isolate plaque-resident DCs to be able to identify unique antigen(s) on their surface. The challenge is to selectively identify regulatory molecules and novel therapies to inhibit DC migration and function during atherogenesis, without affecting normal DC function under physiological conditions.

It is recognized that the development of atherosclerosis involves many elements of an inflammatory process, involving components of both the innate and adaptive immune systems. The presence and roles of macrophages and T-cells in atherogenesis are wellestablished. More recently dendritic cells have been identified in the vasculature and in atherosclerotic lesions. This review summarises our current understanding of the roles of dendritic cells in the development and regression of atherosclerosis.

Naoxintong (NXT), a Chinese Materia Medica standardized product, extracted from 16 various kinds of Chinese traditional herbal medicines including Salvia miltiorrhiza, Angelica sinennsis, Astragali Radix, is clinically effective in treating atherosclerosisrelated diseases. Here, we tested the hypothesis that the anti-atherosclerosis effects of NXT might be mediated by suppressing maturation of dendritic cells (DCs) in a mice model of atherosclerosis. LDLR-/- mice fed a high-fat diet were treated with placebo, NXT (0.7g/kg/d, oral diet) or simvastatin (100mg/kg/d, oral diet) for 8 weeks, respectively. NXT treatment significantly reduced plasma triglyceride (112±18 mg/dl vs. 192±68 mg/dl, P<0.05) and total cholesterol (944±158 mg/dl vs. 1387±208 mg/dl, P<0.05) compared to placebo treatment. Vascular lesions were significantly smaller and macrophage content and amount of DCs in plaques were significantly less in NXT and simvastatin groups than in placebo group (all P<0.05). In addition, expressions of splenic DC membrane molecules (CD40, CD86 and CD80) and the plasma level of IL-12p70 were significantly lower in NXT and simvastatin groups than in placebo group. In conclusion, NXT protects against atherosclerosis through lipid-lowering and inhibiting DCs maturation in this mice model of atherosclerosis.

Atherosclerosis is the leading cause of death in the Western World and has been for decades a field of intense research. Yet, while there is a rich and diverse literature describing in detail the players and mechanisms involved in this complex disease in cell and animal models, we remain today with virtually no reliable markers for early diagnosis and targeted treatments options. This review is centered upon the latter. We summarize the latest studies focused on detecting endothelial dysfunction during the early stages of atherosclerosis, when the disease is asymptomatic and describe strategies recently proposed to image and target advanced plaque.

Atherosclerosis is a chronic disease with a significant inflammatory component. Recent studies indicate a role of extracellular cyclophilins as contributors to endothelial inflammation and pathogenesis of atherosclerosis. In this article, we review current literature on pro-inflammatory activities of extracellular cyclophilins and discuss possible approaches to selectively target this novel proinflammatory factor.

The results of numerous clinical trials with statins and other drugs have demonstrated the principal possibility of the prevention and regression of atherosclerosis by pharmacotherapy. This review describes the use of cultured human arterial cells for the mass screening of anti-atherosclerotic substances, the investigation of the mechanisms responsible for their atherosclerosis-related effects, and the optimization of anti-atherosclerotic and anti-atherogenic drug and dietary therapies. Natural products can be considered promising drugs for anti-atherosclerotic therapy. Our basic studies have shown that cellular lipidosis is the principal event in the genesis of atherosclerotic lesions. Using cellular models and natural products, we have developed an approach to prevent lipid accumulation in arterial cells. Based on our knowledge of atherosclerosis, we developed drugs that possess direct anti-atherosclerotic activity. Two-year treatment with allicor (garlic powder) has a direct anti-atherosclerotic effect on carotid atherosclerosis in asymptomatic men. Inflaminat (calendula, elder, and violet), which possesses anti-cytokine activity, has been shown to cause the regression of carotid atherosclerosis following the treatment of asymptomatic men for one year. The phytoestrogen-rich drug karinat (garlic powder, extract of grape seeds, green tea leaves, hop cones, β-carotene, β-tocopherol, and ascorbic acid) prevents the development of carotid atherosclerosis in postmenopausal women. Thus, our basic findings were successfully translated into clinical practice. Because of this translation, a novel approach to antiatherosclerotic therapy was developed. Our clinical trial confirmed the efficacy of both the novel approach and the novel drugs.

Advances in risk prediction are necessary to stem the tide of the increasing incidence of global cardiovascular disease. Newly discovered biomarkers are needed for primary and secondary prevention and will undoubtedly play a major role in drug development programs and monitoring of treatment efficacy. The combination of improved -omics technologies and the investigation of relatively untapped sources of biomarkers will likely result in risk algorithms that will add value on top of the traditional risk factors. New sources of biomarkers are being explored with encouraging results. These include microvesicles, microRNAs, circulating cells and atherosclerotic plaques. We will review these sources for their potential for new biomarkers. Furthermore, the major impact of advances in genetics on risk prediction and biomarker development programs will be discussed.

Cardiovascular diseases are the leading causes of morbidity and mortality in many industrialized societies. Atherosclerosis is the major risk factor for the development of cardiovascular disease based on arterial endothelial dysfunction caused by the impairment of endothelial-dependent dilation. Atherosclerosis is a complex vascular disease resulted from the harmful interactions between genetic and environmental factors. There is a growing body of evidence in support of a non-redundant role of mitochondrial factors in the pathogenesis of atherosclerosis. Impaired mitochondrial function and structural and qualitative changes in mitochondrial components such as mitochondrial DNA (mtDNA) may be directly involved in the development of multiple atherogenic mechanisms including advanced oxidative stress, abnormalities in glucose and fat metabolism, and altered energy homeostasis. Recent findings showed that the heteroplasmy level of some somatic mtDNA is associated with coronary atherosclerosis. Although this field should further widely elaborated, heteroplasmic mtDNA mutations could represent a new promising molecular biomarker of genetic susceptibility to atherosclerosis and related pathologic conditions. In this review, we critically consider the contribution of mitochondria-related factors to the pathogenesis of the arterial vascular pathology.

Atherogenicity of serum taken from patients with coronary heart disease (CHD) is the ability to induce cholesterol deposition in cultured cells, such as vascular smooth muscle cells (VSMCs) from human aortic intima or blood-derived monocytes/macrophages. The discovery of this phenomenon evoked the series of studies of serum components responsible for atherogenic effects, especially serum lipoproteins. A fraction of circulating negatively charged low density lipoproteins (LDL) enriched with disialylated LDL was found in the blood CHD patients. This LDL fraction was prone to multiple modifications including desialylation and oxidation, and had advanced immunogenic and atherogenic properties, resulting in cholesterol accumulation in cultured intimal VSMCs and formation of circulating immune complexes in blood. The analysis of this proatherogenic LDL helped to understand the mechanisms of subclinical stages of atherogenesis, and pointed out the presence of individual susceptibility to atherosclerosis in humans. The practical application of serum atherogenicity phenomenon is the development of cell-based models for the assessment of cardiovascular drugs. The suitability of these models in pharmacological research was supported by the results of atherosclerosis regression studies, evaluation of antiatherogenic properties of various classes of cardiovascular drugs, and elucidating the ways for further development of drugs for direct anti-atherosclerotic therapy.

With the rising global incidence and burden of atherosclerotic disease, there is a need to identify new molecular targets that may not only serve as biomarkers of clinical risk, but also therapeutic targets. The efficiency of drug-development processes needs to improve in order to allocate finite resources towards the evaluation of the most promising compounds. Atherosclerosis imaging provides key information regarding mechanistic efficacy of novel compounds, as an intermediary endpoint, prior to the consideration of embarking upon a large-scale, multi-center randomized placebo-controlled trial. Coronary intravascular ultrasonography and B-mode sonography of carotid intima-media thickness will continue to play an important role in evaluating the efficacy of novel anti-atherosclerotic compounds. Functional, molecular imaging of atherosclerotic plaque is emerging as an exciting experimental tool, allowing for an even greater understanding of processes that drive the progression and instability of atherosclerotic disease. However, given the complexity and heterogeneity of biological systems, a global, non-targeted, systems biological approach to biomarker discovery is likely to yield the discovery of novel molecular footprints of disease. As such, metabolomic and lipidomic approaches will elucidate metabolite signatures of interest for biomarker evaluation, for clinical risk prediction, as well as safety and toxicity of experimental treatment compounds. Imaging studies will remain important for evaluating novel therapeutic strategies gleaned from omics-based discoveries, as well acting as a triage to further undertake large-scale clinical evaluation of these promising therapeutic strategies. These approaches will provide an opportunity to better understand atherosclerosis, its complications and effective treatments across multiple ethnic populations worldwide.

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by a deficiency of the enzyme α-galactosidase A. FD causes glycolipids, such as globotriaosylceramide (Gb3), to accumulate in the vascular endothelium of several organs (Fig. 2), including the skin, kidneys, nervous system, and heart, thereby triggering inflammation and fibrosis. These processes generally result in organ dysfunction, which is usually the first clinical evidence of FD. Patients with classic FD have various symptoms, eg, acroparesthesias, hypohidrosis, angiokeratomas, corneal opacities, cerebrovascular lesions, cardiac disorders, andrenal dysfunction.However, evolving knowledge about the natural course of disease suggests that it is more appropriate to describe FD as a disease with a wide spectrum of heterogeneously progressive clinical phenotypes. Indeed, most female heterozygotes develop symptoms due to yet undetermined mechanisms and a high percentage of females develops vital organ involvement including the kidneys, heart and/or brain about a decade later than males. Renal failure is a serious complication of this disease. Fabry nephropathy lesions are present and progress in childhood while the disease commonly remains silent by routine clinical measures. Early and timely diagnosis of Fabry nephropathy is crucial since late initiation of enzyme replacement therapy may not halt progressive renal dysfunction. This may be challenging due to difficulties in diagnosis of Fabry disease in children and absence of a sensitive non-invasive biomarker of early Fabry nephropathy. Accurate measurement of glomerular filtration rate and regular assessment for proteinuria and microalbuminuria are useful, though not sensitive enough to detect early lesions in the kidney. The principal clinical manifestations in Fabry disease consist of artery associated complications (such as cerebral disease and nephropathy), but the pathophysiology of this specific vasculopathy is unclear. Several studies indicate that the specific vascular lesions that are present in Fabry disease occur as a result of vascular dysfunction with major components being endothelial dysfunction, alterations in cerebral perfusion and a pro-thrombotic phenotype. Fabry cardiac involvement has several clinical manifestations (Table 10): concentric left ventricular hypertrophy without left ventricular dilation and severe loss of left ventricular systolic function, mitral and aortic valvulopathy, disorders of the atrioventricular conduction or repolarization, and compromised diastolic function. The neurological manifestations of Fabry disease include both peripheral nervous system and CNS involvement, with globotriaosylceramide accumulation found in Schwann cells and dorsal root ganglia together with deposits in CNS neurones. The main involvement of the CNS is attributable to cerebrovasculopathy, with an increased incidence of stroke. The abnormal neuronal accumulation of glycosphingolipid appears to have little clinical effect on the natural history of Fabry disease, with the possible exception of some reported mild cognitive abnormalities. The pathogenesis of Fabry vasculopathy remains poorly understood, but probably relates, in part, to abnormal functional control of the vessels, secondary to endothelial dysfunction as a consequence of α-galactosidase A deficiency. The diagnosis of Fabry disease is made in hemizygous males after the detection of the presence of angiokeratomas (Fig. 19 A, B), irregularities in sweating, edema, scant body hair, painful sensations, and of cardiovascular, gastrointestinal, renal, ophthalmologic, phlebologic, and respiratory involvement. A deficiency of alpha-gal A in serum, leukocytes, tears, tissue specimens, or cultured skin fibroblasts further supports the diagnosis in male patients. Since heterozygous women show angiokeratomas in only about 30% of cases and may have alpha-gal A levels within normal range, genetic analysis is recommended. The resultant storage of undegraded glycolipids leads to the progressive development of potentially life-threatening manifestations affecting multiple organ systems in the body. The Mainz Severity Score Index (MSSI) (Table 12), a scoring system for patients with Fabry disease has been proven to be representative in patients with 'classic' Fabry disease and may be useful for monitoring clinical improvement in patients receiving enzyme replacement therapy. The MSSI of patients with AFD was significantly higher than that of patients with other severe debilitating diseases.

Fabry disease (FD, OMIM 301500) is a rare X-linked lysosomal storage disorder of the glycosphigolipid metabolism caused by total or partial deficiency of the lysosomal enzyme alpha-galactosidase A (α-gal A). Progressive intralysosomal accumulation of neutral glycosphingolipids in a variety of cell types triggers a cascade of pathophysiological events including cellular death, compromised energy metabolism, small vessel injury, K(Ca)3.1 channel dysfunction in endothelial cells, oxidative stress, impaired autophagosome maturation, tissue ischemia and, importantly, development of irreversible cardiac and renal tissue fibrosis, leading to major multisystemic manifestations. Cardiovascular complications of the disease are very frequent and contribute substantially to disease-related morbidity and mortality in men. Cardiovascular involvement is the leading cause of premature death in heterozygous female patients with FD. Left ventricular hypertrophy is the most prominent cardiac manifestation followed by conduction system disease, valve dysfunction, arrhythmias, vessel disease and coronary microvascular dysfunction. The diagnosis of subclinical forms of the disease, before the development of cardiac hypertrophy, using newer techniques (tissue doppler imaging, strain rate and cardiac magnetic resonance) is crucial to the early initation of the treatment. Greatest benefit of the enzyme replacement treatment is achieved when started at an early stage of the disease before extensive fibrosis or other irreversible tissue damage takes place. Fabry disease should be included in the differential diagnosis algorithm of idiopathic hypertrophy. Determination of Alpha-Gal A activity on plasma and peripheral leukocytes in males and genetic testing in females are the diagnostic gold-standards.

Fabry disease is a multisystemic X-linked lysosomal storage disorder, caused by the partial or complete deficiency of alphagalactosidase A activity. The storage of glycosphingolipids in the vascular endothelium and in various tissues can lead to a broad spectrum of clinical manifestations. Renal failure, cardiovascular disease, and strokes are the main causes of morbidity and mortality. Gastrointestinal symptoms, although common, are often under-reported in the literature. This review covers the gastroenterological aspects of Fabry disease.

Characteristic clinical manifestations of AFD such as acroparesthesias, angiokeratoma, corneal opacity, hypo/ and anhidrosis, gastrointestinal symptoms, renal and cardiac dysfunctions can occur in male and female patients, although heterozygous females with AFD usually seem to be less severely affected. The most prominent CNS manifestations consist of cerebrovascular events such as transient ischaemic attacks (TIAs) and (recurrent) strokes. For the most part, CNS complications in AFD have been attributed to cerebral vasculopathy, including anatomical abnormalities. The natural history of Fabry patients includes transitory cerebral ischaemia and strokes, even in very young persons of both genders. The mechanism is partly due to vascular endothelial accumulation of Gb-3. White matter lesions (WML) on occur MRI. Both males and females can be safely treated with enzyme replacement; and thus screening for Fabry disease of young stroke populations should be considered. There are, however, no hard data of treatment effect on mortality and morbidity. Stroke in Anderson-Fabry disease study of 721 patients with cryptogenic stroke, aged 18–55 years, showed a high prevalence of Fabry disease in this group: 5% (21/432) of men and 3% (7/289) of women. Combining results of both sexes showed that 4% of young patients with stroke of previously unknown cause had Fabry disease, corresponding to about 1-2% of the general population of young stroke patients. Cerebral micro- and macro-vasculopathy have been described in Fabry disease. Neuronal globotriaosylceramide accumulation in selective cortical and brain stem areas including the hippocampus has been reported by autopsy studies in FD, but clinical surrogates as well as the clinical relevance of these findings have not been investigated so far. Another Neurologic hallmark of Fabry disease (FD) includes small fiber neuropathy as well as cerebral micro- and macroangiopathy with premature stroke. Cranial MRI shows progressive white matter lesions (WML) at an early age, increased signal intensity in the pulvinar, and tortuosity and dilatation of the larger vessels. Conventional MRI shows a progressive load of white matter lesions (WMLs) due to cerebral vasculopathy in the course of FD. Another study has been conducted to quantify brain structural changes in clinically affected male and female patients with FD. The peripheral neuropathy in Fabry disease manifests as neuropathic pain, reduced cold and warm sensation and possibly gastrointestinal disturbances. Patients with Fabry disease begin having pain towards the end of the first decade of life or during puberty. Children as young as 6 years of age have complained of pain often associated with febrile illnesses with reduced heat and exercise tolerance. The patients describe the pain as burning that is often associated with deep ache or paresthesiae. Some patients also have joint pain. A high proportion of patients with Fabry disease is at increased risk of developing neuropsychiatric symptoms, such as depression and neuropsychological deficits. Due to both somatic and psychological impairment, health-related quality of life (QoL) is considerably reduced in patients with Fabry disease. Targeted screening for Fabry disease among young individuals with stroke seems to disclose unrecognized cases and may therefore very well be recommended as routine in the future. Furthermore, ischemic stroke is related to inflammation and arterial stiffness and no study had addressed this relationship in patients with AF disease and cerebrovascular disease, so this topic could represent a possible future research line.